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.

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.

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.

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.

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.

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.

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.

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.

Estradiol secretion by granulosa cells from rats with four- or five-day estrous cycles: the development of responses to follicle-stimulating hormone versus luteinizing hormone.

The relative importance of LH vs. FSH in stimulating estradiol secretion by granulosa cells during follicular development was assessed in Sprague-Dawley rats with regular 4- or 5-day estrous cycles. At various times during diestrus (D-1200 h, D-2000 h) and proestrus (P-0800 h, P-1400 h, P-2000 h) granulosa cells were isolated from the presumptive preovulatory follicles. The cells were cultured with 0.5 microM testosterone and various doses of LH or FSH (0, 0.1, 1, 10, or 100 ng/ml). Media were collected and replaced daily for 3 days and measured for estradiol by RIA. Estradiol secretion in the absence of gonadotropins (endogenous aromatase activity) increased progressively with stage of the cycle. At earlier times of cell isolation (D-1200, D-2000, P-0800) secretion by cells from 5-day rats was greater relative to 4-day rats. Since estradiol production in the absence of gonadotropins declined progressively over each 3-day culture period, effects of the gonadotropins were most evident on the third day of culture when endogenous aromatase activity was low. On this day FSH consistently increased estradiol secretion above control levels. Sensitivity to FSH, as measured by the 50% maximally effective dose (ED50; approximately 1-3 ng FSH/ml), did not vary with cycle type or stage of the cycle and the response to FSH, in terms of 50% maximal estradiol secretion, was also relatively constant. In contrast, the effects of LH varied with cycle type and time of cell isolation. As follicles developed, cells from both 4- and 5-day rats became more sensitive to LH, as evidenced by a decline in the ED50 from approximately 32 ng LH/ml to approximately 3 ng/ml. This increase in sensitivity to LH is consistent with previous reports that the number of LH receptors on granulosa cells increases progressively during the final stages of follicular development. However, the increase in sensitivity to LH occurred earlier in cells from 5-day rats, relative to the expected next estrus (later, relative to the preceding estrus). Responsiveness to LH (50% maximal estradiol production) was relatively constant, except that it was lower in cells from 4-day rats isolated at D-1200. These results indicate that the LH receptors acquired by granulosa cells during diestrus and proestrus are functionally linked with aromatase activity and may, therefore, be important to the production of estradiol levels sufficient to elicit the LH surge.(ABSTRACT TRUNCATED AT 400 WORDS)

Lengthening the bovine estrous cycle with low levels of exogenous progesterone: a model for studying ovarian follicular dominance.

In cattle the development of ovarian follicles greater than or equal to 5 mm occurs in waves, with either two or three waves per estrous cycle. To increase our understanding of the control of follicular dynamics in cattle, the present study was designed to characterize the pattern of follicular development during artificially lengthened estrous cycles. Cycles were lengthened by intravaginal insertion of Silastic devices containing progesterone [Controlled Internal Drug Release devices (CIDRs)]. Control heifers (group 1) received blank devices, whereas treated heifers received one (group 2) or two CIDRs (group 3) from days 14 to 28 after estrus. In groups 2 and 3, the insertion of CIDRs prevented return to estrus at the normal time and increased cycle length as compared to the control group (30.0 +/- 0.0 and 31.0 +/- 0.3 vs. 21.0 +/- 0.7 days, respectively, P less than 0.05). After natural luteolysis and between days 22 and 28 of cycle, progesterone concentrations were maintained at lower levels in group 2 (range = 0.9-2.1 ng/ml) than in group 3 (range = 3.7-4.9 ng/ml, P less than 0.003). Follicular development and regression were monitored daily by ultrasonography. The number of follicular waves per cycle was identical in groups 1 and 2 (2.7 waves per cycle), despite the significantly longer cycles in group 2. In group 2, the presence of one CIDR altered the normal pattern of follicular development by promoting the prolonged growth of the ovulatory follicle, and associated with it, a complete absence of follicular recruitment. When compared to ovulatory follicles in controls (group 1), ovulatory follicles in group 2 were detected on the ovaries for a longer time (1.8-fold), reached a greater maximal size (1.4-fold), and were dominant for a longer time (3-fold). Heifers in group 3 had significantly more follicular waves per cycle than groups 1 and 2 (3.8 vs. 2.7 waves per cycle, respectively, P less than 0.05), due to the production of additional follicular waves during the lengthened cycle in three of six heifers. The other three heifers in group 3 showed patterns of follicular development similar to those of group 2. All heifers in the control group had normal preovulatory rises in estradiol and LH. During the period of treatment (days 14-28), 17 beta-estradiol concentrations were higher in heifers in group 2 (lower progesterone levels) than in heifers in group 3 (higher progesterone levels; P less than 0.0001). No differences were observed in basal LH concentrations between groups 2 and 3.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of oxytocin on steroidogenesis by bovine theca and granulosa cells.

Oxytocin (OT) is secreted during the final stages of bovine follicular development. To test OT's potential role as a regulator of follicular steroidogenesis, theca and granulosa cells were isolated from bovine preovulatory follicles 48 h after initiation of luteolysis with prostaglandin F2 alpha, and cultured with graded doses of OT (0, 0.5, 5, 50, and 500 mIU/ml). Granulosa cells were cultured with testosterone (0.5 microM) in either defined medium or medium containing 10% fetal bovine serum in the presence or absence of FSH (300 ng/ml); medium was collected and replaced daily for 5 days. In defined medium, oxytocin alone significantly increased progesterone production by granulosa cells (P less than 0.001) in a dose-dependent manner; over 5 days, doses of 0.5, 5, 50, and 500 mIU/ml OT caused 1.7-, 2.0-, 2.2-, and 2.6-fold increases. FSH enhanced progesterone 5-fold, but no dose of OT increased progesterone in the presence of FSH. OT also elevated progesterone in serum-containing medium (P less than 0.005), but the magnitude of its effects was lower (1.07-, 1.1-, 1.2-, and 1.4-fold increases with 0.5, 5, 50, and 500 mIU/ml OT). OT had little effect on estradiol secretion by granulosa cells cultured with or without FSH. To test the specificity of OT's effects on progesterone production by granulosa cells, granulosa cells were treated with graded doses of an OT antagonist (0, 1, 10, 100, and 1000 ng/ml) in the presence or absence of OT (5 and 50 mIU/ml). Progesterone production by granulosa cells in the presence of the antagonist alone was similar to production in control cultures. The stimulatory effects of 5 and 50 mIU OT were completely abolished in the presence of 100 or 1000 ng antagonist, respectively (P less than 0.01). Preparations of theca interna were cultured in defined medium with graded doses of OT (0, 0.5, 5, 50, and 500 mIU/ml) in the presence or absence of LH (300 ng/ml), with collection and replacement of medium at 3, 6, 12, 24, 48, and 72 h. LH alone increased both progesterone (12-fold) and androstenedione (4-fold) production over controls. However, no dose of OT significantly affected either progesterone or androstenedione production. These results show that OT stimulates progesterone production by granulosa cells, and thus, suggest that OT regulates steroidogenesis in bovine granulosa cells in vivo.

Oxytocin/neurophysin-I messenger ribonucleic acid in bovine granulosa cells increases after the luteinizing hormone (LH) surge and is stimulated by LH in vitro.

Bovine granulosa cells express the oxytocin/neurophysin-I (OT/NP-I) gene and secrete OT in vitro. We have shown previously that bovine granulosa cells isolated from the preovulatory follicle after the LH surge secrete 20 times more OT over 5 days in culture than granulosa cells obtained before the surge. LH or FSH stimulates OT secretion in vitro by granulosa cells isolated before the LH surge. We also observed that granulosa cells of preovulatory follicles isolated before the LH surge respond to OT with an increase in progesterone secretion, suggesting that OT may be involved in regulating the follicular/luteal phase shift, or ovulation, in an autocrine fashion. The objective of this study was to determine whether the increase in OT secretion from granulosa cells after the LH surge is regulated at the level of mRNA accumulation, peptide synthesis, and/or peptide secretion. Bovine preovulatory follicles were obtained during the early follicular phase (approximately 36 h before the LH surge), during the midfollicular phase (approximately 12 h before the LH surge), or during the late follicular phase (after the LH surge). Total RNA isolated from granulosa cells and theca interna at the time of cell isolation or after culture with or without LH was subjected to Northern analysis for OT/NP-I mRNA and quantified by densitometry. OT/NP-I mRNA was not detectable or was barely detectable in granulosa cells collected during the early or midfollicular phase (n = 6 and n = 4 follicles, respectively), but a strong hybridization signal was obtained from RNA isolated after the LH surge (n = 5 follicles; P < 0.01). In contrast, OT/NP-I mRNA was not detectable in theca interna before or after the LH surge. Although OT/NP-I mRNA was not detectable in granulosa cells isolated 24 h after prostaglandin F2 alpha injection, after 24 h in culture, a weak OT/NP-I mRNA hybridization signal was observed in RNA from granulosa cells in LH-containing cultures. After 72 h in culture, granulosa cells cultured in control, as well as in LH-containing medium, exhibited a strong signal for OT/NP-I mRNA, but granulosa cells treated with LH exhibited a stronger OT/NP-I hybridization signal than control cultures (P < 0.01). Theca interna did not yield any OT/NP-I hybridization signal initially, and none was induced in culture.(ABSTRACT TRUNCATED AT 400 WORDS)

Proteolysis of insulin-like growth factor binding proteins -4 and -5 in bovine follicular fluid: implications for ovarian follicular selection and dominance.

Dominant follicles are characterized by low levels of low molecular weight IGF binding proteins (IGFBPs) and by proteolytic activity against IGFBP-4 and -5. To examine the hypothesis that proteolysis of IGFBP-4 and -5 plays a critical role in selection of the dominant follicle, we isolated follicles at various stages during the first wave of follicular development during the bovine estrous cycle, using ultrasonography to follow follicular growth. Ovariectomies were performed before divergence in follicular size (group 1; largest follicle, approximately 7 mm in diameter), at about the expected time of size divergence (group 2; largest follicle, approximately 8 mm) or after a dominant follicle was clearly present (group 3; largest follicle, > or =9 mm). The four largest follicles (F1-F4) were dissected and concentrations of steroids, IGFBPs and free IGF-I and levels of proteolytic activity for IGFBP-4 and -5 in the follicular fluid were determined. Follicles in group 1 did not differ significantly in size or estradiol concentrations, but levels of proteolytic activity against IGFBP-4 and -5 were higher in F1-F2 than in F3-F4. However, in group 2 the largest follicle (F1) had higher estradiol, free IGF-I, and IGFBP-4 and -5 proteolytic activity than F2-F4, whereas only slight (dissected) or no (ultrasound) differences in diameters could be detected. Differences between F1 and F2-F4 in diameter, estradiol, free IGF-I, and IGFBP-4 and -5 proteolytic activity were even greater in group 3. In addition, the hormonal regulation of IGFBP-4 and -5 proteolysis was evaluated in vivo by injecting heifers with small doses of recombinant bovine FSH to induce codominant follicles (group 4). The induced codominant follicles were larger and had higher IGFBP-4 and -5 proteolytic activity than subordinate follicles. The results suggest that follicular selection is a progression of changes starting with acquisition of an FSH-inducible IGFBP-4/-5 proteolytic activity, leading to an increase in intrafollicular concentration of free IGF-I that, in turn, synergizes with FSH to promote greater estradiol production by the follicle destined for dominance.

Oxytocin secretion by bovine granulosa cells: effects of stage of follicular development, gonadotropins, and coculture with theca interna.

Oxytocin (OT) has been detected in ruminant preovulatory follicles. Bovine granulosa cells express the oxytocin/neurophysin I (OT/NP-I) gene and secrete OT in vitro. The objective of this study was to determine the developmental pattern of OT secretion by bovine follicle cells as they differentiate during the follicular phase and the preovulatory follicle approaches ovulation. Holstein heifers were injected with prostaglandin F2 alpha in midluteal phase to induce luteal regression and initiate a follicular phase. The ovary bearing the preovulatory follicle was obtained by ovariectomy early in the follicular phase, in midfollicular phase, or late in the follicular phase, after the LH/FSH surge (n = 4 heifers per group). Theca interna and granulosa cells were isolated and cultured for 5 days, individually or in coculture, in defined or serum-containing medium and with or without LH (300 ng/ml) or FSH (300 ng/ml). Media were collected and replaced completely every 24 h, and OT secreted into the media was measured by RIA. Granulosa cells isolated at all three time points during the follicular phase secreted measurable amounts of OT. However, total OT secretion by granulosa cells isolated after the LH/FSH surge was 18.9-fold (defined medium) to 64.8-fold (serum-containing medium) higher than OT secretion by granulosa cells isolated early in the follicular phase, and 14.6-fold (defined medium) to 170-fold (serum-containing medium) higher than OT secretion by granulosa cells isolated in midfollicular phase. Granulosa cells isolated before the LH/FSH surge responded to the addition of LH or FSH to the culture medium with an increase in OT secretion. Cocultures of granulosa cells and theca interna isolated before the LH surge secreted more OT than cultures of granulosa cells alone. When cells were isolated early in the follicular phase the effect of coculture was more than additive, but the effect of coculture was only additive when follicles were obtained in midfollicular phase. OT secretion by granulosa cells isolated after the LH/FSH surge was not affected by gonadotropins or by coculture with theca interna. In contrast to results for granulosa cells, theca interna secreted only small and variable amounts of OT, and responses to LH were inconsistent. These findings suggest that OT detected in cultures of theca interna may be produced by small and variable numbers of granulosa cells contaminating the theca interna preparation.(ABSTRACT TRUNCATED AT 400 WORDS)

Selection of the dominant follicle in cattle occurs in the absence of differences in the expression of messenger ribonucleic acid for gonadotropin receptors.

Mechanisms that allow selection of a dominant ovarian follicle from a cohort of growing follicles are unknown. Large healthy, estrogen-active follicles contain more LH receptors than atretic estrogen-inactive follicles, and levels of messenger RNA (mRNA) for LH receptor increase in the granulosa cells of dominant follicles as growth progresses. The aim of the present study was to test the hypothesis that changes in the temporal pattern of expression of mRNA for LH and FSH receptors are associated with selection of dominant follicles in cattle. Based on size, the dominant and two largest subordinate follicles were collected from the ovaries of heifers on days 2 (n = 3) or 3 (n = 3) of a follicular wave. On day 2, the dominant follicle was 1 mm larger than the largest subordinate follicle, but by day 3 of the wave the dominant follicle was 2-4 mm larger than the largest subordinate. Follicular fluid concentrations of estradiol and estradiol secretion in vitro by pieces of follicle wall (granulosa and theca cells) were greatest by the dominant compared with the subordinate follicles (P < 0.05). These data indicate that selection of a dominant follicle had occurred by the second day of the follicular wave. By in situ hybridization, mRNAs for LH and FSH receptors, P450 aromatase and P450 17alpha-hydroxylase (17alpha-OH) were localized in frozen sections from each follicle. The expression of mRNA for LH receptor in granulosa cells was always at or near background and was not different between days or follicle types (P = 0.63). In contrast, the expression of mRNA for LH receptor in theca cells of the same sections was readily detectable; there was no difference between follicle types on the second day of the follicular wave, but by the third day expression in the subordinate follicles had decreased (P < 0.05). The expression of mRNA for FSH receptor was highest in granulosa cells of dominant follicles collected on day 3 of the follicular wave (P < 0.05) and was not different between dominant and subordinate follicles on day 2 of the wave (P > 0.05). The expression of mRNA for aromatase in granulosa cells was similar (P > 0.05) between the dominant follicles on days 2 and 3 and the largest subordinate follicle on day 2 of the follicular wave and was much lower in the remaining follicles (P < 0.01). On day 2 of the wave, the expression of mRNA for 17alpha-OH was not different between the dominant and subordinate follicles, but by day 3 the dominant follicles had more mRNA for 17alpha-OH than the subordinate follicles (P < 0.05). These data show that the dominant follicle had been selected by the second day of the follicular wave (based on diameter and estradiol secretion) and that selection occurred in the absence of detectable levels of mRNA for LH receptor in the granulosa cells or differences between dominant and subordinate follicles in mRNA for LH receptor in theca cells or FSH receptor in granulosa cells. However, the divergent pattern of growth between dominant and subordinate follicles (after follicle selection) was associated with higher levels of mRNA for gonadotropin receptors and steroidogenic enzymes in dominant compared with subordinate follicles. Therefore, selection of the dominant follicle in cattle does not appear to involve the regulation of expression of mRNA for gonadotropin receptors, although such regulation may be important at other stages of differentiation of the dominant follicle.

Selection of the dominant follicle and insulin-like growth factor (IGF)-binding proteins: evidence that pregnancy-associated plasma protein A contributes to proteolysis of IGF-binding protein 5 in bovine follicular fluid.

Development of a dominant follicle is associated with decreased intrafollicular low molecular weight IGF-binding proteins (namely IGFBP-2, -4, and -5) and increased proteolysis of IGFBP-4 by pregnancy-associated plasma protein A (PAPP-A). In addition to IGFBP-4 proteolytic activity, bovine follicular fluid contains strong proteolytic activity for IGFBP-5, but not for IGFBP-2. Here we show that the IGFBP-5 protease present in bovine follicular fluid is a neutral/basic pH-favoring, Zn(2+) metalloprotease very similar to the previously described IGFBP-4 protease. We hypothesized that immunoneutralization and immunoprecipitation with anti-PAPP-A antibodies would result in abrogation of the IGFBP-4, but not the IGFBP-5, proteolytic activity in follicular fluid. As expected, anti-PAPP-A antibodies were able to neutralize and precipitate the IGFBP-4, but not the IGFBP-5, proteolytic activity of human pregnancy serum, which was used as a positive control for PAPP-A. Surprisingly, immunoneutralization and immunoprecipitation of follicular fluid from bovine preovulatory follicles with anti-PAPP-A antibodies abrogated both IGFBP-4 and IGFBP-5 proteolysis. Quantitative results derived from phosphorimaging revealed a complete inhibition of both IGFBP-4 and -5 proteolysis by follicular fluid incubated for 2 or 5 h in the presence of anti-PAPP-A antibodies. After 18 h of incubation, anti-PAPP-A antibodies still inhibited IGFBP-5 degradation, although with an efficiency lower than that for IGFBP-4 degradation. Both proteolytic activities have identical electrophoretic mobility, and a single band ( approximately 400 kDa) was detected by Western immunoblotting of bovine follicular fluid with anti-PAPP-A antibodies. Proteolysis of IGFBP-5 was readily detectable in follicular fluid from dominant follicles and was negligible in subordinate follicles from the same cohort. These results suggest that an active intrafollicular IGFBP-4/-5 proteolytic system, in which PAPP-A is the major protease involved, is an important determinant of follicular fate.

Androgen production by theca and granulosa isolated from proestrous rat follicles.

Theca and granulosa isolated from proestrous rat ovarian follicles were cultured for 72 h in the presence or absence of highly purified luteinizing hormone (LH, 0.1 microng/ml) and/or follicle-stimulating hormone (FSH, 0.1 microng/ml). Medium was collected and replaced at 3, 6, 12, 24, 48, and 72 h of culture and measured for testosterone by radioimmunoassay. Pieces of isolated theca secreted androgen; androgen production was greatest during the first 12 h of culture. Addition of highly purified LH to the culture medium produced a significant increase (P less than 0.001) in the thecal androgen secretion, while addition of highly purified FSH had no significant effect. Addition of LH + FSH to culture medium produced the same effect as addition of LH alone. The response of the theca to LH was dose-dependent with doses of 0.01 microng/ml or greater eliciting a maximum response. The addition of eoxgenous progesterone (5 x 10(-7)M) to culture medium had no effect on thecal androgen production. Thecal androgen secretion was the same in the presence or absence of fetal calf serum. Since the testosterone antibody used was not entirely specific for testosterone, testosterone and the cross-reacting androgen, 17beta-hydroxy-5alpha-androstan-3-one (DHT), were chromatographically isolated from samples and assayed separately. The androgen measured in culture medium was found to consist primarily of testosterone; DHT was present in much lower concentrations. Granulosa cells isolated from the same follicles as the theca and grown in monolayer culture produced negligible amounts of androgen. It is concluded that the theca is the site of follicular androgen production and that LH regulates androgen secretion by rat ovarian follicles. The results suggest that the theca provides the androgen precursor needed for follicular estradiol-17beta synthesis.

Ovarian follicular dominance in cattle: relationship between prolonged growth of the ovulatory follicle and endocrine parameters.

In cattle, the development of ovarian follicles 5 mm or larger occurs in either two or three consecutive follicular waves per estrous cycle. When the luteal phase is artificially lengthened with an intravaginal progesterone-releasing device (CIDR) that maintains subluteal levels (i.e. levels of progesterone that are below normal luteal levels, but higher than basal follicular phase levels), prolonged development of the ovulatory follicle is observed. To study the endocrinological correlates of prolonged follicular dominance and to test the hypothesis that it is mediated by effects of plasma progesterone on LH pulse frequency, heifers (n = 6/group) were treated with blank CIDRs (no progesterone, control group), with one CIDR for 14 days from day 14 of the cycle (1 CIDR group), or with one CIDR for 14 days from day 14 plus a second CIDR during days 24-28 (2 CIDR group). Cycle length was significantly longer in the 1 and 2 CIDR groups than in the controls (30.2 +/- 0.2 and 31.8 +/- 0.5 vs. 21.6 +/- 0.4 days, respectively; P < 0.0001). Follicular dynamics were normal in the control heifers. In the 1 CIDR group, the ovulatory follicle grew larger than in control or 2 CIDR animals, was maintained as the largest follicle on the ovaries for a much longer time, and ovulated after CIDR removal. In the 2 CIDR group, a similar growth pattern was observed until day 24; after insertion of a second CIDR, however, prolonged dominance was reversed, a new wave was recruited, and the dominant follicle of this wave ovulated after CIDR removal. The size of the ovulatory follicle and the length of the dominance phase in the 2 CIDR group were similar to those in control animals. Reversal of prolonged dominance in the 2 CIDR group was associated with changes in progesterone. Progesterone remained at subluteal levels (1.5-2.3 ng/ml) in both CIDR groups until day 24, when insertion of the second CIDR in the 2 CIDR group restored progesterone concentrations to normal luteal levels (3.5-6 ng/ml). Pulsatile LH secretion was assessed by frequent blood sampling every 12 min for 6 h (0800-1400 h) on selected days of the treatment cycle. LH pulse frequency was not different among groups before treatment started (days 12 and 13). However, LH pulse frequency was significantly higher in the 1 CIDR than in the 2 CIDR group on both day 26 (P < 0.03) and day 28 (P < 0.05), i.e. during the reversal of prolonged dominance in the 2 CIDR group.(ABSTRACT TRUNCATED AT 400 WORDS)