Androgen production in response to LH is impaired in theca cells from nonovulatory dominant follicles in early-postpartum dairy cows.

Most dairy cows develop a dominant follicle within two weeks postpartum, but 60% of these follicles fail to ovulate. In a previous study, we determined that cows destined to ovulate have higher LH pulse frequency and circulating estradiol. The latter characteristic provided a method for distinguishing ovulatory from nonovulatory follicles during development and we found that nonovulatory follicles have lower estradiol and androstenedione in their follicular fluid. We hypothesized that lower LH pulse frequency impairs androgen production by theca cells of nonovulatory cows, reducing their ability to make estradiol. In the present study, we applied our method for predicting follicle fate to collect dominant follicles from predicted ovulatory (n = 7) and nonovulatory (n = 3) follicles. Theca and granulosa cells were separated and cultured in the absence or presence of LH, FSH, and/or testosterone for three days, with daily collection of culture medium for steroid RIAs. Estradiol and progesterone production by granulosa cells were not different between ovulatory and nonovulatory follicles. By contrast, overall androstenedione production by theca cells from ovulatory follicles was significantly higher compared with nonovulatory follicles on all three days of culture and, as culture progressed, theca from nonovulatory follicles had increasingly poorer responses to LH. In the same cultures, the progesterone production by theca cells was similar in ovulatory and nonovulatory groups. In support of our hypothesis, the results show that estradiol production by granulosa cells from nonovulatory follicles is robust when androgen substrate is present, but that thecal androgen production in response to LH is impaired. This suggests that the initial defect in steroidogenesis in dominant follicles that fail to ovulate postpartum is lower production of androgen by theca cells.

Fibroblast growth factor 18 regulates steroidogenesis in fetal bovine ovarian tissue in vitro.

In cattle and other species, the fetal ovary is steroidogenically active before follicular development commences, and there is evidence that estradiol and progesterone inhibit follicle formation and activation. Estradiol levels decline sharply around the time of follicle formation. In the present study, we hypothesized that FGF10 and FGF18, which inhibit estradiol secretion from granulosa cells of antral follicles, also regulate fetal ovarian steroid production. Fetuses were collected at local abattoirs, and age determined by crown-rump length measurements. Real-time polymerase chain reaction assays with RNA extracted from whole ovaries revealed that the abundance of CYP19A1 messenger RNA (mRNA) decreased from 60 to 90 days of gestation, which is consistent with the decline in estradiol secretion previously observed. Immunohistochemistry revealed the presence of FGF18 in ovigerous cords in early gestation and in oocytes later in fetal age (≥150 days). The abundance of FGF18 mRNA increased after Day 90 gestation. Addition of recombinant FGF18 to fetal ovarian pieces inhibited estradiol and progesterone secretion in vitro, whereas FGF10 was without effect. Consistent with these results, FGF18 decreased levels of mRNA for CYP19A1 and CYP11A1 in ovarian pieces in vitro. These data suggest that FGF18 may be an intraovarian factor that regulates steroidogenesis in fetal ovaries.

Anti-Müllerian hormone inhibits activation and growth of bovine ovarian follicles in vitro and is localized to growing follicles.

STUDY QUESTION: Does anti-Müllerian hormone (AMH) inhibit activation (initiation of growth) of primordial follicles and attenuate the growth of primary follicles in cattle, an excellent animal model for human ovarian follicular development? SUMMARY ANSWER: AMH inhibited activation of bovine primordial follicles and attenuated the growth of activated follicles in vitro. WHAT IS KNOWN ALREADY: In mice null mutant for AMH, the pool of primordial follicles is depleted prematurely and AMH inhibits follicle activation in vitro. Results of studies with human ovarian tissue in vitro were inconsistent. Our previous work provided indirect evidence that AMH inhibits follicle activation in bovine ovaries. STUDY DESIGN, SIZE, DURATION: Pieces of fetal bovine ovarian cortex (2 pieces/culture well), obtained during mid or late pregnancy, were cultured in control medium or with graded doses of AMH for 2, 10 or 12 days. Effects of treatment on follicle activation and growth were determined by histological morphometry; follicles in every 20th histological section were staged (primordial or primary), counted, and measured. In addition, AMH was immunolocalized in bovine ovaries obtained at various times during pregnancy (n = 20 ovaries). PARTICIPANTS/MATERIALS, SETTING, METHODS: Bovine fetal ovaries at mid or late gestation were obtained at a commercial abattoir. Pieces of ovarian cortex were cultured without or with AMH and fixed for histological morphometry on Day 0 and at the end of culture. Treatments were applied to duplicate cultures from each of two or three fetuses. In 12-day cultures, addition of AMH was delayed until the third day. Histological analysis provided information about the types, numbers and sizes of follicles in cortical pieces before and after treatments. Ovaries obtained during the second and third trimesters were assessed for the presence of AMH by immunohistochemistry. MAIN RESULTS AND THE ROLE OF CHANCE: AMH (100-500 ng/ml) inhibited follicle activation in response to an activator (insulin) in ovarian cortical pieces from fetal ovaries in late gestation. Dose-dependent inhibitory effects on the diameters of primary follicles and their oocytes were also observed. These results were obtained only when AMH was added to cultures in advance of insulin (presumably because it penetrates tissue more slowly than insulin). Results of experiments with cortical pieces from fetal ovaries at mid-gestation, when follicles are forming, showed that AMH did not inhibit the formation of follicles. Immunohistochemical localization of AMH showed that it is not present in fetal ovaries until the third trimester, when it was localized to the granulosa cells of secondary and small antral follicles. LIMITATIONS REASONS FOR CAUTION: The experiments were performed with fetal ovaries because follicles form and follicle activation begins during fetal life in cattle (as it does in humans), so fetal ovarian cortex of later gestation provides tissue rich in primordial follicles. We assume, but have no experimental evidence, that our findings also apply to post-natal ovaries. WIDER IMPLICATIONS OF THE FINDINGS: Although circulating AMH is used as an indication of the follicular reserve in women, little is known about AMH in human ovaries. Cattle are an excellent non-primate model for human ovarian follicular development and, hence, the findings suggest similar roles for AMH in human follicular development. LARGE SCALE DATA: Not applicable. STUDY FUNDNG/COMPETING INTEREST(S): This research was supported by National Research Initiative Competitive Grants no. 00-35203-9151, 2003-35203-13532, and 2008-35203-05989) from the U.S. Dept. of Agriculture's National Institute of Food and Agriculture to JEF and by an NIH National Research Service Award (F32 HD08264) to RAC. There are no conflicts of interest or competing interests.

Regulation of steroidogenesis in fetal bovine ovaries: differential effects of LH and FSH.

In cattle, primordial follicles form before birth. Fetal ovarian capacity to produce progesterone and estradiol is high before follicle formation begins and decreases around the time follicles first appear (around 90 days of gestation). However, mechanisms that regulate steroid production during this time remain unclear. We hypothesized that LH stimulates progesterone and androgen production and that FSH stimulates aromatization of androgens to estradiol. To test this, we cultured pieces from fetal bovine ovaries for 10 days without or with exogenous hormones and then measured the accumulation of steroids in the culture medium by RIA. LH (100 ng/mL) alone increased the accumulation of progesterone, androstenedione, testosterone and estradiol. FSH (100 ng/mL) alone increased both progesterone and estradiol accumulation, but had no effect on androgens. Exogenous testosterone (0.5 µM) alone greatly increased estradiol accumulation and the combination of testosterone + FSH, but not testosterone + LH, increased estradiol relative to testosterone alone. Interestingly, exogenous testosterone and estradiol decreased progesterone accumulation in a dose-dependent manner. Because the highest dose of estradiol (0.5 µM) decreased progesterone accumulation, but increased both pregnenolone and androstenedione in the same cultures, endogenous estradiol may be a paracrine regulator of steroid synthesis. Together, these results confirm our initial hypotheses and indicate that LH stimulates androgen production in fetal bovine ovaries via the Δ5 pathway, whereas FSH stimulates aromatization of androgens to estradiol. These results are consistent with the two-cell, two-gonadotropin model of estradiol production by bovine preovulatory follicles, which suggests that the mechanisms regulating ovarian steroid production are established during fetal life.

Changes in the transcriptome of bovine ovarian cortex during follicle activation in vitro.

The signals that regulate activation, a key transition in ovarian follicular development, are still not well understood, especially in nonrodent species. To gain insight into the regulation of this transition in cattle, we combined a microarray approach with an in vitro system in which ovarian cortical pieces cultured in control medium are enriched for primordial follicles, whereas pieces cultured with insulin are enriched for primary follicles. Total RNA was extracted from cultured cortical pieces, and then transcripts were identified and analyzed using the Affymetrix Bovine Genome GeneChip array. Around 65% of the transcripts in the bovine GeneChip were detected in cultured cortical pieces. Comparison between pieces cultured with or without insulin generated 158 differentially expressed transcripts. Compared with controls, 90 transcripts were upregulated and 68 were downregulated by insulin. These transcripts are involved in many biological processes and functions, but most are associated with cellular growth or cell cycle/cell death. The transcript encoding ubiquitin-conjugating enzyme E2C (UBE2C) was significantly upregulated during follicle activation, and Ingenuity Pathways Analysis revealed that UBE2C can interact with the tumor suppressor phosphatase and tensin homolog (PTEN). Both PTEN mRNA and protein were lower in cortical pieces cultured with insulin than in controls. In addition, FOXO3a, a downstream effector of PTEN signaling, underwent nuclear-cytoplasmic shuttling during primordial to primary follicle development in bovine fetal ovaries, further suggesting the involvement of the PTEN pathway in follicle activation in cattle. Genes and pathways identified in this study provide interesting candidates for further investigation of mechanisms underlying follicle activation.

Triennial Reproduction Symposium: the ovarian follicular reserve in cattle: what regulates its formation and size?

The ovarian follicular reserve has been linked to fertility in cattle. Young adult cattle with low vs. high numbers of antral follicles ≥ 3 mm in diameter in follicular waves also have fewer preantral follicles and decreased fertility. This underscores the importance of understanding the factors that regulate early follicular development and establish the ovarian follicular reserve, but little is known about how the follicular reserve is first established. In ruminants and humans, follicles form during fetal life, but there is a gap (about 50 d in cattle) between the appearance of the first primordial follicles and the first growing, primary follicles. In this review we present evidence that in cattle, fetal ovarian steroids (i.e., estradiol and progesterone) are negative regulators of both follicle formation and of the acquisition by newly formed follicles of the capacity to activate (i.e., initiate growth). The results indicate that capacity to activate is linked to the completion of meiotic prophase I by the oocyte. The inhibitory effects of estradiol on follicle activation were found to be reversible and correlated with inhibition of the progression of meiotic prophase I. Fetal bovine ovaries produce steroid hormones and production varies considerably during gestation and in a pattern consistent with the hypothesis that they inhibit follicle formation and capacity of newly formed follicles to activate in vivo. However, little was known about how steroid production is regulated. In our studies, both LH and FSH stimulated progesterone and estradiol production by ovarian pieces in vitro. The addition of testosterone to the culture medium enhanced estradiol production, especially when FSH was also present, but inhibited progesterone production, even in the presence of gonadotropins. Evidence is also presented for effects of maternal nutrition and health and for potential effects of estrogenic endocrine-disrupting chemicals on the size of the ovarian follicular reserve established during fetal life. In summary, fetal ovarian steroids may be important regulators of the early stages of follicular development in cattle. Therefore, external factors that alter steroid production or action may affect the size of the ovarian follicular reserve.

Regulation of folliculogenesis and the determination of ovulation rate in ruminants.

The paper presents an update of our 1993 model of ovarian follicular development in ruminants, based on knowledge gained from the past 15 years of research. The model addresses the sequence of events from follicular formation in fetal life, through the successive waves of follicular growth and atresia, culminating with the emergence of ovulatory follicles during reproductive cycles. The original concept of five developmental classes of follicles, defined primarily by their responses to gonadotrophins, is retained: primordial, committed, gonadotrophin-responsive, gonadotrophin-dependent and ovulatory follicles. The updated model has more extensive integration of the morphological, molecular and cellular events during folliculogenesis with systemic events in the whole animal. It also incorporates knowledge on factors that influence oocyte quality and the critical roles of the oocyte in regulating follicular development and ovulation rate. The original hypothetical mechanisms determining ovulation rate are retained but with some refinements; the enhanced viability of gonadotrophin-dependent follicles and increases in the number of gonadotrophin-responsive follicles by increases in the throughput of follicles to this stage of growth. Finally, we reexamine how these two mechanisms, which are thought not to be mutually exclusive, appear to account for most of the known genetic and environmental effects on ovulation rate.

Current achievements and future research directions in ovarian tissue culture, in vitro follicle development and transplantation: implications for fertility preservation.

BACKGROUND Female cancer patients are offered 'banking' of gametes before starting fertility-threatening cancer therapy. Transplants of fresh and frozen ovarian tissue between healthy fertile and infertile women have demonstrated the utility of the tissue banked for restoration of endocrine and fertility function. Additional methods, like follicle culture and isolated follicle transplantation, are in development. METHODS Specialist reproductive medicine scientists and clinicians with complementary expertise in ovarian tissue culture and transplantation presented relevant published literature in their field of expertise and also unpublished promising data for discussion. As the major aims were to identify the current gaps prohibiting advancement, to share technical experience and to orient new research, contributors were allowed to provide their opinioned expert views on future research. RESULTS Normal healthy children have been born in cancer survivors after orthotopic transplantation of their cryopreserved ovarian tissue. Longevity of the graft might be optimized by using new vitrification techniques and by promoting rapid revascularization of the graft. For the in vitro culture of follicles, a successive battery of culture methods including the use of defined media, growth factors and three-dimensional extracellular matrix support might overcome growth arrest of the follicles. Molecular methods and immunoassay can evaluate stage of maturation and guide adequate differentiation. Large animals, including non-human primates, are essential working models. CONCLUSIONS Experiments on ovarian tissue from non-human primate models and from consenting fertile and infertile patients benefit from a multidisciplinary approach. The new discipline of oncofertility requires professionalization, multidisciplinarity and mobilization of funding for basic and translational research.

The earliest stages of follicular development: follicle formation and activation.

The formation of primordial follicles to establish a reservoir of resting follicles and the gradual depletion of that reservoir to provide a succession of growing follicles are key to female fertility, but little is known about the regulation of these early stages of follicular development. This review summarizes the efforts of our laboratory to elucidate these critical processes in cattle. Primordial follicles first appear in fetal ovaries around the end of the first trimester of pregnancy (Day 90), during a decline in fetal ovarian production of estradiol and progesterone. In ovarian cortical pieces from 90 to 120-day-old fetuses, follicles form in vitro and estradiol or progesterone inhibits follicle formation, whereas the non-aromatizable androgen 5alpha-dihydrotestosterone (DHT) does not. Newly formed bovine follicles are not capable of activating within 2 days in vitro, but they can acquire the capacity to activate during a longer culture; estradiol and progesterone inhibit the acquisition of their capacity to activate. When primordial follicles first form in cattle, their oocytes are not yet in meiotic arrest and acquisition of competence to activate is correlated with their progression to meiotic arrest at the diplotene stage of first prophase. After they acquire the competence to activate, bovine primordial follicles can be stimulated to activate in vitro by insulin or kit ligand, whereas anti-Mullerian hormone (AMH) is inhibitory. Although few follicles progress to the secondary stage in vitro, addition of testosterone or vascular endothelial growth factor (VEGF) dramatically increased the incidence of that transition. Regulation of the earliest stages of follicular development is complex and far from understood; better understanding could lead to new interventions to enhance fertility.

The periovulatory period in cattle: progesterone, prostaglandins, oxytocin and ADAMTS proteases.

Ovulation has long been recognized as one of the most dramatic reproductive processes. Decades of research on how the LH/FSH surge leads to ovulation have made it clear that the surge induces a very complex cascade of changes. Studies of genetically modified mice have pointed to progesterone (P4) and its receptor (PGR) and the prostaglandins (PGs) as critical components of the ovulatory cascade. In cattle, the gonadotropin surge also induces oxytocin (OT), which does not appear to increase in rodent periovulatory follicles. This review is an attempt to summarize studies by our laboratory on the temporal patterns, roles, regulation, and interrelationships among P4/PGR, PGs, and OT in bovine periovulatory follicles. Most of these results are based on an experimental model in which the dominant follicle of the first follicular wave of the estrous cycle is induced to develop into a preovulatory follicle by injection of PGF(2α) on Day 6 of the cycle, followed 36 h later by an injection of GnRH to induce the LH/FSH surge. The results suggest that the effects of the gonadotropin surge on PG production by bovine granulosa cells are mediated by the gonadotropin-induced increase in intrafollicular P4 and that P4 acts by binding to its nuclear receptor and increasing the abundance of mRNA for the enzyme PTGS2 (COX-2). Our data thus far also support the hypothesis that PGs, especially PGE(2), can stimulate progesterone secretion by both follicular cell types and suggest a positive feedback relationship between P4/PGR and the PGs. Additional results suggest a positive feedback loop between P4/PGR and OT. The finding that levels of mRNA for several ADAMTS proteases are regulated by the LH/FSH surge in vivo and by P4/PGR and/or PGs in vitro suggests a role for this family of proteases in remodeling the bovine ovulatory follicle in preparation for ovulation and the formation of the corpus luteum. It is important to remember that a process essential for reproduction, such as ovulation, may involve redundant mechanisms and that these mechanisms may have evolved differently from rodents in larger mammalian species, such as ruminants and humans.

The capacity of primordial follicles in fetal bovine ovaries to initiate growth in vitro develops during mid-gestation and is associated with meiotic arrest of oocytes.

In cattle and other species in which the pool of resting, primordial follicles is formed during fetal life, little is known about the regulation of the early stages of ovarian follicular development. We used histological morphometry and a combination of observations in vivo and experiments in vitro to study the timing and regulation of follicle formation and the acquisition of the capacity of primordial follicles to initiate growth in cattle. In vivo, primordial, primary, and secondary follicles were first observed around Days 90, 140, and 210 of gestation, respectively. The long interval between the first appearance of primordial and primary follicles suggests that primordial follicles are not capable of activating when they are first formed, or they are inhibited from activating. This hypothesis was confirmed by the finding that most primordial follicles in pieces of ovarian cortex obtained from fetal ovaries older than 140 days activated (i.e., initiated growth) after 2 days in vitro, whereas follicles in cortical pieces from 90- to 140-day-old fetal ovaries did not. We tested the hypothesis that the oocytes of newly formed primordial follicles are not in meiotic arrest and found that before Day 141, most oocytes ( approximately 73%) were in prediplotene stages of prophase I, whereas after Day 140, the majority of oocytes ( approximately 85%) had arrested at the diplotene stage. This observation was further confirmed by the finding that levels of mRNA for YBX2, a protein associated with meiotic arrest, were 2.3 times higher in ovarian cortical pieces isolated after versus before Day 141. Primordial follicles in cortical pieces from 90- to 140-day-old fetal ovaries did activate during a longer, 10-day culture, but activation could be inhibited by adding estradiol or progesterone, but not dihydrotestosterone (all at 10(-6) M). Fetal ovaries secreted estradiol in vitro, and secretion by ovaries from 83 to 140-day-old fetuses declined precipitously ( approximately 30-fold) with age, consistent with the hypothesis that estradiol inhibits activation of newly formed primordial follicles in vivo. In summary, the results show that newly formed primordial follicles do not activate in vivo or within 2 days in vitro and that capacity to activate is correlated with achievement of meiotic arrest by the oocyte and can be inhibited by estradiol, which fetal ovaries actively produce around the time of follicle formation.

Vascular endothelial growth factor stimulates the primary to secondary follicle transition in bovine follicles in vitro.

Little is known about the mechanisms regulating the growth of early preantral follicles, especially in nonrodent mammalian species. To test the hypothesis that vascular endothelial growth factor (VEGF) promotes the primary to secondary follicle transition, pieces of bovine fetal ovarian cortex were cultured with VEGF (0, 1, 10, or 100 ng/ml) for 0 or 10 days, followed by morphometric analysis. On day 0, cortical pieces contained mostly primordial follicles, but after 10 days in vitro most primordial follicles had activated, differentiating into primary follicles. VEGF had no effect on the numbers of primordial or primary follicles, compared with untreated controls, but all doses increased the number of secondary follicles. In the second experiment, a range of lower doses of VEGF (0.1-10 ng/ml) increased the number of secondary follicles in a dose-dependent manner. Analysis of VEGF transcripts by RT-PCR showed expression of mRNA for three isoforms of VEGF (VEGF121, 165, and 189) in fetal bovine ovarian cortex, with VEGF121 and 165 expressed predominantly and levels of mRNA for VEGF121 and 189 increasing after day 211 of gestation, when the first secondary follicles appear. Expression of mRNA for both VEGF receptors (flt-1 and flk-1) was also detected, but did not change with the development of fetal ovaries. Immunohistochemistry revealed positive staining for VEGF in blood vessels and in follicle cells of secondary follicles, consistent with Western blot analyses showing increases in VEGF protein as ovarian development progressed. Taken together, the results provide support for a role for VEGF in the primary to secondary follicle transition.

Regulation, action and transport of prostaglandins during the periovulatory period in cattle.

Follicular production of prostaglandins (PGs) is essential for mammalian ovulation, but the factors that mediate production and the cell-specific action(s) of PGE and PGF2alpha during the ovulatory cascade remain largely unknown. The aims of these experiments were: (1) to investigate the potential role of oxytocin (OT) in ovulatory PG production, (2) to determine cellular and temporal patterns of expression of mRNA for specific PG receptors during the periovulatory interval, (3) to determine cell-specific effects of PGE2 on progesterone secretion, and (4) to investigate the potential for an active transport mechanism that may regulate the effect of PGs during the ovulatory cascade, using cattle as the animal model. Heifers were treated sequentially with PGF2alpha and GnRH to induce luteal regression, a follicular phase and the LH/FSH surge (ovulation occurs approximately 30 h after GnRH). In experiment 1, OT increased the secretion of PGE and PGF2alpha by granulosa cells collected from preovulatory follicles (before the LH/FSH surge) and OT production by pieces of follicle wall from periovulatory follicles (after the LH/FSH surge) was regulated by progesterone acting through the progesterone receptor. In experiment 2, levels of mRNA for the PGF2alpha receptor and three PGE receptor subtypes were determined by semi-quantitative RT-PCR in theca interna and granulosa cells from pre- and periovulatory follicles collected at 0, 6, 12, 18 and 24 h post-GnRH. Time- and cell-specific patterns of change in mRNA for PG receptors were observed, suggesting multiple effects of both PGE and PGF2alpha in both theca interna and granulosa cells throughout the ovulatory cascade. Cell-specificity of PG action was confirmed in experiment 3; PGE2 increased the secretion of progesterone by theca interna but not granulosa cells collected from late periovulatory follicles. The results of experiment 4 revealed the expression of mRNA for the bovine PG transporter in theca interna and granulosa cells and its regulation during the periovulatory period, thus revealing the presence of a potential transport mechanism that could regulate cellular distribution of PGs throughout the ovulatory cascade. Taken together, these results provide new insight into mechanisms that regulate the production, distribution and site of action of PGE and PGF2alpha during the ovulatory cascade.

Testosterone stimulates the primary to secondary follicle transition in bovine follicles in vitro.

The mechanisms controlling the initiation and early stages of follicular growth are poorly understood. Our laboratory developed a serum-free culture system that supports spontaneous and wholesale activation of primordial follicles in pieces of cortex dissected from the ovaries of fetal calves and fetal baboons. However, very few follicles activated in vitro progressed to the secondary stage. To determine whether androgens can promote the primary to secondary follicle transition, pieces of fetal bovine ovarian cortex were cultured in serum-free medium in the absence or presence of testosterone (T, 10(-7) and 10(-6) M) or estradiol (E(2), 10(-6) M) for 10 days. Cortical pieces were then fixed and embedded in plastic for serial sectioning and morphometric analysis; fresh cortical pieces fixed on Day 0 served as uncultured controls. Freshly isolated cortical pieces contained mostly primordial follicles, whereas after 10 days in vitro, most primordial follicles had activated, differentiating into primary follicles as expected. Neither T nor E(2) affected the number of primordial and primary follicles compared with controls (P > 0.05). However, T (10(-7) and 10(-6) M) increased the number of secondary follicles (P < 0.05), whereas E(2) had no effect, suggesting that the effect of T was not due to conversion of T to E(2). In the second experiment, the optimal concentration of T for preantral follicle growth was determined. A range of lower doses of T (10(-10)-10(-7) M) increased the number of secondary follicles in cultured cortical pieces in a dose-dependent manner, with 10(-7) M T being the most effective (P < 0.05). In the third experiment, addition of a specific androgen receptor blocker, flutamide, inhibited the stimulatory effects of T on the primary to secondary follicle transition (P < 0.05), suggesting a receptor-mediated action of T. Localization of androgen receptors by immunohistochemistry revealed immunostaining for the androgen receptor in ovarian stromal cells and increasing immunoreactivity in follicle cells as follicular development progressed from primordial and primary to secondary to antral follicles, suggesting the involvement of the androgen receptor in bovine folliculogenesis. In summary, our results show that T promotes the growth of bovine follicles activated in vitro and suggest that its stimulatory effect is mediated through androgen receptors in the stroma and/or follicular cells.

Gonadotropin-induced expression of messenger ribonucleic acid for cyclooxygenase-2 and production of prostaglandins E and F2alpha in bovine preovulatory follicles are regulated by the progesterone receptor.

Follicular production of prostaglandins (PGs) is essential for ovulation, but the factors mediating gonadotropin-induced secretion of PGE and PGF(2alpha) remain largely unknown. We tested the hypothesis that gonadotropin-induced changes in progesterone and its receptor (PR) mediate the increase in periovulatory PGs. Heifers were treated with PGF(2alpha) and GnRH to induce luteolysis and the LH/FSH surge (ovulation occurs approximately 30 h after GnRH). Because there are two increases in intrafollicular progesterone/PR mRNA during the bovine periovulatory period, we first examined the temporal pattern of PG production by follicles collected at 0, 3.5, 6, 12, 18, and 24 h after GnRH. Although PGs did not increase in the follicular fluid until 24 h after GnRH, acute secretion of PGs by follicle wall (theca + granulosa cells) was initiated by 18 h and had increased manyfold by 24 h after GnRH. In vitro, FSH and LH induced dramatic transient increases in PG production by follicle wall and granulosa, but not theca, cells isolated from preovulatory follicles (0 h after GnRH). PG accumulation peaked on d 2 of culture, mimicking the secretion pattern after a gonadotropin surge in vivo. In cultures of follicle wall and granulosa cells, the PR antagonist mifepristone (MIFE, 1 microm) inhibited LH-induced PG secretion and the progestin medroxyprogesterone acetate (1 or 10 microm), but not the glucocorticoid dexamethasone (1 or 10 microm), overcame the effect of MIFE on PGs. Semiquantitative RT-PCR revealed that MIFE inhibited LH-induced expression of cyclooxygenase-2 mRNA in granulosa cells in vitro. Again, treatment with medroxyprogesterone acetate overcame the effect of MIFE. Together these results provide strong evidence that periovulatory increases in cyclooxygenase-2 mRNA, PGE, and PGF(2alpha) are mediated by gonadotropin-induced increases in progesterone/PR, indicating that in some species there is an important functional relationship between these pathways in the ovulatory cascade.

Effects of oxygen tension and supplements to the culture medium on activation and development of bovine follicles in vitro.

Our laboratory developed a method for culturing small pieces of bovine and baboon ovarian cortex, rich in primordial follicles, that supports the initiation of follicle growth and development to the primary stage. However, only a few follicles progressed to the secondary stage. The purpose of the current experiments was to determine if changes in culture conditions, specifically oxygen concentration and supplements to the culture medium, would facilitate the primary to secondary follicle transition. In Experiment 1, ovarian cortical pieces from late-gestation bovine fetuses were cultured with 2, 5, 20, or 60% oxygen in Waymouth's medium plus ITS+ (insulin, transferrin, selenium plus linoleic acid and BSA). Although the three lower concentrations of oxygen were generally equivalent in promoting follicle activation and growth, the highest concentration (60%) had deleterious effects on follicle survival after 7 days in culture, reducing the number of healthy follicles to about 35% of the number observed with 20% oxygen (P<0.05). In Experiment 2, bovine ovarian cortical pieces were cultured in the standard gas mixture (5% CO(2) in air) with graded doses of fetal bovine serum (FBS, 2.5, 5, or 10%) in the presence or absence of 0.5 or 1x ITS+. All concentrations of FBS alone were much less effective at maintaining follicular health and supporting the initiation and progression of follicular growth than was ITS+. However, 5 and 10% FBS alone increased the percentage of healthy primordial and primary follicles by about twofold (P<0.05) in the absence of ITS+ and in the presence of 0.5x ITS+, they enhanced the primary to secondary follicle transition by 10- and 9-fold, respectively. Thus, of the culture conditions evaluated, 20% oxygen and medium containing 0.5x ITS+ plus 5% or 10% FBS were the most effective for promoting follicular health and development.

Elevated temperature (heat stress) in vitro reduces androstenedione and estradiol and increases progesterone secretion by follicular cells from bovine dominant follicles.

Dairy cattle are susceptible to heat stress-induced reductions in fertility; however, direct effects of hyperthermia on specific reproductive functions are difficult to determine in vivo. The objective of this experiment was to examine the effect of elevated temperature in vitro on follicular steroidogenesis, to gain insight into specific follicular responses associated with heat stress. Dominant follicles were obtained from Holstein heifers on day 6 post-estrus (luteal phase; n = 4) or day 8, 36 h after an injection with 25 mg PGF(2alpha) to induce regression of the corpus luteum (follicular phase; n = 4). Pieces of follicle wall were isolated from dominant follicles and cultured for 96 h with 0, 2 or 100 ng/ml LH or FSH at 37, 39 or 41 degrees C. Concentrations of androstenedione, estradiol and progesterone were determined in culture media collected every 24h. During the last 48 h of culture, basal secretion of androstenedione and estradiol by pieces of follicle wall was lower at 41 degrees C than at 37 or 39 degrees C (P < 0.05). In contrast, cumulative secretion of progesterone by pieces of follicle wall in medium alone was higher at 41 degrees C than at 37 or 39 degrees C (P < 0.05). Pieces of follicle wall responded to treatment with both low (2 ng/ml) and high (100 ng/ml) doses of gonadotropins at all temperatures. However, gonadotropin-induced secretion of androstenedione and estradiol was generally lower, whereas gonadotropin-induced secretion of progesterone was higher at 41 degrees C and sometimes at 39 than at 37 degrees C. The changes in basal steroidogenesis and in responses to gonadotropins suggest that follicular cells begin to luteinize at elevated temperatures in vitro. Premature luteinization of follicular cells in vivo has been associated with reduced fertility in cattle with persistent follicles, suggesting that the premature differentiation of follicular cells observed in the current study may be responsible, in part, for the reduced fertility of dairy cattle under heat-stressed conditions.

Evidence for a role for anti-Mullerian hormone in the suppression of follicle activation in mouse ovaries and bovine ovarian cortex grafted beneath the chick chorioallantoic membrane.

The first critical transition in follicular development, the activation of primordial follicles to leave the pool of resting follicles and begin growth, is poorly understood, but it appears that the balance between inhibitory and stimulatory factors is important in regulating the exodus of follicles from the resting pool. There is evidence that anti-Mullerian hormone (AMH; also known as MIS) inhibits follicle activation in mice, but whether it plays a similar role in non rodent species is not known. When pieces of bovine ovarian cortex, rich in primordial follicles, are cultured in serum-free medium, most follicles initiate growth, but when cortical pieces are grafted beneath the chorioallantoic membrane (CAM) of chick embryos, follicle activation does not occur. Since embryonic chick gonads of both sexes produce and secrete high levels of AMH, the hypothesis that the AMH in the chick circulation inhibits follicle activation was tested. In Experiment 1, whole newborn mouse ovaries were grafted beneath the CAM (placed "in ovo") or cultured in vitro for 8 days. In vitro (or after 8 days in vivo) follicles activated and proceeded to the primary or secondary stage, but activation was suppressed in ovo. This inhibition was reversed if ovaries were removed from beneath the CAM and cultured in vitro. In contrast, when ovaries from mice null mutant for the AMH type II receptor were CAM-grafted in Experiment 2, follicle activation occurred in a similar fashion to activation in vitro. This finding strongly implicates AMH as the inhibitor of follicle activation in ovo. Since chick embryonic gonads are the source of circulating AMH, chicks were gonadectomized in Experiment 3, prior to grafting of pieces of bovine ovarian cortex beneath their CAMs. Bovine primordial follicles activated in the gonadectomized chicks, similar to the results for mice lacking the AMH type II receptor. Taken together these experiments provide strong evidence that AMH is the inhibitor of mouse follicle activation present in the circulation of embryonic chicks and provide indirect, and hence more tentative, evidence for AMH as an inhibitor of bovine follicle activation.

Follicular development: the role of the follicular microenvironment in selection of the dominant follicle.

The importance of endocrine signals in the regulation of follicular development has long been recognized. However, the follicular microenvironment also plays a critical role in determining follicular fate. This review summarizes our studies on the role of the intrafollicular IGF system in selection of the dominant follicle (DF) in cattle. During the bovine estrous cycle, the largest antral follicles develop in two or three successive waves of follicular recruitment and selection of a DF. High concentrations of estradiol in the follicular fluid are the hallmark of dominant and preovulatory follicles and are associated with lower concentrations of low molecular weight (MW) insulin-like growth factor binding proteins (IGFBP-2, -4, and -5), which can prevent binding of IGF to its receptor. Our studies have shown that dominant and preovulatory follicles also have much higher levels of an IGFBP-4/-5 protease activity, which is the bovine equivalent of the human IGFBP-4 protease, pregnancy-associated plasma protein-A (PAPP-A). Studies of follicles isolated just after the emergence of the DF showed that PAPP-A is present in the follicular fluid of the DF as soon as it can be detected as morphologically dominant. To examine whether higher levels of PAPP-A in one follicle of the cohort (the future DF) precedes morphological dominance, the four largest follicles were isolated from pairs of bovine ovaries obtained before one follicle of the cohort was significantly larger the others, around the time that one follicle was first detected as morphologically dominant and after dominance was well established. Analysis of the temporal sequence of changes in estradiol, low MW IGFBPs, free IGF, and PAPP-A in the follicular fluid suggested that an increase in PAPP-A is the earliest biochemical difference yet detected in the future DF and that follicular selection is the result of a progressive series of changes beginning with the acquisition of PAPP-A, which leads to a decrease in IGFBP-4 and -5 and an increase in free IGF, which synergizes with FSH to increase estradiol production. Co-dominant follicles, induced by injection of small doses of recombinant bovine (rb) FSH, both had levels of PAPP-A similar to the single DF of control heifers, supporting the hypothesized role of FSH in the induction of PAPP-A in the DF. Taken together, these results suggest a critical role for FSH-induced PAPP-A, and thus for free IGF, in the selection of the DF. In contrast, other experiments provided evidence for a deleterious effect of IGF on the initiation of bovine follicular growth and the survival of primordial and primary follicles in vitro. These results underscore the importance of the follicular microenvironment in determining follicular fate and indicate that its effects can be stage-specific.

Characteristics of developing prolonged dominant follicles in cattle.

In cattle, sub-luteal circulating progesterone induces an increase in the frequency of LH pulses, prolonged growth of the dominant follicle, increased peripheral estradiol and reduced fertility. The objective of this study was to examine the earliest stages of development of prolonged dominant follicles, to gain insight into the etiology of this aberrant condition. Heifers were treated with an intravaginal progesterone-releasing device (CIDR) from Day 4-8 post-estrus and PGF2alpha was injected on Day 6 and again 12h later (early prolonged dominant group). Follicular phase (CIDR: Day 4-6, with PGF2alpha) and luteal phase (CIDR: Day 4-8, without PGF2alpha) groups served as controls. As expected, peripheral progesterone in heifers of the early prolonged dominant group was intermediate between luteal and follicular phase groups after luteal regression (P<0.05). On Day 7, the frequency of LH pulses was higher in heifers of the follicular phase and early prolonged dominant groups than the luteal phase group (P<0.05). Dominant follicles (n = 4 per group) were collected by ovariectomy on Day 8 and were similar in size among groups (P>0.05). Estradiol and androstenedione concentrations in the follicular fluid at ovariectomy were higher in the follicular phase and early prolonged dominant groups versus the luteal phase group (P<0.01), whereas progesterone did not differ among groups (P>0.05). Granulosa cells and theca interna isolated from dominant follicles were incubated for 3h with or without gonadotropins or frozen for later analysis of mRNA for steroidogenic enzymes. Luteinizing doses (128 ng/ml) of LH and FSH increased secretion of progesterone (P<0.05) but did not affect secretion of estradiol by granulosa cells in all groups. Low (2 or 4 ng/ml) and luteinizing doses of LH increased secretion of androstenedione by theca interna to a similar extent among groups. Expression of mRNA for P450 side chain cleavage (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), P450 aromatase (aromatase) and Steroidogenic Acute Regulatory (StAR) protein by granulosa cells did not differ among groups (P>0.05). Levels of mRNA for P450scc, 3beta-HSD, 17alpha-hydroxylase (17alpha-OH) and StAR protein in theca interna were similar in the follicular phase and early prolonged dominant groups (P>0.05), but lower in the luteal phase group (P<0.05-0.1). In summary, the premature follicular luteinization observed in previous studies after prolonged periods of sub-luteal progesterone was absent in early prolonged dominant follicles, exposed to sub-luteal progesterone for 36 h, and their characteristics resembled those of control follicles during the follicular phase.