Cumulus Granulosa Research Articles

A standardized extract of Asparagus officinalis stem improves HSP70-mediated redox balance and cell functions in bovine cumulus-granulosa cells

Heat shock (HS) protein 70 (HSP70), a well-known HS-induced protein, acts as an intracellular chaperone to protect cells against stress conditions. Although HS induces HSP70 expression to confer stress resistance to cells, HS causes cell toxicity by increasing reactive oxygen species (ROS) levels. Recently, a standardized extract of Asparagus officinalis stem (EAS), produced from the byproduct of asparagus, has been shown to induce HSP70 expression without HS and regulate cellular redox balance in pheochromocytoma cells. However, the effects of EAS on reproductive cell function remain unknown. Here, we investigated the effect of EAS on HSP70 induction and oxidative redox balance in cultured bovine cumulus-granulosa (CG) cells. EAS significantly increased HSP70 expression; however, no effect was observed on HSP27 and HSP90 under non-HS conditions. EAS decreased ROS generation and DNA damage and increased glutathione (GSH) synthesis under both non-HS and HS conditions. Moreover, EAS synergistically increased HSP70 and HSF1 expression and increased progesterone levels in CG cells. Treatment with an HSP70 inhibitor significantly decreased GSH level, increased ROS level, and decreased HSF1, Nrf2, and Keap1 expression in the presence of EAS. Furthermore, EAS significantly increased progesterone synthesis. Thus, EAS improves HSP70-mediated redox balance and cell function in bovine CG cells.

Mitochondrial cell-free DNA secreted from porcine granulosa cells.

Several studies have proposed that cell-free DNA (cfDNA) is a potential biomarker present in follicular fluid (FF) for oocyte quality. Recently we reported that mitochondria-derived cfDNA (mt-cfDNA) closely reflects the amount of cfDNA in FFs. The present study investigated the mechanism regulating mt-cfDNA secretion from porcine granulosa cells. Oocytes and cumulus cell complexes or granulosa cells (GCs) were cultured in maturation medium for 24 or 48 h respectively. Then, nuclear-derived cell-free DNA (n-cfDNA) or mt-cfDNA contents in the spent medium were examined using real-time polymerase chain reaction. When 10 μM of MG132, a proteasome inhibitor, was added to the culture medium, cellular viability of both COCs and GCs decreased and n-cfDNA significantly increased in the culture medium, whereas mt-cfDNA significantly decreased. Supplementation of the culture medium with GW4869, an inhibitor of intracellular vesicle formation, significantly decreased the mt-cfDNA, whereas no effect was observed on n-cfDNA in the medium of both COCs and GCs. Furthermore, the addition of bafilomycin, an inhibitor of autophagy to the culture medium significantly increased mt-cfDNA in the culture medium. After filtration (0.22 μm) and centrifugation (23,000 g), the mt-cfDNA content of the medium decreased significantly. In conclusion, the proteasomal mitochondrial quality control system is upstream of mt-cfDNA secretion and autophagy plays a role in cellular digestion of mitochondrial DNA in the cytoplasm. It is further suggested that dsDNA is enclosed in certain vesicles or associated with small molecules and secreted into the medium.

Histone demethylase KDM4A and KDM4B expression in granulosa cells from women undergoing in vitro fertilization.

To assess expression of the histone demethylases KDM4A and KDM4B in granulosa collected from women undergoing oocyte retrieval and to determine if expression was related to pregnancy outcome. Cumulus and mural granulosa cells were obtained from women undergoing oocyte retrieval. KDM4A and KDM4B mRNA expression was determined by qRT-PCR. KDM4A and KDM4B proteins were immunohistochemically localized in ovarian tissue sections obtained from archival specimens. KDM4A and KDM4B protein was localized to oocytes, granulosa cells, and theca and luteal cells in ovaries from reproductive-aged women. KDM4A and KDM4B mRNA expression was overall higher in cumulus compared to mural granulosa. When comparing granulosa demethylase gene expression, KDM4A and KDM4B mRNA expression was higher in both cumulus and mural granulosa from not pregnant patients compared to patients in the pregnant-live birth group. Histone demethylases KDM4A and KDM4B mRNA are differentially expressed in cumulus and mural granulosa. Expression of both KDM4A and KDM4B mRNA was lower in cumulus granulosa and mural granulosa from pregnant compared to not pregnant patients. These findings suggest that altered expression of histone demethylases may impact epigenetic changes in granulosa cells associated with pregnancy.

Thyroid peroxidase identified in human granulosa cells: another piece to the thyroid-ovary puzzle?

Thyroid hormones seemingly influence the maturation of the human oocyte. Thyroid hormone receptors have been isolated in granulosa mural and cumulus cells and the mature oocyte of the human ovarian follicle. Thyroid hormones are present in follicular fluid in concentrations similar to those in serum. Most importantly, enzymes involved in the chain that regulate the generation of thyroid hormones have been found in granulosa cells. For the first time, we have isolated thyroid peroxidase by immunocytochemistry in the granulosa cumulus cells of the human ovarian follicle, thereby supporting the hypothesis that the human ovarian follicle may be an independent thyroid-hormone producing unit.

Амінокислотний профіль середовищ співкультивування доімплантаційних ембріонів людини in vitro на моношарі свіжовиділених або кріоконсервованих клітин кумулюса та гранульози

Co-culturing with the cumulus and granulosa cells (CGC) provide a microenvironment which can ensure the normal growth and development of human pre-implantation embryos in vitro . Examining the quantitative and qualitative compositions of this environment is of an important value to produce the systems to culture human embryos in vitro . The paper described the human embryos of the fifth day of development (blastocyst stage), monolayer culture of cryopreserved and freshly isolated CGC. We investigated the influence of culturing on monolayer of freshly isolated and cryopreserved CGC on morphokinetic characteristics of human pre-implantation embryos obtained in vitro as well as the amino acid profile of the culturing media. It has been found that cryopreservation does not affect the ability of CGC to support the development of pre-implantation embryos and improves their quality. There was determined the difference in amino acid composition of the standard medium of those for co-culturing on monolayer of either native or cryopreserved culture and cumulus granulosa. The presence of CGC changes the biochemical profile of culturing medium by increasing the content of the amino acids such as tryptophan, proline, valine, ornithine and glutamine. Probl Cryobiol Cryomed 2016; 26(2):124-132.

187 EXPRESSION AND REGULATION OF THE FIBROBLAST GROWTH FACTOR GENE FAMILY DURING MOUSE FOLLICULAR DEVELOPMENT

Recent studies have shown the critical roles of fibroblast growth factors (FGFs), including FGF8 produced by oocytes, in regulating follicular development. However, the expression and regulation of the FGF gene family, which consists of 22 ligands and 4 receptors, in the mouse ovary have not been well understood. The aim of the present study was to assess the expression and regulation of FGF ligands and receptors in the mouse ovary. Transcript levels of FGF ligands and receptors in immature (3-week-old) and adult (7- to 8-week-old) ovaries as well as other tissues of B6/DBA2F1 mice were analysed with RT-PCR. Furthermore, expression levels of FGF receptors in cumulus cells (CC) and mural granulosa cells (MG) before and after equine chorionic gonadotropin (eCG) treatment were determined with RT-quantitative PCR. Among 21 FGF ligands examined, 12 and 9 transcripts were detectable in immature and adult ovaries, respectively. More FGF ligands were detected in ovary, testis, heart, and brain compared to other tissues, including liver and spleen. Transcripts of all 4 FGF receptors (Fgfr1–4) were detectable in both immature and adult ovaries. Expression levels of Fgfr1 and Fgfr2 were significantly higher in MG compared with CC before and after the eCG treatment. Levels of Fgfr4 were comparable between MG and CC before the eCG treatment, but became significantly different with higher expression levels in MG after the eCG treatment. Fgfr3 transcripts were barely detectable in CC and MG. Overall levels of Fgfr1 in granulosa cells (CC and MG) were downregulated by eCG treatment, whereas those of Fgfr2 and Fgfr4 were upregulated. In summary, many FGF ligands are expressed, at least in mRNA levels, in mouse ovaries. Moreover, the expression levels of Fgfr transcripts in granulosa cells are dynamically regulated during follicular development.

Characterization of Cumulus Expansion-Inhibiting Factor (CEIF) in Goat Follicles1

Studies suggest that oocyte cumulus expansion is regulated by both cumulus expansion-enabling factor (CEEF) and cumulus expansion-inhibiting factors (CEIF). Many reports on CEEF have appeared, but CEIF has rarely been studied. By cumulus expansion assays using mouse cumulus-oocyte complexes (COCs) and oocytectomized complexes, the present study demonstrated that whereas follicular fluid (FF) from medium (diameter, 2-4 mm) goat follicles contained both CEEF and CEIF activities, FF from large (diameter, 5-6 mm) abattoir or large (diameter, 5-7 mm) follicle-stimulating hormone (FSH)-stimulated follicles contained neither. FF from (diameter, 5-7 mm) human chorionic gonadotropin-stimulated follicles showed CEEF but not CEIF activity. Whereas medium conditioned with cumulus or mural granulosa cells from medium goat follicles contained only CEEF activity, theca cell-conditioned medium (CM) showed both CEEF and CEIF activities. Whereas 0.01 mg/ml of heparin efficiently inhibited cumulus expansion of mouse COCs in vitro, FF from large follicles that showed no CEIF activity contained much higher concentrations (0.23-0.25 mg/ml) of heparin. None of the glycosaminoglycans (GAGs) tested inhibited cumulus expansion of goat COCs. Among the follicles observed, only FF from medium goat follicles contained a linoleic acid (LA) level sufficient to inhibit cumulus expansion of both mouse and goat COCs in vitro. CM contained some amount of GAGs but no LA. Taken together, the results suggest that 1) the FSH and luteinizing hormone (LH) surges before ovulation promote cumulus expansion by down-regulating CEIF and up-regulating CEEF activity, respectively; 2) GAGs are not the CEIF in goat follicles; and 3) LA has CEIF activity but additional factors must be involved, because CM that showed high CEIF activity contained no LA.

187 DIFFERENTIAL EXPRESSIONS OF GROWTH AND DIFFERENTIATION FACTOR 9 AND BONE MORPHOGENETIC PROTEIN 15 GENES IN OVARIES OF THE CALF AND COW

It has been reported that prepubertal calf oocytes are less developmentally competent than those obtained from cows. The bone morphogenetic protein (BMP) family of proteins regulate folliculogenesis and the ovulation rate in mammals. Of the members of the BMP family, growth and differentiation factor 9 (GDF9) and BMP15 are oocyte-derived proteins that play critical roles in granulosa cell proliferation and differentiation. In the present study, we characterised the gene expression of bovine GDF9 and BMP15 in calf and adult cow ovaries. The ovaries obtained from 4 calves at 9 to 11 months old and 4 cows at 4 to 6 years old. For quantitative real-time RT-PCR (qPCR), cumulus–oocyte complexes (COC) and mural granulosa cells were collected by aspiration of follicles 2 to 5 mm in diameter from an ovary from each animal. Ovaries from the other side were used for in situ hybridization. The COC and mural granulosa cells were separated and cultured for 22 h according to the protocol for oocyte maturation. Total RNA was isolated from denuded oocytes, cumulus cells, and mural granulosa cells. Bovine glyceraldehyde-3-phosphate dehydrogenase was used to normalise qPCR efficiency. For in situ hybridization, the collected ovaries were immediately fixed with 4% formaldehyde-PBS and embedded in a paraffin block. In situ hybridization was carried out with digoxigenin-labelled RNA probes. We confirmed there was no contamination of oocytes in the collected cumulus and mural granulosa cells by determining the mRNA expression of germ cell–oocyte markers (ZAR1 and VASA). Two, 16, 7, and 10 COC were collected from the ovary on one side of each calf, and 14, 22, 29, and 33 COC were collected from an ovary of each adult cow. Two COC from the calves could not be used for qPCR analysis. Both GDF9 and BMP15 mRNA were detected in oocytes and cumulus cells at the end of maturation culture, whereas only GDF9 mRNA was detected in mural granulosa cells. Quantitative PCR detection revealed that BMP15 and GDF9 mRNA expression of the cumulus cells from adult ovaries was significantly greater than that from calf ovaries. The expression of GDF9 mRNA was significantly greater in calf oocytes than in oocytes from cows. However, BMP15 mRNA expression in the oocytes of calf and adult ovaries was not significantly different. In mural granulosa cells, the intensities of GDF9 mRNA expression were not significantly different between calves and cows. These qPCR results were also ascertained by in situ hybridization. In conclusion, we clarified that the characteristics of bovine GDF9 and BMP15 mRNA expression in oocytes and cumulus cells were different between calves and cows. Our results indicate the possibility that the calf oocytes were less developmentally competent because of excess GDF9 on the oocytes, deficiencies of GDF9 and BMP15 on the cumulus cells, or both.

245 EFFECTS OF MELATONIN ON THE IN VITRO OOCYTE MATURATION OF PORCINE CUMULUS - OOCYTE COMPLEXES

In mammalian species, melatonin is a multifunctional hormone that mediates several circadian and seasonal processes, including reproduction. In addition to its hormonal actions, melatonin has a strong antioxidant effect and acts as a powerful free radical scavenger. Accordingly, the objective of this study was to investigate the local expression of the melatonin-receptor I (Mel-RI) gene and the effect of melatonin as an antioxidant on the in vitro maturation of porcine cumulus–oocyte complexes. Oocytes were obtained from aspiration of slaughterhouse ovaries and then matured in tissue culture medium 199 (TCM199) supplemented with 0.1% polyvinyl alcohol (PVA), 20 ng mL–1 epidermal growth factor (EGF), and 4 IU mL–1 pregnant mare serum gonadotropin (PMSG)/hCG. Expression of Mel-RI gene in cumulus cells, granulosa cells, or oocytes was evaluated by RT-PCR (Exp 1). The effect of various concentrations (0, 10, 50, and 100 ng mL–1) of melatonin on the in vitro maturation in terms of nuclear maturation (polar body extrusion) (Exp 2) and measurement of reactive oxygen species (ROS) by dichlorohydrofluorescein diacetate (Yang et al. 1998 Hum. Reprod. 13, 998–1002) (Exp 3) were investigated. Each experiment was replicated at least four times, and each replication included more than thirty oocytes. For statistical analysis, one-way anova in the Prizm software program (GraphPad, San Diego, CA, USA) was used. The Mel-RI gene was locally expressed in cumulus and granulosa cells but not in oocytes. Melatonin at 10 ng mL–1 had a beneficial effect on in vitro maturation (84.6%) compared to 0 ng mL–1 (75.6%), 50 ng mL–1 (80.9%), and 100 ng mL–1 (76.5%). Levels of ROS were also significantly decreased by melatonin treatment at 10 (14.07), 50 (14.42), and 100 ng mL–1 (13.03) of melatonin (P < 0.001) compared to that of the control group (28.21). The presence of melatonin receptor in cumulus cells and granulosa cells indicates a potentially important role for this hormone in regulating porcine ovarian and reproduction function. In conclusion, the present study demonstrated that melatonin had a beneficial effect on in vitro maturation of porcine cumulus–oocyte complexes, probably through binding a receptor and decreased ROS production. Further studies are required to elucidate the relationship between the appearance of melatonin receptor and mechanisms of melatonin function in the embryo.

Gene Silencing of Cyclooxygenase-2 mRNA by RNA Interference in Bovine Cumulus-Granulosa Cells

Inhibition of specific gene expression using RNA interference (RNAi) is a valuable tool for functional analysis of a target gene. However, there is little information available concerning RNAi for analysis of gene function in relation to the reproductive physiology of follicular cells in ruminants. Thus, the aim of this study was to evaluate the interfering effect of small interference RNA (siRNA) on expression of cyclooxygenase-2 (Cox-2) mRNA and prostagrandin F(2alpha) (PGF(2alpha)) production in bovine cumulus-granulosa (CG) cells. Bovine CG cells were collected from aspirated follicles and cultured. After reaching confluency, two experiments were conducted. In experiment 1, to investigate the effective concentration of siRNA, 0, 100, 250 and 500 pM of Cox-2 siRNA was introduced into the CG cells, respectively. After 24 h, the amount of Cox-2 mRNA expression was measured by RT-PCR and real-time PCR. In experiment 2, to investigate the time required for effective interference of siRNA and Cox-2 activity, 250 pM siRNA was introduced for 0, 3, 6, 12 and 24 h. After culture, the amount of Cox-2 mRNA expression was measured and the culture medium was collected to determine the PGF(2alpha) concentration by enzyme immunoassay. The Cox-2 mRNA expression was not affected by introduction of 100 pM siRNA into CG cells for 24 h, but 250 and 500 pM Cox-2 siRNA significantly reduced the Cox-2 mRNA expression. Moreover, the significant suppressive effect of 250 pM siRNA was observed 6 h after introduction, and the reduction of mRNA expression by RNAi became more obvious over 12 h. On the other hand, the PGF(2alpha) concentration in the culture medium was not significantly different 12 h after siRNA introduction; however, the PGF(2alpha) concentration 24 h after siRNA introduction was significantly decreased compared with the control at the same time point. These results suggest that gene silencing of Cox-2 with siRNA is capable of analyzing the function and expression of specific genes in bovine CG cells.

Effects of epidermal growth factor and hormones on granulosa expansion and nuclear maturation of dog oocytes in vitro

Gonadotropins, steroids and growth factors stimulate or inhibit cumulus expansion, nuclear maturation, or both, of most mammalian oocytes in vitro. The objective was to evaluate the effects of epidermal growth factor (EGF) and various hormone combinations on in vitro granulosa/cumulus (G-C) expansion and nuclear maturation of domestic dog oocytes derived from advanced preantral and early antral follicles. Follicles were collected after enzymatic digestion of ovarian tissue and cultured for 66 h in F-12/DME with 20% fetal bovine serum, 2 mM glutamine and 1% antibiotic–antimycotic (Control). Treatments comprised the following groups; each was cultured both with and without EGF (50 ng/mL): Control, FSH (0.5 μg/mL), LH (5 μg/mL), estradiol-17β (E2, 1 μg/mL), FSH + LH, and FSH + LH + E2. Granulosa/cumulus expansion was scored on a scale of 0 (no expansion) to +3 (maximum expansion). The interaction between EGF and hormone treatment affected ( P = 0.011) maximum G-C expansion. With the exception of the E2 group, EGF increased ( P < 0.05) the proportion of oocytes exhibiting +3 expansion. The synergism of E2 with FSH + LH enhanced maximum G-C expansion; compared to all other treatments, the greatest expansion was observed in the FSH + LH + E2+EGF group (83.5 ± 3.5%). When cultured in EGF alone, oocytes failed to reach metaphase I-II (MI-MII) stages. The interaction between EGF and hormone treatment tended ( P = 0.089) to increase the proportion of oocytes resuming or completing nuclear maturation (GVBD-MII). In addition, supplementing culture media with hormones increased ( P = 0.010) the GVBD-MII rate. Therefore, EGF in combination with FSH and LH enhanced G-C expansion of cultured canine oocytes, with no significant effect on the proportion of oocytes derived from advanced preantral and early antral follicles that reached MI-MII.

253 GENE SILENCING OF CYCLOOXYGENASE-2 mRNA BY RNA INTERFERENCE IN BOVINE CUMULUS - GRANULOSA CELLS

Recently, interference RNA (RNAi), inducing inhibition of the specific gene expression, attracted a lot of attention. Many researchers have reported that the 21-mer small interference RNA (siRNA) is introduced into target cells and then siRNA can suppress the gene expression. RNAi is a useful tool for functional analysis of specific genes. However, there is little information about RNAi for the analysis of gene function in reproductive physiology in ruminants. Thus, this study was aimed at evaluating RNAi for the analysis of cyclooxygenase-2 (Cox-2) mRNA expression in bovine cumulus-granulosa (CG) cells as well as prostaglandin F2� (PGF2�) production. We investigated both the effective concentration of Cox-2 siRNA and the effect of the introduction time of siRNA on Cox-2 mRNA expression. Bovine CG cells were collected at slaughterhouse and cultured in 4-well dishes. After the cells reached confluency, two experiments were conducted. In the present study, Cox-2 siRNA was simply added to culture medium with lipofectamine" 2000 (Invitrogen Japan K. K., Tokyo, Japan) as the transfection reagent. In experiment 1, the concentration of 0, 100, 250, and 500 pM of Cox-2 siRNA was introduced into the CG cells. After 24 h of introduction, the amount of mRNA expression for Cox-2 was measured by reverse transcription polymerase chain reaction (RT-PCR) and real-time PCR. In experiment 2, 250 pM siRNA for Cox-2 was introduced into CG cells for 0, 3, 6, 12, and 24 h. After culture, the amount of mRNA expression for Cox-2 was measured and the culture medium was collected to determine PGF2� concentration by enzyme immunoassay. The Cox-2 mRNA expression was not affected by 100 pM siRNA introduced into CG cells for 24 h, but 250 and 500 pM Cox-2 siRNA significantly reduced the Cox-2 mRNA expression (10% of that of the 0 pM siRNA group). Moreover, the suppressive effect of 250 pM siRNA was observed at 6 h after introduction (60% of that of the 0 pM siRNA group at 0 h) and the reduction of mRNA expression by RNAi became more obvious over 12 h (10% of that of the 0 pM siRNA group at 0 h). On the other hand, the PGF2� concentration in the culture medium was not significantly different at 12 h after siRNA introduction, however, the PGF2� concentration of the RNAi treatment group at 24 h was significantly lower than that of the 0 pM siRNA group at the same time point. These results suggest that gene silencing by Cox-2 siRNA is a means of analyzing the function and expression of specific genes in bovine CG cells. This study was supported by the Japan Society for the Promotion of Science for Young Scientists.

233 TEMPOROSPATIAL EXPRESSION PROFILES OF GROWTH AND DIFFERENTIATION FACTOR-9 AND BONE MORPHOGENETIC PROTEIN-15 GENES IN THE BOVINE OVARY

Bone morphogenetic protein (BMP) family proteins regulate the folliculogenesis and ovulation rate in mammals. Of the members of BMP family, growth and differentiation factor-9 (GDF-9) and BMP-15 are oocyte-derived proteins that play critical roles in follicular development. In the present study, we characterized the temporospatial expression patterns of GDF-9 and BMP-15 in bovine ovaries. Bovine ovaries were obtained from local abattoir. Cumulus–oocyte complexes (COCs) and mural granulosa cells were collected by aspiration of follicles 2–5 mm in diameter. Follicular aspiration was done the day following slaughter. The COC and mural glanulosa cells were separated and cultured for 20 h according to the protocol for oocyte maturation. Total RNA was isolated from COCs, cumulus cells, denuded oocytes, and mural granulosa cells and used for PCR. For in situ hybridization, collected ovaries were immediately fixed with 4% formaldehyde-PBS and embedded in a paraffin block. In situ hybridization was carried out with digoxigenin-labeled probes. In COCs and cumulus cells, the transcripts encoding GDF-9 and BMP-15 were detected immediately after follicular aspiration and still remained at the end of maturation culture. However, in mural granulosa cells, only GDF-9 expression was detected. Germ cell/oocyte markers, ZAR1 and VASA, were not detected in cumulus and mural granulosa cells. Quantitative real-time RT-PCR revealed extensive expressions of both GDF-9 and BMP-15 in oocytes. In situ hybridization study showed that GDF-9 and BMP-15 were co-localized to oocytes in primary, secondary, and antral follicles. The ovular co-expression of GDF-9 and BMP-15 appeared to commence at fetal life. Expression of GDF-9 and BMP-15 was not clear in cumulus cells possibly because of low expression level. In conclusion, our results show that GDF-9 and BMP-15 are co-localized to oocytes from an early stage of folliculogenesis. Since the transcripts encoding GDF-9 and BMP-15 are detected in cumulus cells, it is suggested that the oocyte is not the sole source of them. Additionally, the cumulus and mural granulosa cells are suggested to be functionally differentiated relative to BMP-15 expression.

Meiotic state of bovine oocytes is regulated by interactions between cAMP, cumulus, and granulosa.

Bovine oocytes are arrested at the prophase of first meiotic cell cycle. Meiosis resumes in oocytes of pre-ovulatory follicles upon LH surge. However, oocytes from secondary follicles spontaneously resume meiosis in the absence of hormones if removed from the follicle and cultured in vitro. The nature of meiotic arrestor in bovine follicles is poorly understood. In this study we investigated the role of cell-cell interactions between granulosa and cumulus cells and the oocyte in mediating maintenance of meiotic arrest by cAMP. We sorted oocytes as granulosa-cumulus oocyte complexes (GCOC) if surrounded with cumulus cells attached to a large granulosa investment or cumulus oocytes complexes (COC) if surrounded with cumulus cells only and investigated the role cAMP in maintenance of meiotic arrest in these oocytes under various conditions. In hormone- and serum-free medium both GCOC and COC enclosed oocytes resumed meiosis. When [cAMP](i) was elevated with addition of invasive adenylate cyclase (iAC) GCOC enclosed oocytes were maintained in the prophase with intact germinal vesicle (GV) while COC enclosed oocytes underwent GV breakdown (GVBD). iAC elevated [cAMP](i) in both types of oocytes to the same level. If oocytes were liberated from the cumulus and granulosa cells, they re-initiated meiosis in serum and hormone free medium, but remained in the GV stage if iAC was added to the medium. Untreated GCOC and COC enclosed oocytes extruded first polar body at the same frequency in hormone-supplemented media. GCOC and COC enclosed oocytes but not denuded oocytes (DO) cultured without somatic cells acquired developmental competence if cultured in hormone-containing medium. It is concluded that maintenance of meiotic arrest is regulated by the interplay of [cAMP](i), and cumulus and granulosa cells.

In vitro culture of oocytes with surrounding cumulus complexes and granulosa cells (COCGs) from bovine early antral follicles

AbstractCumulus-oocyte complexes with surrounding granulosa cells (COCGs) in early antral follicles (0·5 to 0·7 mm in diameter) were surgically collected from sections of bovine ovarian cortex under a dissection microscope and subsequently cultured in vitro using follicle stimulating hormone (FSH), epidermal growth factor (EGF), insulin-transferrin-selenium (ITS) and hypoxanthine, singly or in combination, to obtain fully grown matured oocytes. Oocytes cultured in the presence of FSH + hypoxanthine increased (P &lt; 0·05) in diameter from 93 µm on the day of commencement of culture to 106·37±0·34 µm on day 5. Oocytes cultured in the presence of FSH, hypoxanthine or hypoxanthine + ITS + FSH increased (P &lt; 0·05) to mean diameters of 105·40 (s.e. 0·47) µm, 105·50 (s.e. 0·39) µm and 105·35 (s.e. 0·55) µm, respectively. By day 11 of culture, oocyte diameters 110·50 (s.e. 0·35) µm, 110·13 (s.e. 0·39) µm, 109·49 (s.e. 0·46) µm, 109·53 (s.e. 0·58) µm and 109·16 (s.e. 0·43) µm were recorded for treatments FSH + hypoxanthine, hypoxanthine + ITS + FSH, FSH, hypoxanthine and FSH + EGF + hypoxanthine + ITS, respectively. The proportions with COCGs which formed an antrum while cultured in vitro; were categorized as morphologically normal following recovery from the gel; matured in vitro; showed germinal vesicle break down and reached metaphase II were highest (P &lt; 0·05) for the FSH + hypoxanthine treatment (49/60 (81·7%), 48/60 (80·0%), 47/60 (78·3%), 45/60 (75·0%) and 15/60 (25·0%), respectively, followed by hypoxanthine + ITS + FSH (47/60 (78·3%), 44/60 (73·3%), 41/60 (68·3%), 41/60 (68·3%) and 12/60 (20%), respectively), FSH (43/60 (71·7%), 42/60 (70%), 40/60 (66·7%), 39/60 (65·0%) and 9/60 (15%), respectively) and hypoxanthine (41/60 (68·3%), 38/60 (63·3%), 36/60 (60%), 35/60 (58·3%) and 8/60 (13·3%), respectively). In experiment II, the in vitro fertilization and cleavage rates of COCGs were highest (P &lt; 0·05) for FSH + hypoxanthine treatment (17/60; 28·3%) followed by hypoxanthine + ITS + FSH (13/60; 21·6%), FSH (12/60; 20%) and hypoxanthine (11/60; 18·3%) treatments. The results of this study show that COCGs from early antral follicles can be isolated, cultured and grown in vitro. Furthermore, supplements like FSH and hypoxanthine can be used singly or in combination(s) in culture medium to enhance the growth of COCGs.

Ovarian brain-derived neurotrophic factor is present in follicular fluid from normally cycling women

Ovarian brain-derived neurotrophic factor is present in follicular fluid from normally cycling women

Effects of follicle-stimulating hormone, bovine somatotrophin and okadaic acid on cumulus expansion and nuclear maturation of blue fox (Alopex lagopus) oocytes in vitro.

The meiotic competence and meiosis resumption of Blue fox (Alopex lagopus) oocytes from anoestrous animals were followed. Oocyte-cumulus complexes (OCC) were cultured in modified TC 199 medium with or without FSH, recombinant bovine somatotrophin (bST) and okadaic acid (OA). The results showed that oocytes less than 100 microns in diameter did not achieve germinal vesicle breakdown (GFBD) by 72 h of culture, which indicates their meiotic incompetence. Oocytes larger than 100 microns in diameter underwent GVBD after 48 h of culture (27%) and reached metaphase II (MII) after 72 and 96 h (20% and 27%) in control medium. Both bST and OA accelerated resumption of meiosis (bST: 55% GVBD and 42% MII after 48 h; OA: 66% GVBD after 18 h). In contrast, FSH significantly reduced meiosis resumption (only 3% GVBD and MII after 72 h) and induced changes in the shape of cumulus granulosa (CG) cells and F-actin assembly typical for cumulus expansion. However, the innermost layers of CG cells (corona radiata) remained connected with the oocyte via gap junctions until the end of culture. Cumuli of oocytes cultured in control, bST-supplemented or OA-supplemented medium did not expand (changes in cell shape and F-actin redistribution did not occur). Moreover, especially in media with bST and OA an increased detachment and rapid disconnection of their gap junctions with the oocyte were observed. These results suggest that under in vitro conditions FSH stimulates expansion of the CG cells and the attached membrana granulosa cells but in contrast it secures heterologous gap junctions between cytoplasmic processes of the corona radiata cells and oolemma during 3 days of culture. Thus, in agreement with the in vivo situation in which Canidae oocytes are ovulated in the GV stage, the cumulus, mainly corona radiata cells, controls resumption of meiosis in Blue fox oocytes under in vitro conditions also.

Transforming Growth Factor α Gene Expression and Peptide Localization in Porcine Ovarian Follicles1

The present study was undertaken to determine transforming growth factor alpha (TGF alpha) gene expression in various components of medium-sized porcine ovarian follicles by reverse transcription-polymerase chain reaction, and to localize the peptide during folliculogenesis by immunocytochemistry. A strong signal for TGF alpha transcript was detected in cumulus and granulosa cells, whereas the signal in theca cells was very weak but detectable. No TGF alpha mRNA was detected in the oocyte. Immunolocalization studies revealed intense TGF alpha staining in cumulus and granulosa cells of all stages of follicular development. TGF alpha immunostaining was also observed in the oocytes of primordial and primary follicles. In antral follicles, immunostaining was observed only in cumulus and mural granulosa cells, whereas theca cells and oocytes exhibited very little or no detectable staining. TGF alpha immunostaining was absent in follicles undergoing atresia. No TGF alpha immunostaining was observed when the antibody was omitted from the procedure, and the staining was substantially reduced when the primary antibody was preabsorbed with the same mass of TGF alpha peptide, suggesting the specificity of the staining procedure. These results suggest a local follicular production of TGF alpha in the porcine ovary and thus a role for TGF alpha of follicular origin in the regulation of follicular development in an autocrine/paracrine fashion.

Differential secretion of progesterone from cumulus and mural granulosa as a function of ovarian reserve

Differential secretion of progesterone from cumulus and mural granulosa as a function of ovarian reserve

Coexpression of gap junction proteins in the cumulus-oocyte complex.

The connexins constitute a family of proteins that make up the intercellular membrane channels of gap junctions. We had previously reported the presence of two members of this protein family, connexins 32 and 43, in mouse one-cell zygotes (Barron et al., Dev Genet 10:318-323, 1989; Valdimarsson et al., Mol Reprod Dev 30:18-26, 1991), implying that both must be present in the mature oocyte and could be involved in mediating the intercellular coupling that occurs between the oocyte and cumulus granulosa during oogenesis. In the present report we provide evidence for this, based on an analysis of the cumulus-oocyte complex (COC) using reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry with a confocal microscope. Transcripts of both connexin32 (Cx32) and connexin43 (Cx43) were detected by RT-PCR in both components of the COC. Cx32 mRNA in the oocyte declined precipitously following human chorionic gonadotropin (hCG) stimulation of pregnant mare serum gonadotropin (PMSG)-primed ovaries, whereas there was no obvious change in Cx43 mRNA. Peptide-specific antibodies against both connexins provided diffuse cytoplasmic staining of oocytes as well as some punctate staining near the oocyte surface, which could not be unequivocally resolved as cumulus-oocyte gap junctions. However, the two antibodies did provide clear evidence of Cx32 and Cx43 in gap junction-like structures between cumulus cells. We could find no evidence of the incorporation of the oocyte's store of Cx32 into gap junctions during postfertilization development.(ABSTRACT TRUNCATED AT 250 WORDS)