In preovulatory follicles, after the endogenous gonadotropin surge, the oocyte-cumulus complexes (OCCs) produce hyaluronan (HA) in a process called "cumulus expansion". During this process, the heavy chains (HCs) of the serum-derived inter-alpha-trypsin inhibitor (IαI) family bind covalently to synthesized HA and form a unique structure of the expanded cumulus HA-rich extracellular matrix. Understanding the biochemical mechanism of the covalent linkage between HA and the HCs of the IαI family is one of the most significant discoveries in reproductive biology, since it explains basis of the cumulus expansion process running in parallel with the oocyte maturation, both essential for ovulation. Two recent studies have supported the above-mentioned findings: in the first, seven components of the extracellular matrix were detected by proteomic, evolutionary, and experimental analyses, and in the second, the essential role of serum in the process of cumulus expansion in vitro was confirmed. We have previously demonstrated the formation of unique structure of the covalent linkage of HA to HCs of IαI in the expanded gonadotropin-stimulated OCC, as well as interactions with several proteins produced by the cumulus cells: tumor necrosis factor-alpha-induced protein 6, pentraxin 3, and versican. Importantly, deletion of these genes in the mice produces female infertility due to defects in the oocyte-cumulus structure.

The extracellular matrix (ECM) is an essential structure with biological activities. It has been shown that the ECM influences gene expression via cytoskeletal components and the gene expression is dependent upon cell interactions with molecules and hormones. The development of ovarian follicles is a hormone dependent process. The surge in the luteinizing hormone triggers ovulatory changes in oocyte microenvironment. In this review, we discuss how proteolytic cleavage affects formation of cumulus ECM following hormonal stimulation; in particular, how the specific proteasome inhibitor MG132 affects gonadotropin-induced cytoskeletal structure, the organization of cumulus ECM, steroidogenesis, and nuclear maturation. We found that after the inhibition of proteolytic cleavage, gonadotropin-stimulated oocyte–cumulus complexes (OCCs) were without any signs of cumulus expansion; they remained compact with preserved cytoskeletal F-actin-rich transzonal projections through the oocyte investments. Concomitantly, a significant decrease was detected in progesterone secretion and in the expression of gonadotropin-stimulated cumulus expansion–related transcripts, such as HAS2 and TNFAIP6. In agreement, the covalent binding between hyaluronan and the heavy chains of serum-derived the inter-alpha-trypsin inhibitor, essential for the organization of cumulus ECM, was missing.

In the mammalian ovary, the hyaluronan (HA)-rich cumulus extracellular matrix (ECM) organized during the gonadotropin-induced process of oocyte maturation is essential for ovulation of the oocyte-cumulus complex (OCC) and fertilization. Versican is an HA-binding proteoglycan that regulates cell function and ECM assembly. Versican cleavage and function remain to be determined in ovarian follicle. We investigated versican expression in porcine ovarian follicles by real-time (RT)-PCR and western blotting. The aims of the present work were to determine whether 1) versican was produced and cleaved by porcine OCCs during gonadotropin stimulation; 2) these processes were autonomous or required the participation of mural granulosa cells (MGCs); and 3) versican cleavage was involved in the formation or degradation of expanded cumulus ECM. We demonstrate two cleavage products of G1 domain of versican (V1) accumulated in the HA-rich cumulus ECM. One of them, a G1-DPEAAE N-terminal fragment (VG1) of ~70 kDa, was generated from V1 during organization of HA in in vivo and in vitro expanded porcine OCCs. Second, the V1-cleaved DPEAAE-positive form of ~65 kDa was the only species detected in MGCs. No versican cleavage products were detected in OCCs cultured without follicular fluid. In summary, porcine OCCs are autonomous in producing and cleaving V1; the cleaved fragment of ~70 kDa VG1 is specific for formation of the expanded cumulus HA-rich ECM.

Fertilization of the mammalian oocyte requires interactions between spermatozoa and expanded cumulus extracellular matrix (ECM) that surrounds the oocyte. This review focuses on key molecules that play an important role in the formation of the cumulus ECM, generated by the oocyte-cumulus complex. In particular, the specific inhibitors (AG1478, lapatinib, indomethacin and MG132) and progesterone receptor antagonist (RU486) exerting their effects through the remodeling of the ECM of the cumulus cells surrounding the oocyte have been described. After gonadotropin stimulus, cumulus cells expand and form hyaluronan (HA)-rich cumulus ECM. In pigs, the proper structure of the cumulus ECM depends on the interaction between HA and serum-derived proteins of the inter-alpha-trypsin inhibitor (IαI) protein family. We have demonstrated the synthesis of HA by cumulus cells, and the presence of the IαI, tumor necrosis factor-alpha-induced protein 6 and pentraxin 3 in expanding oocyte-cumulus complexes (OCC). We have evaluated the covalent linkage of heavy chains of IαI proteins to HA, as the principal component of the expanded HA-rich cumulus ECM, in porcine OCC cultured in medium with specific inhibitors: AG1478 and lapatinib (both inhibitors of epidermal growth factor receptor tyrosine kinase activity); MG132 (a specific proteasomal inhibitor), indomethacin (cyclooxygenase inhibitor); and progesterone receptor antagonist (RU486). We have found that both RU486 and indomethacin does not disrupt the formation of the covalent linkage between the heavy chains of IαI to HA in the expanded OCC. In contrast, the inhibitors AG1478 and lapatinib prevent gonadotropin-induced cumulus expansion. Finally, the formation of oocyte-cumulus ECM relying on the covalent transfer of heavy chains of IαI molecules to HA has been inhibited in the presence of MG132.

Bone morphogenetic protein-15 (BMP-15), an oocyte-derived growth factor, has been shown to play integral roles in regulation of ovarian follicular function in mammals. Despite the recognition of the physiological importance of the BMP system in regulation of gonadotropin action in the ovary, molecular mechanisms of BMP-15 effect on oocyte and somatic follicular cell functions remain poorly understood. The objective of this study was to determine the effect of BMP-15 on the FSH/LH-stimulated synthesis of hyaluronan (HA) by oocyte cumulus complexes (OCC) and progesterone by OCC and granulosa cells (GC) in the presence or absence of serum using primary porcine cultures. In addition, the effect of BMP-15 on oocyte maturation- and steroidogenesis-related transcripts after 4, 8, 16, and 24 hours of cultivation was evaluated using real-time RT-PCR. We demonstrated that the FSH/LH-induced cumulus expansion was accompanied by a significant increase in CD44, PTGS2, CYP11A1 (at 4 h) and AREG, HAS2, TNFAIP6, STAR (at 8 h) mRNAs. While FSH/LH-stimulated total HA synthesis by OCC was not affected by BMP-15 in serum-supplemented medium, its retention within the complex was significantly increased after the action of BMP-15 in comparison to FSH/LH alone (P < 0.001; 65% versus 35%, respectively). Moreover, we detected a significant increase in the expression of AREG and TNFAIP6 (both at 16 h), and CYP11A1 (at 24 h) in FSH/LH-stimulated OCC due to the action of BMP-15 compared to complexes cultured only with FSH/LH. In the presence of serum, BMP-15 markedly increased FSH/LH-stimulated progesterone secretion by OCC (about 69%) and induced a significant decrease in FSH/LH-induced progesterone release by GC (about 35%) compared to FSH/LH alone. The present results indicate that the addition of BMP-15 to the gonadotropin-stimulated OCC cultured in serum-supplemented medium might improve oocyte-cumulus maturation.

Searchable abstracts of presentations at key conferences on reproductive biology and medicine ISSN 2052-1472 (online)

It has been previously shown that multimeric pentraxin 3 (PTX3) is a key component of the cumulus oophorus extracellular matrix (ECM) in mice. In response to the ovulatory LH surge, the cumulus cells assemble a unique ECM that envelopes the oocyte and cumulus cell complex. Importantly, cumuli from PTX3(-/-) mice were defective in their ECM organization and their fertility was impaired. It has been demonstrated that tumor necrosis factor alpha-induced protein 6 catalyzes the formation of heavy chains of (inter-alpha-trypsin inhibitor) -hyaluronan complexes and these are then cross-linked via PTX3. This process is tightly regulated and requires the proteins to meet/interact in the correct order. Finally, in this way, the above-listed proteins form the cumulus oophorus ECM. We investigated whether PTX3 is expressed in the porcine preovulatory follicle. Porcine oocyte-cumulus complexes (OCC) and mural granulosa cells (MGC) from gilts were obtained either after stimulation in vivo with eCG/hCG (4, 8, 16, 24, and 32 h) or culture in vitro (4, 24, and 44 h) in FSH/LH-supplemented medium. The methods performed were real-time reverse transcriptase-polymerase chain reaction, Western blot analysis, and immunostaining. The expression of PTX3 transcripts was significantly increased 24 h after either in vivo hCG stimulation or in vitro FSH/LH treatment in both OCC and MGC. Western blot analysis with PTX3 antibody revealed that not only matrix extracts from in vivo-stimulated gilts contain high levels of PTX3 protein but also matrix extracts of FSH/LH-stimulated OCC cultured in medium supplemented either with follicular fluid or with porcine serum. The localization of PTX3 in the cumulus oocyte complex was confirmed by immunostaining. In conclusion, PTX3 is produced by porcine OCC and MGC both in vivo and in vitro with gonadotropin stimuli inducing cumulus expansion.

It has been shown that following endogenous gonadotropin surge, oocyte-cumulus complexes (OCC) synthesize hyaluronan (HA) in a process called cumulus expansion. During this process, HA associates with proteins and proteoglycans to form the expanded HA-rich oocyte-cumulus extracellular matrix (ECM), where the heavy chains of the serum derived inter-α-trypsin inhibitor family (IαI) bind covalently to HA. No study has been performed on the occurrence and regulation of this process during oocyte maturation in species other than mouse and pig, although, the heavy chains (of IαI)-HA complex was purified from human amniotic membrane. The present review pointing out that: 1/ formation of expanded HA-rich oocyte-cumulus ECM is dependent on the presence of IαI molecules, 2/ the heavy chains of IαI molecules identified in the serum are covalently linked to HA during cumulus expansion in mouse and pig, 3/ the family of IαI molecules can freely cross the blood-follicle barrier, and the follicular fluid collected at any stage of folliculogenesis can be successfully used instead of serum to form expanded cumulus ECM in pig, and 4/ proteins of the IαI family can affect reproductive process by modulating the expression of a large number of cellular genes during a preovulatory period. Finally, this review provides clear evidence that IαI family members present in the serum or follicular fluid become responsible for cumulus expansion, as without these proteins, expanded cumulus HA-rich ECM is not formed and HA is released into medium.

Searchable abstracts of presentations at key conferences on reproductive biology and medicine ISSN 2052-1472 (online)

This study was designed to determine whether inhibition of either cyclooxygenase-2 (COX-2) by indomethacin or progesterone receptor (PR) by PR antagonist, RU486, affects oocyte maturation, progesterone production, and covalent binding between hyaluronan (HA) and heavy chains of inter-α trypsin inhibitor, as well as expression of cumulus expansion-associated proteins (HA-binding protein, tumor necrosis factor α-induced protein 6, pentraxin 3) in oocyte-cumulus complexes (OCCs). The experiments were based on freshly isolated porcine OCC cultures in which the consequences of PR and COX-2 inhibition on the final processes of oocyte maturation were determined. Granulosa cells (GCs) and OCCs were cultured in medium supplemented with FSH/LH (both 100 ng/mL) in the presence/absence of RU486 or indomethacin. Western blot analysis, (3)H-glucosamine hydrochloride assay, immunofluorescence, and radioimmunoassay were performed. Only treatment with RU486 (25 μM) caused a decrease in the number of oocytes that reached germinal vesicle breakdown and metaphase II stage compared with indomethacin (100 μM) or FSH/LH treatment alone after 44 h. All treated OCCs synthesized an almost equal amount of HA. Heavy chains (of inter-α trypsin inhibitor)-HA covalent complexes were formed during in vitro FSH/LH-stimulated expansion in RU486- or indomethacin-treated OCCs. Follicle-stimulating hormone/LH-induced progesterone production by OCCs was increased in the presence of RU486 after 44 h. In contrast, a decrease of FSH/LH-stimulated progesterone production by GCs was detected in the presence of either RU486 or indomethacin after 72 h. We suggest that the PR-dependent pathway may be involved in the regulation of oocyte maturation. Both PR and COX-2 regulate FSH/LH-stimulated progesterone production by OCCs and GCs.

OBJECTIVE:To determine whether inhibition of epidermal growth factor (EGF) receptor tyrosine kinase with lapatinib affects oocyte maturation, expression of the cumulus expansion-associated genes such as tumor necrosis factor alpha-induced protein 6 (TNFAIP6) and prostaglandin-endoperoxide synthase 2 (PTGS2), and synthesis of hyaluronan (HA) and progesterone (P) by porcine oocyte cumulus complexes (OCC). DESIGN:Our work focuses on lapatinib, an orally active small molecule that selectively inhibits the tyrosine kinase domain of both EGF receptor and human EGF receptor 2, and downstream signaling. SETTING:A reproductive biology laboratory. PATIENT(S):Not applicable. INTERVENTION(S):Porcine OCC were cultured in vitro in a medium with FSH/LH in the presence/absence of lapatinib. MAIN OUTCOME MEASURE(S):Methods performed: real-time reverse transcriptase-polymerase chain reaction (PCR), immunofluorescence, RIA. RESULT(S):In FSH/LH-stimulated and expanded cumulus oophorus extracellular matrix, HA was detected with biotinylated HA-binding proteins. However, weaker HA- and weaker cytoplasmic TNFAIP6 were detected were detected in lapatinib-pretreated OCC. The expression of the two cumulus expansion-associated gene transcripts was significantly decreased and synthesis of HA by cumulus cells was reduced. Lapatinib (10 μM) inhibited FSH/LH-induced oocyte meiotic maturation. Progesterone production increased after OCC stimulation with FSH/LH and was significantly decreased by lapatinib (10 μM). CONCLUSION(S):Lapatinib inhibits oocyte maturation and reduces expression of cumulus expansion-associated transcripts, and synthesis of HA and P in OCC cultured in vitro in FSH/LH-supplemented medium.

UNLABELLED:This review deals with molecular mechanisms controlling three important ovarian follicular processes: 1) expansion of the cumulus, 2) synthesis of the hyaluronan (HA), and 3) production of the progesterone in oocyte cumulus complexes (OCCs). The expansion of the mice cumuli induced by FSH or 8-bromo cAMP is dependent upon a specific factor(s) secreted by the oocyte (called "cumulus expansion enabling factor", CEEF). The porcine oocytes produce at least two factors that have influence on the formation and stability of the preovulatory extracellular cumulus matrix (ECM), although oocytectomy does not alter the ability of the cumulus cells to respond to FSH and forskolin by increased cAMP content, HA synthesis, and subsequent cumulus expansion, The net synthesis of HA, during the FSH-stimulated expansion of the OCCs in the presence of serum, correlates directly with accumulation of glycosaminoglycans in the ECM. In pig, insulin growth factor 1 (IGF1) is a component of the serum that promotes the FSH-stimulated synthesis and retention of HA within the expanded ECM by phosphatidylinositol 3-kinase (PI3K)/v-akt murine thymoma viral oncogene homolog (AKT)- and mitogen-activated kinase 3 and 1 (MAPK3/1)-dependent mechanisms. Mouse, porcine, bovine, and rat oocytes produce CEEF(s). Possible candidate for the CEEF in the mouse is growth differentiation factor 9 (GDF9) secreted by oocytes. In pig, GDF9 mRNA is expressed not only in the oocytes but also in the cumulus and mural granulosa cells of the growing and preovulatory follicles, although the relative abundance of the GDF9 in the somatic cells is approximately 4 times lower than in the oocytes. Cross talk between FSH/ epidermal growth factor receptor (EGFR) and transforming growth factor β (TGFβ)/GDF9 signaling pathways is essential for functional activities of the porcine OCCs, since FSH enhances EGF-induced tyrosine phosphorylation of EGFR, indicating that FSH signaling pathway may stimulate specific EGFR-regulating proteins. Also, FSH-induced synthesis of both HA and progesterone is reduced but not abolished by AG1478 (EGFR tyrosine kinase inhibitor), indicating that other signaling pathways elicited by FSH are operating in parallel. Furthermore, SMAD2/3 signaling pathway is involved in the control of both cumulus expansion and steroidogenesis in porcine OCCs, since SMAD2/3 activation by GDF9/ TGFβ produced by oocyte and/or cumulus cells, significantly affects gonadotropin-induced HA and progesterone synthesis by porcine cumulus cells.KEYWORDS:oocyte-cumulus complex, hyaluronan, progesterone, cumulus expansion.

Porcine oocyte-cumulus complexes (OCCs) form an expanded cumulus extracellular matrix (ECM) in response to gonadotropins during meiotic maturation. Essential components of ECM are hyaluronan (HA), tumor necrosis factor α-induced protein 6 (TNFAIP6) and heavy chains (HC) of interalpha-trypsin inhibitor. To form expanded cumulus ECM, intermediate complexes (TNFAIP6-HC) must bind to HA to allow HC transfer onto HA. Protein turnover by the ubiquitin-proteasome pathway is poorly characterized in this process. It is known that the specific proteasomal inhibitor MG132 prevents cumulus expansion and formation of ECM. To determine whether inhibition of proteasomal proteolysis with MG132 affects cumulus cell steroidogenesis and expression of the cumulus expansion-related components (hyaluronan synthase type 2, HAS2, TNFAIP6) we cultured porcine OCCs and granulosa cells (GCs) in a medium supplemented with FSH/LH. Methods performed included real-time reverse transcription PCR, immunofluorescence and RIAs. The expression of TNFAIP6 and HAS2 transcripts increased significantly after the stimulation of OCCs and GCs with FSH/LH. In contrast, treatment with MG132 reduced the expression of TNFAIP6 and HAS2. Hyaluronan was detected with biotinylated HA-binding proteins within FSH/LH-stimulated expanded OCCs but not in those treated with MG132. Progesterone production, although increased almost three times after OCCs stimulation with FSH/LH, was significantly suppressed by MG132. The FSH/LH-stimulated a 40-fold increase in progesterone secretion by GCs was inhibited in the presence of MG132. In conclusion, MG132 affects progesterone secretion and expression of cumulus expansion-related components by cumulus and GCs, suggesting the requirement of ubiquitin-proteasome pathway-regulated protein turnover for formation of ECM during cumulus expansion in the preovulatory period in the pig.

Several lines of evidence suggest that in mice the activation of SMAD2/3 signaling by oocyte secreted factors, together with epidermal growth factor receptor (EGFR) activation, is essential to induce cumulus expansion. Here we show that inhibition of EGFR kinase in follicle stimulating hormone (FSH)-stimulated porcine oocyte-cumulus cell complex (OCCs) strongly decreases hyaluronan (HA) synthesis and its retention in the matrix, as well as progesterone synthesis. Although porcine cumulus cells undergo expansion independently of oocytes, we use biochemical and gene expression analyses to show that they do require activation of SMAD2/3 for optimal stimulation of HA synthesis and proteins involved in the organization of this polymer in the expanded matrix. Furthermore, FSH-induced progesterone synthesis by porcine cumulus cells was increased by blocking SMAD2/3 activation. In conclusion, these results support the hypothesis that an FSH-EGF autocrine loop is active in porcine OCCs, and provide the first evidence that the SMAD2/3 signaling pathway is induced by paracrine/autocrine factors in porcine cumulus cells and is involved in the control of both cumulus expansion and steroidogenesis.

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.

It has been shown that the activation of both EGF receptor and SMAD2 and SMAD3 in mouse cumulus cells is essential for their preovulatory maturation. In the present study we investigated whether these changes are also required for the final differentiation of pig cumulus cells. Porcine oocyte-cumulus complexes (OCCs) were stimulated in vitro with FSH in the absence or in the presence of the following inhibitors: AG1478 (an EGFR tyrosine kinase inhibitor), SB431542 (a specific inhibitor of SMAD2 and SMAD3 activation) and SIS3 (a specific inhibitor of SMAD3 activation). We found that EGFR inhibitor AG1478 completely blocked FSH-induced cumulus expansion by preventing hyaluronic acid production and its incorporation within the extracellular matrix. In addition, in the presence of AG1478, most of FSH-stimulated OCCs were in germinal vesicle stage (GV) and did not mature. Conversely, AG1478 did not affect the stimulation of progesterone synthesis by FSH. In agreement, EGF was unable to stimulate progesterone synthesis by OCCs cultured in vitro. The SB431542, that inhibits the activation of SMAD2 and SMAD3, only partially inhibited cumulus expansion by slightly decreasing both total production and matrix accumulation of hyaluronic acid. Surprisingly, SIS, that inhibits only SMAD3, produced an inhibitory effect higher than SB431542. A divergent effect of the two inhibitors was observed on steroid synthesis: FSH-induced progesterone synthesis increased in the presence of SB431542 and decreased in the presence of SIS. Both inhibitors did not affect the induction of oocyte meiotic resumption promoted by FSH. These results suggest that in pig, like in mice, FSH stimulates cumulus cells to produce EGF that acting in an autocrine way induces the formation of the expanded matrix and the signaling for oocyte meiotic resumption. The results also suggest that SMAD2 and SMAD3 activation is required for optimal stimulation of hyaluronic acid and proteins involved in the organization of this polymer in the pig OCC matrix. Finally, FSH stimulation of progesterone synthesis seems to be independent from EGF, while it appears to be counteracted by SMAD2, but not SMAD3 activation, thereby suggesting that specific SMAD2 signaling pathway is probably indispensable to prevent luteinization of cumulus cells. Supported by grant GACR 523/08/0111 and VEGA 2/0153/08. (poster)

We show in the present study that freshly isolated pig cumulus-oocyte complexes (COCs) display a limited response to LH, as assessed by the expression of hyaluronan synthase 2 (Has2) mRNA, activation of protein kinase A (PKA), production of hyaluronic acid (HA) and progesterone, cumulus cell expansion and resumption of meiosis. These data indicate that freshly isolated COCs do not possess a sufficient number of functional LH receptors (LHR). However, the expression of Lhr significantly increased during the culture of COCs in vitro in a medium supplemented with FSH. Assuming that the effect of FSH on LHR induction is mediated via cAMP signaling pathways, we developed a new culture system, in which the COCs were pre-cultured for 72 hr in a medium supplemented with dbcAMP. The pre-cultured COCs remained in the germinal vesicle stage, their cumulus investment underwent a dramatic increase in size and gap junctions between the cumulus cells were preserved. The stimulation of such COCs with either FSH or LH led to the resumption and completion of meiosis, activation of PKA, expression of Has2, synthesis of large amounts of HA and progesterone, and extensive expansion of cumulus cells. We conclude that the formation of functional LHR is stimulated in cumulus cells during the culture in vitro in a cAMP-dependent pathway. The dbcAMP-treated COCs thus represent a new model in which the resumption of meiosis and cumulus expansion can be induced exclusively by the action of recombinant LH.

It has been shown previously that tumor necrosis factor alpha-induced protein 6 (TNFAIP6) is essential for formation of the cumulus extracellular matrix and female fertility. Therefore, we studied the expression of TNFAIP6 mRNA in porcine preovulatory follicles. In addition, we asked whether the expression of TNFAIP6 mRNA changes in mural granulosa cells (MGCs) during the periovulatory period or after culture of oocyte-cumulus complexes (OCCs) or MGCs in vitro. Mural granulosa cells obtained from follicles on day 12 (D 12) and day 15 (D 15) of the estrous cycle, eCG-stimulated follicles, follicles at 4-32 h after hCG stimulation and MGCs and OCCs obtained from immature gilts and cultured for 0-44 h in vitro with gonadotropins were used for extraction of total RNA and assessment of the relative abundance (RA) of TNFAIP6 mRNA by reverse transcription-polymerase chain reaction (RT-PCR). The levels of TNFAIP6 mRNA were low in the follicles on D 12 and D 15 of the estrous cycle and at 66 h after eCG stimulation but were significantly increased at 4 h after hCG. The high level of TNFAIP6 expression was maintained until 16 h after hCG stimulation and gradually decreased at 24 and 32 h after hCG. During in vitro culture, FSH/LH-induced TNFAIP6 mRNA was expressed in both OCCs and MGCs in a similar temporal pattern as seen in vivo. We conclude that TNFAIP6 expression in the pig, like other species, increases in preovulatory follicles following the LH (hCG) surge. The OCC and MGC display similar patterns of TNFAIP6 expression under both in vivo and in vitro conditions.

In most mammals, before ovulation, cumulus cells synthesize a large amount of hyaluronan (HA) that is organized into an extracellular matrix (ECM), which provides an essential microenvironment for in vivo oocyte fertilization. This process is called cumulus expansion. The present study assessed effects of selected endocrine disruptors (bisphenol A, BPA; 4-chloro-3-methyl phenol, CMP; di(2-ethylhexyl) phthalate, DEHP; and benzyl butyl phthalate, BBP) in a range of 100 pM–100 μM, on follicle-stimulating hormone (FSH)-induced meiotic maturation and cumulus expansion of porcine oocyte–cumulus complexes (OCC) cultured in vitro. Moreover, FSH-stimulated production of hyaluronic acid (HA) and progesterone by cumulus cells was measured. Both phenols, BPA and CMP (100 μM), significantly affected meiotic maturation of oocytes. The number of oocytes that underwent germinal vesicle breakdown (GVBD) (78.7% and 72.4%, respectively) as well as the rate of oocytes that reached metaphase II stage (MII) (50% and 53.6%, respectively) after 44 h culture were decreased compared to control (89.6% for GVBD and 81.5% for MII). FSH-stimulated expansion of cumulus was altered by the highest concentration of BPA and CMP (70% and 64%, respectively vs. 80.3% in control). Although BPA did not alter FSH-stimulated HA synthesis by cumulus cells, its incorporation within the complex was reduced to a half of control value. Progesterone production by OCC was significantly changed in the presence of BPA or DEHP. Finally, our results provide valuable information that oocyte meiotic progression was adversely affected during in vitro culture with endocrine disruptors.