84 Characterization of agouti-signalling protein expression within the bovine ovary and early embryo

Apoptosis within bovine follicular cells and its effect on oocyte development during in vitro maturation

Changes in the gene expression of adiponectin and adiponectin receptors (AdipoR1 and AdipoR2) in ovarian follicular cells of dairy cow at different stages of development

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.

Characterization of agouti-signaling protein (ASIP) in the bovine ovary and throughout early embryogenesis

Meiosis activating sterol, produced directly by lanosterol 14-alpha-demethylase (CYP51) during cholesterol biosynthesis, has been shown to promote the initiation of oocyte meiosis. However, the physiological significance of CYP51 action on oocyte meiosis in response to gonadotrophins' induction remained to be further explored. Herein, we analyzed the role of CYP51 in gonadotrophin-induced in vitro oocyte maturation via RNA interference (RNAi). We showed that although both luteinizing hormone (LH) and follicle-stimulating hormone (FSH) significantly induced meiotic resumption in follicle-enclosed oocytes (FEOs), the effect of LH on oocyte meiosis resumption in FEOs was weaker than FSH. Moreover, both FSH and LH were able to upregulate CYP51 expression in cultured follicular granulosa cells when examined at 8 h or 12 h posttreatments, respectively. Interestingly, whereas knockdown of CYP51 expression via small interference RNA (siRNA) moderately blocked (23% reduction at 24 h) FSH-induced oocyte maturation [43% germinal vesicle breakdown (GVBD) rate in RNAi vs. 66% in control, P < 0.05] in FEOs, similar treatments showed no apparent effects on LH-induced FEO meiotic maturation (58% GVBD rate in RNAi vs. 63% in control, P > 0.05). Moreover, the results in a cumulus-enclosed oocytes (CEOs) model showed that approximately 30% of FSH-induced CEOs' meiotic resumption was blocked upon CYP51 knockdown by siRNAs. These findings suggest that FSH, partially at least, employs CYP51, and therefore the MAS pathway, to initiate oocyte meiosis.

This study was undertaken to assess the role of follicular cells in the maintenance of meiotic arrest (germinal vesicle [GV] stage). Bovine cumulus-oocyte complexes (COC) were obtained by puncture of ovaries collected at a slaughterhouse. Different monolayers of follicular cells--granulosa cells, theca interna, theca externa, or both types of theca cells together--were cultured in 24-well plates with 1 ml of TCM-199 supplemented with 10% fetal calf serum. Theca cells were obtained by digesting theca layers with collagenase. The medium was renewed 48 h before coculturing selected COC with confluent follicular cell monolayers. Oocytes were maintained in GV stage when cocultured for 12 h directly on monolayers of theca cells. The percentage of oocytes in GV stage was not significantly different between treatments using different types of theca cells (51-66%). Whether COC were cocultured in contact or not in contact with theca cells, the percentage of GV stage was similar (61%). The reversibility of this inhibition was high (85%). However, granulosa cells did not exert meiotic arrest (10%). When oocytes were denuded of their cumulus cells and cocultured with theca cells, only 3% were maintained in GV stage; 59% were maintained in GV stage when COC were not removed before culture. This data provided evidence of the essential role of cumulus cells in maintaining GV stage. In conclusion, we have demonstrated that theca cells, in vitro, maintained bovine oocytes in meiotic arrest. The inhibitory factor(s) produced by theca cells is soluble in the medium and acts through the cumulus cells.

We have investigated the possible role of theca and granulosa cell interaction in the control of the hormone-producing activity and growth of granulosa and theca cells during bovine ovarian follicular development, using a coculture system in which granulosa and theca cells were grown on opposite sides of a collagen membrane. When follicular cells were isolated from small follicles (3-5 mm), theca cells reduced estradiol, progesterone, and inhibin production by granulosa cells to 14 +/- 5%, 64 +/- 6%, and 27 +/- 4%, respectively, of the production by granulosa cells cultured alone. On the other hand, when the cells were isolated from large follicles (15-18 mm), theca cells increased these levels to 253 +/- 34%, 156 +/- 24%, and 287 +/- 45%, respectively. Theca cells did not affect the growth of granulosa cells. Androstenedione production by theca cells was augmented by granulosa cells to 861 +/- 190% (in small follicles) and 1298 +/- 414% (in large follicles), respectively. The growth of theca cells was also augmented by granulosa cells (small follicle, 210 +/- 43%, and large follicle, 194 +/- 24%, respectively). These results indicate that theca cells secrete factor(s) inhibiting the differentiation of immature while promoting that of matured granulosa cells; they also suggest that granulosa cells secrete factor(s) promoting both the differentiation and growth of theca cells throughout the follicular maturation process.

We developed a culture system in which two types of ovarian follicular cells were allowed to attach to opposite sides of a collagen membrane. Using this in vitro cell culture system, we studied the effects of granulosa- and theca-cell interaction on the morphology, structure, and function of bovine ovarian follicular cells. In the first part of the study, we explored how the interaction between theca and granulosa cells affects the morphology and structure of the cells. This study was done using follicular cells collected from bovine ovarian follicles at the early developmental stage. Granulosa cells cultured alone were flattened, and formed a monolayer sheet. By contrast, granulosa cells cultured with theca cells were convex, and formed multilayer sheets. Theca cells cultured alone were thin, flat, and spindle-shaped. Theca cells cultured with granulosa cells were also spindle-shaped; however, they appeared convex and more densely packed when compared with theca cells cultured alone. In the second part of the study, the possible role of the cellular interaction in the control of differentiation and growth of granulosa and theca cells was investigated. When follicular cells were isolated from the early stage of follicular development, theca cells reduced progesterone and inhibin production by granulosa cells and augmented the growth of granulosa cells. When the cells were isolated from the late stage of follicular development, by contrast, theca cells augmented hormonal production by granulosa cells, and did not affect the growth of granulosa cells. The growth and androstenedione production by theca cells were increased by the presence of granulosa cells, irrespective of the origin of follicular cells. These results demonstrated that communication between two types of follicular cells results in reciprocal modulation of their morphology, structure, growth, and function. Cellular interactions seem to be one of the major factors controlling the differentiation and growth of the follicular cells during the follicular maturation process. In contrast to granulosa and theca cells cultured alone, cells in the coculture seemed to possess morphological and functional characteristics more similar to those of cells in the growing follicular wall in vivo. Thus, we speculate that the interaction between these two types of follicular cells is essential for the maintenance of original structure and function of the bovine follicular wall.

Lipid metabolism in ovarian follicular cells supports the preparation of an enclosed oocyte to ovulation. We aimed to compare lipid composition of a dominant large follicle (LF) and subordinated small follicles (SFs) within the same ovaries. Mass spectrometry imaging displayed the differences in the distribution of several lipid features between the different follicles. Comparison of lipid fingerprints between LF and SF by Matrix Assisted Laser Desorption/Ionisation Time-Of-Flight (MALDI-TOF) mass spectrometry revealed that in the oocytes, only 8 out of 468 detected lipids (1.7%) significantly changed their abundance (p < 0.05, fold change > 2). In contrast, follicular fluid (FF), granulosa, theca and cumulus cells demonstrated 55.5%, 14.9%, 5.3% and 9.8% of significantly varied features between LF and SF, respectively. In total, 25.2% of differential lipids were identified and indicated potential changes in membrane and signaling lipids. Tremendous changes in FF lipid composition were likely due to the stage specific secretions from somatic follicular cells that was in line with the differences observed from FF extracellular vesicles and gene expression of candidate genes in granulosa and theca cells between LF and SF. In addition, lipid storage in granulosa and theca cells varied in relation to follicular size and atresia. Differences in follicular cells lipid profiles between LF and SF may probably reflect follicle atresia degree and/or accumulation of appropriate lipids for post-ovulation processes as formation of corpus luteum. In contrast, the enclosed oocyte seems to be protected during final follicular growth, likely due in part to significant lipid transformations in surrounding cumulus cells. Therefore, the enclosed oocyte could likely keep lipid building blocks and energy resources to support further maturation and early embryo development.

Understanding the paracrine events that regulate fertility in the cow is necessary not only because of the agricultural importance of this species, but also its potential use as a model for humans. Fibroblast growth factors (FGF) and their receptors are involved in the paracrine modulation of ovarian function. For example, FGF2, 7 and 10 inhibit estradiol secretion by granulosa cells in vitro. Another member of this family, FGF8, is an oocyte-derived protein that modulates glycolysis in cumulus cells. FGFs are grouped into subfamilies of closely-related proteins, and FGF18 is in the same subfamily as FGF8 and has also been detected in mouse oocytes. The existance of a subfamily of FGFs that are specifically derived from the oocyte are potentially interesting paracrine regulators of fertility. The objectives of the present study were to localize the expression of FGF18 in the bovine follicle, to gain insight into the control of FGF18 expression and to identify potential actions of FGF18 in the follicle. Bovine ovaries were obtained from an abattoir for localizing expression patterns by real-time PCR and immunohistochemistry. FGF18 mRNA was not detected in pooled oocytes by PCR, but was present in isolated theca and granulosa cells (GC), and protein was detected in the follicle wall by immunohistochemistry. Expression during follicle growth was measured by collecting the two largest follicles per cow at defined stages of the first follicle wave. The follicles were classified as dominant and subordinate according to estradiol levels in follicular fluid and aromatase mRNA abundance in GC. FGF18 mRNA abundance in both GC and theca cells was higher in subordinate follicles compared with dominant follicles (P<0.05). To determine regulation and function of FGF18, granulosa cells were isolated from 5-8 mm follicles and employed in an estrogenic, serum-free culture system. In vitro, FGF18 expression was down-regulated by IGF1, insulin, FGF2, FGF10 (P<0.05) in GC. In further experiments, the addition of graded doses of FGF18 (0, 1, 10, 100 ng/ml) to GC in culture inhibited estradiol secretion and abundance of mRNA encoding the estrogenic enzymes aromatase and 17β-hydroxysteroid dehydrogenase (17βHSD), and the FSH receptor. Progesterone secretion was also inhibited, which was associated with decreased abundance of mRNA encoding CYP11, StAR and 3βHSD. Assessment of apoptosis by flow cytometry showed that FGF18 significantly increased the proportion of apoptotic cells, and this was accompanied by decreased abundance of mRNA encoding antiapoptotic and proliferation genes (MIF, RFC4 and GADD45B). In summary, FGF18 is expressed in GC and theca cells but--in contrast to the subfamily prototype, FGF8--not in oocytes in cattle. As FGF18 expression is increased in atretic follicles, and as FGF18 protein inhibits estradiol secretion and induces apoptosis, we conclude that FGF18 plays a role in follicular atresia. Supported by NSERC Canada, CNPq and FAPESP, Brazil. (platform)

The developing ovarian follicle is one of the most rapidly proliferating normal tissues in vivo. Mesenchymal-epithelial cell interactions between theca cells and granulosa cells are essential for this follicular expansion. Ovarian hormones (i.e. estrogen and LH) may promote follicular development by regulating the local production of mesenchymal inducer proteins that mediate theca cell-granulosa cell interactions. Recently, theca cells were shown to produce keratinocyte growth factor (KGF) that can act in a paracrine manner to stimulate granulosa cell growth. In this study, the developmental and hormonal regulation of KGF was examined during follicular development in the bovine ovary. Expression of KGF in theca cells and the KGF receptor (KGFR, or splice variant of the fibroblast growth factor family receptor family, FGFR-2) in granulosa cells was examined using RT-PCR. Both KGF and KGFR were detected throughout follicular development in small (<5 mm), medium (5-10 mm), and large (>10 mm) follicles. Quantitative RT-PCR assays were used to determine steady-state levels of KGF and KGFR messenger RNAs. Developmental regulation of KGF and KGFR was analyzed in freshly isolated theca cells and granulosa cells from small, medium, and large follicles. Observations demonstrated that expression of KGF (in theca cells) and KGFR (in granulosa cells) was highest in large follicles. These results suggest that KGF actions are important for the rapid proliferation of granulosa cells in large follicles. Estrogen and LH are the primary endocrine hormones that regulate theca cell function in vivo. Therefore, hormonal regulation of KGF was analyzed by treating serum-free theca cell cultures with estrogen and human CG (hCG, an LH agonist). Results showed that both estrogen and hCG stimulated KGF gene expression in theca cells. These results suggest that estrogen and LH may promote follicular growth (i.e. granulosa cell proliferation), in part, by stimulating the local production of KGF. Effects of KGF on granulosa cell differentiated functions were examined. Treatment with KGF reduced basal levels and FSH-stimulated levels of aromatase activity in bovine and rat granulosa cells. In addition, KGF inhibited the ability of hCG to stimulate progesterone production by granulosa cells. The inhibition of granulosa cell steroid production by KGF was likely the indirect effect of promoting cellular proliferation. Therefore, KGF directly stimulates granulosa cell proliferation and indirectly inhibits granulosa cell differentiated functions. Combined results suggest that theca cell production of KGF may be important for ovarian folliculogenesis. This is the first report of the regulation of KGF expression in the ovary. The developmental and hormonal regulation of KGF and KGFR during folliculogenesis provides evidence that KGF may be important for hormone-induced granulosa cell proliferation. As a result, KGF may be essential for establishing the microenvironment required for oocyte maturation in the ovary.

The developing ovarian follicle is one of the most rapidly proliferating normal tissues in vivo. Mesenchymal-epithelial cell interactions between theca cells and granulosa cells are essential for this follicular expansion. Ovarian hormones (i.e. estrogen and LH) may promote follicular development by regulating the local production of mesenchymal inducer proteins that mediate theca cell-granulosa cell interactions. Recently, theca cells were shown to produce keratinocyte growth factor (KGF) that can act in a paracrine manner to stimulate granulosa cell growth. In this study, the developmental and hormonal regulation of KGF was examined during follicular development in the bovine ovary. Expression of KGF in theca cells and the KGF receptor (KGFR, or splice variant of the fibroblast growth factor family receptor family, FGFR-2) in granulosa cells was examined using RT-PCR. Both KGF and KGFR were detected throughout follicular development in small (<5 mm), medium (5–10 mm), and large (>10 mm) follicles. Quantitative RT-PCR assays were used to determine steady-state levels of KGF and KGFR messenger RNAs. Developmental regulation of KGF and KGFR was analyzed in freshly isolated theca cells and granulosa cells from small, medium, and large follicles. Observations demonstrated that expression of KGF (in theca cells) and KGFR (in granulosa cells) was highest in large follicles. These results suggest that KGF actions are important for the rapid proliferation of granulosa cells in large follicles. Estrogen and LH are the primary endocrine hormones that regulate theca cell function in vivo. Therefore, hormonal regulation of KGF was analyzed by treating serum-free theca cell cultures with estrogen and human CG (hCG, an LH agonist). Results showed that both estrogen and hCG stimulated KGF gene expression in theca cells. These results suggest that estrogen and LH may promote follicular growth (i.e. granulosa cell proliferation), in part, by stimulating the local production of KGF. Effects of KGF on granulosa cell differentiated functions were examined. Treatment with KGF reduced basal levels and FSH-stimulated levels of aromatase activity in bovine and rat granulosa cells. In addition, KGF inhibited the ability of hCG to stimulate progesterone production by granulosa cells. The inhibition of granulosa cell steroid production by KGF was likely the indirect effect of promoting cellular proliferation. Therefore, KGF directly stimulates granulosa cell proliferation and indirectly inhibits granulosa cell differentiated functions. Combined results suggest that theca cell production of KGF may be important for ovarian folliculogenesis. This is the first report of the regulation of KGF expression in the ovary. The developmental and hormonal regulation of KGF and KGFR during folliculogenesis provides evidence that KGF may be important for hormone-induced granulosa cell proliferation. As a result, KGF may be essential for establishing the microenvironment required for oocyte maturation in the ovary.

Cells from the inner and outer granulosa cell layers of the ovarian follicles differ in function, probably because of their different origins from the surface epithelium and from the rete. This suggestion has not so far been thoroughly investigated in the human ovary. We examined fetal ovaries from the early, middle and late gestational periods, ovaries from fertile women, and preovulatory follicular cells obtained from patients under in vitro fertilization therapy (IVF). Indirect immunohistology and immunocytology were used to detect the presence of cytokeratin (CK)-positive epithelial cells. In fetal ovaries from the early gestational period, prominent rete tubules (sometimes with oocytes) appeared to be fused with the sex cords and primordial follicles. Both showed CK-positively, detected with the pan-CK antibody Lu-5. Cytokeratin 19 was clearly expressed in the fusion area. In the fetal and adult ovaries, CK-positive follicular or granulosa cells were noted in the primordial and primary follicles as well as the preovulatory follicles. Cytokeratin was not detected in the granulosa cells of growing follicles, CK-positive and -negative luteal cells were identified in the developing corpus luteum. We conclude for the human ovary: (1) the heterogeneous morphology of granulosa cells may be explained by their twofold origin from the surface epithelium and the rete, (2) the rete tubules appear to be involved in folliculogenesis, (3) the transient absence of CK expression in growing follicles compared to resting and mature follicles or to the developing corpus luteum indicates a particular role of CK-positive cells at the periovulatory period.

The developmental relationship between granulosa and stromal cells in the fetal ovary was reviewed. The proliferation and expression of basic fibroblast growth factor (bFGF) and luteinizing hormone receptor (LHR) of both the granulosa and theca cells was examined in the adult ovary. Ovarian granulosa cell tumors were then studied under light and electron microscopy and for immunohistochemical expression of bFGF and LHR. In the follicular development of the adult ovary, bFGF expression in the granulosa cells was correlated with theca cell proliferation. In ovarian granulosa cell tumors, estrogen-producing tumors showed a stratified or microfollicular pattern being accompanied by theca cells which had ultrastructural features of steroid-production, and these tumor cells exhibited immunoreactivity for bFGF. In contrast, tumors with no apparent hormone-production showed a solid or gyriform pattern without admixture of theca cells, and were negative for bFGF. LHR was expressed in both types of tumor. Accordingly, bFGF may play an important role in association with the theca cell component in granulosa cell tumors.

CHEMERIN, or RARRES2, is a new adipokine that is involved in the regulation of adipogenesis, energy metabolism, and inflammation. Recent data suggest that it also plays a role in reproductive function in rats and humans. Here we studied the expression of CHEMERIN and its three receptors (CMKLR1, GPR1, and CCRL2) in the bovine ovary and investigated the in vitro effects of this hormone on granulosa cell steroidogenesis and oocyte maturation. By RT-PCR, immunoblotting, and immunohistochemistry, we found CHEMERIN, CMKLR1, GPR1, and CCRL2 in various ovarian cells, including granulosa and theca cells, corpus luteum, and oocytes. In cultured bovine granulosa cells, INSULIN, IGF1, and two insulin sensitizers-metformin and rosiglitazone-increased rarres2 mRNA expression whereas they decreased cmklr1, gpr1, and cclr2 mRNA expression. Furthermore, TNF alpha and ADIPONECTIN significantly increased rarres2 and cmklr1 expression, respectively. In cultured bovine granulosa cells, human recombinant CHEMERIN (hRec, 200 ng/ml) reduced production of both progesterone and estradiol, cholesterol content, STAR abundance, CYP19A1 and HMGCR proteins, and the phosphorylation levels of MAPK3/MAPK1 in the presence or absence of FSH (10(-8) M) and IGF1 (10(-8) M). All of these effects were abolished by using an anti-CMKLR1 antibody. In bovine cumulus-oocyte complexes, the addition of hRec (200 ng/ml) in the maturation medium arrested most oocytes at the germinal vesicle stage, and this was associated with a decrease in MAPK3/1 phosphorylation in both oocytes and cumulus cells. Thus, in cultured bovine granulosa cells, hRec decreases steroidogenesis, cholesterol synthesis, and MAPK3/1 phosphorylation, probably through CMKLR1. Moreover, in cumulus-oocyte complexes, it blocked meiotic progression at the germinal vesicle stage and inhibited MAPK3/1 phosphorylation in both the oocytes and cumulus cells during in vitro maturation.