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Research ArticleThe Increased Expression of Connexin and VEGF in MouseOvarian Tissue Vitrification by Follicle Stimulating Hormone

Yanzhou Yang1 Jie Chen2 Hao Wu1 Xiuying Pei1 Qing Chang1

Wenzhi Ma1 Huiming Ma1 Changchun Hei1 Xiaomin Zheng1 Yufang Cai1

Chengjun Zhao1 Jia Yu1 and Yanrong Wang1

1Key Laboratory of Fertility Preservation and Maintenance of Ministry of Education Key Laboratory of Reproduction andGenetics in Ningxia Department of Histology and Embryology Ningxia Medical University Yinchuan 750004 China2Department of Human Anatomy Inner Mongolia Medical University Hohhot 010010 China

Correspondence should be addressed to Yanrong Wang 4083304163com

Received 17 June 2015 Revised 23 August 2015 Accepted 30 August 2015

Academic Editor Mohammed Rachidi

Copyright copy 2015 Yanzhou Yang et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Ovarian follicular damages were caused by cryoinjury during the process of ovarian vitrification and ischemiareperfusion duringthe process of ovarian transplantation And appropriate FSH plays an important role in antiapoptosis during ovarian follicledevelopment Therefore in this study 03 IUmL FSH was administered into medium during mouse ovarian cryopreservationby vitrification to ascertain the function of FSH on ovarian vitrification and avascular transplantation The results suggestedthat the expressions of Cx37 Cx43 apoptotic molecular caspase-3 and angiogenesis molecular VEGF were confirmed usingimmunohistochemistry western blotting and real-time PCR and the results suggested that the treatment with FSH remarkablyincreased the number of morphologically normal follicles in vitrifiedwarmed ovaries by upregulating the expression of Cx37Cx43 VEGF and VEGF receptor 2 but downregulating the expression of caspase-3 In addition the vitrifiedwarmed ovaries weretransplanted and the related fertility was analyzed and the results suggested that the fertility neoangiogenesis and follicle reservewere remarkably increased in the FSH administrated group Taken together administration of 03 IUmL FSH during ovariancryopreservation by vitrification can maintain ovarian survival during ovarian vitrification and increases the blood supply withavascular transplantation via upregulation of Cx43 Cx37 and VEGFVEGFR2 as well as through its antiapoptotic effects

1 Introduction

Recent studies have suggested that the prevalence of cancerin females has increased by 20 and a trend towardsyoungerwomenhas also been observed [1] Radiotherapy andchemotherapy are the main methods for cancer treatmentand 90 of children and adolescent patients with cancer havehope for a cure [2 3] However the risk of ovarian damageand infertility is present particularly reduction of the primor-dial follicle reserve which may trigger POF (premature ovar-ian failure) [4 5] Thus cryopreserved-thawed ovarian tissueand transplantation act as an important method to preserveovarian function during radiotherapy and chemotherapy andovarian cryopreservation by vitrification is a very effectiveand extensively used method to cryopreserve ovaries [6ndash8]

However due to cryoinjury in ovarian tissue duringvitrification and the possibility of follicular developmentaldelay and partial apoptosis [9ndash11] as well as the fact thatmost ovarian follicles die from ischemiareperfusion injuryin the early stage of transplantation [12] neoangiogenesis isindispensable and increases around the transplanted ovarywithin 48 h [13 14] to protect the survival of the ovarianfollicle Hence the decreasing of follicle die and increasingthe neoangiogenesis are two important objects in ovarianvitrification and transplantation

Previous studies revealed that FSH (follicle-stimulatinghormone) plays an important role in the growth and develop-ment of follicles particularly in the antiapoptosis of the ovar-ian granulosa cell [15ndash17] therefore granulosa cell apoptosisis inevitable in the absence of FSHduring ovarian vitrification

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 397264 13 pageshttpdxdoiorg1011552015397264

2 BioMed Research International

in vitro Previous studies have revealed that granulosa cellsare essential for growth and development of the follicle[18] and that follicular atresia is triggered by granulosa cellapoptosis connexin expression is negatively correlated withfollicle apoptosis [19] during granulosa cell developmentwhich occurs around immature follicle development FSHcan enhance the expression of connexin [20] via indirectinhibition of the activation of the antiapoptotic protein Inaddition ovarian angiogenesis and the expression of VEGFare regulated by FSH [21ndash23]

Our previous studies have suggested that administrationwith 06 IUmLHMG (03 IUmL FSH and 03 IUmLLH) inovarian culture in vitro remarkably improves the blood supplyreconstruction with avascular transplantation and does notcause excessive ovarian follicle activation and depletion [24]

Thus we proposed that FSH might play an importantrole in preserving ovarian survival during cryopreservationby vitrification and avascular transplantation hence in thisstudy 03 IUmL FSH was administrated into vitrificationsolution and the function of FSH was explored in the ovarianvitrification and transplantation

2 Materials and Methods

21 Ethics Statement The Committee for the Ethics onAnimal Care and Experiments in NingxiaMedical Universityapproved the study protocol All operations on animals wereperformed under sodium pentobarbital anesthesia and allefforts were made to minimize suffering

22 Animals and Treatments Four-week-old C57BL6J micewere purchased from Jackson Laboratories (United States IDnumber 000664) and were maintained at 24 plusmn 2∘C in a light-controlled room (12 h light 12 h darkness) with free accessto food and water The estrous cycle was tracked by vaginalsmear according to previous studies [25ndash27] and mice withdiestrus were used for this study

23 Experimental Grouping and Protocol The sacrificedmiceand the collection of ovaries are described below Brieflymice with diestrus were anesthetized with sodium pento-barbital hair on the back was removed with a razor themuscle layer was incised with surgical scissors and theovaries were exposed and collected All procedures wereperformed under aseptic conditions A total of 100 ovarieswere collected from 50 mice and divided into five groupswith 20 whole ovaries in each group The groups weredivided as follows (A) control group (CG) fresh ovarieswere collected from the mice and immediately fixed in4 paraformaldehyde for immunohistochemistry and otherovaries were preserved in liquid nitrogen for RNA andprotein extraction (B) NG-FSH the ovaries underwentvitrifiedwarmed process without any further treatment (C)OG-FSH 03 IUmL FSH was administered into the mediumduring the entire vitrificationwarming process (D) EG-FSH03 IUmL FSH was administered into the medium duringthe early process of vitrified cryopreservation which contains

preculture preequilibrium and osmotic equilibrium (E) LG-FSH 03 IUmL FSH was administered into the mediumduring the late process of vitrified cryopreservation whichconsists of warming and postculture with culture solution

231 Vitrified Cryopreserved Procedure The preparation ofthe basic medium culture freezing and thawing solution aredescribed in supplemental doc1 in Supplementary Materialavailable online at httpdxdoiorg1011552015397264 andthe vitrification procedurewas performed Briefly the processmainly consists of preincubation in culture solution for 1 hat 37∘C with 5 CO

2 The whole ovary was preequilibrated

for 8min with preequilibration solution and then osmoticequilibrium was achieved for 35min with incubation in thevitrified solution The whole ovary was preserved in liquidnitrogen for at least one day warmed for 10min with agradient thawing solution and postcultured for 1 h at 37∘Cwith 5 CO

2with culture solution

24 RNA Extraction and cDNA Synthesis Total RNA wasextracted using TRIzol reagent (TaKaRa Dalian China)according to the manufacturerrsquos instructions Extracted RNAwas dissolved in diethylpyrocarbonate- (DEPC-) treatedwater and the RNA concentration and purity were estimatedat 260 and 280 nm using a spectrophotometer (EppendorfInc Hamburg Germany) The absorption (260280 nm)ratios of all preparations were between 18 and 20 Aliquotsof RNA samples were subjected to electrophoresis using a12 agarose gel with ethidium-bromide staining to confirmRNA integrity The cDNA was reverse-transcribed using thePrimeScipt RT reagent kit (TaKaRaDalian China) accordingto the manufacturerrsquos instructions

25 Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) Real-time quantitative PCR was performed using7500 Software v205 (7500 Fast Real-Time PCR System ABIUSA) The GenBank accession number of the mRNA theprimer sequences and the annealing temperatures are listedin Table 1 The mouse GAPDH gene which was used as areference gene was amplified in parallel with the target geneallowing gene expression normalization and 2minusΔΔCt was usedfor quantificationThe RT-qPCR product was obtained usingSYBR Premix Ex Taq II (TaKaRa Dalian China) accordingto the manufacturerrsquos instructions Each PCR reaction wasperformed in a 200120583L reactionmixture containing 100120583L of2times SYBR Premix Ex Taq II 20 120583L of cDNA (the equivalent of20 ng of total RNA) as the template 08120583L aliquots of eachprimer at 10 120583M and 64 120583L of nuclease-free water The PCRcycling conditions consisted of one cycle at 95∘C for 30 s fol-lowed by 40 cycles at 95∘C for 5 s and finally 60∘C for 30 sTheexperiments were performed in triplicate for each data pointand the mean of these values was used for the final analysis

26 Immunohistochemistry Immunohistochemistry wasperformed according to previous methods [28 29] Brieflyafter dehydration antigen retrieval was performed in citratebuffer (pH 60) by treating the samples twice in a microwaveoven at 100∘C for 15min then the slides were washed three

BioMed Research International 3

Table 1 Primer sequences used for real-time quantitative PCR

Target gene GenBank accession number Primer sequence Product size (bp) Annealing temperature (∘C)

GAPDH XM 0014767073 F 51015840-GGCATTGTGGAAGGGCTC-31015840R 51015840-GACCTTGCCCACAGCCTT-31015840 156 60

Cx43 NM 0102883 F 51015840-CGTCCCACGGAGAAAACCAT-31015840R 51015840-CGGTGGTGGCGTGGTAAG-31015840 151 60

Cx37 NM 0081203 F 51015840-AGCGGTTGCGGCAGAAAG-31015840R 51015840-CCCACGAATCCGAAGACGAC-31015840 137 60

VEGF NM 0010252573 F 51015840-GTAACGATGAAGCCCTGGAGT-31015840R 51015840-TGTTCTGTCTTTCTTTGGTCTGC-31015840 152 60

times with PBS The sections were pretreated with 03(vv) H

2O2in methanol to quench endogenous peroxidase

activity After being washed with PBS the sections wereincubated with 10 goat serum for 30min at 37∘C Followingblocking sections were incubated overnight with rabbit anti-human polyclonal Cx43 antibody (ab11370 1 100 dilutionUK) rabbit anti-mouse polyclonal Cx37 antibody (ab589181 100 dilution UK) rabbit anti-human polyclonal activecaspase-3 antibody (ab2302 1 100 dilution UK) rabbitanti-human polyclonal VEGF (ab46154 1 100 dilution UK)rabbit anti-mouse polyclonal CD31 (ab28364 1 100 dilutionUK) and rabbit anti-mouse polyclonal CD34 (ab812891 100 dilution UK) at 4∘C they were then washed with PBSand incubated with biotinylated anti-rabbit or mouse IgGantibody (Beijing 4A Biotech Co Ltd Beijing China) for 1 hat 37∘C Sections were washed 3 times with PBS and thenincubated with HRP-labeled streptavidin (SA-HRP) for30min at 37∘C Thereafter positive reactions were visualizedwith a diaminobenzidine- (DAB-) peroxidase substrate for30 s and the nuclei were counterstained with hematoxylinAppropriate negative slides were run in parallel without theaddition of primary antibody Slides were imaged using adigital microscope (BA400 Motic Wetzlar Germany)

27 Western Blot Analyses SDS (Sodium Dodecyl Sulfate)gel electrophoresis and western blot analyses were per-formed on homogenates of the ovary samples To stan-dardize the loading samples the protein contents of thecleared supernatant of the homogenates were determinedusing a BCA assay The samples were then separated ona 12 SDS gel with a 5 stacking gel under reducingconditions and transferred onto polyvinylidene difluoride(PVDF) membranes The membranes were blocked for 1 hwith 5 nonfat dry milk and probed with the primaryantibody at a concentration of 1 120583gmLminus1 for 1 h and thenwitha streptavidin-horseradish peroxidase-conjugated anti-rabbitantibody (ZhongBin GOLDEN BRIDGE China) at a dilu-tion of 1 7000 The resulting signal was visualized using theECLDetection Kit (Thermo) according to themanufacturerrsquosinstructions 120573-actin was used as the reference These resultswere analyzed using Image J Software

28 Terminal Deoxynucleotidyl Transferase-Mediated dUTP-Biotin Nick End Labelling Assay (TUNEL) Apoptosis was

determined using the in situ terminal deoxynucleotidyltransferase-mediated nick end labelling (TUNEL) assay(Roche Diagnostics Meylan France) Next the sectionswere washed and double-stained with 1 120583gmL DAPI (410158406-diamidino-2-phenylindole) at room temperature for 20minThe positive ovarian cells were examined using fluorescencemicroscopy and the apoptotic rate was analyzed using IPP60 Software

29 Ovary Orthotopic Transplantation and Quantification ofLitter Size The ovaries from the CG NG-FSH and OG-FSH groups were orthotopically transplanted into 8-week-old allogenic female recipient mice of the C57BL6J-Tyrc-2J-J albino strain There were 10 mice in each group Therecipient female mice underwent oophorectomies prior totransplantation The ovary orthotopic transplantation pro-cedures were performed as previously described [30] Therecipient mice were mated with proven males of the samestrain to induce natural pregnancy at 4-5 days after theemergence of the normal estrous cycle A vaginal smear waschecked on the fifth day eighth day eleventh day fourteenthday and seventeenth day after transplantation the averagelitter size and the average litter size interval which are twoimportant reproductive traits were quantified after delivery

210 Vascular Perfusion Half of a whole ovary (volume of1mm times 1mm times 2mm) was transplanted under the kidneycapsule as previously described [31] 2MD-FITC-Dextran(Sigma FD-2000S)was injected into the tail veins after 48 h oftransplantation and grafts were cut and prepared for frozensections of 60120583m The tissue sections were imaged usinglaser scanning confocal microscopy Other ovarian graftswere collected the ovarian follicle was counted and theneoangiogenesis markers CD31 and CD34 were checked byimmunohistochemistry

211 Follicle Count Paraffin-embedded ovaries were cut intoserial sections and stained using hematoxylin and eosin(HampE) The ovarian follicles were counted according toprevious studies [24 32]

212 Statistical Analyses All experiments were replicated atleast three times for each group The data were presented asthe mean plusmn SEM Data were analyzed with ANOVA followed


SSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSS

PF

S

(a)

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S S

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(c)

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(e)

0102030405060708090

100

CG NG-FSH OG-FSH EG-FSH LG-FSH

Nor

mal

folli

cle (

)

A

A

B

C C

(f)

Figure 1 Quantification of the normal follicle Compared with the CG and NG-FSH groups the morphological structure of the ovariantissues in the OG-FSH group maintained its integrity and the percentage of normal follicles was significantly higher compared to the othergroups (119875 lt 005) (Figure 1) (a)ndash(e) Results of HE staining (a) CG (fresh control group) (b) NG-FSH (no FSH was administered duringovarian vitrification) (c) OG-FSH (FSH was administered during ovarian vitrification) (d) EG-FSH (FSH was administered during the earlyprocess of ovarian vitrification) (e) LG-FSH (FSH was administered during the late process of ovarian vitrification) PF indicates primordialfollicles P indicates primary follicles S indicates secondary follicles (S) (f) Quantification of normal ovarian follicles for the different groupsThe different letters indicate a significant difference the same letters indicate no difference

by Fisherrsquos Least Significant Difference Test (Fisherrsquos LSD)with SPSS software (Version 130 SPSS Inc Chicago IL)Differences were considered significant at 119875 lt 005

3 Results

31 Morphological Observation of the Ovaries Comparedwith the CG and NG-FSH groups the morphological struc-ture of the ovarian tissues in the OG-FSH group showedintegrity and the percentage of morphologically normalfollicles in the OG-FSH group was significantly higher thanthat in the other groups (119875 lt 005) (Figure 1) However

the total number of follicles in each groupwas not statisticallysignificant

32 Detection of Apoptosis during Ovarian Vitrified Cryop-reservation Apoptosis of the ovarian cell was confirmedusing TUNEL and the apoptosis mainly occurred in thegranulosa cell and oocyte of the primary follicle secondaryfollicle and antral follicles Apoptosis was rarely detectedin the primordial follicle These results suggested that theapoptotic rate in the ovarian cell of CG (Figure 2(a))OG-FSH (Figure 2(c)) EG-FSH (Figure 2(d)) and LG-FSH(Figure 2(e)) groups was significantly lower than that in NG-FSH group (Figure 2(b))


(a)

sP

P

PF

(b)

P

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S

(c)

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(d)

P

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S

(e) (f)

012345678


The d

etec

tion

aof ce

llula

r apo

ptos

is

(g)

Figure 2 Apoptosis in the different groups using TUNEL TUNEL results showed that apoptosis mainly occurred in the granulosa cellsand oocytes of the primordial follicle primary follicle secondary follicle and antral follicle with little apoptosis in the primordial follicleTUNEL results suggested that the ovarian apoptotic rates of CG OG-FSH EG-FSH and LG-FSH groups were significantly lower than thatof the NG-FSH group indicates a statistically significant difference compared with the other groups (a) CG (b) NG-FSH (c) OG-FSH(d) EG-FSH (e) LG-FSH (f) Negative control (g) Analysis of apoptotic cell PF indicates primordial follicles P indicates primary follicles Sindicates secondary follicles (S)

Consistent with the results from TUNEL the apoptoticmarker active caspase-3 was also mainly localized in thegranulosa cells and oocytes (Figures 3(a)ndash3(e)) and the levelsof protein (Figure 3(g)) in the CG OG-FSH EG-FSH andLG-FSH groups were significantly lower than those in theNG-FSH group

33 Expression of Cx43 Cx37 and VEGFVEGF Receptor 2(VEGFR2) Localization of Cx43 and Cx37 was confirmedusing immunohistochemistry according to previous studies[28 29] Cx43 was mainly localized in the granulosa cell(Figures 4(a)ndash4(e)) and Cx37 was mainly localized in theoocyte and granulosa cell (Figures 5(a)ndash5(e)) Furthermorethe expression of Cx43 (Figures 4(g)-4(h)) and Cx37 (Figures5(g)-5(h)) mRNA and protein in the OG-FSH EG-FSH and

LG-FSH groups was significantly higher than that in the CGand NG-FSH groups (119875 lt 005)

In addition the expression of VEGF mRNA was con-firmed and the results suggested that the expression ofVEGF in the OG-FSH EG-FSH and LG-FSH groups wassignificantly higher than that in the other groups (119875 lt005) and the protein expression of VEGF and VEGFR2 wasconfirmed and the results suggested that the expression ofVEGF (Figures 6(a)-6(b)) and VEGFR2 (Figure 6(c)) in theOG-FSH groupwas significantly higher than that in the othergroups (119875 lt 005)

34 Morphological Observation of the Ovaries 48 h afterTransplantation The ovaries of CG NG-FSH and OG-FSH were heterotopically transplanted by the kidney capsule(supplemental doc2a) after 48 h the ovarian grafts were


(a) (b) (c)

(d) (e) (f)

005

115

225


A

B

C

A

C

Activ

e cas

pase

-3120573

-act

in

120573-actin

Active caspase-317KD

43KD

(g)

Figure 3 The check of apoptotic marker caspase-3 (a)ndash(f) Localization of the apoptotic marker active caspase-3 using immunohistochem-istry The apoptotic molecular marker active caspase-3 was mainly localized in the granulosa cell and oocyte and specifically in the primaryfollicle secondary follicle and antral follicle (a) CG (b) NG-FSH (c) OG-FSH (d) EG-FSH (e) LG-FSH (f) Negative control (g) Caspase-3protein expression Caspase-3 protein expression in the CG OG-FSH EG-FSH LG-FSH and NG-FSH groups The different letters indicatesignificant differences the same letters indicate no differences

embedded with paraffin and cut into serial sections andstained using hematoxylin and eosin (HampE) and the resultssuggested that the ovarian morphology of CG (Figure 7(a))and OG-FSH (Figure 7(c)) groups maintained its integrityhowever the ovarian morphology of NG-FSH (Figure 7(b))revealed scant follicles and scattered ovarian stroma

The ovarian follicle was counted according to previousstudies [24 32] and the percentage of primordial folliclesprimary follicles and morphologically normal follicles (allstage follicles) in the CG and OG-FSH groups was signifi-cantly higher than that in the NG-FSH group (119875 lt 005)The percentage of atretic follicles in the CG andOG-FSHwas

significantly lower than that in the NG-FSH group (119875 lt 005)(Table 2)

35 Confirmation of Neoangiogenesis Consistent with theexpression of VEGF and VEGFR2 neoangiogenesis in theCGandOG-FSHgroupswas remarkably increased comparedwith the NG-FSH group (Figures 8(a)ndash8(c)) In addition theendothelial cell markers CD31 and CD34 were checked byimmunohistochemistry and the results suggested that theexpression of CD31 (Figures 8(d)ndash8(f)) and CD34 (Figures8(g)ndash8(i)) in the CG and OG-FSH groups was remarkablyincreased compared with the NG-FSH group


(a) (b) (c)

(d) (e) (f)

05

10152025303540

CG NG-FSH OG-FSH EG-FSH LG-FSHA

C

B

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Cx43

GA

PDH

(g)

002040608

112141618


A

C

B

A

C

Cx43

120573-a

ctin

Cx43

120573-actin

43KD

42KD

(h)

Figure 4 Detection of Cx43 expression (a)ndash(f) Localization of Cx43 in the ovarian cell among the different groups (a) CG (b) NG-FSH(c) OG-FSH (d) EG-FSH (e) LG-FSH (f) Negative control (g)-(h) Cx43 mRNA and protein expression (g) Cx43 mRNA expression in theCG OG-FSH EG-FSH LG-FSH and NG-FSH groups (h) Cx43 protein expression in the CG OG-FSH EG-FSH LG-FSH and NG-FSHgroups The different letters indicate significant differences the same letters indicate no differences

36 Quantification of Litter Size of the Ovarian TransplantedMice Ovaries obtained from the CG NG-FSH and OG-FSH groups were orthotopically transplanted (supplementaldoc2b) The pregnant female mice were allowed to litterand the size of each litter was recorded the pups have blackfur (supplemental doc3 a CG b OG-FSH c NG-FSH)After 6 and 12 months the number of pregnant mice thepregnancy rate the average litter size and the average littersize interval of each group were determined (Tables 3 and 4)In addition the number of pregnant mice in the CG NG-FSH and OG-FSH groups was 5 3 and 4 respectively and

the pregnancy rate of the CG NG-FSH and OG-FSH groupswas 50 30 and 40 respectively Moreover the numberof pregnant mice and pregnancy rate of the CG and OG-FSH groups were remarkably increased compared with theNG-FSH group The average litter size in the CG and OG-FSH groups was significantly higher than that in the NG-FSHgroup (119875 lt 005) and the average litter size interval in theCG and OG-FSH groups was lower than that in the NG-FSHgroup (119875 lt 005)

In addition recovery of the estrous cycle after trans-plantation on days 5 8 11 14 and 17 was determined and


(a) (b) (c)

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10152025


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Cx37

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(g)

002040608

112


B

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120573-a

ctin

Cx37

120573-actin37KD42KD

(h)

Figure 5 Detection of Cx37 expression (a)ndash(f) Localization of Cx37 in the ovarian cell among the different groups (a) CG (b) NG-FSH (c)OG-FSH (d) EG-FSH (e) LG-FSH (f) Negative control (g) Cx37 mRNA expression in the CG OG-FSH EG-FSH LG-FSH and NG-FSHgroups (h) Cx37 protein expression in the CG OG-FSH EG-FSH LG-FSH and NG-FSH groups The different letters indicate significantdifferences the same letters indicate no differences

the results suggested that the number of mice with regular4-day estrous cycles in the OG-FSH group was remarkablyhigher than the number of mice in the NG-FSH group(Figure 9)

4 Discussion

There is little damage to the primordial follicle during ovarianvitrification but the damage from the primary to the maturefollicles is caused by granulosa cell apoptosis [33] In additionmost ovarian follicles especiallymost primordial follicles dieas a result of ischemiareperfusion injury in the early stagesof transplantation [12] Therefore antiapoptosis in ovarianvitrification and improving the blood supply in early ovariantransplantation are two main issues for successful birth of

offspring after ovarian vitrification Indeed the existence ofapoptosis in ovarian cells during vitrification was furthersupported by our results from the detection of the apoptoticmarker active caspase-3 and TUNEL analysis Furthermoreour results suggested that administrationwith 03 IUmLFSHsignificantly decreased the apoptosis of granulosa cells andFSH might play a key role in preventing apoptosis duringovarian vitrification Indeed FSH suppressed apoptosis ofovarian granulosa cells [34] and promoted the follicle mat-uration [35] Several studies have suggested that FSH is theearliest discovered hormone to stimulate the growth of theovarian follicle although complete knowledge of the milieufor follicular growth in vivo is still lacking However thereis evidence that adequate levels of specific hormones areessential for the growth of healthy follicles in vitro [36] The


012345678


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b

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c

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FR120573

-act

in

VEGFR2

120573-actin

151KD

42KD

(c)

Figure 6 Detection of VEGF mRNA and VEGF and VEGFR2 protein expression (a) Results of VEGF mRNA expression (b) Results ofVEGF protein expression (c) Results of VEGFR2 protein expression The different letters indicate significant differences the same lettersindicate no differences

(a) (b) (c)

Figure 7 Ovarian morphology 48 h after transplantation The ovarian morphology of CG and OG-FSH maintained its integrity but theovarianmorphology ofNG-FSHdisplayed scant follicles and scattered ovarian stroma (a) CG (b)NG-FSH (c)OG-FSH Red arrow indicatesthe ovarian grafts blue arrow indicates the renal tissue

pituitary gonadotropin FSHmay promote if not be essentialfor follicular growth in culture systems [37] MoreoverFSH administration not only maintained the morphologicalintegrity of caprine preantral follicles but also stimulated theactivation of primordial follicles and the growth of activatedfollicles in culture [38] Indeed additional FSH treatmentmay stimulate excess primordial follicle development thusaccelerating follicular pool consumption and administrationof FSH after ovary transplantation might increase follicular

pool consumption [39] this may be attributed to the con-centration of FSH indeed treatment with 25 IU FSH twicedaily within 1 week following transplantation partly preventsprimordial follicle loss in fresh and frozen-thawed tissues[40] therefore appropriate concentration of FSH plays animportant role in ovarian vitrification

Our results suggested that ovarian tissues treated with03 IUmL FSH during ovarian cryopreservation by vit-rification remarkably protected the ovarian integrity and


Table 2 The count of follicles on 48 h after ovarian transplantation

Group The percentage ofprimordial follicle

The percentage of primaryfollicle

The percentage of normalfollicle

The percentage of atreticfollicle

CG 369 plusmn 64lowast 134 plusmn 54lowast 503 plusmn 63lowast 497 plusmn 63lowast

NG-FSH 257 plusmn 41 27 plusmn 13 289 plusmn 51 715 plusmn 58OG-FSH 349 plusmn 85lowast 86 plusmn 16lowast 435 plusmn 77lowast 565 plusmn 77lowast

lowast indicates statistically significant difference in the same column

(a) (b) (c)

(d) (e) (f)

(g) (h) (i)

Figure 8 Detection of neoangiogenesis using vascular perfusion The green fluorescence indicates neoangiogenesis (a) CG (b) NG-FSH(c) OG-FSH (d)ndash(f) The expression of CD31 in the CG and OG-FSH groups was remarkably increased compared to the NG-FSH group (d)CG (e) NG-FSH (f) OG-FSH (g)ndash(i) The expression of CD34 in the CG and OG-FSH groups was remarkably increased compared to theNG-FSH group (g) CG (h) NG-FSH (i) OG-FSH Red arrow indicates the ovarian grafts blue arrow indicates the renal tissue

decreased the apoptotic rate Consistent with our resultsFriedmanrsquos results also suggested that FSHplays an importantrole in ovarian vitrified cryopreservation [41]

To explore the mechanism of FSH during ovarian cry-opreservation by vitrification the expression of Cx43 and

Cx37 was evaluated and these results suggested that themRNA and protein levels of Cx43 and Cx37 in the OG-FSH group were significantly higher than those in the othergroups Moreover the expression levels of Cx43 and Cx37were upregulated by FSH during ovarian cryopreservation


Table 3 The counts of pregnancy rate and litter sizes of ovarian transplanted mice (6 months)

GroupThe number of

ovarian transplantedmice

The number ofpregnant mice Pregnancy rate () Average of litter sizes The average litter size

interval (days)

CG 10 5 50 581 plusmn 128lowast 4358 plusmn 848lowast

NG-FSH 10 3 30 375 plusmn 071 534 plusmn 1189OG-FSH 10 4 40 527 plusmn 110lowast 405 plusmn 727lowast



GroupThe number of



interval (days)




0123456789

10

The days after transplantation

The n

umbe

r of m

ice w

ith

norm

al es

trous

cycle

5 8 11 14 17

Figure 9 Recovery of the estrous cycle after transplantationRecovery of the estrous cycle after transplantation in the OG-FSHgroup was remarkably higher than mice in the NG-FSH group --indicates the OG-FSH group -◻- indicates the NG-FSH group

by vitrification Indeed Cx43 and Cx37 play importantroles in the development and growth of follicles and Cx43and Cx37 were regulated by FSH [20 42] Thus Cx43 andCx37 represent downstreammolecular targets of FSH duringovarian cryopreservation by vitrification and FSH protectsthe intact ovary via the Cx43 and Cx37 signaling pathwayIndeed previous studies have suggested that the expressionof Cx43 and Cx37 in vitrifiedwarmed ovaries was lowerthan that in normal ovaries [42] Furthermore the signaltransduction by connexin between granulosa cells and theoocytes was disturbed during vitrified cryopreservation [43]

Most ovarian follicles die from ischemiareperfusioninjury in the early stages of transplantation [12] thus neoan-giogenesis and blood supply reconstruction were indispens-able and increased around the transplanted ovary within48 h [13 14] to protect ovarian follicle survival Thereforethe expression of VEGF and VEGFR2 was confirmed andresults suggested that the expression of VEGF and VEGFR2in the OG-FSH group was significantly higher than thatin the other groups Furthermore the expression of VEGF

and VEGFR2 was regulated by FSH during ovarian cryop-reservation by vitrification Indeed previous studies havesuggested that VEGF and VEGFR2 were regulated by FSH[22 44 45] and upregulation of VEGF and VEGFR2 wasbeneficial both for the formation of new blood vessels duringovary transplantation and for increasing the rate of ovarytransplantation In addition VEGF suppressed the apoptosisof ovarian granulosa cells [46] thus upregulation of VEGFand VEGFR2 plays an important role in antiapoptosis of theovarian granulosa cell and the maintenance of the primordialfollicle pool [47] Indeed VEGF protects the granulosa cellfrom apoptosis during the freeze-thaw process [48 49]Accordingly ovarian vascular perfusion was performed at48 h after successful transplantation and these results sug-gested that neoangiogenesis in the OG-FSH and CG groupswas remarkably increased comparedwith theNG-FSHgroupthese data were further validated by immunohistochemicalstaining with the endothelial cell markers CD31 and CD34indeed the expression of CD31 and CD34 was used toanalyze microvessel density [49] Moreover the increasedblood supply protected the primordial follicle from apoptosisunder conditions of ischemia and hypoxia during the earlystage of transplantation [49ndash51] and preserved the ovarianreserve Indeed more primordial follicles died from hypoxiaand delayed revascularization than from freeze-thaw injuries[52]

Furthermore the related index was quantified and ana-lyzed and the number of pregnant mice and the pregnancyrate in the OG-FSH group were remarkably increased com-pared with the NG-FSH group In addition the averagelitter size and the average litter size interval in the OG-FSH group were significantly higher than those in the NG-FSH group In addition the recovery of the estrous cycle inthe OG-FSH group was shorter than that in the NG-FSHgroup Thus FSH remarkably increased the rate of ovarian


transplantation and VEGF and VEGFR2 might be down-stream molecular signals of FSH during ovarian vitrifiedcryopreservation

In conclusion FSH administration during ovarian cry-opreservation by vitrification maintained ovarian survivalspecifically bymaintaining ovarian integrity decreasing ovar-ian apoptosis increasing the blood supply of the transplantedovary and increasing the transplanted rate via upregulation ofthe expression of Cx43 Cx37 and VEGFVEGFR2

Conflict of Interests

All the authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Yanzhou Yang and Jie Chen contributed equally to this work

Acknowledgments

This work was supported by National Natural Science Foun-dation ofChina (81160085 and 30960108) andNatural ScienceFoundation of Ningxia of China (NZ1084)

References

[1] TMaltaris MW Beckmann and R Dittrich ldquoReview Fertilitypreservation for young female cancer patientsrdquo In Vitro vol 23no 1 pp 123ndash130 2009

[2] R Pieters ldquoAcute lymphoblastic leukaemia in children andadolescents chance of cure now higher than 80rdquo NederlandsTijdschrift voor Geneeskunde vol 154 no 35 Article ID A15772010

[3] E Tanzler C G Morris J M Kirwan R J Amdur and WM Mendenhall ldquoOutcomes of WHO grade i meningiomasreceiving definitive or postoperative radiotherapyrdquo Interna-tional Journal of Radiation Oncology Biology Physics vol 79 no2 pp 508ndash513 2011

[4] G Fasano F Moffa J Dechene Y Englert and I DemeestereldquoVitrification of in vitro matured oocytes collected from antralfollicles at the time of ovarian tissue cryopreservationrdquo Repro-ductive Biology and Endocrinology vol 9 article 150 2011

[5] Y Chuai X Xu and A Wang ldquoPreservation of fertility infemales treated for cancerrdquo International Journal of BiologicalSciences vol 8 no 7 pp 1005ndash1012 2012

[6] C A Amorim M-M Dolmans A David et al ldquoVitrificationand xenografting of human ovarian tissuerdquo Fertility and Steril-ity vol 98 no 5 pp 1291ndash1298 2012

[7] C A Amorim S Jacobs R V Devireddy et al ldquoSuccessfulvitrification and autografting of baboon (Papio anubis) ovariantissuerdquoHuman Reproduction vol 28 no 8 pp 2146ndash2156 2013

[8] M Sheikhi K Hultenby B Niklasson M Lundqvist and OHovatta ldquoClinical grade vitrification of human ovarian tissuean ultrastructural analysis of follicles and stroma in vitrifiedtissuerdquo Human Reproduction vol 26 no 3 pp 594ndash603 2011

[9] R Fathi M R Valojerdi and M Salehnia ldquoEffects of differentcryoprotectant combinations on primordial follicle survivabil-ity and apoptosis incidence after vitrification ofwhole rat ovaryrdquoCryo-Letters vol 34 no 3 pp 228ndash238 2013

[10] T Mazoochi M Salehnia M R Valojerdi and S J MowlaldquoMorphologic ultrastructural and biochemical identificationof apoptosis in vitrified-warmedmouse ovarian tissuerdquo Fertilityand Sterility vol 90 no 4 pp 1480ndash1486 2008

[11] M Salehnia M Sheikhi S Pourbeiranvand and M LundqvistldquoApoptosis of human ovarian tissue is not increased by eithervitrification or rapid coolingrdquoReproductive BioMedicineOnlinevol 25 no 5 pp 492ndash499 2012

[12] M Mahmoodi M S Mehranjani S M A ShariatzadehH Eimani and A Shahverdi ldquoEffects of erythropoietin onischemia follicular survival and ovarian function in ovariangraftsrdquo Reproduction vol 147 no 5 pp 733ndash741 2014

[13] F Tang C Zhang X-J Wang D-D Wu and Y ZhouldquoExpression and clinical significance of VEGF and apoptosis infrozen-thawed mouse ovaries after transplantationrdquo ZhongguoYing Yong Sheng Li Xue Za Zhi vol 26 no 2 pp 172ndash176 2010

[14] D Wu Y Lei Y Tong F Tang Y Qian and Y ZhouldquoAngiogenesis of the frozen-thawed human fetal ovarian tissueat the early stage after xenotransplantation and the positiveeffect of Salviae miltiorrhizaerdquo Anatomical Record vol 293 no12 pp 2154ndash2162 2010

[15] D Baird ldquoRole of FSH and LH in follicle developmentrdquo Journalde Gynecologie Obstetrique et Biologie de la Reproduction vol35 no 5 pp 2S24ndash2S29 2006

[16] CWang and S K Roy ldquoExpression of bonemorphogenetic pro-tein receptor (BMPR) during perinatal ovary development andprimordial follicle formation in the hamster possible regulationby FSHrdquo Endocrinology vol 150 no 4 pp 1886ndash1896 2009

[17] C Zhang L Guo B Zhu et al ldquoEffects of 3 5 31015840-triiodothyronine (T3) and follicle stimulating hormone onapoptosis and proliferation of rat ovarian granulosa cellsrdquo TheChinese journal of physiology vol 56 no 5 pp 298ndash305 2013

[18] F H Thomas and B C Vanderhyden ldquoOocyte-granulosa cellinteractions duringmouse follicular development regulation ofkit ligand expression and its role in oocyte growthrdquo Reproduc-tive Biology and Endocrinology vol 4 article 19 2006

[19] D V Krysko S Mussche L Leybaert and K DrsquoHerde ldquoGapjunctional communication and connexin43 expression in rela-tion to apoptotic cell death and survival of granulosa cellsrdquoJournal of Histochemistry and Cytochemistry vol 52 no 9 pp1199ndash1207 2004

[20] M L Johnson D A Redmer L P Reynolds J J Bilski andA T Grazul-Bilskal ldquoGap junctional intercellular commu-nication of bovine granulosa and thecal cells from antralfollicles effects of luteinizing hormone and follicle-stimulatinghormonerdquo Endocrine vol 18 no 3 pp 261ndash270 2002

[21] L K Doyle C AWalker and F X Donadeu ldquoVEGFmodulatesthe effects of gonadotropins in granulosa cellsrdquo DomesticAnimal Endocrinology vol 38 no 3 pp 127ndash137 2010

[22] S-W Kuo F-C Ke G-D Chang M-T Lee and J-J HwangldquoPotential role of follicle-stimulating hormone (FSH) and trans-forming growth factor (TGF1205731) in the regulation of ovarianangiogenesisrdquo Journal of Cellular Physiology vol 226 no 6 pp1608ndash1619 2011

[23] S-Y Shin H-J Lee D-S Ko H-C Lee and W I Park ldquoTheregulators of VEGF expression in mouse ovariesrdquo Yonsei Medi-cal Journal vol 46 no 5 pp 679ndash686 2005

[24] Y Wang Q Chang J Sun et al ldquoEffects of HMG on revascu-larization and follicular survival in heterotopic autotransplantsof mouse ovarian tissuerdquo Reproductive BioMedicine Online vol24 no 6 pp 646ndash653 2012


[25] A-H Zarnani S-M Moazzeni F Shokri M Salehnia and MJeddi Tehrani ldquoAnalysis of endometrial myeloid and lymphoiddendritic cells duringmouse estrous cyclerdquo Journal of Reproduc-tive Immunology vol 71 no 1 pp 28ndash40 2006

[26] H Ni X-J Yu H-J Liu et al ldquoProgesterone regulation ofglutathione S-transferase Mu2 expression in mouse uterineluminal epithelium during preimplantation periodrdquo Fertilityand Sterility vol 91 no 5 pp 2123ndash2130 2009

[27] Y Yang Y Jin P Lin et al ldquoThe expression and localization ofLRF in the female reproductive tract of cyclingmice throughoutthe estrous cyclerdquo Journal of Immunoassay and Immunochem-istry vol 34 no 4 pp 313ndash322 2013

[28] Y Yang P Lin F Chen et al ldquoLuman recruiting factor regulatesendoplasmic reticulum stress in mouse ovarian granulosa cellapoptosisrdquoTheriogenology vol 79 no 4 pp 633ndash639 2013

[29] Y Yang Y Jin A C Martyn et al ldquoExpression pattern impli-cates a potential role for luman recruitment factor in the processof implantation in uteri and development of preimplantationembryos in micerdquo Journal of Reproduction and Developmentvol 59 no 3 pp 245ndash251 2013

[30] J E Marano D Sun A M Zama W Young and M UzumculdquoOrthotopic transplantation of neonatal GFP rat ovary as exper-imental model to study ovarian development and toxicologyrdquoReproductive Toxicology vol 26 no 3-4 pp 191ndash196 2008

[31] X Deng H Zheng X Yu and et al ldquoCryopreserved ovariantissues can maintain a long-term function after heterotopicautotransplantation in ratrdquoReproduction vol 138 no 3 pp 519ndash525 2009

[32] J L Tilly ldquoOvarian follicle countsmdashnot as simple as 1 2 3rdquoReproductive Biology and Endocrinology vol 1 article 11 2003

[33] R Depalo G Loverro and L Selvaggi ldquoIn vitro maturationof primordial follicles after cryopreservation of human ovariantissue problems remainrdquo Medical and Pediatric Oncology vol38 no 3 pp 153ndash157 2002

[34] P Lin andR Rui ldquoEffects of follicular size and FSHon granulosacell apoptosis and atresia in porcine antral folliclesrdquo MolecularReproduction andDevelopment vol 77 no 8 pp 670ndash678 2010

[35] H Han J F Silverman T S Santucci et al ldquoVascular endothe-lial growth factor expression in stage I non-small cell lungcancer correlates with neoangiogenesis and a poor prognosisrdquoAnnals of Surgical Oncology vol 8 no 1 pp 72ndash79 2001

[36] HM Picton S E HarrisWMuruvi and E L Chambers ldquoThein vitro growth and maturation of folliclesrdquo Reproduction vol136 no 6 pp 703ndash715 2008

[37] J Xu M P Bernuci M S Lawson et al ldquoSurvival growthand maturation of secondary follicles from prepubertal youngand older adult rhesus monkeys during encapsulated three-dimensional culture effects of gonadotropins and insulinrdquoReproduction vol 140 no 5 pp 685ndash697 2010

[38] M H T Matos I B Lima-Verde M C A Luque et al ldquoEssen-tial role of follicle stimulating hormone in the maintenance ofcaprine preantral follicle viability in vitrordquo Zygote vol 15 no 2pp 173ndash182 2007

[39] T Maltaris M W Beckmann A Mueller I Hoffmann JKohl and R Dittrich ldquoSignificant loss of primordial folliclesafter prolonged gonadotropin stimulation in xenografts ofcryopreserved human ovarian tissue in severe combinedimmunodeficient micerdquo Fertility and Sterility vol 87 no 1 pp195ndash197 2007

[40] V von Schonfeldt R Chandolia R Ochsenkuhn E NieschlagL Kiesel and B Sonntag ldquoFSHprevents depletion of the restingfollicle pool by promoting follicular number and morphology

in fresh and cryopreserved primate ovarian tissues followingxenograftingrdquo Reproductive Biology and Endocrinology vol 10article 98 2012

[41] O Friedman R Orvieto B Fisch et al ldquoPossible improvementsin human ovarian grafting by various host and graft treatmentsrdquoHuman Reproduction vol 27 no 2 pp 474ndash482 2012

[42] B Sommersberg A Bulling U Salzer et al ldquoGap junctioncommunication and connexin 43 gene expression in a ratgranulosa cell line regulation by follicle-stimulating hormonerdquoBiology of Reproduction vol 63 no 6 pp 1661ndash1668 2000

[43] P Navarro-Costa S C Correia A Gouveia-Oliveira et alldquoEffects of mouse ovarian tissue cryopreservation on granulosacell-oocyte interactionrdquoHuman Reproduction vol 20 no 6 pp1607ndash1614 2005

[44] H Alam J Week E Maizels et al ldquoRole of the phosphat-idylinositol-3-kinase and extracellular regulated kinase path-ways in the induction of hypoxia-inducible factor (HIF)-1activity and the HIF-1 target vascular endothelial growth factorin ovarian granulosa cells in response to follicle-stimulatinghormonerdquo Endocrinology vol 150 no 2 pp 915ndash928 2009

[45] Y Huang K Hua X Zhou et al ldquoActivation of the PI3KAKTpathway mediates FSH-stimulated VEGF expression in ovarianserous cystadenocarcinomardquo Cell Research vol 18 no 7 pp780ndash791 2008

[46] N Kosaka N Sudo A Miyamoto and T Shimizu ldquoVascularendothelial growth factor (VEGF) suppresses ovarian granulosacell apoptosis in vitrordquo Biochemical and Biophysical ResearchCommunications vol 363 no 3 pp 733ndash737 2007

[47] A E Roberts L K Arbogast C I Friedman D E Cohn P TKaumaya and D R Danforth ldquoNeutralization of endogenousvascular endothelial growth factor depletes primordial folliclesin the mouse ovaryrdquo Biology of Reproduction vol 76 no 2 pp218ndash223 2007

[48] S-Y Shin J-Y Lee E Lee et al ldquoProtective effect of vascularendothelial growth factor (VEGF) in frozen-thawed granulosacells is mediated by inhibition of apoptosisrdquo European Journalof Obstetrics Gynecology and Reproductive Biology vol 125 no2 pp 233ndash238 2006

[49] G Irusta D Abramovich F Parborell and M Tesone ldquoDirectsurvival role of vascular endothelial growth factor (VEGF) onrat ovarian follicular cellsrdquo Molecular and Cellular Endocrinol-ogy vol 325 no 1-2 pp 93ndash100 2010

[50] R K-K Lee H-YHo S-L Yu andC-H Lu ldquoBlastocyst devel-opment after cryopreservation and subcutaneous transplanta-tion of mouse ovarian tissuerdquo Journal of Assisted Reproductionand Genetics vol 22 no 2 pp 95ndash101 2005

[51] T Israely N Nevo A Harmelin M Neeman and A TsafririldquoReducing ischaemic damage in rodent ovarian xenograftstransplanted into granulation tissuerdquoHuman Reproduction vol21 no 6 pp 1368ndash1379 2006

[52] R K-K Lee S-H Li C-H Lu H-Y Ho Y-J Chen andH-I Yeh ldquoAbnormally low expression of connexin 37 andconnexin 43 in subcutaneously transplanted cryopreservedmouse ovarian tissuerdquo Journal of Assisted Reproduction andGenetics vol 25 no 9-10 pp 489ndash497 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


in vitro Previous studies have revealed that granulosa cellsare essential for growth and development of the follicle[18] and that follicular atresia is triggered by granulosa cellapoptosis connexin expression is negatively correlated withfollicle apoptosis [19] during granulosa cell developmentwhich occurs around immature follicle development FSHcan enhance the expression of connexin [20] via indirectinhibition of the activation of the antiapoptotic protein Inaddition ovarian angiogenesis and the expression of VEGFare regulated by FSH [21ndash23]

Our previous studies have suggested that administrationwith 06 IUmLHMG (03 IUmL FSH and 03 IUmLLH) inovarian culture in vitro remarkably improves the blood supplyreconstruction with avascular transplantation and does notcause excessive ovarian follicle activation and depletion [24]

Thus we proposed that FSH might play an importantrole in preserving ovarian survival during cryopreservationby vitrification and avascular transplantation hence in thisstudy 03 IUmL FSH was administrated into vitrificationsolution and the function of FSH was explored in the ovarianvitrification and transplantation

2 Materials and Methods

21 Ethics Statement The Committee for the Ethics onAnimal Care and Experiments in NingxiaMedical Universityapproved the study protocol All operations on animals wereperformed under sodium pentobarbital anesthesia and allefforts were made to minimize suffering

22 Animals and Treatments Four-week-old C57BL6J micewere purchased from Jackson Laboratories (United States IDnumber 000664) and were maintained at 24 plusmn 2∘C in a light-controlled room (12 h light 12 h darkness) with free accessto food and water The estrous cycle was tracked by vaginalsmear according to previous studies [25ndash27] and mice withdiestrus were used for this study

23 Experimental Grouping and Protocol The sacrificedmiceand the collection of ovaries are described below Brieflymice with diestrus were anesthetized with sodium pento-barbital hair on the back was removed with a razor themuscle layer was incised with surgical scissors and theovaries were exposed and collected All procedures wereperformed under aseptic conditions A total of 100 ovarieswere collected from 50 mice and divided into five groupswith 20 whole ovaries in each group The groups weredivided as follows (A) control group (CG) fresh ovarieswere collected from the mice and immediately fixed in4 paraformaldehyde for immunohistochemistry and otherovaries were preserved in liquid nitrogen for RNA andprotein extraction (B) NG-FSH the ovaries underwentvitrifiedwarmed process without any further treatment (C)OG-FSH 03 IUmL FSH was administered into the mediumduring the entire vitrificationwarming process (D) EG-FSH03 IUmL FSH was administered into the medium duringthe early process of vitrified cryopreservation which contains

preculture preequilibrium and osmotic equilibrium (E) LG-FSH 03 IUmL FSH was administered into the mediumduring the late process of vitrified cryopreservation whichconsists of warming and postculture with culture solution

231 Vitrified Cryopreserved Procedure The preparation ofthe basic medium culture freezing and thawing solution aredescribed in supplemental doc1 in Supplementary Materialavailable online at httpdxdoiorg1011552015397264 andthe vitrification procedurewas performed Briefly the processmainly consists of preincubation in culture solution for 1 hat 37∘C with 5 CO

2 The whole ovary was preequilibrated

for 8min with preequilibration solution and then osmoticequilibrium was achieved for 35min with incubation in thevitrified solution The whole ovary was preserved in liquidnitrogen for at least one day warmed for 10min with agradient thawing solution and postcultured for 1 h at 37∘Cwith 5 CO

2with culture solution

24 RNA Extraction and cDNA Synthesis Total RNA wasextracted using TRIzol reagent (TaKaRa Dalian China)according to the manufacturerrsquos instructions Extracted RNAwas dissolved in diethylpyrocarbonate- (DEPC-) treatedwater and the RNA concentration and purity were estimatedat 260 and 280 nm using a spectrophotometer (EppendorfInc Hamburg Germany) The absorption (260280 nm)ratios of all preparations were between 18 and 20 Aliquotsof RNA samples were subjected to electrophoresis using a12 agarose gel with ethidium-bromide staining to confirmRNA integrity The cDNA was reverse-transcribed using thePrimeScipt RT reagent kit (TaKaRaDalian China) accordingto the manufacturerrsquos instructions

25 Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) Real-time quantitative PCR was performed using7500 Software v205 (7500 Fast Real-Time PCR System ABIUSA) The GenBank accession number of the mRNA theprimer sequences and the annealing temperatures are listedin Table 1 The mouse GAPDH gene which was used as areference gene was amplified in parallel with the target geneallowing gene expression normalization and 2minusΔΔCt was usedfor quantificationThe RT-qPCR product was obtained usingSYBR Premix Ex Taq II (TaKaRa Dalian China) accordingto the manufacturerrsquos instructions Each PCR reaction wasperformed in a 200120583L reactionmixture containing 100120583L of2times SYBR Premix Ex Taq II 20 120583L of cDNA (the equivalent of20 ng of total RNA) as the template 08120583L aliquots of eachprimer at 10 120583M and 64 120583L of nuclease-free water The PCRcycling conditions consisted of one cycle at 95∘C for 30 s fol-lowed by 40 cycles at 95∘C for 5 s and finally 60∘C for 30 sTheexperiments were performed in triplicate for each data pointand the mean of these values was used for the final analysis

26 Immunohistochemistry Immunohistochemistry wasperformed according to previous methods [28 29] Brieflyafter dehydration antigen retrieval was performed in citratebuffer (pH 60) by treating the samples twice in a microwaveoven at 100∘C for 15min then the slides were washed three




















PF

S

(a)

PPF

S S

(b)

SSS

PF

(c)

S

S

(d)

P

PF

(e)

0102030405060708090

100


Nor

mal

folli

cle (

)

A

A

B

C C

(f)



3 Results





(a)

sP

P

PF

(b)

P

PF

S

(c)

PF

P

(d)

P

PF

S

(e) (f)

012345678


The d

etec

tion

aof ce

llula

r apo

ptos

is

(g)








(a) (b) (c)

(d) (e) (f)

005

115

225


A

B

C

A

C

Activ

e cas

pase

-3120573

-act

in

120573-actin


43KD

(g)







(a) (b) (c)

(d) (e) (f)

05

10152025303540


C

B

A

C

Cx43

GA

PDH

(g)

002040608

112141618


A

C

B

A

C

Cx43

120573-a

ctin

Cx43

120573-actin

43KD

42KD

(h)






(a) (b) (c)

(d) (e) (f)

05

10152025


Ad

cA d

B

C C

Cx37

GA

PDH

(g)

002040608

112


B

CA

A

C

Cx37

120573-a

ctin

Cx37


(h)



4 Discussion




012345678


VEG

FG

APD

H

a

c

c

b

c

(a)

005

115

225

3


b

caa

c

VEG

F120573

-act

in

VEGF

120573-actin

43KD

42KD

(b)

005

115

225

3


b

caa

c

VEG

FR120573

-act

in

VEGFR2

120573-actin

151KD

42KD

(c)


(a) (b) (c)














(a) (b) (c)

(d) (e) (f)

(g) (h) (i)







GroupThe number of



interval (days)





GroupThe number of



interval (days)




0123456789

10


The n

umbe

r of m

ice w

ith

norm

al es

trous

cycle

5 8 11 14 17













Acknowledgments


References






























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




















PF

S

(a)

PPF

S S

(b)

SSS

PF

(c)

S

S

(d)

P

PF

(e)

0102030405060708090

100


Nor

mal

folli

cle (

)

A

A

B

C C

(f)



3 Results





(a)

sP

P

PF

(b)

P

PF

S

(c)

PF

P

(d)

P

PF

S

(e) (f)

012345678


The d

etec

tion

aof ce

llula

r apo

ptos

is

(g)








(a) (b) (c)

(d) (e) (f)

005

115

225


A

B

C

A

C

Activ

e cas

pase

-3120573

-act

in

120573-actin


43KD

(g)







(a) (b) (c)

(d) (e) (f)

05

10152025303540


C

B

A

C

Cx43

GA

PDH

(g)

002040608

112141618


A

C

B

A

C

Cx43

120573-a

ctin

Cx43

120573-actin

43KD

42KD

(h)






(a) (b) (c)

(d) (e) (f)

05

10152025


Ad

cA d

B

C C

Cx37

GA

PDH

(g)

002040608

112


B

CA

A

C

Cx37

120573-a

ctin

Cx37


(h)



4 Discussion




012345678


VEG

FG

APD

H

a

c

c

b

c

(a)

005

115

225

3


b

caa

c

VEG

F120573

-act

in

VEGF

120573-actin

43KD

42KD

(b)

005

115

225

3


b

caa

c

VEG

FR120573

-act

in

VEGFR2

120573-actin

151KD

42KD

(c)


(a) (b) (c)














(a) (b) (c)

(d) (e) (f)

(g) (h) (i)







GroupThe number of



interval (days)





GroupThe number of



interval (days)




0123456789

10


The n

umbe

r of m

ice w

ith

norm

al es

trous

cycle

5 8 11 14 17













Acknowledgments


References






























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a)

sP

P

PF

(b)

P

PF

S

(c)

PF

P

(d)

P

PF

S

(e) (f)

012345678


The d

etec

tion

aof ce

llula

r apo

ptos

is

(g)








(a) (b) (c)

(d) (e) (f)

005

115

225


A

B

C

A

C

Activ

e cas

pase

-3120573

-act

in

120573-actin


43KD

(g)







(a) (b) (c)

(d) (e) (f)

05

10152025303540


C

B

A

C

Cx43

GA

PDH

(g)

002040608

112141618


A

C

B

A

C

Cx43

120573-a

ctin

Cx43

120573-actin

43KD

42KD

(h)






(a) (b) (c)

(d) (e) (f)

05

10152025


Ad

cA d

B

C C

Cx37

GA

PDH

(g)

002040608

112


B

CA

A

C

Cx37

120573-a

ctin

Cx37


(h)



4 Discussion




012345678


VEG

FG

APD

H

a

c

c

b

c

(a)

005

115

225

3


b

caa

c

VEG

F120573

-act

in

VEGF

120573-actin

43KD

42KD

(b)

005

115

225

3


b

caa

c

VEG

FR120573

-act

in

VEGFR2

120573-actin

151KD

42KD

(c)


(a) (b) (c)














(a) (b) (c)

(d) (e) (f)

(g) (h) (i)







GroupThe number of



interval (days)





GroupThe number of



interval (days)




0123456789

10


The n

umbe

r of m

ice w

ith

norm

al es

trous

cycle

5 8 11 14 17













Acknowledgments


References






























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b) (c)

(d) (e) (f)

005

115

225


A

B

C

A

C

Activ

e cas

pase

-3120573

-act

in

120573-actin


43KD

(g)







(a) (b) (c)

(d) (e) (f)

05

10152025303540


C

B

A

C

Cx43

GA

PDH

(g)

002040608

112141618


A

C

B

A

C

Cx43

120573-a

ctin

Cx43

120573-actin

43KD

42KD

(h)






(a) (b) (c)

(d) (e) (f)

05

10152025


Ad

cA d

B

C C

Cx37

GA

PDH

(g)

002040608

112


B

CA

A

C

Cx37

120573-a

ctin

Cx37


(h)



4 Discussion




012345678


VEG

FG

APD

H

a

c

c

b

c

(a)

005

115

225

3


b

caa

c

VEG

F120573

-act

in

VEGF

120573-actin

43KD

42KD

(b)

005

115

225

3


b

caa

c

VEG

FR120573

-act

in

VEGFR2

120573-actin

151KD

42KD

(c)


(a) (b) (c)














(a) (b) (c)

(d) (e) (f)

(g) (h) (i)







GroupThe number of



interval (days)





GroupThe number of



interval (days)




0123456789

10


The n

umbe

r of m

ice w

ith

norm

al es

trous

cycle

5 8 11 14 17













Acknowledgments


References






























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b) (c)

(d) (e) (f)

05

10152025303540


C

B

A

C

Cx43

GA

PDH

(g)

002040608

112141618


A

C

B

A

C

Cx43

120573-a

ctin

Cx43

120573-actin

43KD

42KD

(h)






(a) (b) (c)

(d) (e) (f)

05

10152025


Ad

cA d

B

C C

Cx37

GA

PDH

(g)

002040608

112


B

CA

A

C

Cx37

120573-a

ctin

Cx37


(h)



4 Discussion




012345678


VEG

FG

APD

H

a

c

c

b

c

(a)

005

115

225

3


b

caa

c

VEG

F120573

-act

in

VEGF

120573-actin

43KD

42KD

(b)

005

115

225

3


b

caa

c

VEG

FR120573

-act

in

VEGFR2

120573-actin

151KD

42KD

(c)


(a) (b) (c)














(a) (b) (c)

(d) (e) (f)

(g) (h) (i)







GroupThe number of



interval (days)





GroupThe number of



interval (days)




0123456789

10


The n

umbe

r of m

ice w

ith

norm

al es

trous

cycle

5 8 11 14 17













Acknowledgments


References






























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b) (c)

(d) (e) (f)

05

10152025


Ad

cA d

B

C C

Cx37

GA

PDH

(g)

002040608

112


B

CA

A

C

Cx37

120573-a

ctin

Cx37


(h)



4 Discussion




012345678


VEG

FG

APD

H

a

c

c

b

c

(a)

005

115

225

3


b

caa

c

VEG

F120573

-act

in

VEGF

120573-actin

43KD

42KD

(b)

005

115

225

3


b

caa

c

VEG

FR120573

-act

in

VEGFR2

120573-actin

151KD

42KD

(c)


(a) (b) (c)














(a) (b) (c)

(d) (e) (f)

(g) (h) (i)







GroupThe number of



interval (days)





GroupThe number of



interval (days)




0123456789

10


The n

umbe

r of m

ice w

ith

norm

al es

trous

cycle

5 8 11 14 17













Acknowledgments


References






























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


012345678


VEG

FG

APD

H

a

c

c

b

c

(a)

005

115

225

3


b

caa

c

VEG

F120573

-act

in

VEGF

120573-actin

43KD

42KD

(b)

005

115

225

3


b

caa

c

VEG

FR120573

-act

in

VEGFR2

120573-actin

151KD

42KD

(c)


(a) (b) (c)














(a) (b) (c)

(d) (e) (f)

(g) (h) (i)







GroupThe number of



interval (days)





GroupThe number of



interval (days)




0123456789

10


The n

umbe

r of m

ice w

ith

norm

al es

trous

cycle

5 8 11 14 17













Acknowledgments


References






























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










(a) (b) (c)

(d) (e) (f)

(g) (h) (i)







GroupThe number of



interval (days)





GroupThe number of



interval (days)




0123456789

10


The n

umbe

r of m

ice w

ith

norm

al es

trous

cycle

5 8 11 14 17













Acknowledgments


References






























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



GroupThe number of



interval (days)





GroupThe number of



interval (days)




0123456789

10


The n

umbe

r of m

ice w

ith

norm

al es

trous

cycle

5 8 11 14 17













Acknowledgments


References






























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Acknowledgments


References






























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article the increased expression of connexin and ...cx nm . f: -cgtcccacggagaaaaccat-r:...

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