Sequential exposure of porcine cumulus cells to FSH and/or LH is critical for appropriate expression of steroidogenic and ovulation-related genes that impact oocyte maturation in vivo and in vitro

In this study, we collected follicular fluid, granulosa cells, and cumulus cells from antral follicles at specific time intervals following equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) treatment of gilts. The treatment with eCG increased the production of estrogen coordinately with up-regulated proliferation of granulosa and cumulus cells. eCG also induced the expression of LHCGR and PGR in cumulus cells and progesterone accumulation was detected in follicular fluid prior to the LH/hCG surge. Moreover, progesterone and progesterone receptor (PGR) were critical for FSH-induced LHCGR expression in cumulus cells in culture. The expression of LHCGR mRNA in cumulus cells was associated with the ability of LH to induce prostaglandin production, release of epidermal growth factor (EGF)-like factors, and a disintegrin and metalloprotease with thrombospondin-like repeats 1 expression, promoting cumulus cell oocyte complexes (COCs) expansion and oocyte maturation. Based on the unique expression and regulation of PGR and LHCGR in cumulus cells, we designed a novel porcine COCs culture system in which hormones were added sequentially to mimic changes observed in vivo. Specifically, COCs from small antral follicles were pre-cultured with FSH and estradiol for 10 h at which time progesterone was added for another 10 h. After 20 h, COCs were moved to fresh medium containing LH, EGF, and progesterone. The oocytes matured in this revised COC culture system exhibited greater developmental competence to blastocyst stage. From these results, we conclude that to achieve optimal COC expansion and oocyte maturation in culture the unique gene expression patterns in cumulus cells of each species need to be characterized and used to increase the effectiveness of hormone stimulation.     

Apoptosis in bovine cumulus-oocyte complexes after exposure to polychlorinated biphenyl mixtures during in vitro maturation

Aroclor-1254 (A-1254) is a commercial mixture of coplanar (dioxin-like) and non-coplanar (non dioxin-like) polychlorinated biphenyls (PCBs) affecting bovine oocyte in vitro maturation (IVM) and developmental competence. In the present study, the role of cumulus cell apoptosis in mediating the toxic effects of PCBs during in vitro maturation has been investigated. Results indicate that exposure of cumulus-oocyte complexes (COCs) to A-1254 significantly induced apoptosis of cumulus cells. Furthermore, A-1254 significantly increased the expression of the pro-apoptotic gene, Bax, concomitantly reducing the level of the anti-apoptotic gene, Bcl-2, in the cumulus cell compartment. The effects of pure mixtures of coplanar (PCB 77, 126 and 169) or non-coplanar (PCB 52, 101 and 153) PCBs were examined. Exposure of COCs to coplanar PCBs affected maturation at doses as low as 100.6 pg/ml. Furthermore, a significant increase in apoptosis and in Bax mRNA expression was observed. No variations in maturation or apoptosis were observed in the non-coplanar PCB group. To further analyze the role of cumulus cells, COCs and denuded oocytes (DOs) have been exposed to A-1254 or coplanar PCBs during IVM. Exposure of COCs significantly reduced the percentage of matured oocytes after 24 h of culture in both treatments. In contrast, exposure of DOs significantly decreased the maturation rate only at the highest dose investigated (100-fold greater than that affecting COCs). Taken together, the results indicate a direct role of cumulus cell apoptosis in mediating PCB toxicity on bovine oocytes, and a direct relationship between congener planarity and toxicity in bovine oocytes is suggested.     

Absence of seasonal changes in FSHR gene expression in the cat cumulus-oocyte complex in vivo and in vitro

Domestic cat oocytes are seasonally sensitive to FSH. Compared with those collected during the breeding season, oocytes from the nonbreeding (NB) season require more FSH during in vitro maturation to achieve comparable developmental competence. This study tested the hypothesis that this seasonal variation was due to altered expression of FSH receptors (FSHR) and/or FSH-induced genes. Relative expression levels of FSHR mRNA and FSH-enhanced gene estrogen receptor β (ESR2) were measured by qPCR in whole ovaries and immature cumulus-oocyte complexes (COCs) isolated from cat ovaries during the natural breeding vs NB seasons. Expression levels of FSH-induced genes prostaglandin-endoperoxide synthase 2 (PTGS2), early growth response protein-1 (EGR1), and epidermal growth factor receptor (EGFR) were examined in mature COCs from both seasons that were a) recovered in vivo or b) matured in vitro with conventional (1 μg/ml) or high (10 μg/ml) FSH concentrations. Overall, FSHR mRNA levels were lower in whole ovaries during the NB compared with breeding season but were similar in immature COCs, whereas ESR2 levels did not differ in either group between intervals. We observed changes in PTGS2, EGR1, and EGFR mRNA expression patterns across maturation in COCs within but not between the two seasons. The lack of seasonal differentiation in FSH-related genes was not consistent with the decreased developmental capacity of oocytes fertilized during the NB season. These findings reveal that the seasonal decrease in cat oocyte sensitivity to FSH occurs both in vivo and in vitro. Furthermore, this decline is unrelated to changes in expression of FSHR mRNA or mRNA of FSH-induced genes in COCs from antral follicles.     

Cumulus expansion and glucose utilisation by bovine cumulus-oocyte complexes during in vitro maturation: the influence of glucosamine and follicle-stimulating hormone

Glucose is an important metabolite and its presence during in vitro oocyte maturation (IVM) can have profound effects on the oocyte's developmental capacity. We have demonstrated that glucose uptake increases over a 24 h IVM period, with most accounted for as l-lactate production. However, as maturation proceeds, l-lactate production remains constant, suggesting an alternative role for glucose metabolism. We hypothesised that in the latter stages of oocyte maturation, glucose not accounted for by l-lactate production is utilised for FSH-stimulated extracellular matrix (ECM) synthesis. To examine precursor utilisation for synthesis of ECM, bovine cumulus-oocyte complexes (COCs) were matured in +/- FSH and/or glucosamine (an alternative substrate of matrix components). Measurements included COC diameters, glucose consumption and l-lactate production in spent media and [U-(14)C]glucose incorporation into ECM. FSH significantly stimulated both diameter and glucose consumption during 20-24 h maturation compared with unstimulated complexes, although co-incubation with glucosamine and FSH decreased total glucose consumption 1.7-fold compared with FSH alone (P < 0.05). Furthermore, there was a linear relationship between glucose and l-lactate metabolism in the presence of glucosamine, suggesting that the majority of glucose was being utilised for l-lactate production via glycolysis. In the presence of glucosamine, twofold less [U-(14)C]glucose was incorporated into matrix compared with COCs cultured without glucosamine. These results support the hypothesis that there is a link between glucose and glucosamine uptake in FSH-stimulated ECM synthesis. Furthermore, glucose has multiple fates within the COC during maturation and levels of utilisation are dependent on the composition of the maturation environment.     

Metabolic markers of developmental competence for in vitro-matured mouse oocytes

In vitro maturation of oocytes has enormous potential in assisted reproductive technology, but its use has been limited due to insufficient knowledge of oocyte physiology during this dynamic period and lack of an adequate maturation system. The aim of this study was to characterize the metabolic profiles of three groups of oocytes throughout maturation: cumulus-oocyte complexes (COCs), denuded oocytes, and denuded oocytes co-cultured with cumulus cells. Mouse oocytes were collected from 28-day-old unstimulated females and matured in a defined medium. Oocytes were matured individually and transferred into fresh 0.5 microl drops of medium at 4 h intervals until 16 h. Ultramicrofluorimetry was used to quantitate carbohydrate consumption from and metabolite release into the medium. Glucose consumption and lactate production of COCs increased (P < 0.001) over the maturation interval (0-16 h). Glucose consumption by COCs that subsequently fertilized was higher between 8-12 h of maturation than by COCs that did not fertilize (38 versus 29 pmol/COC per h, respectively; P < 0.01). Lactate production by COCs that subsequently fertilized was higher between 8-16 h of maturation, than by oocytes that did not fertilize (8-12 h, 66 versus 46 pmol/COC per h, P < 0.01; 12-16 h, 56 versus 40 pmol/COC per h, respectively; P < 0.05). These data indicate that the final hours of maturation may hold a unique marker of oocyte competence, as during this time fertilizable COCs take up more glucose and produce more lactate than those not subsequently fertilized.     

Effect of epidermal growth factor-like peptides on pig cumulus cell expansion, oocyte maturation, and acquisition of developmental competence in vitro: comparison with gonadotropins

The aim of this work was to assess the FSH-stimulated expression of epidermal growth factor (EGF)-like peptides in cultured cumulus-oocyte complexes (COCs) and to find out the effect of the peptides on cumulus expansion, oocyte maturation, and acquisition of developmental competence in vitro. FSH promptly stimulated expression of amphiregulin (AREG) and epiregulin (EREG), but not betacellulin (BTC) in the cultured COCs. Expression of AREG and EREG reached maximum at 2 or 4 h after FSH addition respectively. FSH also significantly stimulated expression of expansion-related genes (PTGS2, TNFAIP6, and HAS2) in the COCs at 4 and 8 h of culture, with a significant decrease at 20 h of culture. Both AREG and EREG also increased expression of the expansion-related genes; however, the relative abundance of mRNA for each gene was much lower than in the FSH-stimulated COCs. In contrast to FSH, AREG and EREG neither stimulated expression of CYP11A1 in the COCs nor an increase in progesterone production by cumulus cells. AREG and EREG stimulated maturation of oocytes and expansion of cumulus cells, although the percentage of oocytes that had reached metaphase II was significantly lower when compared to FSH-induced maturation. Nevertheless, significantly more oocytes stimulated with AREG and/or EREG developed to blastocyst stage after parthenogenetic activation when compared to oocytes stimulated with FSH alone or combinations of FSH/LH or pregnant mares serum gonadotrophin/human chorionic gonadotrophin. We conclude that EGF-like peptides do not mimic all effects of FSH on the cultured COCs; nevertheless, they yield oocytes with superior developmental competence.     

Influence of oocyte-secreted factors and culture duration on the metabolic activity of bovine cumulus cell complexes

Intracellular communication between the cumulus cell complex and the oocyte is essential for numerous processes during oocyte maturation. The aim of this study was to determine the interaction between oocyte-secreted factors and the metabolic activity of bovine cumulus cell complexes during in vitro maturation (IVM). Cumulus-oocyte complexes (COCs) were aspirated from ovaries derived from an abattoir and divided into four treatment groups: (i) intact COCs, (ii) oocytectomized complexes (OOX), in which the ooplasm was microsurgically removed, (iii) OOX co-cultured with denuded oocytes (OOX+DO) and (iv) DO. The complexes were cultured individually in IVM media. After 0-4, 10-14 and 20-24 h of culture, the utilization of oxygen, glucose, pyruvate and L-lactate by the complexes was measured. The metabolic activity of the DO was undetectable. There were no significant differences in metabolic measurement among any of the treatment groups, indicating that the metabolism of the cumulus complex is not affected by the presence of the oocyte. When metabolic activity for the complexes was analysed relative to time in culture, there was an approximate twofold increase in the consumption of oxygen, glucose and pyruvate over the 24 h period (P<0.05), although production of L-lactate remained constant. The relationship between total glucose uptake and L-lactate production indicated that the majority of glucose consumed at the start of culture was being utilized via glycolysis, but by the cessation of the maturation period, there was significant utilization of glucose elsewhere, possibly for the formation of cumulus extracellular matrix. These results indicate that metabolism of COC does not reflect biochemical activity of the oocyte. Nevertheless, the metabolic requirements of the COC increase throughout maturation.     

Abilities of cumulus and granulosa cells to enhance the developmental competence of bovine oocytes during in vitro maturation period are promoted by midkine; a possible implication of its apoptosis suppressing effects

We previously reported that when midkine (MK), a heparin-binding growth differentiation factor was used in in vitro maturation (IVM) culture of bovine cumulus-enclosed oocytes (CEOs), their developmental competence to the blastocyst stage after in vitro fertilization (IVF) was enhanced and the effect of MK might be mediated by its action upon mural granulosa cells and cumulus cells that closely surround the oocyte. In the present study, when denuded oocytes (DOs) were matured in IVM medium with or without MK (200 ng/ml) in the presence or absence of isolated cumulus cell masses and subjected to IVF, the enhancing effects of MK on the developmental competence of DOs to the blastocyst stage after IVF were exerted only in the presence of cumulus cells. In addition, we prepared the conditioned media of granulosa cells cultured with or without 200 ng MK/ml (CMMK+ or CMMK- respectively) and examined their effects on the IVM of DOs in terms of their developmental competence to the blastocyst stage after IVF. The supplementation of CMMK+ into IVM medium at 40% (v/v) significantly enhanced the blastocyst development compared with the no additive control and the CMMK- supplemented groups. Furthermore, the effects of MK during IVM of bovine CEOs on the cumulus cell apoptosis were investigated. CEOs were cultured up to 24 h in IVM medium without (control) or with 200 ng MK/ml. The genomic DNA was extracted from CEOs at 0, 6, 12, 18 and 24 h of IVM and subjected to ligation-mediated PCR (LM-PCR) to detect the apoptotic internucleosomal DNA fragmentation. DNA fragmentation was scarcely detected at the start of IVM, whereas it increased time-dependently as the IVM culture progressed. The degree of the fragmentation was significantly lower in the MK-treatment group compared with the control group at 18 and 24 h of IVM. The apoptosis-suppressing effect of MK on cumulus cells was further confirmed in situ by using TUNEL on CEOs. In conclusion, data from the present study further confirmed that MK enhances the developmental competence of bovine oocytes via cumulus and granulosa cells. It was also demonstrated that MK suppresses the apoptosis that occurs in cumulus cells during the period of IVM of bovine CEOs. The putative soluble factor(s) from cumulus cells was suggested from the experiment using CMMK+ . MK may promote the production of such factors in part by its anti-apoptotic effects on cumulus cells.     

Effect of cumulus morphology and maturation stage on the cryopreservability of equine oocytes

Oocyte cryopreservation is a potentially valuable way of preserving the female germ line. However, the developmental competence of cryopreserved oocytes is presently poor. This study investigated whether the morphology of the cumulus complex surrounding an immature equine oocyte and/or the oocyte's stage of maturation affect its cryopreservability. Compact (Cp) and expanded (Ex) cumulus oocyte complexes (COCs) were vitrified either shortly after recovery (germinal vesicle stage, GV) or after maturation in vitro (IVM); cryoprotectant-treated and -untreated non-frozen oocytes served as controls. In Experiment I, oocytes matured in vitro and then vitrified, or vice versa, were examined for maturation stage and meiotic spindle quality. Cp and Ex COCs vitrified at the GV stage matured at similar rates during subsequent IVM (41 vs 46% MII), but meiotic spindle quality was better for Cp than Ex (63 vs 33% normal spindles). Vitrifying oocytes after IVM resulted in disappointing post-warming spindle quality (32 vs 28% normal for Cp vs Ex). In Experiment II, oocytes from Cp and Ex COCs vitrified at the GV or MII stages were fertilized by intracytoplasmic sperm injection (ICSI) and monitored for cleavage and blastocyst formation. Oocytes vitrified prior to IVM yielded higher cleavage rates (34 and 27% for Cp and Ex COCs) than those vitrified after IVM (16 and 4%). However, only one blastocyst was produced from a sperm-injected vitrified-warmed oocyte (0.4 vs 9.3% and 13% blastocysts for cryoprotectant-exposed and -untreated controls). It is concluded that, when vitrification is the chosen method of cryopreservation, Cp equine COCs at the GV stage offer the best chance of an MII oocyte with a normal spindle and the potential for fertilization; however, developmental competence is still reduced dramatically.     

Apoptosis in cumulus cells during in vitro maturation of bovine cumulus-enclosed oocytes

The aim of this study was to investigate whether apoptosis occurs in cumulus cells during in vitro maturation (IVM) of bovine cumulus-enclosed oocytes (CEOs). The bovine CEOs obtained from ovaries from an abattoir were cultured for 24 h in IVM medium in the presence or absence of 10% (v/v) fetal bovine serum. The developmental competence of enclosed oocytes, as assessed by the development of the blastocyst after IVF, was significantly higher in the serum-treated group than in the control group. The morphological features of apoptosis that were analysed by orcein staining were hardly detectable in the cumulus cells at the start (0 h) of IVM, but were evident at the end (24 h) of IVM both in the control and serum-treated groups. Genomic DNA was extracted from CEOs at 0, 6, 12, 18 and 24 h of IVM and subjected to ligation-mediated PCR (LM-PCR) to detect apoptotic internucleosomal DNA fragmentation. DNA fragmentation was hardly detectable at the start of IVM, but increased in a time-dependent manner as the IVM culture proceeded. DNA fragmentation was not observed in the oocytes, indicating that fragmentation occurs in cumulus cells. The degree of fragmentation was lower in the serum-treated group compared with the control group. The LM-PCR analysis of DNA extracted from CEOs at 24 h of IVM, in which the DNA had been pretreated with Klenow enzyme or T4 DNA polymerase, revealed that the characteristic forms of the DNA ends generated during cumulus cell apoptosis were mainly 3'-overhangs and blunt ends. In conclusion, the results of the present study demonstrate that cumulus cells in bovine CEOs spontaneously undergo apoptosis during IVM. The degree of apoptosis may be correlated with the developmental competence of the enclosed oocytes.     

Lysophosphatic acid modulates prostaglandin secretion in the bovine uterus

Lysophosphatidic acid (LPA) modulates prostaglandin (PG) synthesis via LPA receptor 3 (LPAR3) in the murine endometrium. The lack of functional LPAR3 in mice may lead to embryo mortality. In the present study, we examined the role of LPA in the bovine uterus. We confirmed that LPA is locally produced and released from the bovine endometrium. Moreover, there are enzymes involved in LPA synthesis (phospholipase (PL) D(2) and PLA2G1B) in the bovine endometrium during estrous cycle and early pregnancy. Expression of the receptor for LPA (LPAR1) was positively correlated with the expression of PGE(2) synthase (PGES) and negatively correlated with the expression of PGF(2alpha) synthase (aldose reductase with 20 alpha-hydroxysteroid dehydrogenase activity - PGFS) during early pregnancy. In vivo LPA induced P4 and PGE(2) secretion was inhibited by LPAR1 antagonist (Ki16425). The overall results indicate that LPA is locally produced and released from the bovine endometrium. Moreover, LPAR1 gene expression in the endometrium during the estrous cycle and early pregnancy indicates that LPA may play autocrine and/or paracrine roles in the bovine uterus. LPAR1 gene expression is positively correlated with the expression of the enzyme responsible for luteotropic PGE(2) production (PGES) in endometrium. In cow, LPA stimulates P4 and PGE(2) secretion. Thus, LPA in the bovine reproductive tract may indirectly (via endometrium) or directly support corpus luteum action via the increase of P4 synthesis and the increase of PGE(2)/PGF(2)(alpha) ratio. It suggests that LPA may serve as an important factor in the maintenance of early pregnancy in cow.     

Effects of oocyte culture density on meiotic competence of canine oocytes

This study was conducted to determine a suitable ratio of oocytes to medium for in vitro maturation (IVM) of cumulus-oocyte complexes (COCs) collected from bitches at anoestrus and dioestrus and to examine the meiotic competence of COCs cultured singly or in different group sizes. In the first experiment, different numbers of COCs (5, 10, 15 and 20 per drop) were cultured for 72 h in 100 microl drops of maturation medium. The meiotic competence of oocytes from ovaries at anoestrus was affected by the number of COCs incubated, whereas at dioestrus, the incubation number of COCs had no effect. In the second experiment, COCs were cultured singly or in different group sizes for 72 h by suitable oocyte density according to the reproductive cycle of the donor. In the anoestrous group, 1, 5 and 10 COCs were cultured in 10, 50 and 100 microl drops of the medium (10 microl per COC), respectively. In the dioestrous group, 1, 5 and 15 COCs were cultured in 7, 35 and 105 microl drops of the medium (7 microl per COC), respectively. There were no differences in the proportions of oocytes reaching metaphase II among the different group sizes in each stage of the reproductive cycle of the donor. The results indicate that the influence of oocyte density on the meiotic competence of oocytes differs according to the stage of the reproductive cycle of the donor. Moreover, the group sizes have no effect on the meiotic competence of oocytes cultured at suitable oocyte density according to the reproductive cycle of the donor.     

Expression of connexin 43 and gap junctional intercellular communication in the cumulus-oocyte complex in sheep

To evaluate the effects of FSH, LH and/or cAMP on expression of connexin 43 (Cx43) in the ovine cumulus-oocyte complex (COC) and gap junctional intercellular communication (GJIC) of cumulus cells, two experiments were carried out. In experiment 1, Cx43 was immunodetected in the COC, before or after maturation, obtained from non-treated or FSH-treated ewes. The expression of Cx43 in the COC was greater (P < 0.01) on day 16 than on day 15 of the estrous cycle. In vivo FSH treatment decreased (P < 0.02) Cx43 expression on day 16 but not on day 15 of the estrous cycle. In experiment 2, intact COCs or isolated cumulus cells obtained from small and large follicles from FSH-treated ewes were cultured with or without FSH, LH or cAMP agonist and evaluated for GJIC by laser cytometry. For large follicles, the basal rate of GJIC was greater (P < 0.01) for cumulus cells in intact COCs than for isolated cumulus cells. FSH increased (P < 0.04) GJIC in cumulus cells in intact COCs and tended to increase (P < 0.1) GJIC in isolated cumulus cells from small follicles but decreased (P < 0.01) GJIC in cumulus cells in intact COCs from large follicles. LH also increased (P < 0.01) GJIC in isolated cumulus cells from small follicles but decreased GJIC in intact COCs (P < 0.01) and isolated cumulus cells (P < 0.02) from large follicles. cAMP increased (P < 0.01) the GJIC in both intact COCs and cumulus cells from small and large follicles. These results indicate that day of estrous cycle, stage of maturation and duration of FSH treatment affect expression of Cx43 in ovine COCs. In intact COCs, GJIC in cumulus cells was enhanced, probably due to the presence of the oocyte. In addition, the effects of FSH and LH, but not cAMP, on GJIC of cumulus cells depended on the stage of follicular development and on the presence of the oocyte.     

Involvement of connexin 43 in meiotic maturation of bovine oocytes

In ovarian follicles, cumulus cells provide the oocyte with small molecules that permit growth and control maturation. These nutrients reach the germinal cell through gap junction channels, which are present between the cumulus cells and the oocyte, and between the cumulus cells. In this study the involvement of intercellular communication mediated by gap junction channels on oocyte maturation of in vitro cultured bovine cumulus-oocyte complexes (COCs) was investigated. The stages of oocyte maturation were determined by Hoechst 33342 staining, which showed that 90% of COCs placed in the maturation medium for 24 h progress to the metaphase II stage. Bovine COC gap junction communication was disrupted initially using n-alkanols, which inhibit any passage through gap junctions. In the presence of 1-heptanol (3 mmol l(-1)) or octanol (3.0 mmol l(-1) and 0.3 mmol l(-1)), only 29% of the COCs reached metaphase II. Removal of the uncoupling agent was associated with restoration of oocyte maturation, indicating that treatment with n-alkanols was neither cytotoxic nor irreversible. Concentrations of connexin 43 (Cx43), the major gap junction protein expressed in the COCs, were decreased specifically using a recombinant adenovirus expressing the antisense Cx43 cDNA (Ad-asCx43). The efficacy of adenoviral infection was > 95% in cumulus cells evaluated after infection with recombinant adenoviruses expressing the green fluorescence protein. RT-PCR performed on total RNA isolated from Ad-asCx43-infected COCs showed that the rat Cx43 cDNA was transcribed. Western blot analysis revealed a three-fold decrease in Cx43 expression in COCs expressing the antisense RNA for Cx43. Injection of cumulus cells with Lucifer yellow demonstrated further that the resulting lower amount of Cx43 in infected COCs is associated with a two-fold decrease in the extent of coupling between cumulus cells. In addition, oocyte maturation was decreased by 50% in the infected COC cultures. These results indicate that Cx43-mediated communication between cumulus cells plays a crucial role in maturation of bovine oocytes.     

Delay of nuclear maturation and reduction in developmental competence of pig oocytes after mineral oil overlay of in vitro maturation media

Steroid hormones, such as progesterone, oestrogen, androgen and meiosis activating sterols, are secreted from cumulus cells that are stimulated by gonadotrophins during maturation of oocytes in vitro. These steroid hormones may be absorbed by mineral oil or paraffin oil; however, in vitro maturation of pig oocytes is commonly performed using medium covered by oil. In this study, high concentrations of progesterone, oestradiol and testosterone were detected in the culture medium after pig cumulus-oocyte complexes (COCs) were cultured with FSH and LH for 44 h in medium without an oil overlay. However, high concentrations of these steroid hormones were not detected in medium when COCs were cultured with the mineral oil overlay. When high concentrations of these steroid hormones were secreted by COCs, germinal vesicle breakdown (GVBD) and the activation of p34(cdc2) kinase and mitogen-activated protein (MAP) kinase in oocytes occurred earlier in comparison with oocytes cultured in medium covered with mineral oil. Moreover, a decrease in p34(cdc2) kinase activity during meiotic progression beyond metaphase I was observed in oocytes cultured in conditions under which high concentrations of steroid hormones were secreted by COCs. In addition, the rate of development to the blastocyst stage after IVF was higher in oocytes matured in medium without an oil overlay. These adverse effects of oil may be explained by absorption by the oil of cumulus-secreted steroids or by the release of toxic compounds into the medium.     

Epidermal growth factor receptor downregulation in cultured bovine cumulus cells: reconstitution of calcium signaling and stimulated membrane permeabilization

Cumulus cell-oocyte complexes (COCs), cultured in vitro, are competent for maturation and fertilization. Inclusion of epidermal growth factor (EGF) in the COC culture medium enhances in vitro maturation and subsequent embryonic development. It has been shown that isolated COCs exposed to EGF respond with a prolonged and pulsatile release of Ca2+ into the extra-cellular medium and that cumulus cells (CCs) of complexes exhibit both a slow rise in intracellular [Ca2+] ([Ca2+]i) and plasma membrane permeabilization in response to EGF. These unusual signaling responses were examined in isolated, cultured bovine CCs. Few individual CCs showed [Ca2+]i increases; the lack of response was found to be due to decrease of expression of endogenous EGF receptors after dissociation. CCs transfected with a human EGF receptor-GFP fusion protein showed robust, prolonged, EGF-stimulated [Ca2+]i elevations characteristic of CC responses in intact COCs. Many CCs that responded to EGF stimulation with a [Ca2+]i rise also released entrapped fura-2 dye at the peak of the [Ca2+]i response, suggesting that CC permeabilization and death follows activation of the EGF receptor. The [Ca2+]i elevation due to EGF stimulation and subsequent membrane permeabilization was shown to be mediated by the inositol triphosphate signaling pathway.     

Protein kinases influence bovine oocyte competence during short-term treatment with recombinant human follicle stimulating hormone

The aim of this study was to investigate the effect of short-term treatment (first 2 or 6 h) with recombinant human follicle-stimulating hormone (r-hFSH) during in vitro maturation (IVM) on the developmental competence of bovine oocytes. The roles of protein kinase A (PKA) and protein kinase C (PKC) (possibly involved in FSH response), were investigated using activators (Sp-cAMPS, PMA) or inhibitors (Rp-cAMPS, sphingosine) of these two protein kinases, respectively. The developmental competence of bovine oocytes was measured by the rate of blastocyst formation after in vitro fertilization (IVF). Our results showed that when cumulus-oocyte complexes (COCs) were cultured with r-hFSH for the first 6 h, a highly significant (P < 0.0001) improvement is seen in blastocyst development rate as a proportion of oocytes in culture compared with those matured with r-hFSH for the first 2 or 24 h. A transient exposure (6 h) to the highest dose (100 microM) of forskolin (an activator of adenylate cyclase) increased (P < 0.05) the rate of blastocyst formation. But the PKA inhibitors (Rp-cAMPS) did not affect the stimulatory effects of r-hFSH on the blastocyst yield. However, stimulation of PKC by low doses of PMA (0.1-0.5 microM) during short-term treatment, enhanced (P < 0.0001) the developmental capacity of oocytes, while sphingosine (a specific inhibitor of PKC) inhibited (P < 0.05) the stimulatory effects of r-hFSH on the rate of blastocyst formation. Our results indicate that although the developmental capacity of bovine oocytes in vitro can be modulated by both the PKA, and the PKC pathways, the activation of PKC during short-term treatment can mimic the effect of r-hFSH on the cytoplasmic maturation in bovine oocytes in vitro.     

Overcoming poor in vitro nuclear maturation and developmental competence of domestic cat oocytes during the non-breeding season

The domestic cat experiences circannual variations in ovarian activity and intrafollicular oocyte quality. One result is poor nuclear and cytoplasmic maturation during in vitro maturation (IVM) conducted during the annual non-breeding season (July through November). In an attempt to overcome this seasonal phenomenon immature oocytes were collected from July through November and cultured in a conventional IVM medium (IVM1) or in IVM1 supplemented with different FSH concentrations and antioxidant (ascorbic acid or cysteine). Nuclear status of oocytes was assessed after IVM or IVF. Embryo stage and blastocyst quality were evaluated after 7 days of in vitro culture. Although the addition of antioxidant alone had no effect, the presence of 10 microg FSH ml(-1) improved nuclear maturation (75.4+/-4.1% versus 48.7+/-8.8% in IVM1; P<0.05) and fertilization success (47.9+/-3.2% versus 35.0+/-5.1% in IVM1; P<0.05). Furthermore, developmental competence of fertilized oocytes was enhanced (P<0.05) only in the presence of ascorbic acid (30.6+/-6.7%) or cysteine (33.6+/-5.1%) compared with IVM1 (8.1+/-8.8%). Consequently, blastocyst yield (17% of total oocytes cultured) was highest when oocytes were matured in medium containing higher FSH concentration and antioxidants. The results of this study demonstrate that meiotic and developmental competences are inherent to the immature cat oocyte collected during the non-breeding season. However, appropriate mechanisms (perhaps seasonal variation in FSH receptors or lack of antioxidant capacity of the cumulus-oocyte complex) are inadequate during this period of gonadal quiescence. Regardless, this compromised oocyte function during the non-breeding season can be overridden by altering in vitro culture conditions to include supplemental FSH and antioxidant.     

Sonic hedgehog supplementation of oocyte and embryo culture media enhances development of IVF porcine embryos

We investigated the expression of sonic hedgehog (SHH) receptor PTCH1 and its co-receptor smoothened (SMO) in fertilized porcine embryos. Effects of exogenous SHH on embryonic development and expressions of survival- and pluripotency-related genes were also determined. We found that PTCH1 and SMO are expressed from two-cell to blastocyst embryos. When oocytes or fertilized embryos were respectively cultured in the maturation or embryo culture medium supplemented with SHH (0.5?μg/ml), their blastocyst rates and total cell numbers increased (P<0.05) compared with the untreated control. When cultured simultaneously in the in vitro maturation (IVM) and in vitro culture (IVC) media supplemented with SHH, the oocytes gained increased blastocyst rates and total cell numbers in an additive manner, with reduced apoptotic indices (P<0.05). Interestingly, SHH treatment did not affect the expression of the BCL2L1 (BCL-XL) gene, yet reduced BAX expression. Blastocysts cultured with various SHH regimes had similar pluripotency-related gene (POU5F1 (OCT-4) and CDX2) expression levels, but blastocysts derived from SHH treatment during IVM had higher ZPF42 (REX01) expression (P<0.05). The highest ZPF42 expression was observed in the blastocysts derived from SHH-supplemented IVC and from dual IVM and IVC treatments. The levels of acetylated histone 3 (AcH3K9/K14) increased in the two-cell and the four-cell embryos when IVM and/or IVC media were supplemented with SHH (P<0.05). Our findings indicate that SHH conferred a beneficial effect on preimplantation development of porcine embryos, particularly when both IVM and IVC media were supplemented with SHH, and the effects may be further carried over from IVM to the subsequent embryonic development.