Effect of osteopontin (OPN) on in vitro embryo

development in cattle

E. Monaco a,*, B. Gasparrini b, L. Boccia b, A. De Rosa b,L. Attanasio b, L. Zicarelli b, G. Killian a

a John O. Almquist Research Center, Department of Dairy and Animal Science, Pennsylvania State University, University Park, PA, USAb Department of Scienze Zootecniche ed Ispezione degli Alimenti, Faculty of Veterinary Medicine, ‘‘Federico II’’ University,

Via F. Delpino 1, 80137 Naples, Italy

Received 17 November 2007; received in revised form 31 July 2008; accepted 6 August 2008

Abstract

Fertility-related phosphoprotein osteopontin (OPN) is present in the bovine oviduct epithelium and fluid. The objectives were to

determine the effects of OPN on percentages of cleavage and embryo development in vitro in cattle, and to assess the ability of OPN

to induce in vitro capacitation of bovine sperm. In vitro-matured bovine oocytes were fertilized in the presence of 0, 10, 20, or 40 mg/

mL OPN. There were greater percentages (P < 0.01) of cleavage and compact morulae-blastocysts (79.7 and 43.3%, respectively)

with 10 mg/mL OPN than in the control group (without OPN; 71.2 and 32.1%, respectively). Furthermore, percentages of advanced

blastocysts were greater in the group receiving 40 mg/mL OPN versus control (56.4% vs. 42.0%, P < 0.05).

Capacitation was assessed by the ability of sperm to undergo the acrosome reaction after incubation with lysophosphatidylcho-

line. Semen from three bulls was incubated for 2 h in either TALP medium alone (control) or with TALP medium containing

0.01 mM heparin, or with TALP medium containing 10 or 20 mg/mL OPN. Incubation with 10 and 20 mg/mL OPN produced more

(P < 0.01) capacitated sperm (14.4 and 13.6%, respectively) than the untreated control group (8.3%), but both untreated sperm and

those treated with OPN had significantly fewer capacitated sperm than those treated with 0.01 mM of heparin (30.5%). In

conclusion, OPN improved the efficiency of bovine in vitro embryo production and influenced sperm capacitation.

# 2009 Elsevier Inc. All rights reserved.

Keywords: Osteopontin; Embryo development; IVF; Sperm capacitation; Cattle

www.theriojournal.com

Available online at www.sciencedirect.com

Theriogenology 71 (2009) 450–457

1. Introduction

Since 1981, when the first in vitro produced (IVP)

calf was born, remarkable discoveries have been made

to define the metabolic needs of the embryo. There

are differences between in vivo and in vitro produced

* Corresponding author. Present address: Department of Animal

Sciences, University of Illinois at Urbana-Champaign, 1207 West

Gregory Drive, Urbana, IL 61801, USA. Tel.: +1 217 2443150;

fax: +1 217 3338286.

E-mail address: emonaco@uiuc.edu (E. Monaco).

0093-691X/$ – see front matter # 2009 Elsevier Inc. All rights reserved.

doi:10.1016/j.theriogenology.2008.08.012

embryos in metabolic and morphologic profiles at the

cellular level [1–3], as well as in gene expression [4].

The oviduct provides an optimal environment for

sperm capacitation, the acrosome reaction, fertilization,

and early embryo development; oviduct fluid (ODF)

facilitated sperm capacitation, fertilization, and early

embryo development in vitro (reviewed in [5]). More-

over, ODF proteins associated with bovine sperm [6],

ova [7], and embryos [8]. Therefore, formulation of

defined media for in vitro fertilization (IVF) and in vitro

culture (IVC) that mimic the oviductal environment

may improve in vitro embryo production (IVEP) [9–11].

E. Monaco et al. / Theriogenology 71 (2009) 450–457 451

One of the proteins present in bovine oviduct fluid

is osteopontin [12]. Osteopontin was first described

in bone matrix, but has also been detected in epithelial

cells and in secretions of the gastrointestinal tract,

kidneys, thyroid, mammary gland, oviduct, uterus,

placenta, ovary, and testes [13–18]. Structurally, OPN

is an acid single chain phosphorylated glycoprotein that

ranges in length from 264 to 301 amino acids and

undergoes extensive post-translational modifications

that result in molecular weight variations ranging

from 25 to 75 kDa [19]. The resulting multiple

functional protein contains a Gly-Arg-Gly-Asp-Ser

(GRGDS) sequence that binds to cell surface integrins

to promote cell–cell adhesion, cell-extracellular matrix

(ECM) communication, migration of osteocytes and

immune cells [20,21]. It also stimulates immunoglo-

bulin production by B cells, alters intracellular calcium

concentrations, promotes calcium phosphate deposi-

tion in bone, and affects the mineralization of the

tissues (reviewed in [19]).

Osteopontin also appears to be important in

reproduction. Killian et al. [22] and Cancel et al.

[23] reported that more OPN was expressed in the

seminal plasma of high-fertility compared to low-

fertility bulls. Proteomic analyses demonstrated that

high-fertility Holstein bulls had more OPN in their

accessory sex glands fluid than low-fertility bulls

[24]. There are three molecular weight forms (55, 48,

and 25 kDa) of OPN in bovine ODF during the

estrous cycle [12]. These forms are present in both

the ampullary and isthmic regions, with the greatest

amount of OPN found in ampullary non-luteal

oviductal fluid (ANL-ODF) when serum progesterone

concentrations were �1.5 ng/mL [12]. Osteopontin

has been detected on the zona pellucida (ZP) of both

immature and mature bovine oocytes [25]. Goncalves

et al. [26] have recently showed that addition of rabbit

polyclonal IgG antibody against purified bovine milk

OPN to bovine sperm, oocytes, or both, decreased

IVF compared to untreated controls. In swine, Hao

et al. investigated the role of OPN on IVF [27]. They

demonstrated that fertilization rates were improved

when OPN was included in the IVF medium, and the

percentage of acrosome-reacted sperm bound on

the oocytes ZP was increased after 4 h incubation

with 1.0 mg/mL OPN. Based on these observations,

the objectives of our study were to evaluate the effect

of supplementing IVF medium with OPN on bovine

cleavage and post-fertilization embryo development,

and to evaluate the ability of various concentrations

of OPN to induce in vitro capacitation of bovine

sperm.

2. Materials and methods

Unless otherwise stated, all chemicals used were

purchased from Sigma Chemical Co. (St. Louis, MO,

USA). The OPN used in this study was purified (in

our laboratory) from bovine skim milk, as previously

described in Ref. [28].

2.1. Oocyte collection and maturation

Ovaries were harvested at a local abattoir from a

variety of cattle breeds and transported to the laboratory

in saline solution (0.9% NaCl) at 30–35 8C. Immature

cumulus–oocyte complexes (COCs) were aspirated

from follicles of 1 to 6 mm in diameter using an 18-

gauge needle (B-D1, Rutherford, NJ, USA) attached to

a 10 mL disposable syringe (B-D1). The aspirate was

distributed among sterile petri dishes. Oocytes with

uniform cytoplasm and multilayered cumulus cells

were selected, washed twice in TCM aspiration

(Medium 199 supplemented with 25 mM of Hepes,

2 mM sodium bicarbonate, 2 mM sodium pyruvate,

1 mM L-glutamine, 10 mL/mL amphotericin B, and

540 mg/mL heparin) and once in TCM-IVM (Medium

199 supplemented with 15% bovine serum (BS),

0.5 mg/mL FSH, 5 mg/mL LH (Sioux Biochemical1,

Sioux Center, IA, USA), 0.8 mM L-glutamine, and

50 mg/mL gentamicin). The COCs were then placed to

mature, 25/well (NunclonTM, Nunc, Roskilde, Den-

mark), in 400 mLTCM-IVM covered with mineral oil in

the incubator for 22–24 h at 39 8C and 5% CO2 in air.

2.2. Sperm preparation and in vitro fertilization

Mature COCs were washed in Tyrode’s modified

medium [29] without glucose and bovine serum

albumin (BSA), supplemented with 30 mg/mL heparin,

30 mM penicillamine, 15 mM hypotaurine, 0.15 mM

epinephrine, and 1% BS (IVF-TALP). They were

transferred, 25/well, into 300 mL IVF-TALP covered

with mineral oil.

Frozen sperm from one Limousine bull, previously

screened to perform satisfactorily in IVF (percentages

of fertilized oocytes were typically 70% and percen-

tages of expanded blastocysts usually 30%), were

thawed at 37 8C for 40 s and layered carefully into a

15 mL conical tube containing 2 mL 80% Percoll

solution in the bottom and 2 mL 45% Percoll solution

on top of it. Percoll solutions were made in Tyrode’s

modified medium [29] without glucose and BSA

(Sperm-TALP). Semen was centrifuged for 25 min at

300 � g. After centrifugation, the pellet was recon-

E. Monaco et al. / Theriogenology 71 (2009) 450–457452

Fig. 1. Two viable bovine sperm (absence of trypan blue staining); the

sperm on the left has an intact acrosome (intense pink), whereas the

one on the right is acrosome reacted (light pink). (For interpretation of

the references to color in this figure legend, the reader is referred to the

web version of the article.)

stituted into 2 mL of Sperm-TALP and centrifuged

twice (160 and 108 � g). The pellet was diluted with

modified IVF-TALP and added in the fertilization wells

(concentration, 1 � 106 sperm/mL). Gametes were co-

incubated for 20–22 h at 39 8C in 5% CO2 in air.

2.3. In vitro embryo culture

After 20–22 h of co-incubation, presumptive

zygotes were vortexed for 2 min to remove cumulus

cells in Medium 199 supplemented with 25 mM Hepes,

2 mM sodium bicarbonate, 2 mM sodium pyruvate,

1 mM L-glutamine, and 5% BSA (TCM-Hepes).

Presumptive zygotes were then washed twice in the

same medium and transferred, 30–50/well, into 400 mL

synthetic oviduct fluid (SOF) modified medium [30],

supplemented with 30 mL/mL essential amino acids,

10 mL/mL non-essential amino acids, 0.34 mM tri-

sodium citrate, 2.77 mM myo-inositol, and 5% BS.

Zygotes were incubated in a humidified mixture of

5% CO2, 7% O2, and 88% N2 in air at 39 8C. The

percentages of cleaved embryos and oocytes reaching

blastocyst (BL) and advanced blastocyst (expanding

and expanded blastocyst, hatching and hatched

blastocyst, and expanded hatched blastocyst) were

determined at Day 7 of the culture (Day 0 = fertiliza-

tion day).

2.4. Sperm capacitation assay

Frozen sperm, from three Limousine bulls, which

were previously tested to perform well in IVF,

were thawed at 37 8C for 40 s and separated through

Percoll gradients, as described above. Sperm were

then exposed to lysophosphatidylcholine (LPC) as

described by Parrish et al. [31]. After 15 min of

incubation at 38 8C in 5% CO2 in air, sperm were fixed

and stained with Trypan blue-Giemsa [32]. Slides were

then rinsed and left to dry at air covered with a glass

cover slip and sealed with Entellan. Sperm were

counted under differential interference light micro-

scopy and the number of live with acrosome intact,

dead with acrosome intact, live with acrosome reacted,

or dead with acrosome reacted sperm was evaluated

(Fig. 1).

2.5. Experimental design

2.5.1. OPN and IVEP

A total of 1486 COCs were selected and matured in

vitro. At Day 0 of culture, they were randomly assigned

to four experimental groups:

(1) 3

78 oocytes were fertilized in IVF-TALP without

OPN (control group);

(2) 3

72 oocytes were fertilized in IVF-TALP with

10 mg/mL OPN;

(3) 3

72 oocytes were fertilized in IVF-TALP with

20 mg/mL OPN;

(4) 3

64 oocytes were fertilized in IVF-TALP with

40 mg/mL OPN.

At Day 7 of culture, the percentages of cleavage and

oocytes reaching blastocyst were evaluated. The

experiment was repeated six times (range, 25–99

oocytes/replicate/treatment).

2.5.2. OPN and semen capacitation

The ability of various concentrations of OPN (0, 10,

and 20 mg/mL) to induce in vitro capacitation of bovine

sperm was investigated. Capacitation was assessed

indirectly by estimating the percentage of acrosome

reacted sperm after incubation with lysophosphatidyl-

choline (LPC).

For each treatment, one straw of semen was thawed

per bull and two slides were prepared. An aliquot of

semen was taken immediately after thawing, and two

slides were prepared to estimate the percentage of

sperm without acrosomes prior to treatment. A second

aliquot was also prepared for counting after sperm were

incubated for 2 h in either TALP medium alone (control

group) or with TALP medium containing 0.01 mM

heparin or with TALP medium containing 10 or 20 mg/

mL of OPN. The percentage of live and dead sperm

E. Monaco et al. / Theriogenology 71 (2009) 450–457 453

Table 1

Effects of various doses of osteopontin (OPN) on development (%) of IVP bovine embryos to cleavage, compact morulae–blastocysts (CM–BL), and

advanced blastocyst (BL) stages

Embryo stage OPN (mg) SEM P value

0 10 20 40

Cleavage 71.2a 79.7b 77.5b 73.1ab 1.6 <0.01

CM–BL 32.1a 43.3b 36.8ab 29.0a 2.6 <0.01

Advanced BL 42.0ab 52.9bc 40.0a 56.4c 3.8 0.02

a–cWithin a row, percentages without a common superscript differed (P < 0.05).

for each treatment was determined by counting 160–

240 sperm/slide.

2.6. Statistical analyses

Data from both experiments were analysed using

PROC GLM of SAS (Release 8.0; SAS Institute, Cary,

NC, USA) with a repeated statement. The model

included the fixed effect of treatment (three treatments

plus control in both experiments), replicates (n = 6) for

the experiment OPN and IVEP, or bull (n = 3) for the

experiment OPN and semen capacitation, and treat-

ment � replicate or bull interaction. The test for

normality was run using PROC UNIVARIATE NOR-

MAL, and a Shapiro–Wilkins normality test >0.9 was

used as criterion to determine normal distribution of

data. All data were normally distributed; therefore, no

transformation was required prior to MANOVA

analysis. Significant was set at P � 0.05.

3. Results

3.1. Effect of OPN on embryo development

Greater percentages (P < 0.01) of cleaved zygotes

were observed in groups cultured with 10 and 20 mg/mL

of OPN than in the control group (Table 1). Furthermore,

significantly greater percentages of compact morulae–

blastocysts (CM–BL) were obtained after incubation

with 10 mg/mL OPN compared to the control group

Table 2

Effects of various doses of osteopontin (OPN), or 0.01 mM of heparin (EPA

acrosomes relative to total live, and sperm without acrosomes relative to to

Sperm OPN (mg)

0 10

Total live 49.6 54.4

Without acrosome/total live 8.3a 14.5b

Without acrosome/total 16.7a 26.9b

a–cWithin a row, percentages without a common superscript differed (P <

(Table 1). Greater percentages (P < 0.05) of advanced

blastocysts were present in the 40 mg/mL OPN group

versus the control group (Table 1).

3.2. Effect of OPN on sperm capacitation

At time = 0, none of the sperm randomly counted

for each slide showed acrosome lost just after thawing.

The percentages of capacitated sperm were greater

(P < 0.01) in treatments receiving 10 and 20 mg/mL of

OPN than the control group, but both untreated sperm

and those treated with OPN had fewer capacitated

sperm (P < 0.01) than those treated with 0.01 mM of

heparin (Table 2). There were no significant differences

among groups in the percentages of viable sperm.

4. Discussion

In the present study, the addition of OPN signifi-

cantly improved IVEP. Given that the percentages of

cleaved zygotes and transferable embryos produced in

vitro were greatest at the lower concentration of OPN,

we infer that there was a biphasic response pattern to

incremental doses of OPN. However, the percentages of

advanced blastocysts were greatest in the 40 mg/mL

group. Perhaps 10 mg/mL OPN is optimal for the

development of oocytes to CM-BL stage, but a higher

concentration is needed for advanced blastocyst

formation. In vivo, an accumulation of OPN on the

embryo is possible from both ODF [33] and the

), on percentages of live sperm relative to total sperm, sperm without

tal sperm

EPA SEM P value

20

53.4 65.4 5.2 0.26

13.7b 30.5c 1.4 <0.01

27.3b 47.4c 2.5 <0.01

0.05).

E. Monaco et al. / Theriogenology 71 (2009) 450–457454

endometrium [34], as it moves from the oviduct to the

uterus. Uterine OPN expression has been detected

during peri-implantation in humans [35,36], pigs [34],

and rabbits [37]. Perhaps with 40 mg/mL OPN there is a

carry-over effect of the protein that is not evident until

the latest stage of embryo development in vitro.

Our results were not consistent with those of Hao

et al. [27] for swine, where an overall increase in the

fertilization efficiency was obtained with 0.01 and

0.1 mg/mL OPN. In preliminary studies, we tested 0.1

and 1 mg/mL of OPN, but these concentrations were

ineffective. The different responses between porcine

and bovine to similar doses of OPN may be due to

species differences, or to the source of the OPN. We

used OPN that was from purified skim bovine milk,

whereas Hao et al. [27] used recombinant rat OPN.

Furthermore, in our study, the end point measured for

the effect of OPN on IVF was the percentage of

cleavage, whereas in the previous study [27], penetra-

tion rates, polyspermic fertilization rates, male pro-

nuclear formation rate, normal fertilization efficiency

(number of oocytes with one male and one female

pronucleus/total number of oocytes inseminated), and

number of sperm penetrated per oocyte were deter-

mined.

The ability of osteopontin to positively affect IVEP

may be due to direct interactions with both sperm and

oocyte. In a previous study, we detected OPN on the

zona pellucida (ZP) and oolemma of both immature and

mature bovine oocytes [25,33]. We also detected OPN

on the post-equatorial segment, acrosomal cap, and

midpiece of ejaculated bull sperm [33], and its distri-

bution on sperm changed after incubation in bovine

oviduct fluid [33].

The interaction of OPN with sperm or ova may occur

through its GRGDS sequence and CD44 [26,38]. The

GRGDS peptide is known to interact with integrin cell

receptors [15,39], which have been identified on the

surface of several cells. Different integrins have been

described on the oolemma of bovine fertilized oocytes

[40] and human sperm [33,41]. Integrins are known to

promote cell–cell adhesion and may facilitate fertiliza-

tion in mammalian species by promoting oocyte–sperm

binding [42]. Goncalves et al. [26] reported that

addition of OPN antibodies to fertilization media

reduced both the number of sperm bound to oocytes and

the percentage of fertilized oocytes in cattle. Further-

more, treatment of sperm or oocytes with an RGD

peptide, or with a5 and av integrin antibodies, reduced

the number of sperm bound to the ZP and fertiliza-

tion rates similar to what was found using antibodies

against OPN [26]. Perhaps OPN participates in sperm–

oocyte interaction (with potential involvement of

integrins).

Gabler et al. [12] detected three distinct molecular

weight forms of OPN protein in the bovine ODF from

both the ampullary and isthmic regions during the

estrous cycle. The greatest amount of OPN was in

ampullary non-luteal oviductal fluid, whereas the

minimum amount was present in both isthmus and

ampullary luteal fluid. Coincidently, the expression of

av e b1 integrin subunits in the oviduct during the luteal

phase was the lowest among the estrous cycle phases.

Moreover, Gabler et al. [12] observed that the quantity

of oviductal cells mRNA for the integrin subunit b3

significantly increased, starting from the latest luteal

phase until just before ovulation. Integrin beta (3)

mRNA content increased significantly from the lowest

level during the late luteal phase to the highest level

before ovulation. Taken together, the Gabler et al. [12]

work suggests the potential role of an osteopontin–

integrin interaction during normal oviduct physiology,

including early embryo development.

Previously, we suggested that the interaction of OPN

with sperm and oocyte may occur in two ways [33]. One

possibility is that OPN binds to sperm and the resulting

sperm–OPN complex adheres to the ZP of the oocyte.

Alternatively, the sperm–OPN complex may adhere to

the ovary/oviduct derived OPN, previously bound to the

ZP, since OPN forms bonds with other OPN molecules

[43,44]. However, in either case, once sperm entered the

perivitelline space, OPN may also mediate the

interaction of sperm with the oolemma, through

integrins and/or CD44. Numerous intracellular signals

are activated within the egg after the fusion with the

sperm, which are believed to have important roles in the

resumption of meiosis and consequent development of

the zygote [45,46]. We suggest that OPN, adhering to

the oolemma, triggers intracellular signals via interac-

tion with integrins and/or CD44. It is also likely that

OPN activates zygote genes involved in promoting/

accelerating development. This would explain not only

the improved percentages of IVEP after treatment with

OPN, but also the highest percentage of advanced

blastocysts obtained in this study. Perhaps the IVEP

effect of OPN may be derived from sperm which were

capacitated in the presence of OPN and heparin.

Although our results confirmed that heparin was the

best capacitating agent in vitro, they also showed that

OPN promoted in vitro sperm capacitation without

the negative effect on sperm viability associated with

heparin.

Sperm capacitation and the acrosome reaction are

dependent on increases in sperm intracellular calcium.

E. Monaco et al. / Theriogenology 71 (2009) 450–457 455

Because osteopontin influences intracellular calcium

concentrations [39], perhaps the positive effect of OPN

on bovine sperm capacitation is through its ability to

influence calcium transients.

Mammalian sperm capacitation is a maturation step

that enables sperm to achieve an acrosome reaction and

ova penetration. For capacitation and an acrosome

reaction to occur, calcium is necessary in the extra-

cellular environment. During sperm capacitation and

acrosome reaction, there is an increase in intracellular

Ca2+ concentration ([Ca2+]i) that finally leads to the

exocytose of the acrosomal vesicle and to the fusion of

the two gametes. Evidence suggests that ligand binding

to various integrins triggers an increase in [Ca2+]i

[47,48]. Rat osteoclasts or mouse osteoclasts-like cells

had a transient [Ca2+]i increase when perfused with

osteopontin and other integrin ligands [49]. This rise in

[Ca2+]i came from intracellular stores, although extra-

cellular Ca2+ was necessary to produce the [Ca2+]i

changes. During sperm capacitation and acrosome

reaction, the presence of extracellular Ca2+ is necessary

for an increase in [Ca2+]i and believed to be due to the

known Ca2+ requirement for ligand binding. However,

the mechanism by which OPN triggers an increase in

intracellular calcium is unknown.

Zimolo et al. [49] reported that binding of avb3-

ligands to the osteoclasts triggers cellular changes that

have second messenger characteristics. Both a- and b-

chains have relatively short intracellular domains that

lack kinase activity or typical domains known to

interact with G proteins. Nevertheless, both intracellular

domains contained tyrosine residues that, if phosphory-

lated, may be the regions for Src homology (SH) region

2 binding shown to modulate interactions in signal

transduction pathway [50,51].

Heparin is routinely used to induce sperm capacita-

tion in vitro; the heparin-induced capacitation in cattle

is mediated by heparin binding to sperm membrane

[31], with consequent Ca2+ uptake and an increase in

intracellular free calcium [52]. Perhaps heparin inter-

action with the sperm membrane occurs through OPN.

In that regard, OPN contains a calcium binding site and

two heparin binding domains. In a recent study, it was

highlighted that the phosphorylation of many bull sperm

proteins was important for the heparin induction of

sperm capacitation [53]. Remarkably, we showed OPN

to be present on the bovine sperm membrane [33] and it

is known that OPN undergoes extensive post-transla-

tional modifications, including phosphorylation [19].

Our hypothesis is that OPN present on the sperm surface

at ejaculation binds to heparin through its heparin

domain and that this complex OPN-heparin guides

calcium transients and sperm capacitation. In this

regard, it would be interesting to test the combined

effect of OPN and heparin on sperm capacitation in

vitro, since in vivo, the OPN present on the sperm at

ejaculation is supplemented with OPN from the ODF.

In conclusion, fertilization medium containing

10 mg/mL OPN improved in vitro embryo production

and OPN positively influenced sperm capacitation in

vitro. On a practical basis, addition of OPN or other

oviductal proteins to IVF systems may significantly

improve the percentage of live embryos produced in

vitro. However, the specific functions of OPN, as well

as, the molecular mechanisms underlying its action,

remain to be investigated.

Acknowledgements

The assistance of the staff at the Department of

Scienze Zootecniche ed Ispezione degli Alimenti

‘‘Federico II’’ University, Naples, Italy is greatly

appreciated.

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