Effects of oestradiol-17beta and testosterone on progesterone production in the cultured granulosa cells of Japanese quail

In order to study the effects of steroid hormones on steroidogenesis in the avian ovary, quail granulosa cells were cultured with follicle stimulating hormone (FSH), oestradiol-17beta or testosterone. The progesterone content of the medium during the culture period of 66 h and the following 3 h of incubation with luteinising hormone (LH), was measured by radioimmunoassay. When FSH, oestradiol-17beta or testosterone were added during the 66 h culture, subsequent progesterone production by the cells during 3 h of incubation with LH was significantly increased. However, testosterone also stimulated progesterone production in the medium during the 66 h culture period, whereas FSH oroestradiol-17beta did not. Addition of staurosporine during culture inhibited both LH-stimulated progesterone production and testosterone-stimulated progesterone production. These results indicate that the processes during which granulosa cells acquired responsiveness to LH, and testosterone stimulates progesterone production might both be mediated by a staurosporine-sensitive protein kinase C-dependent pathway in quail granulosa cells.     

Gonadotropin-releasing hormone inhibition of LH stimulated progesterone secretion by porcine granulosa cells in vitro

GnRH has several direct actions on rat granulosa cells. Specific receptors for GnRH have been demonstrated on rat and human ovaries. Whether the porcine ovary has specific receptors for GnRH is still debated and the physiological actions of GnRH on porcine granulosa cells have not yet been clarified. Consequently, we have examined the actions of a GnRH agonist (GnRHa) on basal and LH stimulated progesterone secretion by porcine granulosa cells. GnRHa inhibited both basal and LH stimulated progesterone secretion by granulosa cells from medium (3-5 mm) and large (6-10 mm) antral follicles during 3 day incubations. LH stimulated progesterone secretion was more sensitive to inhibition than basal progesterone secretion. Studies on the time course for GnRHa inhibition of progesterone secretion indicated that the decrease in progesterone secretion occurred 48 to 72 hr after first exposure to GnRHa. Earlier inhibition occurred in only a fraction of the experiments. GnRHa did not have to be present during the time when inhibition occurred. Incubations of 2 days with GnRHa were just as effective as 3 day incubations at inhibiting progesterone secretion on day 3. Furthermore, a 30 min exposure to GnRHa on day 1 was just as inhibitory as a full 2 day incubation with GnRHa in inhibiting LH stimulated progesterone secretion on day 3. Incubation of the cells for 3 days prior to exposure of the cells to GnRHa did not alter the time course for GnRHa action. GnRHa did not alter the DNA content of the cultures in up to 6 day incubations or the number of viable cells attached to the wells in up to 3 day incubations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of FSH and LH on Steroid Production by Buffalo (Bubalus bubalis) Granulosa Cells Cultured In Vitro Under Serum‐Free Conditions

The objective of this study was to examine the effects of FSH and LH on oestradiol‐17β and progesterone production by buffalo granulosa cells cultured under serum‐free conditions. Granulosa cells (3 × 10⁵) from small (≤5 mm diameter) follicles were cultured for up to 4 days in 48‐well plates coated with 3.3 μg/cm² fibronectin in Dulbecco's modified Eagle's medium (DMEM) : nutrient mixture F‐12 Ham (1 : 1 ratio) supplemented with 10^?7 m androstenedione, 5 μg/ml human apo‐transferrin and 0.1% bovine serum albumin, in the presence or absence of FSH or LH (0, 1, 2, 4, 8, 16, 32 or 64 ng/ml each). Basal oestradiol‐17β production by granulosa cells from small follicles reduced (p < 0.01) from days 1 to 2 of culture and became undetectable by day 3 and basal progesterone production increased (p < 0.05) from day 1 through day 4 of the culture. Although there was no effect of FSH on day 1 of the culture, FSH at 2, 4, 8 and 16 ng/ml increased (p < 0.05) oestradiol‐17β production by granulosa cells from small follicles on day 2. Progesterone secretion was increased (p < 0.05) by all doses of FSH on all days of culture. All doses of LH had no effect on oestradiol‐17β or progesterone production by granulosa cells from small follicles on any day of the culture. The results of this study demonstrate a serum‐free culture system for buffalo granulosa cells and stimulatory effect of FSH but not LH on steroid hormone production by buffalo granulosa cells under these conditions.     

The effects of phospholipase C on oestradiol and progesterone secretion in porcine granulosa cells cultured in vitro

Granulosa cells play important roles in the regulation of ovarian functions. Phospholipase C is crucial in several signalling pathways and could participate in the molecular mechanisms of cell proliferation, differentiation and ageing. The objective of this study was to identify the effects of phospholipase C on the steroidogenesis of oestradiol and progesterone in porcine granulosa cells cultured in vitro. Inhibitor U73122 or activator m‐3M3FBS of phospholipase C was added to the in vitro medium of porcine granulosa cells, respectively. The secretion of oestradiol decreased after 2 hr, 8 hr, 12 hr, 24 hr and 48 hr of treatment with 500 nM U73122 (p < .05) and decreased after 2 hr of treatment in the 500 nM m‐3M3FBS addition group (p < .05). The secretion of progesterone increased after 4 hr of treatment with 500 nM U73122 (p < .05) and increased after 2 hr and 8 hr of treatment in the 500 nM m‐3M3FBS addition group (p < .05). The ratio of oestradiol to progesterone decreased at each time point, except 8 hr after the addition of 500 nM U73122 (p < .05). The ratio of oestradiol to progesterone decreased after 2 hr (p < .05) of treatment with 500 nM m‐3M3FBS. In genes that regulate the synthesis of oestradiol or progesterone, the mRNA expression of CYP11A1 was markedly increased (p < .05), and the mRNA expression of other genes did not change significantly in the U73122 treatment group, while the addition of m‐3M3FBS did not change those genes significantly despite the contrary trend. Our results demonstrated that phospholipase C can be a potential target to stimulate the secretion of oestradiol and suppress progesterone secretion in porcine granulosa cells cultured in vitro, which shed light on a novel biological function of phospholipase C in porcine granulosa cells.     

Cumulus and oocyte maturation and in vitro and in vivo fertilization of oocytes in relation to follicular steroids,prolactin, and glycosaminoglycans throughout the estrous period in superovulated heifers with a normal LH surge,no detectable LH surge,and progestin inhibition of LH surge

Crossbred heifers (n = 103) were synchronized to estrus with prostaglandin (PGF_2α) and superovulated with follicle stimulating hormone (FSH-P). Animals were ovariectomized every 12 hr after the PGF_2α injection (n = 7 to 9/time) up to 108 hr to monitor the follicular, hormonal, and oocyte changes associated with follicular development and ovulation. Twenty-eight animals were implanted with Norgestomet implants 12 hr before PGF_2α and ovariectomized at 72, 84, 96, and 108 hr post PGF_2α injection to monitor effects of progesterone and suppression of the luteinizing hormone (LH) surge on oocyte maturation and quality. Follicular fluid was collected and analyzed for progesterone, estradiol, prolactin, and glycosaminoglycan content in conjunction with cumulus maturation and nuclear stage of oocyte maturation. Analysis of in vivo matured oocytes by in vitro fertilization was carried out at 60, 72, 84, and 96 hr post PGF_2α and in vitro matured oocytes at 12 to 108 hr post PGF_2α. No developmental changes in cumulus cells surrounding the oocyte of small follicles was noted (≤ 4 mm dia) indicating a static population. Medium (> 4 ≤ 8 mm) and large size (> 8 mm) follicles developed to the corona radiata and loose cumulus stages in animals in which an LH surge was detected but cumulus status remained primarily in the tight cumulus stage for animals without an LH surge. The estradiol-to-progesterone ratio for tight cumulus (TC), corona radiata (CR), and loose cumulus (LC) stages was 1.8 ± .1, 1.0 ± .1, and .4 ± .2, respectively (P < .01). Nuclear maturation of oocytes in small follicles from animals without a detectable LH surge seem to indicate early maturation (48 to 72 hr post PGF_2α) in conjunction with a high percent of degenerate oocytes not seen in animals exhibiting an LH surge. Oocytes from medium size follicles matured to germinal vesicle breakdown (GVBD) and early meiosis (metaphase I; MI) stages of development in all treatments. Most oocytes were degenerate in Norgestomet-implanted animals. Oocytes from large follicles (> 8 mm dia) from animals exhibiting an LH surge were in MI and metaphase II (MII) stages (48 to 84 hr post PGF_2α) in preparation of ovulation whereas oocytes from animals not exhibiting an LH surge had oocytes that early matured to MII (48 to 72 hr post PGF_2α), later regressing to degenerate oocytes (84 to 108 hr). Follicular progesterone, estradiol, and prolactin increased with oocyte maturation, particularly in medium and large follicles. In vivo matured oocytes for fertilization (60, 72, 84, and 96 hr post PGF_2α) were nude (from the oviduct) and primarily CR from follicles. Tubal oocytes (37%) were fertilized more frequently by a single sperm than follicular oocytes (14.3%; P < .01) and single sperm penetration peaked at 72 hr post PGF_2α. Follicular hormone concentrations were not related to sperm penetration. Oocytes (n = 101) matured in vivo had lower fertilization potential from ovaries producing < 14 or > 50 follicles (39.3%) as compared to 21 to 45 aspirated follicles (68.2%; P < .05), with a peak penetration at 32 follicles (86.7% penetration). No treatment differences (LH surge or no detectable LH surge) were noted in relation to in vivo matured oocytes. Oocytes with single sperm penetration had the lowest estradiol/progesterone ratio of 2.2 vs polyspermic penetration of 13.7.     

Association Between Leptin, LH and its Receptor and Luteinization and Progesterone Accumulation (P4) in Bovine Granulosa Cell In Vitro

A full understanding of the cellular events that occur during in vitro luteinization of bovine granulosa cells, stimulated by LH and by leptin, is a complex goal that has not been completely achieved. The aim of this work was to study the effects of leptin, LH and leptin + LH on progesterone accumulation (P4) and on the expression of LH receptors (LHR) in bovine granulosa cells in culture. The results confirm that this in vitro model is representative of functional and morphological luteinization/differentiation. The pattern of expression of LHR with time of incubation was an important marker of in vitro luteinization, with 50–90% of cells expressing LHR by 96 h in culture. Cytoplasmic lipidic droplets were highly abundant in granulosa cells, suggesting a sufficient source of precursors for steroid hormone synthesis: P4 accumulation ranged between 40 and 550 ng/ml. In addition, a positive correlation ( r  = 0.58, p < 0.05) between the expression of LHR and accumulation of P4 throughout the time of incubation was observed. The expression of LHR was inhibited by LH and leptin + LH treatment. In conclusion, we found an inverse modulation between the expression of LHR and the concentration of LH, and the expression of LHR could be regulated by P4 produced by the luteinized granulosa cells. These findings are contributing to elucidate further the panoply of interactions during the differentiation of granulosa cells into luteal cells in vitro .     

Follicular development,oocyte viability and recovery in relation to follicular steroids,prolactin and glycosaminoglycans throughout the estrous period in superovulated heifers with a normal LH surge,no detectable LH surge,and progestin inhibition of LH surge

Estrous cycles of heifers (n = 137) were synchronized with prostaglandin (PGF_2α) and follicular development stimulated with follicle stimulating hormone. Twenty-eight animals were administered Norgestomet implants 12 hr prior to the initial PGF2α injection to suppress the LH surge that initiates ovulation. Animals were ovariectomized every 12 hr after the initial PGF2α (7–9/time, 12–108 hr and at 192 and 240 hr post PGF2α) and divided into three treatment groups to consist of: 1) animals exhibiting a normal luteinizing hormone (LH) surge (n = 86), 2) animals in which no LH surge was detected (n = 23), and 3) suppression of the LH surge via Norgestomet implants (72–108 hr, n = 28). Follicular diameter was measured and follicular fluid was collected for analysis of prolactin, estradiol, progesterone and glycosaminoglycan concentrations. Progesterone concentrations were increased in animals exhibiting an LH surge as compared to animals in which no LH surge was detected; primarily in large follicles (> 8 mm diameter) after the LH surge. Animals not exhibiting an LH surge also had increased follicular progesterone concentrations compared to Norgestomet-implanted animals (242.3 ± 36.3 vs 86.7 ± 6.4 ng/ml, respectively, P < .01), indicating some LH stimulation. Follicular estradiol in animals exhibiting an LH surge increased up to the time of LH surge detection and then declined whereas animals with no LH surge detected had follicular estradiol concentrations that declined after the PGF_2α injection. No differences were noted between those that did not exhibit an LH surge or in which the LH surge was suppressed with Norgestomet in relation to follicular estradiol concentrations. Follicular estradiol concentrations increased with follicular size in all treatment groups (P < .01). Follicular concentrations of prolactin were increased in small follicles (P < .05; ≤ 4 mm diameter) and follicular prolactin increased from 12 to 36 hr post PGF2α injection, then declined after the LH surge. Follicular glycosaminoglycan concentrations decreased with increases in follicular size (P < .01) and were higher in animals that did not exhibit an LH surge (P < .01). No differences in follicular glycosaminoglycans were noted between Norgestomet-implanted animals and those not exhibiting an LH surge. In the animals representing days 4 and 6 of the subsequent estrous cycle (192 and 240 hr post PGF2α), numbers of small-sized follicles were increased. Follicular progesterone and estradiol concentrations were related to atretic large follicles unovulated from the prior estrus and a wave of growth in small and medium follicles. Follicular prolactin and glycosaminoglycans increased with time of the new estrous cycle and were increased in smaller follicles (P < .01). Suppression of LH with progestin implants (Norgestomet) may relate to early effects of progesterone, which may not be totally eliminated at target tissues and subsequently alters the LH surge, steroidogenesis of the follicle, and ovulation. Oocytes were predominantly found in the follicular fluid from animals in which an LH surge was detected and in the buffer wash of follicles in which no LH surge was detected. Oocyte viability was higher in animals exhibiting an LH surge (75% viable) whereas the oocytes of Norgestomet-implanted animals were 75% degenerate.     

Expression of adiponectin and its receptors (AdipoR1 and AdipoR2) in chicken ovary: potential role in ovarian steroidogenesis

Adiponectin and its receptors (AdipoR1 and AdipoR2) mRNAs are expressed in various chicken tissues including ovary. However, the cellular expression and the role of adiponectin system have never been investigated in chicken ovary. Here, we have shown that the level of adiponectin mRNA is about 10- to 30-fold higher (p < 0.001) in theca cells than in granulosa cells from each hierarchical yellow follicle studied (F4–F1). In contrast, the level of AdipoR1 mRNA expression was about two-fold lower in theca cells than in granulosa cells (p < 0.05) whereas those of AdipoR2 was similar in both ovarian cells. Whereas expression of adiponectin mRNA increased with follicular differentiation in theca cells, it decreased in granulosa cells. In contrast, mRNA expression of AdipoR1 and AdipoR2 in both theca and granulosa cells remained stable during yellow follicle development. To determine whether adiponectin is involved in the ovarian steroidogenesis, LH (100 ng/ml)-, FSH (100 ng/ml)- and IGF-1 (100 ng/ml)-induced progesterone production was measured in absence or presence of human recombinant adiponectin (10 μg/ml) for 36 h in cultured granulosa cells from F1, F2 and mixed F3 and F4 follicles. In absence of LH, FSH and IGF-1, adiponectin treatment had no effects on progesterone production whatever vitollegenic follicle studied. However, it increased by about two-fold IGF-1-induced progesterone secretion in F2 and F3/4 follicles whereas it halved progesterone production in response to gonadotropins (LH and FSH) in F3/4 follicles. Thus, in chicken, adiponectin, mainly expressed in theca cells, could exert paracrine or autocrine effect on the ovarian steroidogenesis.     

Involvement of high-density lipoprotein in stimulatory effect of hormones supporting function of the bovine corpus luteum

The hypothesis that epinephrine (noradrenaline, NA) enhances utilisation of high-density lipoproteins (HDL) by bovine luteal cells and that this process involves phospholipase (PL) C and protein kinase (PK) C intracellular pathway was tested. Luteal cells from days 2-4, 5-10 or 11-17 of the oestrous cycle were preincubated for 20 h. Subsequently DMEM/Ham's F-12 medium was replaced by fresh medium and the cells were treated for 6 h as follows: In Experiment I with HDL (5-75 micrograms cholesterol per ml), NA, isoprenaline (ISO) or luteinising hormone (LH). In Experiment II cells were incubated for further 24 h in deficient medium (without FCS) and next treated as in Experiment I. In Experiment III cells were stimulated with NA, ISO or LH alone and together with HDL. In Experiment IV cells were treated with PLC inhibitor (U-73122) or with PKC inhibitor (staurosporine) or stimulator (phorbol 12-myristrate 13-acetate) and with either NA, insulin or LH. Only luteal cells from days 5-10 of the cycle responded on HDL and beta-mimetics (P < 0.05). LH stimulated progesterone secretion from the luteal cells during all stages of the cycle (P < 0.001). Cells incubated in deficient medium and supplemented with HDL secreted as much progesterone as those stimulated by LH in all stages of the cycle. Beta-mimetics were unable to enhance the stimulatory effect of HDL. Blockade of PLC had no influence on progesterone secretion from cells treated with either NA or LH, but this did impair the stimulatory effect of insulin (P < 0.05). Similarly, blockade of PKC by staurosporine impaired (P < 0.05) the effect of insulin only but not that observed after LH or NA treatment. We suggest that: (a) noradrenergic stimulation does not enhance utilisation of cholesterol from HDL for progesterone secretion; (b) the fasting of luteal cells seems to activate enzymes responsible for the progesterone synthesis; (c) effect of NA on progesterone secretion from luteal cells does not involve the PLC-PKC pathway.     

Mechanistic target of rapamycin is activated in bovine granulosa cells after LH surge but is not essential for ovulation

The LH surge induces functional and morphological changes in granulosa cells. Mechanistic target of rapamycin (mTOR) is an integrator of signalling pathways in multiple cell types. We hypothesized that mTOR kinase activity integrates and modulates molecular pathways induced by LH in granulosa cells during the preovulatory period. Cows were ovariectomized and granulosa cells collected at 0, 3, 6, 12 and 24 hr after GnRH injection. While RHEB mRNA levels increased at 3 and 6 hr, returning to basal levels by 12 hr after GnRH treatment, RHOA mRNA levels increased at 6 hr and remained high thereafter. Western blot analyses revealed increased S6K phosphorylation at 3 and 6 hr after GnRH injection. Similarly, mRNA levels of ERK1/2, STAR and EGR‐1 were higher 3 hr after GnRH treatment. Rapamycin treatment inhibited mTOR activity and increased AKT activity, but did not alter ERK1/2 phosphorylation and EGR1 protein levels in cultured bovine granulosa cells. Rapamycin also inhibited LH‐induced increase in EREG mRNA abundance in granulosa cells in vitro. However, intrafollicular injection of rapamycin did not suppress ovulation. These findings suggest that mTOR is involved in the control of EREG expression in cattle, which may be triggered by LH surge stimulating RHEB and S6K activity.     

Factors Modulating Apoptosis: an in-vitro Study in Swine Granulosa Cells

The aim of this study was to investigate the effects of some endocrine and intra‐ovarian factors on the activation/inhibition of apoptosis in swine granulosa cells. Upon incubation in a 10% FCS‐supplemented M199, granulosa cells from small (< 3 mm) follicles programmed their death after 24–48 h of culture; in the absence of FCS, apoptosis was reduced after 24 h of culture. Cells cultured in the presence of FCS were treated with db‐cAMP, LH, FSH, Insulin‐like Growth Factor‐I IGF‐I or PMSG to verify the role of these substances in apoptotic death: all these molecules inhibited apoptosis after 48 h of incubation. A further aim of the study was to investigate the possible involvement of nitric oxide (NO), an intra‐ovarian modulator, in the regulation of granulosa cell apoptosis and its possible role in the modulation of steroidogenesis. After a 48 h incubation with a substrate of NO synthesis ( l ‐arginine, 0.1 and 1 m m ), a NO donor [S‐nitroso‐N‐acetyl‐penicillamine (SNAP) , 0.2 and 1 m m ] or a NO synthase inhibitor [N^ω‐nitro‐ l ‐arginine‐methyl‐ester (NAME, 1 and 5 m m )], the onset of apoptotic death was evaluated: l . arginine and NAME did not induce any significant variation of apoptosis, whereas 1 m m SNAP exerted a protective action. A significant stimulatory effect of l ‐arginine on NO production, associated with a suppressive action on estradiol 17β concentrations was observed; NAME exerted an inhibitory effect on NO production, associated with an increase in estradiol secretion; estradiol 17β production was markedly inhibited by SNAP. In summary, the depletion of FCS could induce a cell cycle arrest in G₀ whereas apoptosis could be the consequence of cell cycle progression mediated by FCS; gonadotropins and IGF‐I could also act as survival factors. NO appeared to represent a ‘trophic’ signal for the follicle, whose involvement in the regulation of ovarian function is substantiated by its modulatory action on steroidogenesis.     

Utilization of substrates for testosterone and estradiol-17β production by small follicles of the chicken ovary

A study was conducted to characterize steroidogenesis in small ovarian follicles (1–10 mm in diameter) of the hen. The aims of our study were: 1) to determine basal estradiol-17β (E₂) production by different sizes of small follicles; 2) to determine the ability of intact small follicles to utilize exogenous substrates for testosterone (T) and E₂ production; and 3) to investigate the preferred steroidogenic pathway in small follicles. Small follicles which had not entered the hierarchy were isolated from ovaries obtained 2 hr after oviposition and divided into three groups: small white follicles (SWF; 1–2 mm in diameter), large white follicles (LWF; 2–4 mm in diameter), and small yellow follicles (SYF; 5–10 mm in diameter). Yolk and granulosa cells were removed from LWFs and SYFs and the remaining theca layer was called a follicle shell. Intact follicles or follicle shells (4/4 ml/tube) were incubated in avian Ringer's buffer supplemented with 10 mM HEPES and 0.1% BSA at 37°C for 3 hr with various treatments. Testosterone and E₂ were measured in the medium. The SYFs and their corresponding follicle shells produced the greatest amount of E₂ when E₂ production was expressed per follicle. Addition of 2 mM 8-Br-cAMP to the incubation medium stimulated E₂ production by all sizes of follicles and follicle shells. However, follicle shells produced lower basal- and 8-Br-cAMP-stimulated E₂ secretion compared to corresponding intact follicles. There was no significant difference in E₂ production in response to various concentrations of 25-hydroxycholesterol (25-OH-CHOL; 0–100 μM) by intact follicles and follicle shells. On the contrary, intact follicles and follicle shells produced T and E₂ in a dose-dependent manner in response to increasing concentrations (0–100 μM) of pregnenolone (P₅). Intact follicles also used progesterone (P₄) and dehydroepiandrosterone (DHEA) as substrates for T and E₂ production. DHEA was the preferred substrate for steroid production compared to P₄. In summary, we found that: 1) steroidogenesis in small follicles is regulated by a cAMP-dependent protein kinase A second messenger system; 2) adequate amounts of endogenous cholesterol are available for steroidogenesis; and 3) both Δ⁵ and Δ⁴ pathways are functional in small follicles and the Δ⁵ pathway may be the preferred steroidogenic pathway. pathway.     

Direct Effects of Luteal Regression on Anterior Pituitary Response To GnRH

The preovulatory period of the ewe is marked by a dramatic decrease in concentrations of progesterone in serum during the late luteal phase, followed by elevated luteinizing hormone (LH) secretion, final follicular maturation and ovulation. This experiment was designed to ascertain the extent to which removal of endogenous progesterone negative feedback at the anterior pituitary gland, independent of effects at the hypothalamus, promotes increased secretion of LH in the hours immediately after induction of luteolysis. Estrus was synchronized in ovary-intact ewes with two injections of prostaglandin F₂α (PGF₂α) analog given 10 d apart (Day 0 = second day after the second PGF₂α injection). Ewes were subjected to hypothalamic-pituitary disconnection (HPD; n = 6) on Day 3 and were pulsed with gonadotropin-releasing hormone (GnRH). Ewes were used during the estrous cycle or received approximately 400 IU pregnant mare serum gonadotropin (PMSG) on Day 2 to stimulate ovulation; there was no difference (P < 0.10) in ovulation rate or progesterone production between these two groups. Luteal regression was induced by injection of PGF₂α analog on approximately Day 10 of the estrous cycle. Blood samples were collected around exogenous GnRH pulses before and at 2- or 4-hr intervals after PGF₂α administration and concentrations of LH and progesterone determined. At 4, 12 and 24 hr after PGF₂α administration, mean serum progesterone levels in all ewes had decreased by 54.7%, 66.2% and 89.4%, respectively (P < 0.05) from pre-injection levels. The decrease in progesterone was associated with an increase (P < 0.01) in LH pulse amplitude with means at 4-hr post-PGF₂α ranging from 190% to 288% of pre-PGF₂α values. Mean serum LH levels were also increased (P < 0.01) within 4 hr of PGF₂α administration and remained elevated at all but the 24-hr time point. The timing of this increase (within 4 hr) indicates that it is independent of changes in serum estradiol concentrations, which do not increase for at least 16 hr after induction of luteolysis. Thus, removal of endogenous progesterone negative feedback at the anterior pituitary gland in the hours immediately after induction of luteolysis seems to play a role in facilitating LH release independently of hypothalamic action.     

Effects of adding luteinizing hormone to a medium containing follicle stimulating hormone on progesterone-induced differentiation of cumulus cells during meiotic resumption of porcine oocytes

In the present study, we investigated the effects of adding luteinizing hormone (LH) to a medium containing follicle stimulating hormone (FSH) on the shift in expression of progesterone receptor (PR) isoforms (PR‐A and PR‐B) and the roles in function of cumulus cells of cumulus‐oocyte complexes (COC). The level of PR‐B mRNA in cumulus cells was up‐regulated by FSH during the first 16‐h cultivation but the level was significantly decreased at 20 h. The decrease of PR‐B mRNA was accelerated when COC were cultured with FSH and LH. Still, a high level of total PR mRNA was maintained in cumulus cells cultured with or without the addition of LH up to 20 h, suggesting that the expression of PR isoforms was shifted from PR‐B to PR‐A in cumulus cells. The reduction of PR‐B was also induced by addition of progesterone to FSH‐containing medium. The addition of LH or progesterone to FSH‐containing medium stimulated cumulus expansion of COC as compared with that of COC cultured with FSH. In the expanded COC, ADAMTS‐1 which is expressed in granulosa cells and cumulus cells in rodent follicles through LH‐induced progesterone‐ and PR‐dependent pathway, was more accumulated within the COC matrix. These results suggest that the addition of LH or progesterone to FSH‐containing medium is required for the differentiation of cumulus cells, such as cumulus expansion, mediated by the shift from PR‐B to PR‐A in them.     

Role of ghrelin in regulating rabbit ovarian function and the response to LH and IGF-I

The aim of these in vivo and in vitro studies was to examine the role of ghrelin in the control of plasma hormone concentrations, the proliferation, apoptosis and secretory activity of ovarian granulosa cells and the response of these cells to hormonal treatments. Female rabbits were injected with ghrelin (10 μg/animal/day for one week before ovulation induced by 25 IU PMSG and 0.25 IU LHRH). On the day of ovulation, blood samples were collected and analyzed for concentrations of progesterone (P₄), testosterone (T), estradiol (E₂), estrone-sulphate (ES), insulin-like growth factor I (IGF-I) and leptin (L) by RIA. Some control and ghrelin-treated animals were killed in the periovulatory period, their ovaries were weighed and granulosa cells were isolated and cultured for 2 d. Cell proliferation (expression of PCNA) and apoptosis (expression of TdT) were evaluated by immunocytochemistry and TUNEL respectively. Secretion of P₄, T, E₂, IGF-I, and prostaglandin F (PGF) by granulosa cells cultured with and without LH or IGF-I (1, 10 or 100 ng/ml medium) was assessed by RIA. The remaining control and treated animals were kept until parturition, while the number, viability and body weight of pups were recorded.     

Activin-A differentially regulates steroidogenesis by sheep granulosa cells

Intra-ovarian factors, such as activin, are implicated in multiple aspects of follicular development in mammalian ovaries. This study was conducted to investigate a possible effect of activin-A on steroidogenesis in sheep granulosa cells in vitro. Sheep granulosa cells were obtained from medium antral follicles and cultured in a chemically defined RPMI -1640. Oestradiol and progesterone production, secreted by the cultured cells, was evaluated by enzyme-linked immunosorbent assay. In order to determine the dose effect of activin-A on steroidogenesis, granulosa cells were cultured in the presence of increasing concentrations of activin-A (0, 0.5, 5 and 50 ng ml(-1)) for 48 hours. The results revealed that activin-A exerts a differential effect on steroidogenesis in granulosa cells in such a way that it significantly (P < 0.05) suppressed progesterone production and enhanced oestradiol production. These results were confirmed by the time effect of activin-A on oestradiol and progesterone production in granulosa cells. In the absence of activin-A treatment, granulosa cells showed enhanced capacity to produce progesterone, but not oestradiol, as the time progressed from 12 to 48 hours. Treatment of sheep granulosa cells with 25 ng ml(-1)activin-A for 12, 24 and 48 hours significantly stimulated oestradiol production but inhibited progesterone production. These results suggest that activin-A is a local regulator of sheep folliculogenesis that might act to support differentiation in granulosa cells and suppress luteinisation.     

Characterization of the LH peak after short and long fixed‐time artificial insemination protocols in sheep raised in the tropics

The aim of this study was to evaluate the peak in luteinizing hormone (LH) and the pregnancy rate of sheep (Texel × Santa Inês) in the tropics using short‐ (6 days) and long‐term (12 days) progesterone protocols followed by artificial insemination (AI) both in and out of the breeding season. Experiment 1 was conducted within (IN) the breeding season (autumn, n = 36), and experiment 2 was conducted outside (OUT) of the breeding season (spring, n = 43). In each experiment, the sheep were divided into two groups (6 or 12 days) according to the duration of treatment with a single‐use progesterone release vaginal device (CIDR^®, Pfizer, São Paulo, SP, Brazil), and blood samples were collected from 10 animals per group every 4 hr to measure the LH and progesterone concentrations. In the spring, the characteristics of the LH peak did not differ between groups; but in the autumn, there were differences between groups at the beginning (G‐6 IN: 36.44 ± 5.46 hr; G‐12 IN: 26.57 ± 4.99 hr) and end of the LH peak (G‐6 IN: 46.22 ± 7.51 hr; G‐12 IN: 34.86 ± 8.86 hr). The results showed alterations in the LH peak during the breeding season only in the sheep undergoing the short‐term protocol.     

The Content of β-endorphin-like Immunoreactivity in Porcine Corpus Luteum and the Potential Roles of Progesterone, Oxytocin and Prolactin in the Regulation of β-endorphin Release from Luteal Cells in vitro

The amount of β‐endorphin‐like immunoreactivity (β‐END‐LI) in porcine corpora lutea from several stages of the oestrous cycle and the effects of progesterone, oxytocin, and prolactin on β‐END‐LI secretion in vitro by luteal cells were studied. Porcine corpora lutea obtained on days 1–5, 6–10, 11–13, 14–18, and 19–21 of the cycle were used to prepare extracts for β‐END‐LI determination. Additionally, corpora lutea from days 11–13 and 14–18 were enzymatically dissociated and isolated luteal cells were used for further study of β‐endorphin secretion in vitro. Cells were cultured in serum‐free defined M 199 medium (10⁶ cells/ml) at 37°C under 5% CO₂ in air, for 12 h. The influences of the following factors on β‐END‐LI secretion by luteal cells were tested: progesterone (10^–9, 10^–7 and 10^–5M ), oxytocin (0.01, 0.1, 1 and 10 ng/ml), and prolactin (0.1, 1, 10 and 100 ng/ml). The β‐END‐LI contents in extracts and media were measured by radioimmunoassay. The tissue concentration of β‐END‐LI was lowest on days 1–5 of the cycle (0.35 ± 0.03 ng/g wet tissue). Subsequently, it constantly increased to the highest value on days 14–18 (16.58 ± 0.52 ng/g wet tissue) and on days 19–21 it declined (11.10 ± 0.52 ng/g wet tissue). Progesterone at a low dose (10^–9 M ) resulted in significant (p < 0.05) increases and decreases in β‐END‐LI secretion by luteal cells from days 11–13 and 14–18, respectively. Higher doses of progesterone (10^–7 and 10^–5 M ) had no effect on β‐END‐LI release, compared with the control group. All dose‐levels of oxytocin used decreased β‐END‐LI secretion by luteal cells on days 11–13 and 14–18 of the cycle. Prolactin at doses of 0.1 and 1 ng/ml on days 11–13, and all doses tested on days 14–18 resulted in decreases in β‐END‐LI release from luteal cells. These results document evident changes in β‐END‐LI content in the pig corpus luteum during its development and indicate the potential roles of progesterone, oxytocin, and prolactin in luteal cell secretion of β‐END‐LI.     

IGF-1 receptor signaling pathways and effects of AMPK activation on IGF-1-induced progesterone secretion in hen granulosa cells

IGF-1 plays a key role in the proliferation and differentiation of granulosa cells. However, the molecular mechanism of IGF-1 action in avian granulosa cells during follicle maturation is unclear. Here, we first studied IGF-1 receptor (IGF-1R) expression, IGF-1-induced progesterone production and some IGF-1R signaling pathways in granulosa cells from different follicles. IGF-1R (mRNA and protein) was higher in fresh or cultured granulosa cells from the largest follicles (F1 or F2) than in those from smaller follicles (F3 or F4). In vitro, IGF-1 treatment (10(-8)M, 36h) increased progesterone secretion by four-fold in mixed F3 and F4 (F3/4) granulosa cells and by 1.5-fold in F1 granulosa cells. IGF-1 (10(-8)M, 30min)-induced increases in tyrosine phosphorylation of IGF-1R beta subunit and phosphorylation of ERK were higher in F1 than in F3/4 granulosa cells. Interestingly, IGF-1 stimulation (10(-8)M, 10min) decreased the level of AMPK Thr172 phosphorylation in F1 and F3/4 granulosa cells. We have recently showed that AMPK (AMP-activated protein kinase) is a protein kinase involved in the steroidogenesis in chicken granulosa cells. We then studied the effects of AMPK activation by AICAR (5-aminoimidazole-4-carboxamide ribonucleoside), an activator of AMPK, on IGF-1-induced progesterone secretion by F3/4 and F1 granulosa cells. AICAR treatment (1mM, 36h) increased IGF-1-induced progesterone secretion, StAR protein levels and decreased ERK phosphorylation in F1 granulosa cells. Opposite data were observed in F3/4 granulosa cells. Adenovirus-mediated expression of dominant negative AMPK totally reversed the effects of AICAR on IGF-1-induced progesterone secretion, StAR protein production and ERK phosphorylation in both F3/4 and F1 granulosa cells. Thus, a variation of energy metabolism through AMPK activation could modulate differently IGF-1-induced progesterone production in F1 and F3/4 granulosa cells.     

Mechanisms by which a phorbol ester and a diacylglycerol analog inhibit hen granulosa cell steroidogenesis

We have previously reported that treatment of hen granulosa cells with the tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), or the diacylglycerol analog, 1-oleoyl-2-acetylglycerol (OAG), attenuates the steroidogenic response to luteinizing hormone (LH) at sites both prior and distal to the formation of cyclic 3',5'-adenosine monophosphate (cAMP). The present study was designed to determine the site(s) of inhibition within the steroidogenic pathway by evaluating the effects of OAG and PMA on key enzyme systems involved in hen granulosa cell steroidogenesis: adenylyl cyclase, phosphodiesterase, the cholesterol-side-chain-cleavage (CSCC) complex and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD). The adenylyl cyclase activator, forskolin (0.1 mM), stimulated a 3.3-fold increase in granulosa cell cAMP formation, and this increase was inhibited by the presence of OAG (2.5, 25 and 63 microM) in a dose-dependent manner. By contrast, a 1.8-fold increase in cAMP accumulation induced by the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX; 1.0 mM), was not altered by OAG at any dose (2.5, 25 and 63 microM). Inclusion of 25-hydroxycholesterol (2500 ng/tube) in the incubation medium in the presence of 1.0 microM cyanoketone resulted in a 10-fold increase in pregnenolone production. Increasing concentrations of OAG (2.5, 25 and 63 microM) caused a dose-dependent suppression of the conversion of 25-hydroxycholesterol to pregnenolone. On the other hand, granulosa cells incubated with 200 ng/tube pregnenolone increased progesterone production 100-fold, but this increase was not inhibited by either PMA (3.2, 32, 8.1 and 162 nM) or OAG (2.5, 25 and 63 microM). The results indicate that activation of protein kinase C can suppress the function of at least two key enzymes involved in hen granulosa cell steroidogenesis. Inhibition of adenylyl cyclase greatly reduces the steroidogenic response of granulosa cells to endocrine factors that act via increasing levels of cAMP (i.e. LH). Furthermore, a reduction in CSCC activity limits the availability of precursor required for progesterone production. These data provide additional evidence of a role for protein kinase C in modulating ovarian function in the domestic hen.