Effects Of Ovarian Cortex Cell Co-culture During In Vitro Maturation On Porcine Oocytes Maturation, Fertilization And Embryo Development

Animal Reproduction Science 99 (2007) 306–316

Effects of ovarian cortex cell co-culture during in vitro maturation on porcine oocytes maturation, fertilization and embryo development Xiao-Yu Chen a , Qing-Wang Li a,b,∗ , Shu-Shan Zhang a , Zeng-Sheng Han a , Rui Zhao b , Shu-Yun Wu a , Jing Huang a a

College of Animal Science, Northwest Agriculture & Forestry University, Yangling, Shannxi Province 712100, People’s Republic of China b Department of Biological Engineering, College of Enviroment & Chemical Engineering, Yanshan University, Qinhuangdao, Hebei Province 066004, People’s Republic of China Received 22 January 2006; received in revised form 17 April 2006; accepted 11 May 2006 Available online 19 June 2006

Abstract The objective of the experiments was to evaluate the effects of porcine ovarian cortex cells (pOCCs) during in vitro maturation (IVM) of porcine oocytes on IVM of porcine oocytes, in vitro fertilization (IVF) parameters and subsequent embryo development. The pOCCs was cultured in the 500 ␮l TCM199 without hormone until the confluence, and then cultured in 500 ␮l TCM199 supplemented with hormone for 12 h before the oocytes added. Porcine oocytes were co-cultured with the pOCCs monolayers in the co-culture system for 44 h, following fertilized in the mTBM for 6 h. Finally, the presumptive zygotes were cultured for 144 h in the NCSU-23 supplemented with 0.4% BSA. The results showed that matured M II oocytes in the co-culture group were higher than that in the control group (P < 0.05). Although penetration did not differ between the co-culture and control groups (P = 0.481), polyspermy declined in the co-culture group (P < 0.05), whereas male pronucleus (MPN) formation was improved in the co-culture group compared with the control group (P < 0.05). More blastocysts developed in the co-culture group than that in the control group (P < 0.05); however, the cleavage rates and the mean number cells per blastocyst showed no significant difference between the treated group and the control group (P = 0.560 and 0.873, respectively). In conclusion, the presence of the pOCCs monolayers during IVM enhanced the maturation quality of the porcine oocytes,

∗ Corresponding author at: College of Animal Science, Northwest Agriculture & Forestry University, Yangling, Shannxi Province 712100, People’s Republic of China. E-mail address: [email protected] (Q.-W. Li).

0378-4320/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2006.05.007

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reduced the polyspermy, increased the percentages of MPN formation and blastocyst, but the blastocyst quality was not improved. © 2006 Elsevier B.V. All rights reserved. Keywords: Porcine; Ovarian cortex cells; Monolayers; IVM; IVF

1. Introduction In vitro production (IVP) technology in domestic animals has been considered as an important technology in agriculture and the biomedical field. However, there are still many inadequacies in IVP of the pig compared with other technologies currently used in domestic animals. Incomplete cytoplasmic maturation, polyspermy, low percentage of MPN formation and week development capability of the blastocyst have not been overcome completely until today (Day, 2000; Abeydeera, 2002). In order to improve the quality of matured porcine oocytes and embryos in vitro, all kinds of methods were attempted in IVP of pig. Supplement of IVM medium with follicular cellconditioned medium (Mattioli et al., 1988; Ding and Foxcroft, 1994) or co-culture with follicular shell pieces (Abeydeera et al., 1998) have positive effects on nuclear maturation, subsequent fertilization and porcine embryo development in vitro. Addition of porcine oviductal epithelial cells (pOECs) during the IVM resulted in more blastocysts formation as well as the blastocyst quality is improved (Bureau, 2000; Kidson et al., 2003; Qian et al., 2005). These studies indicated the IVM system at present is still suboptimal; therefore, the co-culture with the homologous cell during IVM is an efficient method which can improve the quality of porcine matured oocytes and embryos in vitro. Most mammalian oocytes enter meiosis during the fetal stage, arrest at the dictyotene stage of the first meiotic division until re-initiation of meiosis under the surge of gonadotropins or degradation because of follicles atresia. During the follicles growth, oocytes gradually gain more development capacity until arriving at Prophase II (M II) stage after undergoing germinal vesicle breakdown (GVBD) and Prophase I (M I). Synchronization of nuclear and cytoplasmic maturation of oocytes is regarded as pivotal precondition in the following development. How to perfect the maturation condition in vitro and improve the synchronization of nuclear and cytoplasmic maturation has become a focus during porcine embryos IVP (Day, 2000; Niemann and Rath, 2001; Abeydeera, 2002). Clearly, ovarian cortex performs important function during the reproductive life span of the female mammalian as the best optimal environment of oocytes maturation or follicles growth in vivo. In order to find out the positive effect of ovarian cortex on the follicles growth in vitro, the ovarian cortex was co-cultured successfully with follicles in vitro (Fortune et al., 1998; Parrot and Skinner, 1999; Oktay et al., 2000; Cushman et al., 2002; Silva et al., 2004). Moreover, the effects of oviductal epithelial, granulosa and cumulus monolayers co-cultured with oocytes on the meiotic resumption competence were also studied during IVM (Teotia et al., 2001; Abeydeera, 2002; Kidson et al., 2003; Romar et al., 2003). Now, we wonder whether the maturation and subsequent development capacity of porcine oocytes could be improved when porcine ovarian cortex cells (pOCCs) are directly co-culture with oocytes during whole IVM. In the present study, we investigated the effects of pOCCs co-cultured during IVM on the maturation, fertilization parameters, embryo development capacity and resultant blastocyst quality of the pig in vitro.

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2. Materials and methods 2.1. Culture medium Unless otherwise stated, all chemicals used in this study were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The maturation medium was modified TCM-199 medium (TCM199; Gibco, Grand Island, NY; Cat. No. 31100-035) supplemented with 0.1% PVA (Cat. No. P8136), 3.05 mM glucose (Ameresco, Cat. No. 0188), 0.91 mM sodium pyuvate (Cat. No. P5280), 0.57 mM/ml l-cysteine (Cat. No. C7352), 10 IU/ml PMSG (Cat. No. G4877), 10 IU/ml hCG (Cat. No. C1063), 50 IU/ml penicillin G and 50 IU/ml streptomycin sulfate. The fertilization medium was a modified Tris-buffered medium (mTBM, pH 7.2) (Abeydeera and Day, 1997b) containing 113.1 mM NaCl, 3 mM KCl, 7.5 mM CaCl2 ·2H2 O, 20 mM Tris, 11 mM d(+)-glucose, 5 mM sodium pyuvate, 1 mg/ml BSA (Ameresco, Cat. No. 0332, pH 7.0) and 2 mM caffeine (Cat. No. C0750). The embryo culture medium was NCSU-23 (Petters and Wells, 1993) supplemented with 0.4% BSA. The cell culture medium was TCM-199 supplemented with 2.2 mg/ml NaHCO3 , 20% fetal calf serum (FCS, Hyclone, Cat. No. SH30406.02), 1 mg/ml glutamine (Cat. No. G8540), 1% (v/v) non-essential amino acids (Cat. No. M7145), 150 IU/ml penicillin, and 100 ␮g/ml streptomycin. 2.2. Ovaries selection, oocytes collection and in vitro maturation Porcine ovaries were obtained from prepubertal swine within 5 min after slaughter. The selected ovaries were in the follicular phase with no apparent corpus luteum as previously described (Parrott and Skinner, 1998a,b) and were transported to the laboratory in d-PBS containing 150 IU/ml penicillin, and 100 ␮g/ml streptomycin at 35 ◦ C within 2 h. In the laboratory, cumulus–oocyte complexes (COCS ) were collected from non-atretic follicles (2–6 mm in diameter) by aspiration with an 18 G needle fixed to a 10 ml disposable syringe. Only oocytes with compact cumulus cells showing a homogeneous and granulated cytoplasm were selected and rinsed twice in TL-HEPESPVA (Funahashi et al., 1997), at last the COCS was washed three times with maturation medium that previously equilibrated in incubator for 3 h under 5% CO2 in air at 38.5 ◦ C. About 40–50 washed COCS were placed into each well of the 4-well multidish (Nunclon, Roskilde, Denmark, Cat. No. 176740) containing 500 ␮l of IVM medium which had previously been covered with mineral oil (Cat. No. M8410) and equilibrated for 12 h with the pOCCs monolayers at 38.5 ◦ C under 5% CO2 in air prior use. The COCS were co-cultured at the same medium with pOCCs monolayers for 44 h. 2.3. Porcine ovarian cortex cells (pOCCs) monolayers preparation The pOCCs monolayers were prepared beforehand for IVM. The same ovaries as above described were prepared. After aspiration, the fat tissue and ligaments of ovaries were trimmed off carefully. The ovaries were chipped into small tissues, then the medulla and visible antral follicles were removed. Following this, the ovarian cortex was micro-dissected in fragments approximately 0.5 mm × 0.5 mm (1 mm thick) for next digestion as previously described (Silva et al., 2004). The pieces of ovarian cortex were digested with 1 mg/ml collagenase (Cat. No. C2674), 2.5 mg/ml trypsinase (Ameresco, Cat. No. 0458) and 1 mg/ml hyaluronidase (Cat. No. H2126) for 1 h at 37 ◦ C. After terminated the digestion, the digested cells clusters were dissociated by gentle, repeated pipetting followed by centrifugation at 750 × g for 4 min. The supernatant was

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removed before the pellet was re-suspended with D-Hank’s medium. The procedure above was repeated thrice. After the final centrifugation, discarded the resultant supernatant, re-suspended the pellet in fresh cell culture medium, then seeded in the 4-well multidish at a density of 0.5 × 106 to 1 × 106 cells/ml, the cell was maintained in cell culture medium at 38.5 ◦ C under 5% CO2 in air for 48 h and the medium was changed after 48 h. Once the pOCCs monolayers arrived to 80% of confluence stage, changed the cell culture medium with the maturation medium and incubated at 38.5 ◦ C under 5% CO2 at least 12 h before oocytes added for IVM. 2.4. Glutathione (GSH) assay The concentration of intracellular GSH was determined as described previously (Brian and James, 2004). Basically, denuded matured oocytes in each group were washed three times in stock buffer (0.2 M sodium phosphate containing 10 mM EDTA, pH 7.2). Groups of 30 oocytes from each group were transferred with 5 ␮l stock buffer to a 1.5 ml Eppendorf tube and stored at −80 ◦ C until the day of the assay. On the day of the assay, 5 ␮l of 1.25 M H3 PO4 was added to every tube and the oocytes were ruptured using a blunt glass rod. The intracellular concentration of GSH in oocytes was determined using the dithionitrobenzonic acid-glutathione disulphide (DTNB-GSSH) reductase recycling assay using spectrophotometer. 2.5. In vitro fertilization and embryo culture When maturation was completed, the expanded cumulus cells were removed by pipetting in NCSU-23 containing 0.1% hyaluronidase. The denuded oocytes with first polar (Pb I) were washed three times in 100 ␮l drops of mTBM equilibrated for 24 h in an incubator at 38.5 ◦ C under 5% CO2 in air. About 40–50 oocytes were stored in 50 ␮l drops of mTBM under mineral oil then equilibrated in a 35 mm × 10 mm Petri dish (Nunc, Roskilde, Denmark, Cat. No. 153066) at 38.5 ◦ C under 5% CO2 in air at least 0.5 h. A frozen semen straw was prepared for IVF as described previously (Brian and James, 2004). Briefly, the frozen–thawed semen were thawed in 8 ml of d-PBS supplemented with 0.1% BSA, 0.10 g/l CaCl2 ·2H2 O, 150 IU/ml penicillin, and 100 ␮g/ml streptomycin at 40 ◦ C in 50 ml polypropylene conical tube. The washed semen was centrifuged at 73 × g for 5 min to remove dead spermatozoa in the subnatant. The supernatant was transferred to a new tube and the d-PBS was added to 15 ml. The semen was centrifuged at 1052 × g for 5 min to collect viable spermatozoa in the pellet. The pellet was re-suspended with mTBM and the spermatozoa density was regulated to 1.5 × 105 spermatozoa/ml. Then 50 ␮l of the spermatozoa was added to each drop, mixed, and the oocytes and spermatozoa co-incubated at 38.5 ◦ C in an atmosphere of 5% CO2 for 6 h. Then the fertilized oocytes were washed three times with embryos culture medium and cultured for another 18 h in the same medium. After IVF, zygotes were washed three times in 100 ␮l drops of embryo culture medium and transferred to the 4-well multidish which containing 500 ␮l embryo culture medium under warm mineral oil. The zygotes were cultured at 38.5 ◦ C in a mixture of 5% CO2 in air. After 48 h postIVF, embryos were placed in fresh embryo culture medium in the same way as described above. 2.6. Assessment of maturation, fertilization results and development of oocytes After IVM culture, about 50–60 denuded oocytes from every treatment were mounted on slides with a cover slip, secured by two lines of vaseline, and then fixed with acetic acid:ethanol (1:3,

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v/v) for 48–72 h. The fixed oocytes were stained with acetic–orcein (Cat. No. O7380) (1% orcein in 45% acetic acid) then sealed with nail polish. Maturation degree of oocytes was evaluated under a phase-contrast microscope. At the end of IVF, about 50–60 fertilized oocytes from each treatment were stained with acetic–orcein as above method. Oocytes were considered to be polyspermic that had more than one swollen sperm head or male pronuclei with corresponding sperm trail. Oocytes were considered to be monospermic that had only one swollen sperm head. MPN formation was considered with a visual identification of a MPN. Oocytes were considered to be cleaved that the embryos with two to eight blastomers 48 h after IVF. When the embryos culture period (144 h post-IVF) was completed, the percentages of the blastocyst in every group were calculated. The blastocystes were placed in 110 ␮l PBS containing 10 ␮l of 1 mg/ml bis-benzimide H33342 (Hoechst 33342, Cat. No. B2261) staining for 10 min, then the embryos were de-stained in PBS three times for 5 min and the cell number of the blastocyst was counted under a fluorescent microscope. 2.7. Experimental design Experiment 1 investigated the effect of pOCCs monolayers co-culture during IVM on maturation of porcine oocytes. Before IVM, all of the selected COCS were assigned to two treatment groups randomly and equally. One group consisted of COCS that maturated in vitro with the pOCCs monolayers for 44 h, the other group, as the control group, in which the COCS were cultured in vitro for 44 h alone. At termination of IVM, one-quarter of denuded oocytes from every group were stained with orcein to evaluate the meiotic competence of the oocytes, one-quarter of denuded oocytes from every group were prepared for GSH assay, the other oocytes were prepared for IVF. This experiment consisted of six replicates. Experiment 2 investigated the effect of pOCCs monolayers co-culture during IVM on the penetration, polyspermy and MPN formation rates of porcine oocytes after IVF. After IVF, half of the fertilized oocytes from both of the treatment groups were removed and stained with orcein respectively, and then the percentages of the penetration, the polyspermy and the MPN formation were counted. This experiment consisted of six replicates. The other fertilized oocytes were cultured in NCSU-23 supplemented with 0.4% BSA for continuous development. Experiment 3 investigated the effect of pOCCs monolayers co-culture during IVM on the cleavage and embryo development rates of fertilized porcine oocytes. The rates of the cleavage and the blastocyst of the embryos were counted respectively at 48 and 144 h after IVF. Finally, the cell number in each blastocyst was recorded. This experiment consisted of six replicates. 2.8. Statistical analysis Each experiment was repeated six times. Data are presented as means ± S.E.M. All rates were modeled according to binomial model of parameters and were analyzed by one-way ANOVA. The percentages of oocytes reaching each stage of meiosis, polyspermy, MPN, as well as the cleavage and blastocyst formation rates, and the mean number of cells per blastocyst were analyzed by Tukey test in SPSS 11.0 soft. Differences among treatments were considered significant when the P-value < 0.05.

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Table 1 Effect of pOCCs mono layers co-culture during IVM on in vitro matured oocytes after IVM Treatment

Co-culture Control

No. of total oocytesa

463 452

Percentage of oocytes at each meiotic stage (mean ± S. E. M.) GVb

M Ic

AI/TId

M IIe

2.8 ± 0.7 a 3.3 ± 0.3 a

3.3 ± 1.6 a 6.4 ± 1.9 b

8.7 ± 1.l a 9.7 ± 1.4 a

85.2 ± 3.6 a 80.5 ± 4.1 b

Values with different letters (a and b) within columns are significantly different (P < 0.05). a The experiment was replicated six times. b GV: germinal vesicle. c M I: Metaphase I. d AI/TI: Anaphase I or Telophase I. e M II: Metaphase II.

3. Results 3.1. Experiment 1 Effect of pOCCs monolayers co-culture during IVM on maturation of porcine oocytes is shown in Table 1. The presence of the pOCCs monolayers during IVM significantly increased percentage of oocytes in M II stage compared to the control group (P = 0.023, and P < 0.05, respectively). Although the percentages of oocytes in GV and Anaphase I/Telophase I (AI/TI) stage were lower than that in the co-culture group, the percentages of oocytes in the stages above showed no significant decrease in the co-culture group after IVM (P = 0.116 and 0.705, respectively). More oocytes in M I stage were found in the control group than co-culture group (P = 0.036, and P < 0.05, respectively). After maturation (Table 2), it was found that GSH concentration per oocyte was higher than the oocyte before IVM (P = 0.024 and 0.004, and P < 0.05, respectively) and GSH concentration per oocyte in co-culture group was higher than that in the control group (P = 0.031, and P < 0.05, respectively). 3.2. Experiment 2 Effect of the pOCCs monolayers cell co-culture during IVM on penetration, polyspermy and MPN formation rates of porcine oocytes after IVF are presented in Table 3. The total penetration rates were similar in both groups (P = 0.481), with the proportion of total penetration ranging from 88.01 to 90.45%. It was found that the polyspermy rate showed significant decrease in the co-culture group compared with that in the control group (P = 0.039 and P < 0.05, respectively), but the mean number of spermatozoa per penetrated oocyte showed no significant decrease in the Table 2 Effect of pOCCs mono layers co-culture during IVM on intracellular GSH concentration of porcine oocytes IVM group

No. of oocytes examineda

GSH concentration (pmol/oocyte; mean ± S. E.M.)

Before maturation in vitro Co-culture group Control group

96 96 96

4.1 ± 0.3 a 8.1 ± 1.3 b 6.3 ± 1.1 c

Values with different letters (a–c) within columns are significantly different (P < 0.05). a The experiment was replicated six times.

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Table 3 Effect of pOCCs mono layers co-culture during IVM on fertilization parameters 6 h post-IVF of porcine oocytes Treatment

Co-culture Control

No. of total fertilized oocytesa

354 341

Percentage of fertilized oocytes (% ± S.E.M.) Total penetration

Polyspermyb

MPNc

S/Od

88.0 ± 2.4 a 90.4 ± 3.6 a

39.4 ± 1.5 a 45.0 ± 2.8 b

97.5 ± 3.2 a 90.0 ± 3.9 b

1.9 ± 0.1 a 1.6 ± 0.5 a

Values with different letters (a and b) within columns are significantly different (P < 0.05). a The experiment was replicated six times. b Percentage of the number of penetrated oocyte. c MPN: male pronucleus. d S/O: spermatozoa/penetrated oocyte.

Table 4 Effect of pOCCs mono layers co-culture during IVM on embryo development and mean number cells per blastocyst Treatment

Co-culture Control

No. of total fertilized oocytesa

373 374

Percentage of embryo at different development stage of fertilized oocytes (% ± S.E.M.) Cleavage

Blastocystb

78.5 ± 2.3 a 80.1 ± 2.1 a

25.6 ± 2.7 a 21.9 ± 2.2 b

Mean number of cells/ blastocyst (% ± S.E.M.)

35.7 ± 2.3 a 33.6 ± 2.0 a

Values with different letters (a and b) within columns are significantly different (P < 0.05). a The experiment was replicated six times. b Percentage of the number of cleavage oocytes.

co-culture group (P = 0.766). Co-culture of the pOCCs monolayers during IVM showed positive effect on the MPN formation of oocytes after IVF, the proportion of the fertilized oocytes with MPN was significantly increased in the co-culture group compared with that in the control group (P = 0.013, and P < 0.05, respectively). 3.3. Experiment 3 Effect of the pOCCs monolayers co-culture during IVM on the cleavage rate and embryo development rate of fertilized porcine oocytes are presented in Table 4. The pOCCs monolayers co-cultured during IVM had a positive effect on the blastocyst rate (P = 0.024, and P < 0.05, respectively), although the cleavage rate showed no significant increase in the co-culture group (P = 0.560). At the end of 144 h after IVF, it was found that the mean number of cells per blastocyst showed no significant increase in the co-culture groups (P = 0.861). 4. Discussion The results indicated that we first used pOCCs monolayers during IVM and were successful in developing a co-culture system. Porcine oocytes nuclear and cytoplasm maturation degree, fertilization parameters and blastocyst development were promoted significantly in the present co-culture system. The ovary cortex is poorly vascularized in vivo (Van Wezel and Rodgers, 1996; Herrman and Spanel-Borowski, 1998), in which atresia follicles constantly occur. When oocytes co-cultured

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with pOCCs monolayers, there will be a new environment that is richer in hormones, nutrients and/or oxygen during IVM because the distribution of those will not be influenced by microcirculation. Ovarian cell cultured in vitro could secret keratinocyte growth factor (KGF) which is a 28-kilodalton (kDa) protein and belongs to the fibrobkast growth factor family (FGF7) (Parrott et al., 2000). It has been reported that KGF is primarily produced by stromal- or mesenchymal-derived cells in many tissues and acts as an epithelial cell-specific mitogen to proliferate granulose/cumulus cell growth in vitro (Jewgenow, 1996). Furthermore, the feasible progesterone concentration produced by cumulus cells is responsible for an acceleration of GVBD in porcine oocytes (Shimada et al., 2002; Yamashita et al., 2003). It seemed that the presence of pOCCs monolayers during IVM resulted a higher percentage of oocytes completing M I synchronally to reach the M II stage after IVM in the co-culture group compared with the control group. Oocytes matured in vivo keep cytoplasmic contact with cumulus cells by gap junctions until the M I stage and then lose the contact during progression towards the M II stage. But, in vitro, oocytes progressively lose contact with surrounding cumulus cells immediately from the onset of maturation (Motlik et al., 1986). Lower rate of oocytes at GV stage and higher GSH content in the co-culture group also shown that the pOCCs monolayers co-culture system could provide a better microenvironment for cumulus cells keeping the gap junction from GV stage to M I stage. It has been reported that gap junctions between oocytes and cumulus cells are related to GSH content of porcine oocytes cultured in vitro through the synthesis of GSH in cumulus cells or oocytes (Mori et al., 1998). When porcine oocytes were cultured in the co-culture system of the present study, secretions by the pOCCs monolayers probably participated in the regulation mechanism and facilitated maturation progress of oocytes by modulating the intercellular cooperation during IVM. In mammals, sperm penetration triggers oocyte activation, premature migration and partial exocytosis of cortical granule (CGs). The lower polyspermy in the co-culture group showed that the pOCCs monolayers may have contributed to the normal distribution of intracellular organelles (mitochondria and CGs) during IVM. It has been reported that normal distribution of CGs during IVM may play an important role in preventing polyspermy (Day, 2000), and the occurrence of polyspermy of porcine matured oocytes from in vitro may be due to a delay in CGs exocytosis (Wang et al., 1997). The ability to form a male pronucleus after IVF is correlated with cytoplasmic maturation (Funahashi and Day, 1993) and intracellular GSH concentration in pigs (Yoshida et al., 1993). GSH participates in sperm decondensation in parallel with oocyte activation after fertilization, as well as in the transformation of the penetrated sperm head into the MPN. In the present study, the high concentration of GSH in the co-culture group were mainly attributable to the two reasons below: (1) the microenvironments containing the pOCCs monolayers was propitious to synthesis GSH utilizing the cysteine by reduced the oxygen stress; (2) the presence of pOCCs monolayers during IVM maintained availably the gap junctions from the GV stage to the M I stage, which promoted intracellular cumulation of the extracellular GSH. Intracellular GSH was beneficial to normal male MPN formation presumably by reduction of oxidative stress (Yoshida et al., 1992, 1993). It seems probable that the presence of pOCCs monolayers induces a lower oxygen tension in vitro culture environment, as reported for oviduct cell in culture (Bavister, 1988), so that oocytes may effectively utilized cysteine to synthesis GSH by preventing the oxidation of cysteine to cystine. The rates of total penetration showed no significant difference between the both of two groups, which due to suboptimal fertilization time in vitro and suboptimal semen concentration. Generally, porcine sperm–oocytes incubation time changes during 3–12 h, and the number of polyspermic oocytes increases with incubation time when oocytes were fertilized in vitro (Abeydeera and Day, 1997a). In our study, although the percentages of cleavage in the two

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groups showed no significantly different, the rate of blastocyst showed higher in the co-culture group compared with the control group. The pOCCs monolayers co-culture of oocytes enhanced the subsequent percentage of oocytes developed to blastocysts, which showed the homologous cells co-culture with porcine oocytes during IVM may become a potential method to increase the maturation (nuclear and cytoplasm) degree in porcine embryos IVP. It has been evaluated that the blastocyst development and the hatchability of the blastocyst were enhanced, when the pOECs was co-cultured with porcine oocytes during IVM (Bureau et al., 2000; Kidson et al., 2003). When the in vivo-derived porcine embryos cultured in vitro, distinct ultrastructural differences was observed from the 2- and 4-cell stage to the blastocyst stage compared with their in vivo counterparts. These deviations were related to the quality of porcine embryos cultured in vitro (Hyttel and Niemann, 1990). It has been evaluated that abnormal cleavage and low cell numbers in blastocyst produced in vitro were due to apparent deficiencies in actin filament distribution within the cytoplasm and inadequate cytoplasmic maturation of oocytes (Wang et al., 1999). Low rate of blastocyst was correlated with the cytoplasmic maturation degree of porcine oocytes in the control group compared with the co-culture group. In the present study, the presence of pOCCs monolayers had no influence on the mean number of cells per blastocyst, which was different from the effect of the pOEC co-culture during the IVM (Kidson et al., 2003). Perhaps, the diversity between the both was caused by the secretion of the two types of cells. Although the NCSU23 supplemented BSA was considered the best medium at present of porcine embryos culture in vitro (Wang and Day, 2002), suboptimal embryo culture medium here may be responsible for poor mean number of cells per blastocyst cultured in vitro when compare with the embryos derived in vivo (Machat et al., 1998; Wang and Day, 2002). In summary, the present study demonstrated that the pOCCs monolayers co-culture during IVM enhanced the quality of nuclear and cytoplasmic maturation degree of the oocytes. Furthermore, the co-culture of the pOCCs monolayers with oocytes during IVM reduced the percentages of polyspermy, but improved rate of MPN after fertilization. Although the co-culture seemed have no positive effect on both the total penetration and mean number sperm per oocyte here, the rate of the blastocyst increased at the presence of the pOCCs monolayers during IVM. Finally, it seemed that the presence of pOCCs monolayers during the IVM had no effect on the mean cell number of the blastocyst at 144 h after IVF. Acknowledgement This research was supported by the Institute of Science and Technology of Qin Huangdao City of Hebei Province of China (No. D08). References Abeydeera, L.R., 2002. In vitro production of embryos in swine. Theriogenology 57, 257–273. Abeydeera, L.R., Day, B.N., 1997a. Fertilization and subsequent development in vitro of pig oocytes inseminated in modified tris-buffered medium with frozen–thawed ejaculated spermatozoa. Biol. Reprod. 57, 729–734. Abeydeera, L.R., Day, B.N., 1997b. In vitro penetration of pig oocytes in a modified tris-buffered medium: effect of BSA, caffeine and calcium. Therigenology 48, 537–544. Abeydeera, L.R., Wang, W.H., Cantly, T.C., Rieke, A., Prather, R.S., Day, B.N., 1998. Co-culture with follicular shell pieces can enhance the developmental competence of pig oocytes after in vitro fertilization: relevance to intracellular glutathione. Biol. Reprod. 58, 213–218. Bavister, B.D., 1988. Role of oviductal secretions in embryonic growth in vivo and in vitro. Theriogenology 29, 143– 154.

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