Differential responses of granulosa cells from small and large follicles to follicle stimulating hormone (FSH) during the menstrual cycle and acyclicity: effects of tumour necrosis factor-alpha

This study determined effects of follicle stimulating hormone (FSH) alone and in combination with tumour necrosis factor (TNF), on granulosa cells from small (5-10 mm diameter) and large (>10-25 mm) follicles during follicular and luteal phases of the cycle and during periods of acyclicity. Granulosa cells were collected from ovaries of premenopausal women undergoing oophorectomy. The cells were cultured with human FSH (2 ng/ml) and testosterone (1 microM) in the presence or absence of human TNF-alpha (20 ng/ml). Media were removed at 48 and 96 h after culture and progesterone, oestradiol and cAMP in media were measured by radioimmunoassays. FSH stimulated the accumulation of oestradiol from granulosa cells of small follicles during the follicular and luteal phases but not during acyclicity; and TNF reduced oestradiol accumulation in the presence of FSH. Interestingly, in granulosa cells from small follicles, progesterone and cAMP secretion increased in response to FSH and neither was affected by TNF. Thus, TNF specifically inhibited the conversion of testosterone to oestradiol in granulosa cells from small follicles. FSH stimulated oestradiol production by granulosa cells of large follicles obtained only during the follicular phase of the cycle and TNF inhibited the FSH-induced oestradiol secretion. Granulosa cells obtained from large follicles during the luteal phase and during acyclicity did not accumulate oestradiol in response to FSH. However, FSH increased progesterone and cAMP secretion by granulosa cells obtained from large follicles during the follicular and luteal phases. During the luteal phase alone, TNF in combination with FSH increased progesterone accumulation above that of FSH alone. FSH did not increase progesterone, oestradiol or cAMP secretion by granulosa cells obtained from large follicles during acyclicity. Thus, FSH increases progesterone, oestradiol and cAMP secretion by granulosa cells of small follicles during the follicular and luteal phases and TNF appears to inhibit FSH-induced oestradiol secretion specifically in those cells. In large follicles, FSH-stimulated granulosa cell secretion of oestradiol is limited to the follicular phase and this effect can be inhibited by TNF. In addition, when granulosa cells of large follicles do not increase oestradiol secretion in response to FSH, TNF stimulates progesterone secretion.     

Apoptosis occurs in granulosa cells but not cumulus cells in the atretic antral follicles in pig ovaries

The porcine antral follicles, 3-6 mm in diameter, were dissected from the ovaries of mature pigs, and then granulosa and cumulus cells were isolated from each follicle. In atretic follicles, high activity of neutral Ca2+/Mg2+-dependent endonuclease and DNA ladder formation, estimated by electrophoresis, were noted in granulosa cells but not in cumulus cells. Extremely low activity of the endonuclease and no DNA ladder formation were observed in both types of cells obtained from healthy follicles. Moreover, apoptotic cells were observed histochemically among granulosa cells only. A good correlation (r = 0.987) between the endonuclease activity of granulosa cells and the progesterone/estradiol ratio of follicular fluid in each follicle was found. These results suggest that apoptosis occurs in granulosa cells but not cumulus cells in the atretic antral follicles in pigs.     

Circulating and ovarian IGF binding proteins: potential roles in normo-ovulatory cycles and in polycystic ovarian syndrome

IGFs function as co-gonadotropins in the ovary, facilitating steroidogenesis and follicle growth. IGFBP-1 to -5 are expressed in human ovary and mostly inhibit IGF action in in vitro ovarian cell culture systems. In the clinical disorder of polycystic ovarian syndrome (PCOS), which is characterized by hyperandrogenemia, polycystic ovaries and anovulation, follicles have a higher androgen: estradiol (A : E2) content and growth is arrested at the small antral stage. In the PCOS follicle, follicle stimulating hormone (FSH) and IGF levels are in the physiologic range, and even in the face of abundant androstenedione (AD) substrate, aromatase activity and E2 production are low. When PCOS granulosa are removed from their ovarian environment, they respond normally or hyperrespond to FSH. It has been postulated that an inhibitor of IGF's synergistic actions with FSH on aromatase activity may be one (or more) of the IGFBPs, which contributes to the arrested state of follicular development commonly observed in this disorder. High levels of IGFBP-2 and IGFBP-4 are present in follicular fluid (FF) from androgen-dominant follicles (FFa) from normally cycling women and in women with PCOS. This is in marked contrast to the near absence of these IGFBPs in estrogen-dominant FF (FFe), determined by Western ligand blotting. Regulation of granulosa-derived IGFBPs is effected by gonadotropins and insulin-like peptides. In addition, an IGFBP-4 metallo-serine protease is present in FFe, but not in FFa in ovaries from normally cycling women and those with PCOS, although the IGFBP-4 protease is present in PCOS follicles hyperstimulated for in vitro fertilization. Recent studies demonstrate that IGF-II in FFe is higher than in FFa' whereas IGF-I, IGFBP-3 and IGFBP-1 levels do not differ, underscoring the importance of local IGF-II production by the granulosa and the importance of IGFBP-4 and IGFBP-2 in regulation of IGF-II action within the follicle during its developmental pathway as an E2- or A-dominant follicle. In the androgen-treated female-to-male transsexual (TSX) model for PCOS, IGF-I, IGF-II, IGFBP-3 and IGFBP-1 levels do not differ.     

Local control of ovarian steroidogenesis

A number of putative paracrine factors are now thought to interact with FSH in the control of ovarian steroidogenesis. The relative importance of these factors remains to be determined, but the presence of the insulin-like growth factors and their binding proteins and the mechanism of control of the latter through the local production of proteases suggests a role for this system in folliculogenesis. We have demonstrated over-production of steroid hormones in tissue from women with polycystic ovaries. Theca cells in monolayer culture produced excessive amounts of progesterone and androstenedione and granulosa cell oestradiol production was considerably enhanced in response to FSH. Recent evidence points to a genetic defect in the expression or translation of steroidogenic hormones as a cause of excess androgen production, but the gene or genes involved has not been established. Data from our group suggest that granulosa cells from anovulatory polycystic ovaries are prematurely matured and we hypothesize that this is due to the interaction of the raised circulating insulin levels with LH in these follicles, an interaction that results in arrested follicular growth.     

Dietary fat and body composition

Vascular endothelial growth factor (VEGF) is a cytokine that induces angiogenesis. Angiogenesis is a prominent histologic component of the luteinization process. Luteinization is also characterized by granulosa cell progesterone secretion in response to the luteinizing hormone (LH) surge. Local VEGF production in human pre-ovulatory follicles, induced by LH, was postulated to be a luteinization mediator in women. To investigate this hypothesis, serum and fluid from the dominant follicle of 31 healthy regularly cycling multiparous women undergoing laparoscopic sterilization were obtained. VEGF was measured by enzyme-linked immunosorbent assay, and LH and progesterone were measured by radioimmunoassay. Follicle aspiration was performed at a median of 13 days from the last menstrual period (range 11-17 days). The median pre-ovulatory follicle diameter was 16 mm (range 11-23 mm). Follicle fluid VEGF concentrations (mean 6900 pg/ml, range 1200-17 100 pg/ml) were correlated positively with follicle fluid progesterone concentrations (mean 10 176 nmol/l, range 636-66780 nmol/l, r=0.62, P=0.002). This correlation was even tighter (r=0.87, P < 0.0001) when only samples from the 22 women in the earliest stages of follicle luteinization were considered. In these women serum LH concentrations were also correlated with follicle fluid VEGF concentrations (r=0.51, P=0.02). Our findings demonstrate the close dynamic relationship between VEGF production and early luteinization in human follicles during normal non-stimulated cycles.     

Follicle-stimulating hormone and luteinizing hormone/chorionic gonadotropin stimulation of vascular endothelial growth factor production by macaque granulosa cells from pre- and periovulatory follicles

Granulosa cells in the ovulatory follicle express messenger ribonucleic acid encoding vascular endothelial growth factor (VEGF), an agent that may mediate the neovascularization of the developing corpus luteum, but it is not known whether luteinizing granulosa cells synthesize and secrete VEGF during the periovulatory interval. Studies were designed to evaluate the effects of an in vivo gonadotropin surge on VEGF production by macaque granulosa cells (study 1) and to test the hypothesis that gonadotropins act directly on granulosa cells to regulate VEGF production (study 2). Monkeys received a regimen of exogenous gonadotropins to promote the development of multiple preovulatory follicles. Nonluteinized granulosa cells (i.e. preovulatory; NLGC) and luteinized granulosa cells (i.e. periovulatory; LGC) were aspirated from follicles before and 27 h after an ovulatory gonadotropin bolus, respectively. Cells were either incubated for 24 h in medium with or without 100 ng/mL hCG (study 1) or cultured for 6 days in medium with or without 100 ng/mL hCG or 0.1, 1, 10, and 100 ng/mL of recombinant human LH (r-hLH) or r-hFSH (study 2). Culture medium was assayed for VEGF and progesterone. In study 1, LGC produced 8-fold greater levels of VEGF than NLGC (899 +/- 471 vs. 111 +/- 26 pg/mL, mean +/- SEM; P < 0.05). In vitro treatment with hCG increased (P < 0.05) VEGF production by NLGC to levels that were not different from the LGC incubated under control conditions. In vivo bolus doses of r-hCG (100 and 1000 IU) and r-hFSH (2500 IU) were equally effective in elevating granulosa cell VEGF production. In study 2, in vitro treatment with r-hFSH, r-hLH, and hCG markedly increased (P < 0.05) VEGF and progesterone production by the NLGC in a dose- and time-dependent manner. By comparison, the three gonadotropins (100 ng/mL dose) only modestly increased VEGF and progesterone production by LGC. These experiments demonstrate a novel role for the midcycle surge of gonadotropin (LH/CG or FSH) in primates to promote VEGF production by granulosa cells in the periovulatory follicle. Further, the data demonstrate that FSH-like as well as LH-like gonadotropins directly stimulate VEGF synthesis by granulosa cells.     

Potent inhibition of human ovarian steroidogenesis by insulin-like growth factor binding protein-4 (IGFBP-4)

Several studies have now documented the existence of IGFBPs in follicular fluid and their correlation with the health of the follicle. In particular, increased levels of IGFBP-4 have been reported in androgen-dominant atretic follicles and those from polycystic ovaries. The aim of this study was to elucidate the role of IGFBP-4 in ovarian steroidogenesis. Granulosa cells and theca tissue were incubated with or without LH or FSH in the presence or absence of IGFBP-4 (0.5-50 ng/ml). Inhibition by IGFBP-4 of estradiol production in the presence of testosterone alone was seen in three of four experiments. IGFBP-4 completely inhibited FSH-stimulated estradiol production in three experiments and caused 67% inhibition in a fourth. Similar results were obtained for theca, in which concurrent incubation with IGFBP-4 completely negated the stimulatory effects of LH on androstenedione production. The mechanism by which IGFBP-4 exerts these potent effects and the possibility that this may by IGF-independent are currently being investigated.     

Development of a long-term bovine granulosa cell culture system: induction and maintenance of estradiol production, response to follicle-stimulating hormone, and morphological characteristics

The objectives of this study were to develop a serum-free bovine granulosa cell culture system in which FSH-responsive estradiol production could be induced and maintained, and to use this system to evaluate the effects of FSH, insulin, and IGF-I on steroidogenesis and proliferation of bovine granulosa cells from different follicle size categories (< 4-, 4-8, and > 8-mm diameter). In the presence of FSH, granulosa cells from small follicles differentiated in vitro, and estradiol secretion increased with time (p < 0.01) so that by the end of the culture period it was similar to that of cells from large follicles. Granulosa cells from medium and large follicles secreted estradiol throughout the culture period. Cells cultured in plasma-coated culture wells had an increased proliferative response but had lower estradiol production compared to cells cultured under serum-free conditions (p < 0.01). Insulin promoted proliferation and estradiol production by granulosa cells from the three follicle-size categories (p < 0.01). Physiological concentrations of FSH induced proliferation and estradiol secretion (p < 0.01) by granulosa cells in a dose-responsive manner. The inclusion of IGF-I in the culture system enhanced proliferation and estradiol production (p < 0.01), even in the absence of gonadotropic support, demonstrating the gonadotropic characteristics of this growth factor. These results demonstrate the development of a relevant physiological culture system for bovine granulosa cells. This system will permit the detailed study of the key factors controlling the differentiation and proliferation of bovine granulosa cells.     

Influence of serum, estrogen, and gonadotropins upon growth and progesterone secretion by cultures of granulosa cells from small porcine follicles

Granulosa cells from small (less than 2mm) antral porcine follicles were grown in culture to study the effects of various hormones on growth, morphology and progesterone secretion. Culture medium 199D + 4% serum was found to be most suitable since it maintained a fairly constant cell population. Estradiol (1mug/ml) and human FSH stimulated cell growth. LH and FSH stimulated progesterone secretion and induced morphological changes associated with luteinization. Estradiol (0.1 mug/ml) inhibited progesterone secretion by granulosa cells.     

Modulation of ovarian cytochrome P450 17 alpha-hydroxylase and cytochrome aromatase messenger ribonucleic acid by prolactin in the domestic turkey

The effect of exogenous ovine prolactin (oPRL) on preovulatory follicle P450 17 alpha-hydroxylase (C17) and aromatase (ARO) mRNA abundance was investigated in turkeys. Ovine PRL (124 IU/hen per day) was injected i.m. into four sets (n = 8) of laying turkeys for 2, 4, 8, or 14 days. Vehicle was injected into control hens for 8 days (n = 8). Blood samples were collected and serum was assayed for LH, progesterone (P), testosterone (T), and estradiol (E). Theca layers from the largest (F1) and the third (F3), fifth (F5), and seventh (F7) largest preovulatory follicles and from small white follicles (SWF) were examined for C17 and ARO mRNA contents. The number of atretic follicles increased from 0 (vehicle-injected controls) to 9 (14-day-oPRL-injected hens). Serum E, T, and LH levels decreased, while P levels remained unchanged. There was a transient increase in theca C17 mRNA abundance of 2- and 4-day-oPRL-treated hen follicles. Cytochrome P450 ARO mRNA levels were reduced in SWF and F7 in response to oPRL. Thecal C17 and ARO mRNA content was reduced during follicular maturation in laying hens. ARO mRNA was not detectable in granulosa cells. The progressive decline in C17 and ARO mRNA content associated with follicular maturation as well as the absence of ARO mRNA in granulosa cells is consistent with the secretory activity of P, T, and E in preovulatory follicles. These findings suggest that reduced circulating E may be a consequence of suppressed ARO gene expression whereas the oPRL suppression of T secretion may not be coupled to C17 gene expression.     

Hormone-induced proliferation and differentiation of granulosa cells: a coordinated balance of the cell cycle regulators cyclin D2 and p27Kip1

The proliferation and terminal differentiation of granulosa cells are critical for normal follicular growth, ovulation, and luteinization. Therefore, the in situ localization and hormonal regulation of cell cycle activators (cyclin D1, D2, and D3) and cell cycle inhibitors (p27Kip1 and p21Cip1) were analyzed in ovaries of mice and rats at defined stages of follicular growth and differentiation. Cyclin D2 mRNA was specifically localized to granulosa cells of growing follicles, while cyclin D1 and cyclin D3 were restricted to theca cells. In hypophysectomized (H) rats, cyclin D2 mRNA and protein were increased in granulosa cells by treatment with estradiol or FSH and were increased maximally by treatment with both hormones. In serum-free cultures of rat granulosa cells, cyclin D2 mRNA was rapidly elevated in response to FSH, forskolin, and estradiol, indicating that estradiol as well as cAMP can act directly and independently to increase cyclin D2 expression. The levels of p27Kip1 protein were not increased in response to estradiol or FSH. In contrast, when ovulatory doses of human CG (LH) were administered to hormonally primed H rats to stimulate luteinization, cyclin D2 mRNA and protein were rapidly decreased and undetectable within 4 h, specifically in granulosa cells of large follicles. Also in response to LH, the expression of the cell cycle inhibitor p27Kip1 was induced between 12 and 24 h (p21Cip1 was induced within 4 h) and remained elevated specifically in luteal tissue. A critical role for cyclin D2 in the hormone-dependent phase of follicular growth is illustrated by the ovarian follicles of cyclin D2-/- mice, which do not undergo rapid growth in response to hormones, but do express markers of FSH/LH action, cell cycle exit, and terminal differentiation. Collectively, these data indicate that FSH and estradiol regulate granulosa cell proliferation during the development of preovulatory follicles by increasing levels of cyclin D2 relative to p27Kip1 and that LH terminates follicular growth by down-regulating cyclin D2 concurrent with up-regulation of p27Kip1 and p21Cip1.     

Characteristics of prolonged dominant versus control follicles: follicle cell numbers, steroidogenic capabilities, and messenger ribonucleic acid for steroidogenic enzymes

Cattle with low (subluteal) levels of plasma progesterone develop a persistent dominant follicle; plasma estradiol and LH pulse frequency are elevated, and fertility subsequent to the ovulation of a prolonged dominant follicle is compromised. The hypotheses were 1) that prolonged dominant follicles produce more estradiol because they have theca and granulosa cells with an enhanced capacity to produce androgen and estradiol, respectively, and 2) that these changes in steroidogenic capacity are paralleled by concomitant changes in mRNA for the appropriate steroidogenic enzymes. Prolonged dominant follicles were induced by treating Holstein heifers with exogenous progesterone via an intravaginal controlled internal drug-release device (CIDR) from Day 14 to 28 of the cycle. Prolonged dominant follicles were collected just before (CIDRb, Day 28; n=4) or 24 h after (CIDRa, Day 29; n=4) CIDR removal, and their steroidogenic capacity was compared to that of growing, control dominant follicles obtained just before (CONTb, n=4) or 24 h after (CONTa, n=4) a luteolytic injection of prostaglandin F2alpha during the late luteal phase. After natural luteolysis, CIDR heifers maintained subluteal concentrations of progesterone (1-2 ng/ml) and had higher estradiol and LH pulse frequency than control heifers, as expected. In CIDR heifers, prolonged dominant follicles were present on the ovary for a longer time, reached a larger diameter, and had more granulosa cells and a larger mass of theca than dominant follicles from control heifers (p < 0.05). Concentrations of steroids in follicular fluid, estradiol secretion by granulosa cells in vitro, and levels of mRNA for steroidogenic enzymes in theca and granulosa cells provided no evidence for greater capacity of theca and granulosa cells of CIDR follicles to produce androgen and estradiol. In fact, follicular fluid estradiol and mRNA for P450 aromatase were higher after luteolysis than before in control animals (p < 0.05) but not after CIDR removal in treated animals. Therefore, the data do not support the hypotheses. Rather it is suggested that prolonged dominant follicles produce more estradiol because they have more granulosa cells and a larger mass of theca than control dominant follicles. In contrast, progesterone concentrations in the follicular fluid increased in CIDRa relative to CIDRb follicles (p < 0.05), a change that did not occur in control follicles; and granulosa cells from CIDRa follicles secreted more progesterone than granulosa cells from any other group. The increased capacity of CIDRa follicles to secrete progesterone suggests premature luteinization, which could contribute to decreased fertility in cattle that ovulate a prolonged dominant follicle.     

Expression of messenger ribonucleic acid for inhibin subunits and ovarian secretion of inhibin and estradiol at various stages of the sheep estrous cycle

The relationship between expression of inhibin mRNA and ovarian secretion of estradiol (E2) and immunoactive inhibin was investigated at midluteal phase and throughout the follicular phase of the sheep estrous cycle. At laparotomy, timed samples of ovarian blood were collected and ovaries were removed from 39 Scottish Blackface ewes (ovulation rate 1.3 +/- 0.1) on Day 10 of the luteal phase or 24, 48, 60, 72, or 84 h after injection of cloprostenol (PG; 100 micrograms) on Days 10-12. Ovaries were removed and fixed for in situ hybridization using 35S-labeled antisense riboprobes transcribed from inhibin alpha, beta A, and beta B cDNAs. LH, E2, and inhibin concentrations were determined by RIA. On the basis of peripheral LH levels and the presence of estrogen-active follicles (E-A; > or = 3 mm in diameter secreting > 1 ng/min E2) or recent ovulations, animals were grouped as follows: presurge (24 or 48 h post-PG; LH < 5 ng/ml; n = 7), midsurge (with E-A; LH > 5 ng/ml; n = 6), late surge (large follicle not E-A; LH > 5 ng/ml; n = 4), postsurge (large follicle not E-A; LH < 5 ng/ml; n = 7), and postovulation (n = 10). As expected, E2 secretion by the "active" ovary (containing preovulatory follicle) tended to increase with follicular development such that secretion was maximal at midsurge and then declined. E2 secretion by the "inactive" ovary was low at all stages. Immunoactive inhibin, in contrast, was secreted in substantial quantities by both ovaries, although secretion from active ovaries was higher at all stages (p < 0.05). Effects of stage on secretion were not significant, but immunoactive inhibin secretion from active ovaries was high in postsurge animals when E2 secretion was very low. Hybridization for inhibin mRNA was specific for granulosa cells of antral follicles. While most sheep in the luteal (4 of 5), presurge (2 of 3), and midsurge groups (5 of 5) had at least one inhibin-positive large follicle (expressing both alpha- and beta-subunit mRNA), none were present between the LH surge and ovulation (late and postsurge groups). Inhibin mRNA was undetectable in midcycle CL, but 4 of 10 recent ovulations hybridized weakly with the alpha probe and one very weakly with the beta A probe. The mean number of inhibin-positive large follicles per animal (in those having at least one) was 1.3 +/- 0.15 (n = 15 ewes).(ABSTRACT TRUNCATED AT 400 WORDS)     

Growth and cellular proliferation of antral follicles throughout the follicular phase of the estrous cycle in Meishan gilts

Meishan gilts were ovariectomized 2 h after an i.v. injection of 5'-bromo-2'-deoxyuridine (BrdU, a thymidine analogue; 5 mg/kg body weight) on Days 15-19 of the estrous cycle or 24-30 h after observed estrus (post LH, PLH). All antral follicles > or = 3 mm from one ovary were fixed in Carnoy's solution. Granulosa and thecal cell labeling indexes (LI; percentage of nuclei staining for BrdU) as well as LI of cells within the basal, middle, and antral thirds of the granulosa cell layer were estimated for each follicle. In addition, antral and granulosa cell layer volume, granulosa cell layer thickness, granulosa cell density, number of granulosa cells, and number of S-phase cells per hour were estimated for each follicle. Mean follicular diameter increased linearly (p < 0.01) from Day 15 to PLH, with a growth rate of 0.77 mm/day. Granulosa and thecal cell LI decreased (p < 0.01) from Day 15 to PLH; however, granulosa cell LI was greater (p < 0.01) than thecal cell LI on Days 15 and 16 but less (p < 0.05) than thecal cell LI on Day 19. Follicles collected from PLH gilts contained no labeled granulosa cells. Cells within the basal third of the granulosa cell layer contained fewer (p < 0.01) labeled nuclei than did cells within the middle or antral thirds. In addition, LI within the basal and middle thirds of the granulosa cell layer decreased (p < 0.01) from Days 15 to 18 and from Days 15 to 17, respectively, whereas LI within the antral third remained constant from Days 15 to 18. Granulosa cell layer thickness was greatest (p < 0.01) on Day 15, then decreased (p < 0.01) and was similar from Day 16 to PLH. Granulosa cell density was similar from Days 15 to 19, then decreased (p < 0.01) for PLH gilts. Antral and granulosa cell layer volumes increased linearly (p < 0.01) from Days 15 to 19 and Day 15 to PLH, respectively, resulting in 2.8 and 1.9 volume doublings and doubling times of 1.4 and 2.7 days, respectively. Number of granulosa cells per follicle increased linearly (p < 0.01) from Day 15 to PLH, resulting in 1.5 cell doublings and a doubling time of 3.3 days. Number of S-phase cells per follicle per hour was similar from Days 15 to 18 and then decreased (p > 0.01) from Day 18 to PLH. In summary, the percentages of proliferating granulosa and thecal cells decreased throughout the final stages of antral follicular development. Differentiation of granulosa cells occurred from the basal to the antral area as follicles matured. We proposed that, during the latter stages of follicular development, the rapid increase in follicular diameter resulted primarily from expansion of the antral cavity, whereas increases in the granulosa cell layer volume and number of granulosa cells per follicle maintained a constant granulosa cell layer thickness.     

Developmental changes in marmoset granulosa cell responsiveness to insulin-like growth factor-I: interactions with follicle-stimulating hormone and estradiol

The biological actions of insulin-like growth factor-I (IGF-I) on granulosa cell steroidogenesis at defined stages of preovulatory follicular development in the marmoset monkey were examined. Studies were carried out by primary cell culture of granulosa cells derived from small antral (0.5-1.mm diameter) and large preovulatory (2-3.mm diameter) follicles collected during the mid-late follicular phase of the ovarian cycle. IGF-I (0.3-100 ng/ml) had no effect on progesterone accumulation or aromatase activity during 48-h culture of granulosa cells from small follicles. Progesterone accumulation by cells from large follicles was also unaffected by IGF-I over the same time period, although aromatase activity was stimulated in a dose-dependent manner (18-fold increase over basal levels with a maximally stimulatory dose of 30 ng IGF-I/ml). In contrast, granulosa cells from small and large follicles responded to IGF-I in terms of both progesterone accumulation and aromatase activity after longer periods of culture (4 days for progesterone; 6 days for aromatase). Concurrent treatment of granulosa cells from small follicles with estradiol (10(-7) M) enhanced the dose-dependent actions of IGF-I on both indices of steroidogenesis and advanced the time at which IGF-I stimulated activity was first detectable. The effects of estradiol on granulosa cell IGF-I responsiveness were independent of cell number. A synergistic action of IGF-I on FSH-stimulated granulosa cell steroidogenesis was not apparent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in ovarian function and gonadotropin secretion preceding the onset of nutritionally induced anestrus in Bos indicus heifers

The aim of this study was to monitor endocrine and ovarian changes immediately preceding the onset of nutritionally induced anestrus. Daily blood samples were obtained from 14 postpubertal heifers for one estrous cycle (initial estrous cycle). Subsequently, heifers designated "restricted" were given a limited diet (n = 9), and daily blood samples were obtained for approximately 21 days preceding onset of anestrus (anovulatory cycle). Controls were allowed ad libitum dietary intake (n = 5), and daily blood samples were collected for a complete estrous cycle during a time period that coincided with that preceding onset of anestrus in restricted heifers. Plasma samples were assayed for LH, FSH, progesterone, and estradiol-17 beta. The ovaries of all heifers were examined daily using transrectal ultrasonography from the initial until the final or anovulatory estrous cycles to determine changes in growth of follicles and corpora lutea. Anestrus was defined as failure of ovulation of the dominant follicle following luteolysis. When anovulatory and initial estrous cycles in restricted heifers were compared, mean concentrations of LH were lower (p < 0.01), and diameters of dominant follicles were smaller (p < 0.01); mean concentrations of estradiol-17 beta were also lower in the three days following luteolysis (p = 0.06), but concentrations of FSH appeared to be higher (p = 0.003); maximum diameters of corpora lutea were smaller (p < 0.001), but duration of luteal phases and concentrations of progesterone preceding luteolysis were similar (p > 0.10). In controls, no differences were found between estrous cycles for any of these variables. It is concluded that failure of ovulation, following reduced dietary intake, resulted from insufficient circulating LH to stimulate maturation of the ovulatory follicle.     

Steroidogenesis in luteinized granulosa cell cultures varies with follicular priming regimen

During follicular development, a co-ordinated gonadotrophin and endocrine environment is believed to be essential for normal function of the resulting corpus luteum. Whether differences in the gonadotrophins used to promote follicular development can have lasting effects on granulosa cells after they have undergone luteinization and culture, remains to be studied. We measured steroid production under basal and human chorionic gonadotrophin (HCG) stimulation in short and long term cultures of luteinizing granulosa cells obtained from normal ovulatory women undergoing assisted folliculogenesis with either human menopausal gonadotrophin (HMG) or follicle stimulating hormone (FSH). Basal progesterone and oestradiol production by luteinized granulosa cells obtained from follicles stimulated to develop with FSH was significantly greater than that from HMG derived follicles (P < 0.001). In short term cultures, treatment with 10 IU HCG caused a 10-fold increase in progesterone release by cells from FSH stimulated follicles, whereas cells of HMG origin produced only 5-fold more progesterone (P < 0.0001). In cultures that were maintained for 2 weeks, progesterone secretion was reduced, but a similar trend in HCG responsiveness was observed. These experiments demonstrate that the composition of the gonadotrophins used to promote follicular development in vivo leads to differences in granulosa cell steroidogenesis which are evident after luteinization and culture. They additionally support the notion that the environment of follicular development will be reflected in the resulting corpus luteum.     

Mid-follicular phase pulses of inhibin B are absent in polycystic ovarian syndrome and are initiated by successful laparoscopic ovarian diathermy: a possible mechanism regulating emergence of the dominant follicle

The hypothalamic pulse generator of GnRH is recognized to be central to ovulatory function as evidenced by the anovulation of women with hypogonadotrophic hypogonadism due to Kallmann's syndrome or severe anorexia nervosa. LH is released from the anterior pituitary in pulses, the frequency of which is closely entrained with those of GnRH. In contrast, secretion of FSH is influenced by a number of regulatory molecules, including GnRH, estradiol, inhibin, and activin. The close temporal relationship between changes in levels of inhibin B and FSH in the mid-follicular phase suggests that the release of inhibin B by the preovulatory follicle critically regulates pituitary FSH secretion. Polycystic ovarian syndrome (PCOS) is one of the most common endocrine disorders affecting ovulation, and abnormal ovarian morphology as detected by ultrasonography remains the most sensitive diagnostic marker for this disorder. The etiology of PCOS is unclear, but its effective treatment by both anti-estrogens and by exogenous FSH suggests that a primary disorder of FSH regulation may be central. To investigate the possible role of inhibin B in the pathology of PCOS, serum inhibin B levels were measured in 10 women with PCOS on cycle day 5 of a spontaneous or progestrogen-provoked bleed and compared with levels on cycle day 5 of 10 women with regular ovulatory cycles. The mean serum inhibin B levels in the PCOS patients were significantly higher at 248 (+/- 43.4) pg/mL compared with normal controls, 126 (+/- 18.6) pg/mL (P < 0.01). Ten women with clomiphene resistant PCOS and 5 normal controls consented to undergo serial blood sampling on cycle day 5. Time Series Analysis using a Fourier Transformation to analyze the power spectrum of the data revealed that in normal women there is a distinct periodicity in inhibin B levels with a clear peak detectable every 60-70 min (P < 0.05), whereas in women with ovulatory dysfunction due to PCOS, no such pattern of regular pulsatility was seen. Four women with PCOS whose anovulation was successfully treated with laparoscopic ovarian diathermy (LOD) underwent repeat venous sampling following LOD. Their serum inhibin B levels fell to the upper limit of the normal range (160 +/- 38.5) pg/mL, and pulsatility was initiated. It is possible that inhibin B pulses are being generated directly by the ovary in response to pulses of GnRH in the peripheral circulation, or indirectly in response to FSH pulses arising in the pituitary. The function of inhibin B pulses in the mid-follicular phase of the normal cycle remains to be elucidated, but the absence of the normal pulsatile pattern in women with PCOS, in conjunction with high basal levels of inhibin B arising from the multiple small follicles characteristic of the PCOS ovary, appears to reinforce the development of a large cohort of small, developmentally arrested, and ultimately atretic follicles in these patients. Initiation of normal inhibin B pulsatility by LOD in patients with polycystic ovaries appears to correlate with the post-operative onset of ovular cycles.     

Follicular growth and maturation in hyperprolific and large white sows

The mechanisms whereby hyperprolific sows achieve their increased ovulation rate (+5 oocytes on average) compared with normal Large White sows were explored in this study. The following specific questions were asked. Is increased ovulation rate related to 1) increased follicular populations within the ovaries or 2) alterations in terminal follicular growth and maturation? The population of antral follicles in six sows of each genotype was studied using histological techniques on ovaries obtained at the preovulatory stage. No difference between the total number of antral follicles, number of healthy or atretic follicles in specific size classes, and in size of the preovulatory follicles could be detected. The number of granulosa cells contained in preovulatory follicles was also similar between genotypes. Terminal follicular growth and maturation were studied in 15 sows of each genotype killed at d 1, 3, or 5 (n = 5.d-1.genotype-1) after the end of Regumate administration (d 0). Small (< or = 3.5 mm) follicles were counted, and follicles > 3.5 mm were dissected, measured, and incubated in vitro. Steroid concentrations (estradiol and testosterone) in culture medium were then measured. The two genotypes differed in the patterns of growth of their ovulatory follicles, because at d 3 all ovulatory follicles were obvious in Large White sows. In contrast, between d 3 and 5, seven additional ovulatory follicles grew in hyperprolific sows. Differences in follicular maturation between genotypes were also detected. Whereas testosterone concentrations in culture medium were similar in the two genotypes, estradiol concentrations were markedly (P < .01) increased in hyperprolific follicles. This indicates that these follicles may have an increased aromatizing ability. How this generates the altered pattern of follicular growth and the increased ovulation rate of hyperprolific sows remains to be established.