development of artificial insemination technique in pig ... · to be developed in artificial...

AGri-Bioscience Monographs, Vol. 6, No. 2, pp. 59–83 (2016) www.terrapub.co.jp/onlinemonographs/agbm/

Received on August 28, 2014Accepted on December 15, 2014Online published on

December 13, 2016Keywords• pig• reproductive technology• ovulation• sperm• frozen semen

© 2016 TERRAPUB, Tokyo. All rights reserved.doi:10.5047/agbm.2016.00602.0059

pean countries, more than 70% of pig farms carry outartificial insemination using liquid storage semen, andthis rate reaches more than 98% in the Netherlands(Feitsma 2009). However, the diffusion of this tech-nique has been still less than half in other countriesincluding Japan. The improvements of both insemina-tion technique and diluent extender of boar semen

AbstractReproductive technology in the animal production field not only improves the reproduc-tive performance of swine but also reduces the production costs. However, the diffusionof this technique has been limited in most countries including Japan due to the reproduc-tive characteristics of pig, such as multiple ovulations in female and large volume ofsemen in male. In females, the key mechanisms of oocyte maturation in pig are not dif-ferent as compared with that of the rodent model. However, the timing and regulation ofgene expression and enzyme activity are characterized in pig, which results in the longerduration of oocyte maturation and ovulation. In semen, seminal plasma has dual roles insperm quality and successful implantation. The contaminating of bacteria in semen in-creases endotoxin activity and reduces the sperm quality. On the other hand, the seminalplasma contains factors to regulate immune activity to reduce the number of leukocytesto make an optimal condition for implantation and to maintain the pregnancy. Based onbasic scientific information, we developed novel techniques for oocyte in vitro matura-tion, freezing and thawing sperm and artificial insemination. These technologies will beexpanded to commercial farms to increase the production-efficiency all over the world.

Development of Artificial Insemination Techniquein Pig Production Based on the Evidence ofPhysiology and Molecular Biology inReproductive Organs

Masayuki Shimada1,2*, Yasuhisa Yamashita3 and Tetsuji Okazaki4

1Laboratory of Reproductive Endocrinology Graduate School of Biosphere Science Hiroshima University 1-4-4, Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8528, Japan2The Research Center for Animal Science Hiroshima University 1-4-4, Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8528, Japan3Faculty of Life and Environmental Sciences Prefectural University of Hiroshima 562 Nanatuka-cho, Shobara 727-0023, Japan4Smaller Livestock and Environment Section Livestock Research Institute Oita Prefectural Agriculture Forestry and Fisheries Research Center Bungo-ono, Oita 879-7111, Japan*e-mail: [email protected]

1. Introduction

Artificial insemination technique is useful in the pigproduction field because this technique not only solvesthe infection problem due to the contact of boar andswine during mating but also improves utilization rateof boar due to the dilution of semen. In western Euro-

60 M. Shimada et al. / AGri-Biosci. Monogr. 6: 59–83, 2016

doi:10.5047/agbm.2016.00602.0059 © 2016 TERRAPUB, Tokyo. All rights reserved.

would increase the reproductive performance of artifi-cial insemination. The low number of pups per deliv-ery by this technique as compared with that by naturalmating and the limited storage period of semen are themain problems to expand artificial insemination in thepig production field all over the world.

The prolificacy that is major reproductive charac-teristics of pig is caused by multiple ovulations. In re-cent improved breeds, more than 20 oocytes are ovu-lated and then fertilized, which provides the increasednumber of pups per delivery. The ovulation processesin each oocyte are induced by lutenizing hormone (LH)surge secreted from the pituitary gland of the brain,but don’t simultaneously proceed (Soede et al. 1995).Additionally, the fertilization ability in each ovulatedoocyte is limited to within a few hours after ovulation(Soede et al. 1995; Almeida et al. 2000). Thus, to clearthe timing of insemination by artificial inseminationis a key factor to lead to maximum reproductive per-formance. The studies to identify how to induce ovu-lation in each follicle and how to maintain fertiliza-tion ability of oocytes after ovulation are required forthe decision of insemination timing.

In cattle production field, artificial insemination us-ing frozen-thawed semen is commonly used all overthe world. Cryopreservation of sperm offers an effec-tive means for long-term storage of important geneticmaterial. This technique solves the problem of trans-porting animals or fresh semen over long distances.However, in the pig production field, cryopreservationof sperm is not available for artificial inseminationsince the conception rate, farrowing rate and litter sizeusing this method spermatozoa have remained at lowlevels (Johnson 1981; Johnson et al. 2000). In the fro-zen-thawed sperm, the motility, membrane integrityand acrosome integrity are injured during freezingprocess (Curry 2000; Chauhan et al. 1994; Royerez etal. 1996; Courtens et al. 1989); it has been speculatedthat intracellular ice crystal formation is the major fac-tor responsible for the freezing damage to spermato-zoa (Johnson et al. 2000; Watson 2000). Thus, the liq-uid storage semen that is kept under 15–18∞C for up toa week is mainly used in artificial insemination in pig.The dramatic improvement of frozen technique is es-sential to expand this technique in pig production.

Because the total number of pups per swine in a yearis related to efficiency management in each pig farm,the methods to know which insemination timing is goodand to improve the quality of frozen-thawed sperm haveto be developed in artificial insemination techniques.In this monograph, we introduced the mechanisms ofovulation and oocyte fertilization ability. Additionally,the fertilization ability of sperm was also mentioned.Finally, we introduced the novel artificial insemina-tion technique based on above basic biology.

2. The basic biological study to know how to in-duce ovulation

The surge of luteinizing hormone (LH) acts on granu-losa cells of preovulatory follicles to induce luteiniza-tion, cumulus cell-oocyte complex (COC) expansion,oocyte maturation and follicle rupture (Richards 2005).During these dramatic changes, LH markedly inducesthe expression of genes (Cyp11a1, Star) regulating pro-gesterone biosynthesis in mouse (Richards 1994), rat(Henderson et al. 1981; Ronen-Fuhrmann et al. 1998),pig (Ainsworth et al. 1980; Conely et al. 1994) andhuman (Duncan et al. 1999). In progesterone receptor(Pgr) knockout (PRKO) mice, follicle rupture is com-pletely suppressed (Lydon et al. 1995; Mulac-Jericevicet al. 2000). Prostaglandin E2 (PGE2) that is derivedfrom arachidonic acid by the rate-limiting enzymeprostaglandin synthase 2 (PTGS2; also known ascyclooxygenase 2, COX-2), is also increased by LHstimulation in mouse (Downs and Longo 1983), rat(Plas-Roser et al. 1985), rabbit (LeMarie et al. 1976),ewe (Murdoch et al. 1986), pig (Evans et al. 1983)and human (Gelety and Chaudhuri 1992). In Ptgs2 KOmice, or the PGE2 receptor (EP2) null mice, ovulationis impaired, and oocytes resume meiosis and reach themetaphase II stage in both in vitro and in vivo(Matsumoto et al. 2001; Segi et al. 2003). Thus, theproduction of progesterone and PGE2 is essential forthe induction of the ovulation process.

In the LH-stimulated granulosa cells, the increase ofcAMP level was markedly detected (Osterman et al.1978; Hamberger et al. 1978, 1979), and then the tar-geted gene expression including Cyp11a1, Star andPtgs2 was induced by PKA-CREB dependent manner(Sayasith et al. 2005; Sher et al. 2007; Manna et al.2009). Although the signaling pathway was transientlyactivated by LH in granulosa cells due to the degrada-tion of LH receptor (Richards et al. 1976; Rao et al.1977), the gene expressions were maintained in thecells during the ovulation process. The results suggestthat other signaling pathway(s) is (are) also involvedin granulosa cell differentiation during ovulation proc-ess. To identify the key signaling pathway(s), severalgroups generated mutant mice models that disruptedthe signaling molecules activated in granulosa cellsduring the ovulation process. From these studies,ERK1/2 pathway is identified as signaling cascade inthe change of the follicular development stage to theovulation stage via the suppression of granulosa cellproliferation, the induction of final differentiation(luteinization) of granulosa cells and cumulus cell ex-pansion (Fan et al. 2009).

ERK1/2 is activated by the EGFR-RAS-cRAF-MEK1 pathway in both garanulosa cells and cumuluscells after LH surge (Fan et al. 2009; Richards andPangas 2010). When COCs were cultured with the

M. Shimada et al. / AGri-Biosci. Monogr. 6: 59–83, 2016 61


EGFR tyrosine kinase inhibitor (AG14789) or MEK1inhibitor (PD98059 or U0126), the phosphorylation ofERK1/2 in cumulus cells was blocked dramatically(Yamashita et al. 2007, 2009). In granulosa cells, theseinhibitors also suppress the induction of Cyp11a1 andStar expression (Shimada et al. 2006), and progester-one production (Puttabyatappa et al. 2013), suggest-ing that the EGFR-dependent signaling pathway regu-lates ERK1/2 pathway, which is required forluteinizaion of granulosa cells and differentiation ofcumulus cells at least in in vitro culture. To clarify thefunctional roles of ERK1/2 in in vivo, Fan et al. (2009)generated granulosa cell- and cumulus cell-specificERK1/2 mutant mice using the Cre/LoxP technique.In ERK1/2 mutant mice in which both kinases are de-pleted in granulosa cells and cumulus cells, not onlycumulus cell expansion and granulosa cell luteiniza-tion but also oocyte meiotic resumption are completelysuppressed, suggesting that the ERK1/2 pathway ingranulosa cells and cumulus cells works as a rate-lim-iting factor on the ovulation process.

As ligands for EGFR to activate the downstreamsignaling pathway, the EGF-like factors, amphiregulin(Areg), betacellulin (Btc) and epiregulin (Ereg) aretransiently expressed after LH stimulation in granu-losa cells and act on both granulosa cells and cumuluscells (Park et al. 2004; Ashkenazi et al. 2005; Shimadaet al. 2006). Especially, Areg and Ereg are rapidly ex-pressed within 1 hr after the onset of LH surge (Parket al. 2004). In mouse Areg gene promoter region, aputative cAMP responsible element (CRE) site is ob-served (Qin and Partridge 2005). The mutation of thisregion decreases the promoter activity in the luciferasepromoter assay using primary culture of mouse granu-losa cells, and CRE sequence binds to phosphorylatedCREB (CRE binding protein) after LH stimulation (Fanet al. 2010). However, there is no CRE site in mouseEreg promoter region, and the important role of Sp1binding sites has been reported in the increase of itspromoter activity in granulosa cells (Sekiguchi et al.2002). The transcription factor, Sp1 has multiple phos-phorylated sites including the target sites by PKA andthe phosphorylation induces to transfer Sp1 to nucleiand to bind it to the consensus sequence of promoterregion (Rohiff et al. 1997). Thus, the expressions ofAreg and Ereg are directly regulated by the cAMP-PKA-CREB or cAMP-PKA-Sp1 cascades in granulosacells during the ovulation process.

EGF like factors are transmembrane proteins thatconsist a signal sequence, a transmembrane domain andat least one EGF domain. To work as a ligand for EGFR,metalloprotease activity is critical for the release ofthe EGF domain. Several studies implied the existenceof proteolytic enzyme that cleaved EGF domain ofEGF-like factor to stimulate its own receptor duringovulation process (Ashkenazi et al. 2005; Jamnongjitet al. 2005; Conti et al. 2006). Firstly, Sahin et al.

Fig. 1. Expression of Tace/Adam17 mRNA (A), TACE/ADAM17 protein (B) and TACE/ADAM17 enzyme activity(C) in cumulus cells during in vitro maturation of porcineCOCs. A: Expression of Tace/Adam17 mRNA in cumuluscells of cultured COCs. B: Expression of TACE/ADAM17protein in cumulus cells of cultured COCs. C: TACE/ADAM17 enzyme activity in cumulus cells of culturedCOCs. Republished with permission of The Endocrine So-ciety, from Endocrinology, 148, Yamashita et al., Hormone-induced expression of TACE/ADAM17 protease impacts por-cine cumulus cell oocyte complex expansion and meioticmaturation via ligand activation of the EGF receptor, 6164–6175, Figs. 1 and 2, „ 2007; permission conveyed throughCopyright Clearance Center, Inc.

(2004) showed that tumor necrosis factor a-convert-ing enzyme (TACE) was the main sheddase of AREGand EREG in mouse embryonic cells. The expressionof TACE has been observed in many tissues, includingheart (Sahin et al. 2004; Horiuchi et al. 2005), brain(Käkkäinen et al. 2000; Hurtado et al. 2001), lung(Dijkstra et al. 2009), liver (Bourd-Boittin et al. 2009),muscle (Ohtsu et al. 2006), kidney (Göoz et al. 2006;Schramme et al. 2008) and testis (Lizama et al. 2010;



inhibitor, the enzyme activity of TACE was signifi-cantly decreased, which resulted in the reduction ofERK1/2 phosphorylation and target gene expressionincluding Cyp11a1 (Fig. 2). Moreover, the knockdownof TACE by the specific siRNA suppressed the phos-phorylation level of ERK1/2 and its target gene ex-pression, and the down-regulations were overcome bythe addition of AREG to culture media. Thus, the cen-tral dogma of the ovulation process is the sequentialinduction of EGF like factor, PGE2 and progesteronein LH-stimulated granulosa cells. The increase of PGE2and progesterone to maximum levels is required forgene expressions directly induced granulosa cell lutei-nization, cumulus expansion and follicular wall rup-ture (Fig. 3).

3. The ovulation model in pig—The differencesbetween pig and rodents—

The matured oocytes that resume meiotic from GVstage to progress to the metaphase II (MII) stage arereleased to oviducts at 36 to 44 hr after LH stimula-tion in pig (Ten Haff et al. 2002; Wongkaweewit et al.2012). On the other hand, the ovulation is observedwithin 14 h after ovulation stimuli in mice (Fig. 4A).To clear which mechanisms are similar to and whichare different as compared with those in mice model,we collected follicular fluid, granulosa cells, cumuluscells and oocytes from the hormone-stimulated swineat different time points after hCG injection to induceovulation following eCG priming to induce folliculardevelopment (Kawashima et al. 2008). The expressionof Areg, Ereg and Ptgs2 in granulosa cells, the phos-phorylation of ERK1/2 and the secretion of progester-one in follicular fluid were detected in pig as similarto those in mice. However, the starts of cumulus ex-pansion and oocyte meiotic resumption were delayedin pig model to 20 h after hCG injection. Interestingly,Areg and Ereg have already expressed in granulosacells before hCG injection, however, the phosphoryla-tion of ERK1/2 and Ptgs2 expression were induced at12 hr after hCG injection (Fig. 4B). Thus, the resultssuggested that the enzyme activity of TACE to releaseEGF domain of EGF like factors works as a key regu-lator to initiate the ovulation process in pig. Strikingly,the expression of TACE was induced by hCG injec-tion in porcine granulosa cells, and then activated byCa2+-PKC-c-Src dependent mechanism (Yamashita etal. 2014) (Fig. 2A). In mice model, since the expres-sion level of TACE is consistent during follicular de-velopment and ovulation process (Hernandez-Gonzalezet al. 2006), the enzyme activity is increased immedi-ately by signaling pathway without transcription. Thus,it is estimated that the time lag to progress ovulationprocess in pig as compared with that in mice is causedby the time of transcription and translation of TACEin granulosa cells. The graduate increase of TACE en-

Fig. 2. The effect of PKC inhibitor (CalC) or Src inhibitor(PP2) on TACE/ADAM17 enzyme activity (A), phosphor-ylation of ERK1/2 (B) and Cyp11a1 mRNA expression incultured granulosa cells. A: Effect of CalC or PP2 on TACE/ADAM17 enzyme activity in cultured granulosa cells. B:Effect of CalC or PP2 on phosphorylation of ERK1/2 in cul-tured granulosa cells. C: Effect of Cyp11a1 mRNA expres-sion in granulosa cells in cultured granulosa cells. Repub-lished with permission of The Endocrine Society, fromEndocrinology, 155, Yamashita et al., Protein kinase C (PKC)increases TACE/ADAM17 enzyme activity in porcine ovar-ian somatic cells, which is essential for granulosa cell lutei-nization and oocyte maturation, 1080–1090, doi:10.1210/en.2013-1655, Fig. 4, „ 2014; permission conveyed throughCopyright Clearance Center, Inc.

Inoue et al. 2014). We revealed that TACE was ex-pressed in granulosa cells and the enzyme activity wasincreased in granulosa cells during the ovulation proc-ess (Yamashita et al. 2007, 2014) (Fig. 1). The in-creased level of TACE enzyme activity was inducedby the binding of TACE with PKC-induced phosphor-ylation of c-Src in granulosa cells. When the granu-losa cells were cultured with PKC inhibitor or c-Src



zyme activity in granulosa cells after hCG injectionwould be a reason for the different time course of ovu-lation process in each follicle (Figs. 4A, B). Therefore,to synchronize the timing of ovulations and to judgethe best timing of insemination, we have to examinethe expression and activation of TACE in granulosacells in each commercial breed of pig.

4. In vitro maturation technique of porcineoocytes

The positive roles of cumulus cells in oocyte matu-ration in vitro have been well known for the past fourdecades (Cross 1973; Thibault et al. 1975; Hillensjöet al. 1982; Eppig and Downs 1984). Recent microarraystudies have identified novel activators including EGFlike factors as mentioned above secreted from granu-

losa cells that induce the differentiation of cumuluscells (Hernandez-Gonzalez et al. 2006; Shimada et al.2006). The gene targeting technology has clearly shownwhich signaling pathways are required in cumulus cellsfor oocyte maturation (Shimada et al. 2007; Lui et al.2009). Most of them are only expressed in granulosacells but not in cumulus cells during the ovulation proc-ess. Additionally, some of them are secreted from cu-mulus cells as well as granulosa cells in vivo, howeverin in vitro culture condition, the expression is not in-duced in cumulus cells of COCs cultured with anyagonists. More importantly, their receptors are ex-pressed in cumulus cells during follicular developmentprocess to preovulatory follicles. Because the COCsare collected from small antral follicles, with a diam-eter of 3 to 5 mm, of ovary of gilts collected fromslaughterhouse, the preculture of COCs is required to

Fig. 3. Schematic diagram shows the simplified model of oocyte maturation and luteinization process. LH stimulus inducesmRNA expression of EGF-like factor (Areg, Ereg and Btc) and Tada/Adam17 via cAMP-PKA-CREB or cAMP-PKA-SP1dependent manner. Expressed TACE/ADAM17 protein is activated by LH-induced PKC-Src pathway, and sheds EGF-likefactor. Soluble form of EGF-like factor stimulates EGFR-RAS-RAF-MEK1-ERK1/2 pathway in granulosa cells and cumuluscells. Activated ERK1/2 phosphorylates CEBP and AP1 transcriptional factor, and phosphorylated CEBP or AP1 induce granu-losa cells specific genes, Ptgs2 and progesterone production-related genes (Star, Cyp11a1 and Hsd3b1), which results ininduction of granulosa lutenization and cumulus expansion.



acquire the ability to respond to EGF like factors andothers that are secreted from granulosa cells during theovulation process (Shimada et al. 2003; Kawashima etal. 2008).

The number of COCs cultured in 300 ml of matura-tion medium has significant correlation with the rateof oocyte maturation (Yamashita et al. 2003). When 1

COC was cultured in maturation medium, the rate ofmeiotic resumption was significantly lower than thatwhen more than 20 COCs were cultured in the samevolume of medium for 20 hr. Another group showedthat, using condition medium where porcine cumuluscells or denuded porcine oocytes had cultured for 24hr, the rate of meiotic resumption was increased when

Fig. 4. Expressional and temporal differences between mouse (A) and pig (B) in EGF-like factor (Areg and Ereg), Tace/Adam17 and Ptgs2 mRNA during follicular development and ovulation process. A: Temporal expression of EGF-like factor(Areg and Ereg), Tace/Adam17 and Ptgs2 mRNA in granulosa cells obtained from immature female mice (day 23 after birth)during follicular development and ovulation process. Expression of EGF-like factor is transiently induced after hCG injectionwithin 4 hr. Tace/Adam17 mRNA expression is not changed by any stimulation and constitutively expressed in granulosacells, whereas the enzyme activity was induced by LH(hCG)-PKC-Src dependent manner, which results in induction of cleav-age and secretion of EGF domain after hCG injection. Thus, in mouse, expression of EGF-like factor and activation of TACE/ADAM17 enzyme activity in LH-stimulated granulosa cells is the key step to induce the ovulation process. B: Temporalexpression of EGF-like factor (Areg and Ereg), Tace/Adam17 and Ptgs2 mRNA in granulosa cells obtained for immaturefemale pig (day 180 after birth) during follicular development and ovulation process. Expression of EGF-like factor is alreadyinduced after eCG injection 72 hr and the expression level is maintained just after hCG injection. Before and after eCGinjection, Tace/Adam17 mRNA expression is low level, however, the expression is increased by hCG injection within 5hr ingranulosa cells. Furthermore, TACE/ADAM17 enzyme activity was induced by LH(hCG)-PKC-Src dependent manner, whichresults in induction of cleavage and secretion of EGF domain after hCG injection. Thus, in pig, expression and activation ofTACE/ADAM17, but not EGF-like factor expression, in LH-stimulated granulosa cells is the key step to induce the ovulationprocess.



COCs were cultured in condition medium where cu-mulus cells were cultured, but not oocyte (Procházkaet al. 1998). The results suggested that the secretionfactor(s) from cumulus cells play(s) an important rolein oocyte maturation. The number of cumulus cells at-tached oocyte is increased during follicular develop-ment stage (Buccione et al. 1990; Okazaki et al. 2003).The proliferation of cumulus cells is dependent on theexpression of Ccnd2 that is induced by FSH andestrogen (Robker and Richards 1998; Kawashima etal. 2008). In small antral follicles, the androgen isdominant and the level of estrogen is increased by FSHstimuli during follicular development (Leung andArmstrong 1980; Armstrong et al. 1991). Thus, pre-incubation with FSH and estrogen is required for cu-mulus cell proliferation before cumulus cell differen-tiation to acquire the responding ability to EGF likefactors. Our study (Kawashima et al. 2008) revealedthat the switch to change proliferation stage to differ-entiation is regulated by the ration of estrogen per pro-

gesterone. The decrease of the ration leads to the ex-pression of EGF receptor and LH receptor in cumuluscells. The information should provide new methods forimproving in vitro oocyte maturation. We have alreadyreported a new system of porcine COC maturationwhich we briefly describe below and illustrate in Fig.5.

1. COCs are recovered from small antral follicles (3–5 mm in diameter).

2. The recovered COCs are pre-cultured with 2 ng/ml FSH and 100 ng/ml estradiol 17b for 10 hr to in-duce cell proliferation.

3. At 10 hr, 20 ng/ml of progesterone is added to theFSH- and estradiol-containing medium to suppress cellproliferation and induce Egfr and Lhcgr mRNA expres-sion.

4. After 20 hr of culture, COCs are moved to freshmedium with 1 mg/ml LH, 1 ng/ml EGF and 100 ng/mlprogesterone for an additional 24 hr.

Using this novel culture system based on in vivo

Fig. 5. A schematic with regard to the novel culture system of porcine COCs when compared with conventional culturesystem. Novel culture system: porcine COCs were cultured with FSH and E2 for 10 h. At 10 h point, progesterone was addedto the culture medium, and then the COCs were further cultured for additional 10 h. The precultured COCs were moved tonew culture medium containing LH, EGF and P4 and then cultured for additional 24 h.



changes in hormones and growth factor production, thematured porcine COCs exhibit full expansion, the cu-mulus cells remain healthy (low number of apoptoticcells) and when oocytes obtained from these COCs areused for in vitro fertilization, developmental compe-tence to the blastocyst stage is significantly improvedas compared with the conventional FSH+LH culturesystem (Kawashima et al. 2008). Although the success-ful rate of embryo transfer is still limited, the combi-nation with in vitro maturation technique and embryofreezing will be contributed for conservation of geneticresources. In cattle production field, the system hasbeen used in the commercial field. The numerous ba-sic researches in this field promote the next genera-tion reproduction technique to be available for pig com-mercial field.

5. Fertilization ability of oocytes

The matured oocytes are ovulated to oviduct and arethen arrested at the MII stage until the penetration ofsperm. The ability of oocytes to prevent the resump-

tion of meiosis from the MII stage or the induction offragmentation is called cytostatic factor (Masui et al.1977), however the function is limited within severalhours after ovulation (Blandau et al. 1952; Marstonand Chang 1964). Our recent study revealed that theactivity of cytostatic factor of matured oocytes is de-cided by granulosa cell-secreted neuregulin 1 (NRG1)during the ovulation process (Noma et al. 2011;Kawashima et al. 2014). NRG1 is one of the membersof EGF like factor but does not act on EGFR as similarto AREG or EREG (Riese et al. 1995) (Figs. 6A, B).The ErbB3 and ErbB2 heterodimer that are expressedin granulosa cells and cumulus cells (Noma et al. 2011)are worked as receptor for NRG1 (Wallasch et al.1995). The induction of Nrg1 mRNA in granulosa cellsbut not in cumulus cells was started within 2 hr afterhCG injection (Fig. 6C). Western blot analyses showedthat at least two high intensity immuno-reactive bandswere detected in whole ovarian extracts after hCGstimulation (Fig. 6D). However the small molecularweight band was less intense in granulosa cells. In re-sponse to hCG, granulosa cells express specific cleav-

Fig. 6. The expression of Neuregulin 1 (NRG1) in granulosa cells during ovulation process. (A) The image of murine Nrg1gene; Because Nrg1 gene has three different transcription sites, three different types of transcripts are potentially expressed.(B) The functional mechanism of NRG1; EGF domain of NRG1 is released by Adam19 and then acts on ErbB2 and ErbB3heterodimer. (C) The expression of Nrg1 in granulosa cells and cumulus cells during ovulation process. (D) The expressionand modification of NRG1 in pre-ovulatory and peri-ovulatory follicles. Republished with permission of The EndocrineSociety, from Mol. Endocrinol., 25, Noma et al., LH induces Neuregulin 1 (NRG1) type III specific transcripts that interactwith EGF-like factor pathways to control granulosa cell differentiation and oocyte maturation, 104–116, Fig. 3, „ 2011;permission conveyed through Copyright Clearance Center, Inc. Republished with permission of The Endocrine Society, fromMol. Endocrinol., 28(5), Kawashima et al., Targeted disruption of Nrg1 in granulosa cells alters the temporal progression ofoocyte maturation, 706–721, Fig. 1, „ 2014; permission conveyed through Copyright Clearance Center, Inc.



age enzymes, including Adam19 that processes extra-cellular growth factors (Shirakabe et al. 2001). Becausethe full length of type III NRG1 is more than 110 kDa(Hu et al. 2006; Savonenko et al. 2008), the presentresults suggest that the 75 kDa band is a secreted formincluding EGF domain and is worked as active ligandform. In fact, the specific antibody of EGF domain ofNRG1 recognized both bands but the anti C-terminaldomain of NRG1 antiserum revealed only 110 kDa.Thus, the small form is presumed to include the EGFdomain converted by an enzyme that recognizes the Nterminal region of type III NRG1. The positive signalsby anti-EGF domain of NRG1 antibody were detectedat granulosa cell layers, follicular fluid and cumuluscell layers (Kawashima et al. 2014).

To clear the role of NRG1 in in vivo, we tried to makean analysis of the oocyte maturation using Nrg1 mu-tant mice model (Kawashima et al. 2014). Whole bodyNrg1 KO mice are lethal at day 10 during embryogen-esis (Meyer and Birchmeier 1995). To overcome thissevere limitation, we generated granulosa cell (GC)-specific Nrg1gc–/– (gcNrg1KO) by mating Nrg1flox/flox

mice to mice expressing Cre recombinase driven byCyp19a1 (GC specific) promoter (Yang et al. 2001;Fan et al. 2008). Mating test with wild type male micehas been done to check the fertility in the mutant fe-male mice. The average number of pups per deliverywas significantly decreased in gcNrg1KO as comparedwith that in Nrg1flox/flox (WT) female mice due to theincrease of abnormal fertilization. Although the ferti-lization rates of oocytes recovered at 14 and 16 h fromgcNrg1KO were similar to those in Nrg1flox/flox (WT)female mice, the successful fertilization (2 pronucleiand sperm tail) rate was significantly lower in oocytesrecovered at 18 h (Fig. 7A). Additionally, the rate ofsuccessful fertilization in in vivo was linearly decreased

by delaying time points when plug was formed ingcNrg1KO mice (Fig. 7B). Thus, the reason for de-creasing number of pups per delivery in gcNrg1KO iscaused by the limiting time window of oocytes acquirefertilization competence.

In pig granulosa cells, Nrg1 is also expressed by hCGinjection and the receptor, ErbB2 and ErbB3 is detectedin both cumulus cells and granulosa cells ofpreovulatory follicles (Tabata et al., unpublished data).Moreover, the activation of ErbB3 was induced in cu-mulus cells when porcine COCs were cultured withNRG1, suggesting that NRG1 also plays an importantrole in fertilization ability of porcine oocytes. TheNRG1-induced fertilization ability permits late timingof fertilization in in vivo, suggesting that further analy-sis of Nrg1 gene in pig leads to get a novel geneticmarker to select a high reproductive performance line.

From the above study, we revealed that the induc-tion of Nrg1 was observed in mouse and pig ovaryduring ovulation process. Although both species weremembers of prolificacy animals, we also detect the highlevel of Nrg1 expression in human granulosa cells col-lected from ovarian-stimulated cycles of infertilitypatients (Tabata et al., unpublished data). The prelimi-nary data suggested that not only in multiple ovula-tion species such as pig and mouse but also in singleovulation species including human, NRG1 would playan important role in oocyte fertilization competence.

6. The fertilization ability of sperm

In ovulated COCs, a hyaluronan rich matrix was ac-cumulated among cumulus cells called as cumulus ex-pansion (Richards 2005). The expansion is essential,not only for ovulation but also for in vivo fertilization,and perhaps more specifically for sperm capacitation

Fig. 7. The fertilization ability of oocytes in granulosa cell specific Nrg1KO mice. (A) In vitro fertilization rate of WT andgcNrg1KO mice oocytes recovered form oviducts at 14, 16 or 18 hr after hCG injection. (B) The relationship between in vivofertilization rate and timing of plug formation in WT and gcNrg1KO mice. Republished with permission of The EndocrineSociety, from Mol. Endocrinol., 28, Kawashima et al., Targeted disruption of Nrg1 in granulosa cells alters the temporalprogression of oocyte maturation, 706–721, Fig. 3, „ 2014; permission conveyed through Copyright Clearance Center, Inc.



(Gutnisky et al. 2007). During fertilization process, thematrix is broken down by sperm-secreted hyaluroni-dase, which induces chemokine secretion from cumu-lus cells (Shimada et al . 2008). The secretedchemokine, such as CCL5 acts on sperm via the spe-cific receptors (CCR1, CCR3 and CCR5) to inducehyperactivation status (Shimada et al. 2008, 2013). Thehigh progressive motility and high frequency of spermhead are associated with the ability to pass through zonapellucida to detach the surface of oocyte cytoplasmicmembrane (Suarez and Ho 2003a, b). Thus, the accu-mulation of hyaluronan rich matrix and the degrada-tion of it by sperm-secreted hyaluronidase are an ini-tial step of successful fertilization.

Hyaluronidase storages in sperm head region is calledacrosome (Kim et al. 2008). The release of hyaluroni-dase is induced by follicular fluid-containing factor,such as progesterone that is emitted from follicles withmatured oocytes (Calogero et al. 2000). Progesteroneacts on sperm to increase Ca2+ level in sperm cyto-plasm, which induces exocytosis system that openssecret vesicle to release hyaluronidase (Roldan andVazquez 1996; Publicover et al. 2008; Witte andSchäfer-Somi 2007). The loss or mutation of acrosome

lost the fertilization ability, but the rate of acrosomeintegrity was very high, about 80% in boar and murinesperm recovered from epididymis (Lusignan et al.2007; Yamashita et al. 2007) (Fig. 8). However, therate was dramatically increased in bacteria-infectedsemen (Zan et al. 2008).

Bacterial infections of the genital tract increase therisk of male infertility and economical loss in domes-tic animal production (Althouse and Lu 2005; Maes etal. 2008; Goldberg et al. 2013). Because leukocytesare contaminated in bacteria-infected semen (El Fekyet al. 2009; Gdoura et al. 2008) (Fig. 9), and becauseleukocytes express the Toll-like receptor (TLR) that isactivated by bacteria-released endotoxin, LPS (Takedaand Akira 2005; Poltorak et al. 1998), the activationof leukocyte TLRs by LPS can induce the secretion ofTumor Necrosis Factor (TNF)-a thereby causing de-creased sperm motility (Liu et al. 2012; Aggarwal etal. 2003; Perdichizzi et al. 2007). However, our previ-ous study revealed that TLR2 and TLR4 were expressedin pig, mouse and human sperm (Fujita et al. 2011).TLR2 and TLR4 were localized at the acrosome re-gions and mid piece of pig, mouse and human sperm.

The function(s) of TLRs in sperm were then analyzed

Fig. 8. The intact acrosome in boar sperm stained by FITC-peanut agglutinin. Some of sperm are FITC positive-acrosomeintact sperm (fi), but other sperm are FITC-negative sperm that lost acrosome region (Æ).



in more detail by determining the effects of LPS onsperm in specific mutant mouse models (Fujita et al.2011). Because LPS had high affinity binding forTLR4, and a high dose of LPS also activated TLR2,

Tlr4–/– or Tlr2–/–; Tlr4–/– male mice were used for thisstudy. Sperm motility decreased significantly in wildtype (WT) mice exposed to 1.0 mg/ml of LPS for 6 hr,whereas LPS had little or no effect on sperm motility

Fig. 9. The leukocytes in collected boar semen stained by Giemsa staining. Leukocyte was not detected in boar A semen, butwas observed in boar B and C.



in Tlr4–/– mice or Tlr2/4 double knockout mice. De-creased acrosome integrity and the number of TUNELpositive and cleaved caspase-3 positive (apoptotic)sperm were increased in WT mice exposed to LPS butnot in the Tlr mutant mice. These results in the Tlr4and Tlr2/4 mutant mice, provide strong evidence thatthe bacterial endotoxin LPS mediates its apoptotic ef-fects in sperm via activating the TLR pathways. Be-cause apoptosis can be activated in sperm by the LPS-TLR4 pathways, we conclude that sperm respond tobacterial infection (LPS) in semen via a direct TLR4-dependent mechanism and do not require immune cell-secreted cytokines (Fig. 10).

Fertilization competence of mouse sperm was alsoimpaired by bacterial release of LPS in both in vivoand in vitro and via a TLR4-dependent manner (Fujitaet al. 2011). When the sperm samples were pre-cul-tured with or without bacteria and then injected intothe uterus of female mice, the fertilization rate in con-trol mice was about 60% whereas the fertilization ratein mice injected with sperm exposed to bacteria wasless than 20%. However, the presence of polymyxin B(PMB) that neutralized LPS activity reversed the nega-tive effects of bacteria and increased fertilization ratesto 40%, suggesting that bacteria-secreted LPS reducedthe in vivo fertilization ability of mouse sperm (Fujitaet al. 2011).

In conclusion, sperm express some innate immunefunctions, including TLRs and functional responses toLPS. In semen samples infected with bacteria, spermrecognized the bacterial endotoxins via TLR-depend-ent pathways, inducing an apoptotic process. Theapoptotic response induced by LPS may be required toensure the removal of poor sperm that would be detri-mental to fertilization and embryo development.

7. How to improve the quality of frozen-thawedboar sperm

7-1. Bacteria infection in boar semen decreasesthe frozen-thawed sperm motility and fertili-zation ability

Bacteria are frequently detected in boar semen andaffect detrimental effects on sperm motility orsurvivability. In our recent study, the presence of bac-teria in pig semen was determined first by collectingsemen samples from 22 individual boars (pigs), andthen culturing the seminal plasma in agar without an-tibiotics. Bacterial colonies were observed in 19 (13gram-negative; 3 gram-positive; double positive 3) ofthe 22 samples but were absent in 3 samples (Table1).

To examine the effects of bacteria on sperm motil-

Fig. 10. A schematic of the expression and function of TLR4 in sperm.



ity, we incubated semen at 37∞C for 1, 3 or 6 hr andthen analyzed sperm motility rates. Before and 1 hrafter incubation, sperm motility was not significantlycorrelated with the number of bacteria (Fig. 11). How-ever, by 3 hr of incubation a significant negative cor-relation (r2 = 0.279, P < 0.05) was observed betweensperm motility and the number of bacteria (Fig. 11). Ithas been thought that the increasing number of bacte-ria in seminal plasma changes pH, metabolized source,or free radical due to their proliferation and metabolicactivity (Althouse et al. 2000; Fraczek et al. 2007).Because sperm has a high responsibility to the envi-ronmental changes mentioned above, the motility andfertilization ability are decreased in the presence ofbacteria. Additionally, the gram negative bacteria re-lease lipopolysaccharide (LPS) that acts as endotoxinand sperm recognize the endotoxin by the specificreceptor, TLRs to decrease sperm quality. Thus, to re-duce the bacteria infection problem, Penicillin G thatweakens the cell wall of the bacterium, or Amikamycinthat suppresses protein synthesis in bacteria have beenwell-used for boar semen treatments (Okazaki et al.2010). However, when the antibiotics 1,000 U/ml ofPenicillin G and 100 mg/mL of Amikamycin were addedto the pig semen samples containing high titers of bac-teria (more than 5000 CFU/ml), neither Penicillin Gnor Amikamycin significantly improved sperm motil-ity 3 hr after incubation (Fig. 12). This is why the treat-

ment with the antibiotics releases LPS from gram nega-tive bacteria to semen due to the antibiotics-inducedbacteriolysis. LPS directly bound to the sperm headregion (Fig. 13) to reduce motility and induce apoptoticprocess as mentioned above. The negative effects ofLPS on sperm function quickly started even if thesperm was only treated with LPS for 5 min and thenremoved. Because PMB neutralizes LPS activity, wesought to develop and improve the techniques for longstorage of boar sperm by testing the beneficial effectsof PMB.

In our study, the LPS activity in seminal plasma wasincreased by the addition of Penicillin G compared tothat in seminal plasma without Penicillin G (endotoxinactivity; 0.05 vs. 0.17 EU/mL). The addition of 100mg/ml PMB to seminal plasma completely inactivatedPenicillin G-increased LPS activity in boar semen. Theinhibitory effect of PMB on the binding of LPS tosperm was examined using FITC-conjugated LPS. Theresults showed that in control (without PMB), the posi-

Table 1. The number of gram positive and negative bacteriain each boar semen.

Fig. 11. The correlation between the motility of sperm ateach culture point and the number of bacteria in the semenjust after ejaculation.



tive signals of LPS were localized at the head regionand mid piece of sperm, and most of sperm were posi-tive (Fig. 13). However, the binding was dramaticallysuppressed by PMB treatment in a dose dependentmanner, and the maximum effect was observed at 100mg/ml.

7-2. Removing and/or neutralizing endotoxin is(are) important for long storage and freez-ing of boar sperm

Because pig semen frequently contains high titers ofbacteria and the treatment with antibiotics releases LPSto semen, which reduces sperm quality (Okazaki et al.2009a), we reasoned that LPS released from bacteriawould decrease sperm motility during long-term stor-age or freeze-thaw processes and consequently wouldaccount for the low success rate of artificial insemina-tion in the pig. In fact, the treatment of sperm withPMB has been used to improve assisted reproductivetechnology protocols for domestic animals (Okazakiet al. 2010; Okazaki and Shimada 2012). However, onlya few papers have documented the positive effects ofPMB on sperm motility during short-term storage(Hakimi et al. 2006; Hosseinzadeh et al. 2003). There-fore to document this, a sperm-rich fraction was col-lected weekly from each of 22 boars and then treatedwith or without antibiotics. During long-term storageof pig sperm at 15∞C for 2 days, sperm motility dra-matically decreased in antibiotic free conditions. De-creased sperm motility was also observed when Peni-cillin G alone was present during a similar test period.However, when Penicillin G was combined with PMB,sperm motility improved significantly after 6 days ofstorage as compared with that in the Penicillin G group(Fig. 14). When sperm were stored for 10 days andthen used for artificial insemination, the conception

rate was significantly higher in the combined treatmentgroup as compared to Penicillin G alone (Fig. 14).When pig sperm were treated immediately after ejacu-lation with both Penicillin G and PMB, and then fro-zen under liquid nitrogen, the motility of sperm afterthawing was significantly higher than that of spermtreated with penicillin G alone (Fig. 15).

Gram positive bacteria were also detected in 14%(3/22 boars) of semen (Table 1). Gram positive bacte-ria releases peptidoglycan or lipoprotein, and thesefactors stimulate TLR2 (Tsan and Gao 2004). Whensperm was incubated with TLR2 ligand, Pam3Cys, themotility was markedly decreased in human (Fujita etal. 2011) and pig (Okazaki et al. 2009a) due to thedecrease of membrane integrity. Therefore, it is nec-essary step to remove both LPS and gram positive bac-terial factors from sperm incubation medium just afterejaculation.

Based on these findings, we designed the followingnovel handling method before the cooling and freez-ing process. Firstly, seminal plasma is removed fromthe sperm by centrifugation at room temperature im-mediately after semen collection. Secondly, the spermpellets are resuspended in pre-treatment solution con-taining common antibiotics (e.g. Penicillin G and/orAmikamycin) and PMB. When semen was handled bynovel extender before the freezing procedure, the suc-cess rate (borderline for preservation; post-thaw spermmotility >60%) of cryopreservation was significantlyhigher (88%) than that by conventional method.

7-3. Optimal combination of osmolality and glyc-erol in the freezing extender

In the conventional freezing method of boar sperm,the ejaculated semen is diluted to pretreatment solu-tion and then kept at 15∞C for at least 2 hrs. During the

Fig. 12. The effects of antibiotics on sperm motility when semen containing high number of bacteria was incubated at 37∞C.



first step, we added the additional process to removebacteria-released endotoxin by centrifugation and thecombined treatment with antibiotics as mentionedabove. The incubation at 15∞C reduces the metabolicactivity of sperm. After 2-hr incubation, the dilutionsolution is removed by centrifugation, and then spermis resuspended by the cooling extender containing eggyolk, and cooled from 15 to 5∞C. At 5∞C, the glycerolis added as cryoprotectant agents (CPAs) to sperm dilu-ents to remove the surplus water from sperm or bindthe water inside sperm. After 20-min of glycerol equi-libration, the sperm diluents are put into straws, andare frozen under liquid nitrogen.

Glycerol is known to be good CPAs in freezing ex-tenders to protect sperm from the freezing damages. Ithas been reported that the addition of glycerol improves

motility and acrosomal integrity of frozen-thawedspermatozoa (Johnson et al. 2000; Almilid and Johnson1988; Fiser and Fairfull 1984). However, because boarsperm are highly susceptible to glycerol (Almilid andJohnson 1988), more than 4% of glycerol affects themembrane integrity and reduces the fertilization rateafter artificial insemination (Wilmut and Polge 1974).The concentration of glycerol for sperm freezing ismuch lower in pig as compared with that in other spe-cies, such as cattle (8 to 10%) and human (10 to 12%).Therefore, in pigs, it is necessary to develop a novelextender containing a lower concentration of glycerolthan that used in other species to protect sperm fromfreeze-injury and maintain motility and fertilizationcompetence after thawing.

The positive effects of glycerol for sperm freezing

Fig. 13. The binding of LPS on boar sperm, modified from Okazaki et al. 2010, Theriogenology LPS bound to some of spermsurface, but the treatment with PMB completely suppressed the binding of LPS to sperm. A; control bright, b; control immun-ofluorescent microscope, c; 100 mg/mL PMB bright, d; 100 mg/mL PMBl immunofluorescent microscope. Reprinted withpermission from Theriogenology, 74, Okazaki et al., Polymyxin B neutralizes bacteria-released endotoxin and improves thequality of boar sperm during liquid storage and cryopreservation, 1691–1700, Fig. 3, „ 2010, Elsevier.



are thought to reduce the damage caused by water icecrystals. Sperm have little cytoplasm, but nonethelessa sufficient volume of water to form intracellular iceduring the freezing procedure, estimating that the de-crease of water in cytoplasm before the freezing proc-ess might improve the quality of frozen-thawed sperm.Liu et al. (1998) showed that the exposure of bull sper-matozoa to hyperosmotic solution caused sperm shrink-age and dehydration, which prevented intracellular icecrystal nucleation during the freezing process. Thepositive effects of the treatment with hyperosmoticextender before freezing on motility and membraneintegrity have also been reported in ram sperm (Fiseret al. 1981; Watson and Duncan 1988) and boar sperm(Zeng et al. 2001).

To determine the optimal combination ofhyperosmotic extender and glycerol treatment, boarsperm is divided to 3 ¥ 4 (total 12) treatment groupswith varying osmolalities (300 to 500 mOsm/kg) dur-ing cooling process and glycerol concentration (0.5 to3%) treating before freezing in 3 boars. The ANOVAsanalysis showed that significant difference was not de-tected in sperm motility among 3 boars, however inparameter, osmolality and glycerol concentration, sig-nificant differences are detected (Table 2). More than2% glycerol is required to keep the sperm high motil-ity after thawing. 400 mOsm is optimal osmolality fortreatment of sperm during cooling process. The novelhyperosmotic (400 mOsm/kg) and low-glycerol (finalconcentration 2%) freezing extender not only improvedthe motility of sperm just after thawing but also main-tained the high motility after long incubation at 37∞C.The continuous motility after thawing in vitro predicts

high motility of sperm in uterus after artificial insemi-nation. Additionally, the novel combinational treatmentincreased acrosome integrity of frozen-thawed sperm.The integrity will be contributed for high fertilizationability in vivo as described above.

7-4. Ca2+ uptake in sperm during thawing proc-ess damages sperm quality

The frozen-thawed sperm has exhibited abnormalcapacitation and lost acrosome region due to an in-crease of Ca2+ level in sperm (Bailey and Buhr 1993).When the frozen semen was made by our novel tech-nique to suppress the risk of bacteria released endo-toxin and formation of ice crystal during freezing proc-ess, the acrosome integrity and motility of sperm afterlong incubation were still lower than those in spermjust after ejaculation. Because the inductions were ob-served in a time dependent manner after thawing, wehypothesized that the induction of abnormal capacita-tion and the loss of acrosome would be caused by thedamage during the thawing process but not the cool-ing and freezing process. The capacitated sperm

Fig. 14. The additional effects of polymyxin B on spermmotility during liquid storage at 15∞C. Reprinted with per-mission from Theriogenology, 74, Okazaki et al., PolymyxinB neutralizes bacteria-released endotoxin and improves thequali ty of boar sperm during l iquid storage andcryopreservation, 1691–1700, Fig. 4, „ 2010, Elsevier.

Fig. 15. The effects of pre-treatment with polymyxin B be-fore cooling and freezing process on the motility of frozen-thawed sperm. Reprinted with permission fromTheriogenology, 74, Okazaki et al., Polymyxin B neutral-izes bacteria-released endotoxin and improves the qualityof boar sperm during liquid storage and cryopreservation,1691–1700, Fig. 5, „ 2010, Elsevier.

Table 2. The effects of individuals of boar, osmolality ofextender and glycerol concentration of extender on the mo-tility of frozen-thawed sperm (from Okazaki et al., 2009a).



showed high motility but was not maintained for a longtime culture after thawing. The capacitation is inducedby several extracellular factors, however in sperm cy-toplasm, the increased level of Ca2+ is a trigger of ca-pacitation (Arnoult et al. 1999; Baldi et al. 2000). Ithas been known that during the thawing process, themembrane integrity is decreased due to release of cho-lesterol and phospholipid from cell membrane (DeLeeuw et al. 1990; Plummer and Watson 1985; Mülleret al. 1999; Ollero et al. 1998; Pena et al. 2003; Bwangaet al. 1991). From these reports, we estimated thatunstabilization of cell membrane during thawing proc-ess would allow the uptake of Ca2+ in sperm cytoplasmto induce the abnormal capacitation and the loss ofacrosome cap. To clear this hypothesis, we investigatedthe level of Ca2+ in sperm after thawing and the addi-tional effects of Ca2+ chelator, EGTA to thawing solu-tion on [Ca2+]i level and post-thawed sperm functions(Okazaki et al. 2011).

Strikingly, the intercellular level of Ca2+ in spermwas dramatically increased in frozen-thawed spermafter incubation. Firstly, the increased signals weredetected in the sperm mid piece and the signals wereexpanded to sperm head region. Capacitation of spermis referred to be the change of metabolic activity, andthe ATP production was dramatically increased in ca-pacitated sperm. The induction of Ca2+ to activate TCAcycle and electronic chain transfer in mitochondria thatlocalize on the mid piece of sperm. The loss of acro-some cap is caused by the exocytosis of hyaluronidaseand other secreted factors, which is induced by Ca2+-dependent formation of SNAP25, Synapototagmin, andsynaptobrevin (Schulz et al. 1998; Tomes et al. 2002).Thus, the increase of Ca2+ after thawing is thought tobe a trigger of abnormal capacitation and loss of acro-some cap.

The freezing extender and thawing solution don’tcontain Ca2+, however it is very difficult to make Ca2+

free solution without any contaminations. We used Ca2+

chelator, EGTA to suppress the uptake of Ca2+ to spermafter thawing. The addition of EGTA (10 mM) to thaw-ing solution completely suppressed the increase of Ca2+

in frozen-thawed sperm. The tyrosine phosphorylationof sperm protein that was a marker of capacitation wasalso significantly suppressed by the addition of EGTA.Moreover, the treatment increased the acrosome integ-rity and the thawed sperm motility similar to those insperm just after ejaculation.

8. The novel artificial insemination using frozen-thawed boar sperm

8-1. Seminal plasma improves uterus conditionafter artificial insemination

It is known that the injection of seminal plasma intothe female genital tract suppressed the increase of poly-morphonuclear neutrophilic granulocytes in the uterus(uterine clearance) and enhanced the rate of disappear-ance of uterine inflammation (Matthijs et al. 2000;Rozeboom et al. 1998, 2001). Moreover, O’Leary etal. (2004) reported that differentiation of endometrialcells was induced by seminal plasma and that thesecells expressed cytokine and chemokine mRNA forembryo development and implantation preparation inpigs. Taken together, these data indicate that seminalplasma plays an important role in the uterine environ-ment conducive to the success of conception and preg-nancy. However, when sperm was injected into theuterus in pig artificial insemination, the factors in-cluded in seminal plasma were not or less taken intouterus because the diluted semen or frozen-thawed

Fig. 16. The procedure of frozen boar sperm.



sperm were injected to uterus. Thus, it is possible thatlow reproductive performance in artificial insemina-tion as compared with that in natural mating is causedby the dilution of semen or removing seminal plasmaduring the process of preparing frozen semen.

To clear this hypothesis, we identified the factors inseminal plasma and then developed a novel pig artifi-cial insemination method using cryopreserved sperm(Okazaki et al. 2009b) as shown in Fig. 16. The sperm-rich fraction was collected weekly from each boar us-ing the gloved-hand technique. The seminal plasma wasremoved just after collection by centrifuge and thenwas frozen as described in above section. The frozensperm was thawed using the thawing solution with orwithout 10% (v/v) of seminal plasma. The addition ofseminal plasma significantly increased the conceptionrate as compared with that of frozen-thawed spermwithout the addition of seminal plasma to the thawingsolution. The increased rate was comparable to that offresh semen (Table 3). When frozen-thawed sperm wasartificially inseminated into natural estrus sows, theconception rate was 67%, and the total number of foe-tuses was 9.7+/–3.5. The rates were not significantlydifferent from those for artificial insemination usingfresh semen (Table 3).

8-2. Chemicaly defined solution to improve uteruscondition and increase the conception rateafter artificial insemination

Since it has been known that seminal plasma con-tains various bacteria and/or virus (e.g. PRRS, AD,PCV2, PPV etc.) (Kim et al. 2003; Guérin and Pozzi2005; Althouse and Rossow 2011), it is estimated thatthe use of seminal plasma as thawing solution increasesthe risk of infection. Furthermore, the quality of semi-nal plasma depends on seasonal or individual charac-teristics, suggesting that it is necessary to develop thethawing solution without animal derived materials(without seminal plasma). The treatment with EGTAincreased the fertilization rate in in vivo artificialinseminations, similar to that in sperm with seminalplasma. The same number of blastocyst embryo werecollected from the uterus by artificial inseminationusing post thawed sperm with EGTA, however thenumber of leukocytes in uterus was increased and im-

plantation was prevented. Because the co-injection ofseminal plasma with frozen-thawed sperm decreasedthe number of leukocytes in uterus, it is suggested thatthe factors to repress inflammation in the uterus arecontained in seminal plasma.

Cortisol is well known to be the factor to reduce in-flammation and decrease the number of leucocytes(Whitehouse 2011). Although it is no report to men-tion cortisol in seminal plasma, it has been reportedthat the circular level of cortisol is increased at the timeof mating (Borg et al. 1991). Thus, we examined thelevel of cortisol in seminal plasma and the effects ofinjection of cortisol in uterus condition after artificialinsemination. The results showed that cortisol was de-tected in seminal plasma (Table 3). When the sows ofnatural estrus were twice artificially inseminated withor without cortisol, the injection of cortisol into theuterus with sperm significantly decreased the numberof leukocytes in the uterus and endometrium at 24–36h after artificial injection. The low number ofleukocytes in the uterus was similar to that in the uterusinjected with fresh semen. The cortisol injection sig-nificantly increased the implantation rate and litter sizeof sows as compared to artificial insemination withoutcortisol (implantation rate; 83% vs. 51%, litter size;10.6 vs. 7.3). From these results, we concluded thatthe injection of 5 mg cortisol with frozen-thawed sper-matozoa by EGTA-containing solution was a novelmethod of pig artificial insemination usingcryopreserved spermatozoa.

9. Discussion

The rodent model is commonly used to analyze howto induce ovulation and oocyte maturation (Richards2005). The characteristic of multiple ovulations aresimilar to that in pig and most factors to regulate ovar-ian function in mice are also expressed in pig ovaries.However, it takes a long time to induce ovulation inpig compared with mice, and especially the synchro-nization of ovulations is not observed in pig even ifunder hormone-stimulation condition. To clear the rea-sons why the ovulation process is not synchronized andtakes a long time to induce the ovulation process inpig compared with those in rodent model, we exam-ined the kinetic changes of gene expression and acti-

Table 3. The reproductive performance of artificial insemination using fresh semen and frozen-thawed semen (from Okazakiet al., 2011).



vation of signaling pathway in granulosa cells duringthe ovulation process. In mice, the pattern of gene ex-pression was sequentially induced in granulosa cellsafter hCG (ovulation stimuli) injection. Firstly, LH-targeted genes were expressed within an hour after hCGinjection via LH-induced cAMP-PKA-CREB orcAMP-PKA-Sp1 dependent mechanisms (Fan et al.2010; Sekiguchi et al. 2002; Rohiff et al. 1997). EGFlike factors, AREG and EREG were included in theinitial expression genes, and activated the expressionof the next group of genes, such as Ptgs2 and Cyp11a1(Shimada et al. 2006; Yamashita et al. 2007). Thesegenes are involved in PGE2 production and progester-one production, respectively, which also induce thegene expressions involved in cumulus expansion,granulosa cell luteinization and follicular wall rupture(Richards 1994; Matsumoto et al. 2001; Segi et al.2003). In porcine granulosa cells, the initial expres-sion of genes that was observed in mice just after hCGinjection was also induced within a few hours(Kawashima et al. 2008). However, the phosphoryla-tion of ERK1/2 that was a target signaling pathway ofEGF like factor and the expression of Ptgs2 were de-layed for 12 hr after hCG injection (Kawashima et al.2008; Yamashita et al. 2009). The different time courseswere associated with the different mechanism of therelease of EGF like factor (Yamashita et al. 2007;Kawashima et al. 2008). Thus, the wide variation toinduce ovulation in each follicle and different individu-als is a logical consequence of characteristic induc-tion of ovulation in pig. Therefore, it is very difficultto know the timing of ovulation and to synchronizethe timing of ovulation for efficient artificial insemi-nation technique in pig.

To solve the above problem of artificial insemina-tion in pig, we think of two different approaches; oneis to prolong the period that ovulated oocytes can besuccessfully fertilized in the oviduct, the other is toprolong the period that injected sperm is alive in uterusand oviduct, and can be fertilized in oviduct. In thefirst strategy, the limited period of fertilization abilityof oocytes is caused by the spontaneous resumption ofoocyte meiosis from the MII stage (Komar 1982). Thearrested period at the stage is thought to be less than 5to 10 hours, and the ovulation is observed from 30 to44 hours after hCG injection (Branden and Austin1954). Thus, several times of injection are required to

penetrate sperm to all of the oocytes arrested at theMII stage. A recent study using a mutant mouse modelrevealed that the ability to arrest at the MII stage wasdecided to be the granulosa cell-secreted NRG1 dur-ing ovulation process (Kawashima et al. 2014). NRG1acted on cumulus cells to regulate the AREG-EGFR-induced PKC activity to a moderate level. The superbregulation system controls the timing of closing gapjunctional communication between cumulus cells andoocyte, which induces meiotic resumption after oocytegets the ability to be arrested at the MII stage. It isestimated that the period between LH surge and theonset of meiotic resumption is required forpolyadenylation of maternal RNA that is stably accu-mulated in oocyte during follicular development stage(Goldman et al. 1987; Mutter et al. 1987). In granu-losa cell-specific Nrg1 mutant mice, the stabilizationof phosphorylated ERK1/2 was limited in the MIIoocytes, suggesting that Mos protein synthesis duringmeiotic progression was low level in oocytes due tothe limited polyadenylation of Mos RNA. Thepolyadenylation in oocytes is initiated after EGF likefactors act on cumulus cells, and is stopped at the MIstage (Chen et al. 2013). In other words, the tight con-trol of the level of polyadenylatoin of maternal mRNAis regulated by the timing of EGF like factors andNRG1 expressions. In our unpublished data, NRG1 andits receptor (ErbB3) were expressed in porcine granu-losa cells and cumulus cells. However, there is no re-port about the differences of their expressions amongthe breeds, individuals and follicles. It has been knownthat the genes encoding Nrg1 and ErbB3 have a nu-merous single nucleotide polymorphism (SNP) in notonly promoter region but also exon to change the ex-pression levels or function of their translation prod-ucts in the human genome (Li et al. 2006; Garcia-Barcelo et al. 2009; Betcheva et al. 2009). Currentgenome project of pig finds SNP using the next gen-eration sequencer, and challenges to clear the relation-ship between each SNP and function. The genomeproject of pig and molecular study about porcine ovarycoordinately progress to breeding to select the highreproductive performance.

Another strategy to reduce the times of artificial in-semination is to prolong the period that sperm is aliveand has the fertilization ability in uterus and oviduct.The survival of sperm in uterus and oviduct is des-tined by sperm themselves and the condition of femalereproductive tracts (Katila 2001; Rodríguez-Martínezet al. 2005). In artificial insemination, especially us-ing frozen-thawed sperm, the morphological integritysuch as acrosome and tail before injection predicts themotility in uterus and oviducts (Roca et al. 2006; Zengand Terada 2000). We developed the method to freezeand thaw boar semen and described it in this mono-graph. Briefly, the sperm was separated from seminal

Table 4. The concentration of cortisol in boar seminal plasma.



plasma just after ejaculation and then treated with an-tibiotics to remove the risk of bacteria-released endo-toxin. The sperm was re-suspended by hyperosmoticextender to remove the free water from cytoplasm ofsperm to reduce the risk of ice crystal formation dur-ing the freezing process. During the thawing process,to suppress the uptake of Ca2+ to sperm cytoplasm, Ca2+

chelater, EGTA was added to thawing solution. Thefrozen-thawed sperm can be alive for at least 6 hoursafter thawing in vitro, however the conception rate byartificial insemination has still been limited.

We also revealed the increase number of leukocytesin the uterus after the injection of frozen-thawed sperm.Leukocytes are accumulated to the region of inflam-mation, and then excludes non-self or repair the wound(Borregaard et al. 2005). Because sperm is non-selffor female, a factor is required for the reduction of in-flammation status in uterus when semen is injected notonly by artificial insemination but also natural mat-ing. We cleared that cortisol that is a component ofseminal plasma reduced the number of leukocytes tomake an optimal condition for implantation and main-tain the pregnancy. The combinational approach to keepthe good morphology of sperm and to induce the opti-mal condition in uterus improved the conception rateand litter size by artificial insemination using frozen-semen. In the current data, the conception rate wasmore than 85% and litter size was more than 10, when3 to 5 billion sperm were injected twice per estrus. Thereproductive performance by frozen-thawed semen wassimilar to that by fresh semen and natural mating. Ifwe induced synchronization of ovulation, the one-timeinjection of frozen-thawed sperm would predict goodreproductive performance. The genetic breeding andthe method to induce ovulation by pharmacologicalapproach will progress to develop a more efficient tech-nique for artificial insemination.

In conclusion, the basic biological approaches toidentify the mechanisms of ovulation, oocyte matura-tion and sperm fertilization ability are required to de-velop a novel technique of reproduction. Each specieshas common mechanisms but also has a unique systemto survive and leave offspring. In our model to developreproductive technology of pig, we examined the mo-lecular mechanisms using knockout mice model andcomprehensive gene expression analysis. The basicdata from rodent model is adjusted to pig model todevelop the reproductive technology. Our novel artifi-cial insemination technique using frozen-thawed spermwould be expanded to commercial pig farm to improvethe genetics and increase the production-efficiency allover the world.

ReferencesAggarwal BB. Signalling pathways of the TNF superfamily:

a double-edged sword. Nat. Rev. Immunol. 2003; 3: 745–747.

Ainsworth L, Tsang BK, Downey BR, Marcus GJ, ArmstrongDT. Interrelation between follicular steroid levels, gona-dotropic st imuli , and oocyte maturation duringpreovulatory development of porcine follicles. Biol.Reprod. 1980; 23: 621–627.

Almeida FR, Noack S, Foxcroft GR. The time of ovulationin relation to estrus duration in gilts. Theriogenology 2000;53: 1389–1396.

Almilid T, Johnson LA. Effects of glycerol concentration,equilibration time and temperature of glycerol additionon post-thaw viability of boar spermatozoa frozen instraws. J. Anim. Sci. 1988; 66: 2899–2905.

Althouse GC, Lu KG. Bacteriospermia in extended porcinesemen. Theriogenology 2005; 63: 573–584.

Althouse GC, Rossow K. The potential risk of infectiousdisease dissemination via artificial insemination in swine.Reprod. Domest. Anim. 2011; 46 Suppl 2: 64–67.

Althouse GC, Kuster CE, Clark SG, Weisiger RM. Field in-vestigations of bacterial contaminants and their effects onextended porcine semen. Theriogenology 2000; 53: 1167–1176.

Armstrong DT, Zhang X, Vanderhyden BC, Khamsi F. Hor-monal acitions during oocyte maturation influence fertili-zation and early embryonic development. Ann. NY Acad.Sci. 1991; 626: 137–158.

Arnoult C, Kazam IG, Visconti PE, Kopf GS, Villaz M,Florman HM. Control of the low voltage-activated cal-cium channel of mouse sperm by egg ZP3 and by mem-brane hyperpolarization during capacitation. Proc. Natl.Acad. Sci. USA 1999; 96: 6757–6762.

Ashkenazi H, Cao X, Motola S, Popliker M, Conti M, TsafririA. Epidermal growth factor family members: endogenousmediators of the ovulatory response. Endocrinology 2005;146: 77–84.

Bailey JL, Buhr MM. Ca2+ regulation by cryopreserved bullspermatozoa in response to A23187. Cryobiology 1993;30: 470–481.

Baldi E, Luconi M, Bonaccorsi L, Muratori M, Forti G. In-tracellular events and signaling pathways involved insperm acquisition of fertilizing capacity and acrosomereaction. Front Biosci. 2000; 5: E110–123.

Betcheva ET, Mushiroda T, Takahashi A, Kubo M,Karachanak SK, Zaharieva IT, Vazharova, RV, Dimova II,Milanova VK, Tolev T, Kirov G, Owen MJ, O’DonovanMC, Kamatani N, Nakamura Y, Toncheva DI. Case-con-trol association study of 59 candidate genes reveals theDRD2 SNP rs6277 (C957T) as the only susceptibility fac-tor for schizophrenia in the Bulgarian population. J. Hum.Genet. 2009; 54: 98–107.

Blandau RJ. The female factor in fertility and infertility. I.Effects of delayed fertilization on the development of thepronuclei in rat ova. Fertil. Steril. 1952; 3: 349–365.

Borg KE, Esbenshade KL, Johnson BH. Cortisol, growthhormone, and testosterone concentrations during matingbehavior in the bull and boar. J. Anim. Sci. 1991; 69: 3230–3240.

Borregaard N, Theilgaard-Mönch K, Cowland JB, Ståhle M,Sørensen OE. Neutrophils and keratinocytes in innateimmunity—cooperative actions to provide antimicrobialdefense at the right time and place. J. Leukoc. Biol. 2005;77: 439–443.

Bourd-Boittin K, Basset L, Bonnier D, L’helgoualc’ A, Théret



N. CX3CL/fractalkine shedding by human hepatic stellatecells: contribution to chronic inflammation in the liver. J.Cell Mol. Med. 2009; 13: 1526–1535.

Braden AWH, Austin CR. The fertile life of mouse and rateggs. Science 1952; 120: 610–611.

Buccione R, Schroeder AC, Eppig JJ. Interactions betweensomatic cells and germ cells throughout mammalian oog-enesis. Biol. Reprod. 1990; 43: 543–547.

Bwanga CO, Einarsson S, Rodriguez-Martinez H.Cryopreservation of boar semen. II: Effect of cooling rateand duration of freezing point plateau on boar semen fro-zen in mini- and maxi-straws and plastic bags. Acta Vet.Scand. 1991; 32: 455–461.

Calogero AE, Burrello N, Barone N, Palermo I, Grasso U,D’Agata R. Effects of progesterone on sperm function:mechanisms of action. Hum. Reprod. 2000; 15 Suppl 1:28–45.

Chauhan MS, Kapila R, Gandhi KK, Anand SR. Acrosomedamage and enzyme leakage of goat spermatozoa duringdilution, cooling and freezing. Androgia 1994; 26: 21–26.

Chen J, Torcia S, Xie F, Lin CJ, Cakmak H, Franciosi F,Horner K, Onodera C, Song JS, Cedars MI, Ramalho-Santos M, Conti M. Somatic cells regulate maternal mRNAtranslation and developmental competence of mouseoocytes. Nat. Cell Biol. 2013; 15: 1415–1423.

Conely AJ, Howard HJ, Slanger WD, Ford JJ. Steroidogen-esis in the preovulatory porcine ffolicle. Biol. Reprod.1994; 51: 655–661.

Conti M, Hsieh M, Park JY, Su YQ. Role of the epidermalgrowth factor network in ovarian foll icles. Mol.Endocrinol. 2006; 20: 715–723.

Courtens JL, Paquignon M, Blaise F, Ekwall H, Plöen L.Nucleus of the boar spermatozoon: Structure and modifi-cations in frozen, frozen-thawed, or sodium dodecylsulfate-treated cells. Mol. Reprod Dev. 1989; 1: 264–277.

Cross PC. The role of cumulus cells and serum in mouseoocyte maturation in vitro. J. Reprod. Fertil. 1973; 34:241–245.

Curry MR. Cryopreservation of semen from domestic live-stock. Rev. Reprod. 2000; 5: 46–52.

De Leeuw FE, Chen HC, Colenbrander B, Verkleij AJ. Cold-induced ultrastructural changes in bull and boar spermplasma membranes. Cryobiology 1990; 27: 171–183.

Dijkstra A, Postma DS, Noordhoek JA, Lodewijk ME,Kauffman HF, ten Hacken NH, Timens W. Expression ofADAMs (“a disintegrin and metalloptotease”) in the hu-man lung. Virchows Arch. 2009; 454: 441–449.

Downs SM, Longo FJ. Prostaglandins and preovulatoryfollicular maturation in mice. J. Exp. Zool. 1983; 228(1):99–108.

Duncan WC, Cowen GM, Illingworth PJ. Steroidogenic en-zyme expression in human corpora lutea in the presenseand absence of exogenous human chorionic gonadotropoin(HCG). Mol. Hum. Reprod. 1999; 5: 291–298.

El Feky MA, Hassan EA, El Din AM, Hofny ER, Afifi NA,Eldin SS, Baker MO. Chlamydia trachomatis: methods ofidentification and impact on semen quality. Egypt J.Immunol. 2009; 16: 49–59.

Eppig JJ, Downs SM. Chemical signals that regulate mam-malian oocyte maturation. Biol. Reprod. 1984; 30: 1–11.

Evans G, Dobias M, King GJ, Armstrong DT. Production ofprostaglandins by porcine preovulatory follicular tissues

and their roles in intrafollicular function. Biol. Reprod.1983; 28: 322–328.

Fan HY, Shimada M, Liu Z, Cahill N, Noma N, Wu Y, GossenJ, Richards JS. Selective expression of KrasG12D ingranulosa cells of the mouse ovary causes defects in folli-cle development and ovulation. Development 2008; 135:2127–2137.

Fan HY, Lui Z, Shimada M, Sterneck E, Hohnson PF, HedrickSM, Richards JS. MAPK3/1 (ERK1/2) in ovarian granu-losa cells are essential for female fertility. Science 2009;324: 938–941.

Fan HY, O’Connor A, Shitanaka M, Shimada M, Liu Z,Richards JS. Beta-catenin (CTNNB1) promotespreovulatory follicular development but represses LH-mediated ovulation and luteinization. Mol. Endocrinol.2010; 24: 1529–1542.

Feitsma H. Artificial insemination in pigs, research and de-velopments in The Netherlands, a review. Acta Sci. Vet.2009; 37 Suppl 1: s61–s71.

Fiser PS, Fairfull RW. The effect of glycerol concentrationand cooling velocity on cryosurvival of ram spermatozoafrozen in straws. Cryobiology 1984; 21: 542–551.

Fraczek M, Szumala-Kakol A, Jedrzejczak P, KamienicznaM, Kurpisz M. Bacteria trigger oxygen radical release andsperm lipid peroxidation in in vitro model of semen in-flammation. Fertil. Steril. 2007; 88 Suppl 4: 1076–1085.

Fujita Y, Mihara T, Okazaki T, Shitanaka M, Kushino R,Ikeda C, Negishi H, Liu Z, Richards JS, Shimada M. Toll-like receptors (TLR) 2 and 4 on human sperm recognizebacterial endotoxins and mediate apoptosis. Hum. Reprod.2011; 26: 2799–2806.

Garcia-Barcelo MM, Tang CS, Ngan ES, Lui VC, Chen Y,So MT, Leon TY, Miao XP, Shum CK, Liu FQ, Yeung MY,Yuan ZW, Guo WH, Liu L, Sun XB, Huang LM, Tou JF,Song YQ, Chan D, Cheung KM, Wong KK, Cherny SS,Sham PC, Tam PK. Genome-wide association study iden-tifies NRG1 as a susceptibility locus for Hirschsprung’sdisease. Proc. Natl. Acad. Sci. USA 2009; 24: 2694–2699.

Gdoura R, Kchaou W, Znazen A, Chakroun N, Fourati M,Ammar-Keskes L, Hammami A. Screening for bacterialpathogens in semen samples from infertile men with andwithout leukocytospermia. Andrologia 2008; 40: 209–218.

Gelety TJ, Chaudhuri G. Prostaglandins in the ovary andfallopian tube. Baillieres. Clin. Obstet. Gynaecol. 1992;6: 707–729.

Goldberg AM, Argenti LE, Faccin JE, Linck L, Santi M,Bernardi ML, Cardoso MR, Wentz I, Bortolozzo FP. Riskfactors for bacterial contamination during boar semen col-lection. Res. Vet. Sci. 2013; 95: 362–367.

Goldman DS, Kiessling AA, Millette CF, Cooper GM. Ex-pression of c-mos RNA in germ cells of male and femalemice. Proc. Natl. Acad. Sci. USA 1987; 84: 4509–4513.

Göoz M, Göoz P, Luttrell LM, Raymond JR. 5-HT2Areceptor induces ERK phosphorylation and proliferationthrough ADAM-17 tumor necrosis factor-alpha-convert-ing enzyme (TACE) activation and heparin-bound epider-mal growth factor-like growth factor (HB-EGF) sheddingin mesangial cells. J. Biol. Chem. 2006; 281: 21004–21012.

Guérin B, Pozzi N. Viruses in boar semen: detection andclinical as well as epidemiological consequences regard-ing disease transmission by artificial insemination.



Theriogenology 2005; 63: 556–572.Gutnisky C, Dalvit GC, Pintos LN, Thompson JG, Beconi

MT, Cetica PD. Influence of hyaluronic acid synthesis andcumulus mucification on bovine oocyte in vitro matura-tion, fertilisation and embryo development. Reprod. Fertil.Dev. 2007; 19(3): 488–497.

Hakimi H, Geary I, Pacey A, Eley A. Spermicidal activityof bacterial lipopolysaccharide is only partly due to lipidA. J. Androl. 2006; 27: 774–779.

Hamberger L, Nordenströn K, Rosberg S Sjögren A. Acuteinfluence of LH and FSH on cyclic AMP formation in iso-lated granulosa cells of the rat. Act. Endocrinol. (Copenh.)1978; 88: 567–579.

Hamberger L, Nilsson L, Nordenström K, Sjögren A. LH-stimulated cAMP formation in rat granulosa cells duringfollicular maturation—a non-refractory response. Adv.Exp. Med. Biol. 1979; 112: 105–112.

Henderson KM, Gorban AM, Boyd GS. Effect LH factorsregulating ovarian cholesterol metabolism and progester-one synthesis in PMSG-primed immature rat. J. Reprod.Fertil. 1981; 61: 373–380.

Hernandez-Gonzalez I, Gonzalez-Robayna I, Shimada M,Wayne CM, Ochsener SA, White L, Richards JS. Geneexpression profiles of cumulus cells oocyte complexesduring ovulation reveal cumulus cells express neuronaland immune-related genes: dose this expand their role inthe ovulation process? Mol. Endocrinol. 2006; 20: 1300–1321.

Hillensjö T, Magnusson C, Ekholm C, Billig H, Hedin L.Role of cumulus cells in oocyte maturation. Adv. Exp. Med.Biol. 1982; 147: 175–188.

Horiuchi K, Zhou HM, Kelly K, Blobel CP. Evaluation ofthe contributions of ADAMs 9, 12, 15, 17, and 19 to heartdevelopment and ectodomain shedding of neuregulinsbeta1 and beta2. Dev. Biol. 2005; 283: 459–471.

Hosseinzadeh S, Pacey AA, Eley A. Chlamydia trachomatis-induced death of human spermatozoa is caused primarilyby lipopolysaccharide. J. Med. Microbiol. 2003; 52: 193–200.

Hu X, Hicks CW, He W, Wong P, Macklin WB, Trapp BD,Yan R. Bace1 modulates myelination in the central andperipheral nervous system. Nat. Neurosci. 2006; 9: 1520–1525.

Hurtado O, Cárdenas A, Lizasoain I, Boscá L, Lorenzo P,Moro MA. Up-regulation of TNF-alpha convertase (TACE/ADAM17) after oxygen-glucose deprivation in ratforebrain slices. Neuropharmacology 2001; 40: 1094–1102.

Inoue S, Tomasini R, Rufini A, Elia AJ, Agostini M, AmelioI, Cescon D, Dinsdale D, Zhou L, Harris IS, Lac S, Sil-vester J, Li WY, Sasaki M, Haight J, Brüstle A, WakehamA, McKerlie C, Jurisicova A, Melino G, Mak TW. TAp73is required for spermatogenesis and the maintenance ofmale fertility. Proc. Natl. Acad. Sci. USA 2014; 111: 1843–1848.

Jamnongjit M, Gill A, Hammes SR. Epidermal growth fac-tor receptor signaling is required for ovarian steroidogen-esis and oocyte maturation. Proc. Natl. Acad. Sci. USA2005; 102: 16257–16267.

Johnson LA, Weitze KF, Fiser P, Maxwell WM. Storage ofboar semen. Anim. Reprod. Sci. 2000; 62: 143–172.

Johnson RK. Crossbreeding in swine: Experimental results.

J. Anim. Sci. 1981; 52: 906–923.Katila T. Sperm-uterine interactions: a review. Anim. Reprod.

Sci. 2001; 68: 267–272.Käkkäinen I, Rybnikova E, Pelto-Huikko M, Huovilla AP.

Metalloproteinase-disintegrin (ADAM) genes are widelyand differentially expressed in the adult CNS. Mol. CellNeurosci. 2000; 15: 547–560.

Kawashima I, Okazaki T, Noma N, Nishibori M, YamashitaY, Shimada M. Sequential exposure of porcine cumuluscells to FSH and/or LH is critical for appropriate expres-sion of steroidogenic and ovulation—related genes thatimpact oocyte maturation in vivo and in vitro. Reproduc-tion 2008; 136: 9–21.

Kawashima I, Umehara T, Noma N, Kawai T, Shitanaka M,Richards JS, Shimada M. Targeted disruption of Nrg1 ingranulosa cells alters the temporal progression of oocytematuration. Mol. Endocrinol. 2014; 28: 706–721.

Kim E, Yamashita M, Kimura M, Honda A, Kashiwabara S,Baba T. Sperm penetration through cumulus mass and zonapellucida. Int. J. Dev. Biol. 2008; 52: 677–682.

Kim J, Han DU, Choi C, Chae C. Simultaneous detectionand differentiation between porcine circovirus and por-cine parvovirus in boar semen by multiplex seminestedpolymerase chain reaction. J. Vet. Med. Sci. 2003; 65: 741–744.

Komar A. Fertilization of parthenogenetically activatedmouseeggs I. Behaviour of sperm nuclei in the cytoplasmof parthenogeneticallyactivated eggs. Exp. Cell Res. 1982;139: 361–367.

LeMaire WJ, Davies PJ, Marsh JM. The role ofprostaglandins in the development of refractoriness to LHstimulation by Graafian follicles. Prostaglandins 1976; 12:271–279.

Leung PC, Armstrong DT. Interactions of steroids andgonadotropins in the control of steroidogenesis in the ovar-ian follicle. Annu. Rev. Physiol. 1980; 42: 71–82.

Li D, Collier DA, He L. Meta-analysis shows strong posi-tive association of the neuregulin 1 (NRG1) gene withschizophrenia. Hum. Mol. Genet. 2006; 15: 1995–2002.

Liu C, Liu H, Wang X, Xinbo S. Clinical significance andexpression of PAF and TNF-alpha in seminal plasma ofleukocytospermic patients. Mediators Inflamm. 2012;2012: 639–735.

Liu Z, Foote RH, Brockett CC. Survival of bull sperm fro-zen at different rates in media varying in osmolarity. Cryo-biology 1998; 37: 219–230.

Lizama C, Rojas-Benítez D, Antonelli M, Ludwig A,Bustamante-Marín X, Brouwer-Visser J, Moreno RD.TACE/ADAM17 is involved in germ cell apoptosis dur-ing rat spermatogenesis. Reproduction 2010; 140: 305–317.

Lui Z, Matos DG, Shimada M, Palmer S, Richards JS.Interleukin-6: an autocrine regulator of the mouse cumu-lus cell-oocyte complex expansion process. Endocrinology2009; 150: 3360–3368.

Lusignan MF, Bergeron A, Crête MH, Lazure C, ManjunathP. Induction of epididymal boar sperm capacitation by pB1and BSP-A1/-A2 proteins, members of the BSP proteinfamily. Biol. Reprod. 2007; 76: 424–432.

Lydon JP, DeMayo FJ, Funk CR, Mani SK, Hughes AR,Montgomery CA Jr, Shyamala G, Connely OM, O’MallyBW. Mice lacking progesterone receptor exhibit pleio-



tropic reproductive abnormalities. Genes Dev. 1995; 9:2266–2278.

Maes D, Nauwynck H, Rijsselaere T, Mateusen B, Vyt P, deKruif A, Van Soom A. Diseases in swine transmitted byartificial insemination: an overview. Theriogenology 2008;70: 1337–1345.

Manna PR, Huhtaniemi IT, Stocco DM. Mechanism of pro-tein kinase C signaling in the modulation of 3¢,5¢-cyclicadenosine monophosphate-mediated steroidogenesis inmouse granulosa cells. Endocrinology 2009; 150: 3308–3317.

Marston JH, Chang MC. The fertilizable life of ova and theirmorphology following delayed insemination in mature andimmature mice. J. Exp. Zool. 1964; 155: 237–251.

Masui Y, Markert CL. Cytoplasmic control of nuclear be-haviour during meiotic maturation of frog oocytes. J. Exp.Zool. 1977; 177: 129–146.

Matsumoto H, Ma W, Smalley W, Trazaskos J, Breyer RM,Dey SK. Diversification of cyclooxygenase-2-derivedprostaglandins in ovulation and implantation. Biol. Reprod.2001; 64: 1557–1565.

Matthijs A, Harkema W, Engel B, Woelders H. In vitro phago-cytosis of boar spermatozoa by neutrophils from periph-eral blood of sows. J. Reprod. Fertil. 2000; 120: 265–273.

Meyer D, Birchmeier C. Multiple essential functions ofneuregulin in development. Nature 1995; 378: 386–390.

Mulac-Jericevic B, Mullinax RA, DeMayo FJ, Lydon JP,Connely OM. Subgroup of reproductive function of pro-gesterone mediated by progesterone receptor-B isoform.Science 2000; 289: 1751–1754.

Murdoch WJ, Peterson TA, Van Kirk EA, Vincent DL,Inskeep EK. Interactive roles of progesterone,prostaglandins, and collagenase ion the ovulatory mecha-nism of the ewe. Biol. Reprod. 1986; 35: 1187–1194.

Müller K, Pomorski T, Müller P, Herrmann A. Stability oftransbilayer phospholipid asymmetry in viable ram spermcells after cryotreatment. J. Cell Sci. 1999; 112: 11–20.

Mutter GL, Wolgemouth DJ. Distinct developmental patternsof c-mos protooncogene expression in female and malemouse germ cells. Proc. Natl. Acad. Sci. USA 1987; 84:5301–5305.

Noma N, Kawashima I, Fan HY, Fujita Y, Kawai T, TomodaY, Mihara T, Richards JS, Shimada M. LH-inducedneuregulin 1 (NRG1) type III transcripts control granu-losa cell differentiation and oocyte maturation. Mol.Endocrinol. 2011; 25: 104–116.

Ohtsu H, Dempsey PJ, Frank GD, Brailoiu E, Higuchi S,Suzuki H, Nakashima H, Eguchi K, Eguchi S. ADAM17mediates epidermal growth factor receptor transactivationand vascular smooth muscle cell hypertrophy induced byangiotensin II. Arterioscle. Thromb. Biol. 2006; 26: e133–137.

Okazaki T, Shimada M. New strategies of boar spermcryopreservation: development of novel freezing and thaw-ing methods with a focus on the roles of seminal plasma.Anim. Sci. J. 2012; 83: 623–629.

Okazaki T, Nishibori M, Yamashita Y, Shimada M. LH re-duces proliferative activity and accelerates GVBD of por-cine oocytes. Mol. Cell Endocrinol. 2003; 209: 43–50.

Okazaki T, Abe S, Shimada M. Improved conception ratesin sows inseminated with cryopreserved boar spermato-zoa prepared with a more optimal combination of osmo-

lality and glycerol in the freezing extender. Anim. Sci. J.2009a; 80: 121–129.

Okazaki T, Abe S, Yoshida S, Shimada M. Seminal plasmadamages sperm during cryopreservation, but its presenceduring thawing improves semen quality and conceptionrates in boars with poor post-thaw semen quality.Theriogenology 2009b; 71: 491–498.

Okazaki T, Mihara T, Fujita Y, Yoshida S, Teshima H,Shimada M. Polymyxin B neutralizes bacteria-releasedendotoxin and improves the quality of boar sperm duringliquid storage and cryopreservation. Theriogenology 2010;74: 1691–1700.

Okazaki T, Akiyoshi T, Kan M, Teshima H, Shimada M.Artificial insemination trial of frozen-thawed boar sper-matozoa with thawing solution containing seminal plasma.Nihon Yoton Gakkaishi 2011; 48: 164–168.

O’Leary S, Jasper MJ, Warnes GM, Armstrong DT,Robertson SA. Seminal plasma regulates endometrialcytokine expression, leukocyte recruitment and embryodevelopment in the pig. Reproduction 2004; 128: 237–247.

Ollero M, Bescós O, Cebrián-Pérez JA, Muiño-Blanco T.Loss of plasma membrane proteins of bull spermatozoathrough the freezing-thawing process. Theriogenology1998; 49: 547–555.

Osterman J, Demers LM, Hammond JM. Gonadotropinstimulation of procine ovarian ornithine decarboxylase invitro: the role of 3 ¢,5¢-adenosine monophosphoate.Endocrinology 1978; 103: 1718–1724.

Park JY, Su YQ, Ariga M, Law E, Jin SL, Conti M. EGF-like growth factors as mediator of LH action in the ovula-tory follicle. Science 2004; 303: 682–684.

Pena FJ, Johannisson A, Wallgren M, Rodriguez-MartinezH. Assessment of fresh and frozen-thawed boar semenusing an annexin-V assay: a new method of evaluatingsperm membrane integrity. Theriogenology 2003; 60: 677–689.

Perdichizzi A, Nicoletti F, La Vignera S, Barone N, D’AgataR, Vicari E, Calogero AE. Effects of tumour necrosis fac-tor-alpha on human sperm motility and apoptosis. J. ClinImmunol. 2007; 27: 152–162.

Plas-Roser S, Kauffmann MT, Aron C. Prostaglandins in-volvement in the formation of luteinized unruptured folli-cles in the cyclic female rat. Prostaglandins 1985; 29: 243–253.

Plummer JM, Watson PF. Ultrastructural localization of cal-cium ions in ram spermatozoa before and after cold shockas demonstrated by a pyroantimonate technique. J. Reprod.Fertil. 1985; 75: 255–263.

Poltorak A, He X, Smirnova I, Liu MY, Van Huffel C, Du X,Birdwell D, Alejos E, Silva M, Galanos C et al. DefectiveLPS signaling in C3H/HeJ and C57BL/10ScCr mice: mu-tations in Tlr4 gene. Science 1998; 282: 2085–2088.

Procházka R, Nagyová E, Brem G, Schellander K, Motlík J.Secretion of cumulus expansion-enabling factor (CEEF)in porcine follicules. Mol. Reprod. Dev. 1998; 49: 141–149.

Publicover SJ, Giojalas LC, Teves ME, de Oliveira GS,Garcia AA, Barratt CL, Harper CV. Ca2+ signalling in thecontrol of motility and guidance in mammalian sperm.Front Biosci. 2008; 1: 5623–5637.

Puttabyatappa M, Brogan RS, Vandevoort CA, Chaffin CL.EGF-like ligands mediate progesterone’s anti-apoptotic



action on macaque granulosa cells. Biol. Reprod. 2013;88: 18.

Qin L, Partridge NC. Stimulation of amphiregulin expres-sion in osteoblastic cells by parathyroid hormone requiresthe protein kinase A and cAMP response element-bindingprotein signaling pathway. J. Cell Biochem. 2005; 96: 632–640.

Rao MC, Richards JS, Midgley AR Jr, Reichert LE Jr. Regu-lation of gonadotropin receptors by luteinizing hormonein granulosa cells. Endocrinology 1977; 101: 512–523.

Richards JS. Hormonal control of gene expression in theovary. Endocr. Rev. 1994; 15: 725–751.

Richards JS. Ovulation: new factors that prepare the oocytefor fertilization. Mol. Cell Endocrinol. 2005; 234: 75–79.

Richards JS, Pangas SA. The ovary: basic biology and clini-cal implications. J. Clin Invest. 2010; 120: 963–973.

Richards JS, Ireland JJ, Rao MC, Bernath GA, Midgley ARJr, Reichert LE Jr. Ovarian follicular development in therat: hormone receptor regulation by estradiol, folliclestimulating hormone and luteinizing hormone.Endocrinology 1976; 99: 1562–1570.

Riese DJ, van Raaij TM, Plowman GD, Andrews GC, SternDF. The cellular response to neuregulins is governed bycomplex interactions of the erbB receptor family. Mol. CellBiol. 1995; 15: 5770–5776.

Robker RL, Richards JS. Hormone-induced proliferation anddifferentiation of granulosa cells: a coordinated balanceof the cell cycle regulator cyclin D2 and p27kip1. Mol.Endocrinol. 1998; 12: 924–940.

Roca J, Rod´riguez-Martínez H, Vázquez JM, Bolarín A,Hernández M, Saravia F, Wallgren M, Martínez EA. Strat-egies to improve the fertility of frozen-thawed boar se-men for artificial insemination. Soc. Reprod. Fertil. Suppl.2006; 62: 261–275.

Rodríguez-Martínez H, Saravia F, Wallgren M, Tienthai P,Johannisson A, Vázquez JM, Martínez E, Roca J, Sanz L,Calvete JJ. Boar spermatozoa in the oviduct.Theriogenology 2005; 63: 514–535.

Rohiff C, Ahmad S, Borellini F, Lei J, Glazer RI. Modula-tion of transcription factor Sp1 by cAMP-dependent pro-tein kinase. J. Biol. Chem. 1997; 272: 21137–21141.

Roldan ER, Vazquez JM. Bicarbonate/CO2 induces rapidactivation of phospholipase A2 and renders boar sperma-tozoa capable of undergoing acrosomal exocytosis in re-sponse to progesterone. FEBS Lett. 1996; 396: 227–232.

Ronen-Fuhrmann T, Timberg R, King SR, Hales DB, StoccoDM, Orly J. Spatio-temporal expression patterns of ster-oidogenic acute regulatory protein (StAR) during folliculardevelopment in the rat ovary. Endocrinology 1998; 139:303–315.

Royerez D, Barthelemy C, Hamamah S, Lansac J.Cryopreservation of spermatozoa: a 1996 review. Hum.Reprod. Update 1996; 2: 553–559.

Rozeboom KJ, Troedsson MH, Crabo BG. Characterizationof uterine leukocyte infiltration in gilts after artificial in-semination. J. Reprod. Fertil. 1998; 114: 195–199.

Rozeboom KJ, Troedsson MH, Rocha GR, Crabo BG. Thechemotactic properties of porcine seminal componentstoward neutrophils in vitro. J. Anim. Sci. 2001; 79: 996–1002.

Sahin U, Weskamp G, Kelly K, Zhou HM, Higashiyama S,Peschon J, Hartmann D, Saftig P, Blobel CP. Distinct role

for ADAM10 and ADAM17 in ectodomain shedding ofsix EGFR ligands. J. Cell Biol. 2004; 164: 769–779.

Savonenko AV, Melnikova T, Laird FM, Stewart KA, PriceDL, Wong PC. Alteration of BACE1-dependent NRG1/ErbB4 signaling and schizophrenia-like phenotypes inBACE1-null mice. Proc. Natl. Acad. Sci. USA 2008; 105:5585–5590.

Sayasith Km Lussier JG, Sirois J. Role of upstream stimula-tory factor phosphorylation in the regulation of the pros-taglandin G/H synthease-2 promoter in granulosa cells. J.Biol. Chem. 2005; 280: 28885–28893.

Schramme A, Abdek-Bakky MS, Kämpfer-Kolb N,Pfeilschifter J, Gutwein P. The role of CXCL16 and itsprocessing metalloproteinases ADAM10 and ADAM17 inthe proliferation and migration of human mesangial cells.Biochem. Biophys. Res. Commun. 2008; 370: 311–316.

Schulz JR, Sasaki JD, Vacquier VD. Increased associationof synaptosome-associated protein of 25 kDa with syntaxinand vesicle-associated membrane protein followingacrosomal exocytosis of sea urchin sperm. J. Biol. Chem.1998; 273: 24355–24359.

Segi E, Haraguchi K, Sugimoto Y, Tsuji M, Tsunekawa H,Tamba S, Tsuboi K, Ichikawa A. Expression of messengerRNA for prostaglandin E receptor subtype EP2/4 andcyclooxygenaseisozymes in mouse periovulatory folliclesand oviducts during superovulation. Biol. Reprod. 2003;68: 804–811.

Sekiguchi T, Mizutani T, Yamada K, Yazawa T, Kawata H,Yoshino M, Kajitani T, Kameda T, Minegishi T, MiyamotoK. Transcritional regulation of the epiregulin gene in therat ovary. Endocrinology 2002; 143: 4718–4729.

Sher N, Yivgi-Ohana N, Orly J. Transcriptional regulationof the cholesterol side chain cleavage cytochrome P450gene (CYP11A1) revisited: binding of GATA, cyclic ad-enosine 3¢,5¢-monophosphate response element-bindingprotein and activation protein (AP)-1 proteins to a distalnovel cluster of cis-regulatory elements potentiates AP-2and steroidogenic factor 1-dependent gene expression inthe rodent placenta and ovary. Mol. Endocrinol. 2007; 21:948–962.

Shimada M, Nishibori M, Isobe N, Kawano N, Terada T.Luteinizing hormone receptor formation in cumulus cellssurrounding porcine oocytes and its role during meioticmaturation of porcine oocytes. Biol. Reprod. 2003; 68:1142–1149.

Shimada M, Hernandez-Gonzalez I, Gonzalez-Robayna I,Richards JS. Paracrine and autocrine regulation of epi-dermal growth factor-like factors in cumulus oocyte com-plexes and granulosa cells: key roles for prostaglandinsynthase 2 and progesterone receptor. Mol. Endocrinol.2006; 20: 1352–1365.

Shimada M, Yanai Y, Okazaki T, Yamashita Y, Sriraman V,Wilson MC, Richards JS. Synaptosomal-associated pro-tein 25 gene expression is hormonally regulated duringand is involved in cytokine/chemokine exocytosis fromgranulosa cells. Mol. Endocrinol. 2007; 21: 2487–2502.

Shimada M, Yanai Y, Okazaki T, Noma N, Kawashima I,Mori T, Richards JS. Hyaluronan fragments generated bysperm-secreted hyaluronidase stimulate cytokine/chemokine production via the TLR2 and TLR4 pathwayin cumulus cells of ovulated COCs, which may enhancefertilization. Development 2008; 135: 2001–2011.



Shimada M, Mihara T, Kawashima I, Okazaki T. Anti-bac-terial factors secreted from cumulus cells of ovulatedCOCs enhance sperm capacitation during in vitro fertili-zation. Am. J. Reprod. Immunol. 2013; 69: 168–179.

Shirakabe K, Wakatsuki S, Kurisaki T, Fujisawa-Sehara A.Roles of Meltrin beta/ADAM19 in the processing ofneuregulin. J. Biol. Chem. 2001; 276: 9352–9358.

Soede NM, Noorhdhuizen JP, Kemp B. The duration of ovu-lation in pigs, studied by transrectal ultrasonography, isnot related to early embryonic diversity. Theriogenology1992; 38: 653–666.

Soede NM, Wetzeis CC, Zondag W, de Koning MA, KempB. Effects of time of insemination relation to ovulation,as determined by ultrasonography, on fertilization rate andaccessory sperm count in sows. J. Reprod. Fertil. 1995;104: 99–106.

Suarez SS, Ho HC. Hyperactivated motility in sperm.Reprod. Domest. Anim. 2003a; 38: 119–124.

Suarez SS, Ho HC. Hyperactivation of mammalian sperm.Cell Mol. Biol. 2003b; 49: 351–356.

Takeda K, Akira S. Toll-like receptors in innate immunity.Int. Immunol. 2005; 17: 1–14.

Ten Haff W, Thacker PA, Kirkwood RN. Effect of injectinggonadotropins during the luteal phase of the estrous cycleon the inter-estrus intervals of gilts. Can. J. Amin. Sci.2002; 82: 457–459.

Thibault C, Gerard M, Menezo Y. Preovulatory and ovula-tory mechanism in oocyte maturation. J. Reprod. Fertil.1975; 45: 604–610.

Tomes CN, Michaut M, De Blas G, Visconti P, Matti U,Mayorga LS. SNARE complex assembly is required forhuman sperm acrosome reaction. Dev. Biol. 2002; 243:326–338.

Tsan MF, Gao B. Endogenous ligands of Toll-like receptors.J. Leukoc. Biol. 2004; 76: 514–519.

Wallasch C, Weiss FU, Niederfellner G, Jallal B, Issing W,Ullrich A. Heregulin-dependent regulation of HER2/neuoncogenic signaling by heterodimerization with HER3.EMBO J. 1995; 14: 4267–4275.

Watson PF. The causes of reduced fert i l i ty withcryopreserved semen. Anim. Reprod. Sci. 2000; 60–61:481–492.

Watson PF, Duncan AE. Effect of salt concentration andunfrozen water fraction on the viability of slowly frozenram spermatozoa. Cryobiology 1988; 25: 131–142.

Whitehouse MW. Anti-inflammatory glucocorticoid drugs:reflections after 60 years. Inflammopharmacology 2011;19: 1–19.

Wilmut I, Polge C. The fertilizing capacity of boar semenstored in the presence of glycerol at 20, 5 and –79 degreesC. J. Reprod. Fertil. 1974; 38: 105–113.

Witte TS, Schäfer-Somi S. Involvement of cholesterol, cal-cium and progesterone in the induction of capacitation andacrosome reaction of mammalian spermatozoa. Anim.Reprod. Sci. 2007; 102: 181–193.

Wongkaweewit K, Prommachart P, Raksasub R,Burannammuay K, Techakumphu M, De Rensis F,Tummaruk P. Effect of the administration of GnRH or hCGon time of ovulation and the onset of estrus-to-ovulationintercal in sows in Thailand. Trop. Anim. Health Prod.2012; 44: 467–470.

Yamashita M, Yamagata K, Tsumura K, Nakanishi T, BabaT. Acrosome reaction of mouse epididymal sperm onoocyte zona pellucida. J. Reprod. Dev. 2007; 53: 255–262.

Yamashita Y, Shimada M, Okazaki T, Maeda T, Terada T.Production of progesterone from de novo-synthesized cho-lesterol in cumulus cells and its physiological role duringmeiotic resumption of porcine oocytes. Biol. Reprod. 2003;68: 1193–1198.

Yamashita Y, Kawashima I, Yanai Y, Nishibori M, RichardsJS, Shimada M. Hormone-induced expression of tumornecrosis factor alpha-converting enzyme/A disintegrin andmetalloprotease-17 impacts porcine cumulus cell oocytecomplex expansion and meiotic maturation via ligand ac-tivation of the epidermal growth factor receptor.Endocrinology 2007; 148: 6164–6175.

Yamashita Y, Hishinuma M, Shimada M. Activation of PKA,p38 MAPK and ERK1/2 by gonadotropins in cumulus cellsis critical for induction of EGF-like factor and TACE/ADAM17 gene expression during in vitro maturation ofporcine COCs. J. Ovarian Res. 2009; 24: 2: 20.

Yamashita Y, Okamoto M, Ikeda M, Okamoto A, Sakai M,Gunji Y, Nishimura R, Hishinuma M, Shimada M. Proteinkinase C (PKC) increases TACE/ADMA17 enzyme activ-ity in porcine ovarian somatic cells, which is essential forgranulosa cell luteinization and oocyte maturation.Endocrinology 2014; 155: 1880–1890.

Yang X, Arber S, William C, Li L, Tanabe Y, Jessell TM,Birchmeier C, Burden SJ. Patterning of muscle acetylcho-line receptor gene expression in the absence of motor in-nervation. Neuron 2001; 30: 399–410.

Zan Bar T, Yehuda R, Hacham T, Krupnik S, Bartoov B.Influence of Campylobacter fetus subsp. fetus on ramsperm cell quality. J. Med. Microbiol. 2008; 57(11): 1405–1410.

Zeng W, Terada T. Freezability of boar spermatozoa is im-proved by exposure to 2-hydroxypropyl-beta-cyclodextrin.Reprod. Fertil. Dev. 2000; 12: 223–228.

Zeng WX, Shimada M, Isobe N, Terada T. Survival of boarspermatozoa frozen in diluents of vrying osmolality.Theriogenology 2001; 56: 447–458.

development of artificial insemination technique in pig ... · to be developed in artificial...

Documents