Action of protein kinase A regulators on secretory activity of porcine granulosa cells in vitro

To understand the role of protein kinase A (PKA) in the control of ovarian secretory activity, we examined effects of stimulators (db-cAMP, 6-Phe-cAMP, Sp-cDBIMPS) or inhibitors (Rp-cAMPS, KT5720) of PKA on the release of insulin-like growth factor I (IGF-I), progesterone (P) and estradiol (E) by cultured porcine granulosa cells using RIA. All the PKA stimulators db-cAMP (10-10000 ng/ml), 6-Phe-cAMP (10-10000 pmol) or Sp-cDBIMPS (1-10000 pmol) increased IGF-I almost at all doses tested. P release was stimulated by db-cAMP (at doses 100-10000 ng/ml), Sp-cDBIMPS (at 10-1000 pmol) and 6-Phe-cAMP (at 1000 and 10000 pmol). The release of E was stimulated by Sp-cDBIMPS (1-100 pmol), db-cAMP (1000 and 10000 ng/ml) and 6-Phe-cAMP (1000 and 10000 pmol). Since Sp-cDBIMPS, which activates preferentially PKA isozyme type II, showed stimulating effects at doses lower than those of 6-Phe-cAMP, a preferential activator of both, type I and II of PKA, it is assumed that PKA type II is more important for the control of ovarian steroidogenesis than type I. A PKA inhibitor Rp-cAMPS inhibited release of IGF-I (10000 pmol), P (1000 pmol) and E (1000 and 10000 pmol), whereas Rp-cAMPS, at doses higher than 1000 pmol, tended to reverse this inhibitory effect. Other PKA inhibitor KT5720 suppressed P (at 10-1000 ng/ml), but not IGF-I or E release.The stimulation of growth factor and sex steroid release by PKA activators, and suppression of the secretion some of these substances by PKA inhibitors may indicate the implication of PKA (probably site B) in up- and down-regulation of ovarian IGF-I and steroid release.     

Some endocrine traits of transgenic rabbits. II. Changes in hormone secretion and response of isolated ovarian tissue to FSH and ghrelin

In the present in vitro experiments we examined FSH- and ghrelin-induced changes in ovarian hormone secretion by transgenic rabbits. Fragments of ovaries isolated from adult transgenic (carrying mammary gland-specific mWAP-hFVIII gene) and non-transgenic rabbits from the same litter were cultured with and without FSH or ghrelin (both at 0, 1, 10 or 100 ng/ml medium). The secretion of progesterone (P4), estradiol (E2) and insulin-like growth factor I (IGF-I) was assessed by RIA. It was observed that ovaries isolated from transgenic rabbits secreted much less P4, E2 and IGF-I than the ovaries of non-transgenic animals. In control animals FSH reduced E2 (at doses 1-100 ng/ml medium) and IGF-I (at 1-100 ng/ml), but not P4 secretion, whereas ghrelin promoted P4 (at 1 ng/ml) and IGF-I (at 100 ng/ml), but not E2 output. In transgenic animals, the effects were reversed: FSH had a stimulatory effect on E2 (at 100 ng/ml) and ghrelin had an inhibitory effect on P4 (at 10 ng/ml). No differences in the pattern of influence of FSH on P4 and IGF-I and of ghrelin on E2 and IGF-I were found between control and transgenic animals. The present observations suggest that 1) both FSH and ghrelin are involved in rabbit ovarian hormone secretion, 2) transgenesis in rabbits is associated with a reduction in ovarian secretory activity, and 3) transgenesis can affect the response of ovarian cells to hormonal regulators.     

Effects of genistein and lavendustin on reproductive processes in domestic animals in vitro

The aim of our experiments was to study the influence of genistein [tyrosine kinase (TK) inhibitor with estrogenic activity] and lavendustin A (TK inhibitor without estrogenic activity) on female reproductive processes in domestic animals in vitro. It was found that genistein (0.001–1 μg/ml) increased IGF-I release by cultured bovine and porcine granulosa cells, but decreased its secretion by rabbit granulosa cells (0.01–10 μg/ml). Genistein stimulated progesterone secretion by bovine and rabbit granulosa cells (at 0.01–10 μg/ml), estradiol output by rabbit granulosa cells (at 1 μg/ml) and porcine ovarian follicles (at 10 μg/ml), as well as cAMP production by bovine (at 0.001–1 μg/ml) and rabbit (at 1 μg/ml) granulosa cells. No effects of genistein (at 10 μg/ml) on PGF-2 alpha and progesterone release by porcine ovarian follicles were observed. Genistein significantly (P < 0.05) stimulated the reinitiation and completion of nuclear maturation of porcine oocytes (at 5 μg/ml), as well as the preimplantation development of rabbit zygotes (at 1 μg/ml). Lavendustin A (0.001–1 μg/ml) increased IGF-I release by bovine (but not by porcine) granulosa cells, cAMP release by bovine granulosa cells, and PGF-2 alpha output by porcine ovarian follicles (at 10 μg/ml). Lavendustin (at 1 μg/ml) had no significant effect on IGF-I release by porcine granulosa cells, on estradiol and cAMP output by rabbit granulosa cells, or on progesterone secretion by porcine follicles (at 10 μg/ml). Inhibitory actions of lavendustin (at 10 μg/ml) on estradiol secretion by porcine follicles were also found. Furthermore, lavendustin, like genistein, promoted the reinitiation and completion of meiosis in porcine oocytes. The present study demonstrates a predominantly stimulatory effect of TK inhibition on endocrine and generative processes in domestic animals. The majority of these effects are similar for both compounds, indirectly suggesting that their action is due to tyrosine kinase inhibition and protein kinase A-stimulation, rather than estrogenic activity.     

Different action of ovine GH on porcine theca and granulosa cells proliferation and insulin-like growth factors I- and II-stimulated estradiol production

Growth hormone (GH) and insulin-like growth factors (IGFs) are recognized as regulators of ovarian function. This study was designed to compare the effect of GH and IGFs added alone or together on porcine theca interna and granulosa cells proliferation and steroidogenesis. Moreover, the effect of GH on IGF-I secretion was examined. Cells were isolated from medium size follicles and cultured in vitro for 48 h in serum free medium. Estradiol and IGF-I medium concentrations were determined by radioimmunoassays. Proliferation was evaluated by alamar blue assay and by radiolabelled thymidine incorporation. GH increased IGF secretion by granulosa cells while decreased its secretion by theca cells. Proliferation of both cell types was stimulated by IGF-I and IGF-II (30 ng/ml) and modestly inhibited by GH (100 ng/ml). Insulin-like growth factor II increased, in a statistically significant manner, estradiol secretion by both cell types, while IGF-I stimulated estradiol secretion to a greater extent by granulosa then by theca cells. The synergistic action of GH and IGFs on estradiol secretion was stimulatory in theca cells and inhibitory in granulosa cells. These data demonstrate that despite its direct action on estradiol secretion by granulosa and theca cells, GH also modulated estradiol secretion induced by IGFs. Differences in the estradiol production in response to GH alone and the effect of the synergistic action of GH and IGFs suggest that different cellular mechanisms for these hormones are triggered in each cell type.     

Effects of insulin-like growth factor-II on proliferation and differentiation of ovarian granulosa cells.

The effects of insulin-like growth factor-II (IGF-II) on the proliferation and differentiation of ovarian granulosa cells were studied in cultured human and porcine granulosa cells. IGF-II significantly increased basal progesterone secretion in granulosa cells at concentrations of 1-100 ng/ml. A stimulatory effect was also observed in gonadotropin-stimulated porcine granulosa cells treated with IGF-II. The secretion of estradiol by basal and gonadotropin-stimulated porcine granulosa cells was also significantly increased by adding IGF-II. IGF-II led to dose-dependent increases in [3H]thymidine incorporation into DNA and in the number of granulosa cells. To further characterize the cellular mechanisms underlying the stimulatory effects of IGF-II on the proliferation and differentiation of granulosa cells, we investigated the intermediary roles of cyclic AMP and intracellular Ca2+ concentration ([Ca2+]i). Treatment with 100 ng/ml IGF-II produced a significant increase in the basal accumulation of cyclic AMP in porcine granulosa cells. However, no change of [Ca2+]i by IGF-II was noted. IGF-II produced effects in accumulation that were similar to those of IGF-I. Our findings suggest that IGF-II may be a general stimulator in the proliferation and differentiation of granulosa cells, and that cyclic AMP may be a second messenger for the effects of IGF-II in ovarian granulosa cells.     

Effect of follicle-stimulating hormone on steroid secretion and messenger ribonucleic acids encoding cytochromes P450 aromatase and cholesterol side-chain cleavage in bovine granulosa cells in vitro

We determined 1) whether the previously observed induction of estradiol secretion in bovine granulosa cells cultured in serum-free conditions is associated with an increase in cytochrome P450 aromatase (P450(arom)) mRNA abundance and 2) whether P450(arom) mRNA levels are responsive to FSH in vitro. Granulosa cells from small (2-4-mm) follicles were cultured in serum-free medium. Estradiol secretion increased with time in culture and was correlated with increased P450(arom) mRNA abundance. Progesterone secretion also increased with time in culture, but P450 cholesterol side-chain cleavage (P450(scc)) mRNA abundance did not. FSH stimulated estradiol secretion and P450(arom) mRNA abundance; the effect was quadratic for both estradiol and P450(arom) mRNA. Estradiol secretion and P450(arom) mRNA levels were correlated. FSH stimulated progesterone secretion and P450(scc) mRNA abundance, although the minimum effective dose of FSH was lower for estradiol (0.1 ng/ml) than for progesterone (10 ng/ml) production. Insulin alone stimulated estradiol secretion and P450(arom) mRNA levels but not progesterone or P450(scc) mRNA abundance. We conclude that this cell culture system maintained both estradiol secretion and P450(arom) mRNA abundance responsiveness to FSH and insulin, whereas P450(scc) mRNA abundance and progesterone secretion were responsive to FSH but not insulin.     

Effects of interferon on the steroidogenic functions and proliferation of immature porcine granulosa cells in culture.

Recent studies suggest the relevance of several cytokines to the growth and differentiation of granulosa cells. In the present study, we investigated the effects of interferon (IFN) on the steroidogenic functions and proliferation of immature porcine granulosa cells. Human IFN-alpha inhibited FSH-induced progesterone secretion in a concentration-dependent manner. The effect of IFN-alpha was significant at a concentration as low as 10 pg/ml. Maximal inhibitory concentrations (10-50 ng/ml) of IFN-alpha reduced FSH-induced progesterone secretion by 70%. In contrast, estradiol secretion induced by FSH was significantly enhanced by relatively high concentrations (1-50 ng/ml) of IFN-alpha. IFN-alpha (0.1-10 ng/ml) reduced cAMP generation in response to FSH by as much as 80%, although its effect was not concentration-dependent. The proliferation of cultured granulosa cells was inhibited by IFN-alpha in a concentration-dependent manner. Human IFN-gamma did not affect granulosa cell functions. The stimulation of estradiol secretion and the inhibition of cell proliferation induced by IFN-alpha in cultured porcine granulosa cells in this study are in contrast with the effects of IL-1, which, as we reported previously, inhibited both progesterone and estradiol secretion and stimulated cell growth in these cell cultures. Such differences in the mode of action of cytokines may contribute to the regulation of granulosa cell functions under physiological or pathological conditions.     

Leptin directly controls secretory activity of human ovarian granulosa cells: possible inter-relationship with the IGF/IGFBP system

AIMS: The aim of our in vitro studies was to understand the role of leptin and the insulin-like growth factor I/insulin-like growth factor protein (IGF/IGFBP) system in controlling human ovarian function. METHODS: We studied the action of leptin (0, 1, 10, or 100 ng/ml) and immunoneutralization of IGF-I using specific antiserum (0.1%) on the release of progesterone (P), estradiol (E), oxytocin (OT), IGF-I, IGFBP-3, and prostaglandins F (PGF) by these cells using radioimmunoassay/immunoradiometric assay. RESULTS: It was found that leptin stimulated the secretion of OT, IGFBP-3, and PGF. It suppressed the secretion of E and IGF-I, but not P, into the medium. The addition of antiserum against IGF-I decreased IGF-I output, increased P, OT, IGFBP-3, and PGF secretion, and had no effect on E release. Immunoneutralization of IGF-I also prevented or reversed the effects of leptin on P, E, IGF-I, IGFBP-3, PGF, but not on OT. CONCLUSIONS: These observations (1) demonstrate that leptin directly controls the secretory activity of human ovarian cells, (2) confirm the involvement of IGF-I in the regulation of ovarian cells, and (3) suggest an inter-relationship between leptin and the IGF/IGFBP system in the control of these functions and the involvement of IGF/IGFBP system in mediating leptin action on the ovary.     

The effect of obestatin on porcine ovarian granulosa cells

The aim of our in vitro experiments was to investigate the role of obestatin, a newly discovered metabolic hormone produced in the stomach and other tissues, in the direct control of ovarian cell proliferation, apoptosis and secretion. Porcine granulosa cells were cultured in the presence of obestatin (0, 1, 10 and 100ng/ml medium). The expression of intracellular peptides associated with proliferation (PCNA, cyclin B1, MAP kinase), as well as markers of apoptosis (Bax, p53, Caspase 3), were detected using immunocytochemistry and Western immunoblotting. Secretion of progesterone (P(4)), testosterone (T) and estradiol (E(2)) was measured by EIA. Addition of obestatin (1-100ng/ml) to the culture medium significantly stimulated the expression of PCNA and resulted in an increase in expression of cyclin B1 and MAPK. It also significantly increased the percentage of cells containing the apoptotic and anti-proliferating peptides p53, Caspase 3 and Bax. At 10 and 100ng/ml, obestatin promoted the secretion of P(4), but not T or E(2). Our results are the first demonstration that obestatin directly controls porcine ovarian cell functions: it can stimulate proliferation (accumulation of rPCNA, cyclin B1 and MAPK), apoptosis (expression of p53, Caspase 3 and Bax) and the secretion of progesterone.     

Luteal macrophage conditioned medium affects steroidogenesis in porcine granulosa cells

The purpose of the study was to examine the effect of luteal macrophage conditioned medium (LMCM) on progesterone and estradiol production by cultured granulosa cells. Porcine granulosa cells were cultured for 48 h with or without LMCM in the absence or presence of 100 ng/ml LH, FSH or prolactin. Progesterone and estradiol concentrations were measured by radioimmunoassay. Granulosa cells were analyzed histochemically and immunocytochemically for the activity and presence of Δ5, 3β-hydroxysteroid dehydrogenase (3β-HSD), respectively. LMCM stimulated basal and LH-, FSH- or prolactin-induced progesterone secretion. Similarly, LMCM augmented basal and stimulated activity of 3β-HSD in the examined cells. In contrast, LMCM decreased LH- and prolactin-induced estradiol secretion but increased FSH-induced estradiol secretion. These data demonstrate the clear stimulatory effect of LMCM on granulosal progesterone production. It is concluded that substances secreted by macrophages modulate gonadotropin effect on follicular progesterone secretion in a paracrine manner via 3β-HSD activity.     

Effects of angiogenin on granulosa and theca cell function in cattle

Angiogenin is a member of the ribonuclease A superfamily of proteins that has been implicated in stimulating angiogenesis but whether angiogenin can directly affect ovarian granulosa or theca cell function is unknown. Therefore, the objective of these studies was to determine the effect of angiogenin on proliferation and steroidogenesis of bovine granulosa and theca cells. In experiments 1 and 2, granulosa cells from small (1 to 5 mm diameter) follicles and theca cells from large (8 to 22 mm diameter) follicles were cultured to evaluate the dose-response effect of recombinant human angiogenin on steroidogenesis. At 30 and 100 ng/ml, angiogenin inhibited (P<0.05) granulosa cell progesterone production and theca cell androstenedione production but did not affect (P>0.10) granulosa cell estradiol production or theca cell progesterone production, and did not affect numbers of granulosa or theca cells. In experiments 3 and 4, granulosa and theca cells from both small and large follicles were cultured with 300 ng/ml of angiogenin to determine if size of follicle influenced responses to angiogenin. At 300 ng/ml, angiogenin increased large follicle granulosa cell proliferation but decreased small follicle granulosa cell progesterone and estradiol production and large follicle theca cell progesterone production. In experiments 5 and 6, angiogenin stimulated (P<0.05) proliferation and DNA synthesis in large follicle granulosa cells. In experiment 7, 300 ng/ml of angiogenin increased (P<0.05) CYP19A1 messenger RNA (mRNA) abundance in granulosa cells but did not affect CYP11A1 mRNA abundance in granulosa or theca cells and did not affect CYP17A1 mRNA abundance in theca cells. We conclude that angiogenin appears to target both granulosa and theca cells in cattle, but additional research is needed to further understand the mechanism of action of angiogenin in granulosa and theca cells, as well as its precise role in folliculogenesis.     

Effect of growth hormone and insulin-like growth factor-I on spontaneous apoptosis in cultured luteal cells collected from early, mature, and regressing porcine corpora lutea

The aim of the present study was to test the hypothesis that growth hormone (GH) and insulin-like growth factor-I (IGF-I) act at a local level to inhibit luteal cell apoptosis. Luteal cells collected from the corpora lutea at different stages of the luteal phase were cultured for 24 h in M 199 medium supplemented with 5% of calf serum to cause attachment cells to the plastic. After 24 h, the media were changed and various concentrations of GH (10, 100 or 200 ng/ml) or IGF-I (30, 50 or 100 ng/ml) were added to the culture medium. Twenty-four hours later, cells were fixed for morphological assessment of apoptotic cells utilising a Hoechst staining technique. To support morphological observations, measurements of caspase-3 activity in cultured porcine luteal cells were performed. Increased incidence of apoptotic bodies and caspase-3 activity accompanied luteal regression and was associated with a decreased progesterone (P4) secretion by luteal cells. GH stimulated P4 secretion by luteal cells collected from developing (ELP) and mature (MLP) corpora lutea but had no effect on its secretion by cells collected from regressing corpora lutea (LLP). Moreover, it had no effect on the incidence of apoptotic bodies in all types of corpora lutea. However, suppression of caspase-3 activity was observed with 100 and 200 ng of GH/ml in all types of corpora lutea. IGF-I had a stimulatory effect on P4 secretion by ELP and MLP, decreased the incidence of apoptotic bodies and suppressed caspase-3 activity in cultures treated with all doses used. In conclusion, our results indicate that both GH and IGF-1 trigger anti-apoptotic effects either indirectly, by increasing progesterone secretion, or directly, through the inhibition of caspase-3 activity and subsequent prevention of apoptotic body formation.     

In vitro assessment of iron effect on porcine ovarian granulosa cells: secretory activity, markers of proliferation and apoptosis

It would be desirable to expand the existing general knowledge concerning direct action of metals on the ovary. Nevertheless, the results of testing of iron compound on porcine ovarian cells should be interpreted carefully because iron is an essential element which could also induce changes in cellular processes. The aim of this in vitro study was 1) to examine dose-dependent effects of iron on the secretory activity of porcine ovarian granulosa cells, and 2) to outline the potential intracellular mediators mediating these effects. Specifically, we evaluated the effect of iron sulphate on the release of insulin-like growth factor I (IGF-I) and progesterone, as well as the expression of markers of proliferation (cyclin B1) and apoptosis (caspase-3) in porcine ovarian granulosa cells. Concentrations of IGF-I and progesterone were determined by RIA, cyclin B1 and caspase-3 expression by immunocytochemistry (ICC). Our results show a significantly decreased IGF-I secretion by ovarian granulosa cells after iron sulphate addition at the doses 0.5 and 1.0 mg/ml. The iron sulphate additions at doses 0.17 and 1.0 mg/ml had no effect on progesterone secretion. In contrast, iron sulphate addition at doses 0.17-1.0 mg/ml resulted in stimulation of cyclin B1 and caspase-3 expression. In conclusion, the present results indicate a direct effect of iron on 1) secretion of growth factor IGF-I but not steroid hormone progesterone, 2) expression of markers of proliferation (cyclin B1), or 3) apoptosis (caspase-3) of porcine ovarian granulosa cells. These results support an idea that iron could play a regulatory role in porcine ovarian function: hormone release, proliferation and apoptosis.     

Development of a long-term serum-free culture system for immature granulosa cells from diethylstilboestrol-treated prepubertal rabbits: influence of androstenedione and fibronectin on FSH-induced cytodifferentiation

Granulosa cells from diethylstilboestrol-treated prepubertal rabbits were cultured for 6 days in M199 with FSH (1-100 ng ml(-1)) in uncoated or fibronectin-coated plates with or without androstenedione to define the time course profile of oestradiol and progesterone secretion, and the possible modulator role of androstenedione and fibronectin during FSH-induced rabbit granulosa cell differentiation. Every 48 h, cultures were photographed and samples of medium were collected and assayed by ELISA for oestradiol and progesterone. FSH increased oestradiol secretion in a dose-dependent manner. Androstenedione augmented FSH-stimulated oestradiol secretion, and led to a decrease in secretion of oestradiol with time in culture. FSH stimulated progesterone secretion in a dose-dependent manner. This was increased by androstenedione with 10 ng FSH ml(-1) (0-96 h) and 1 ng FSH ml(-1) (96-144 h). FSH-stimulated (100 ng ml(-1)) progesterone secretion decreased at 48-96 h. Fibronectin prevented this decrease, without affecting oestradiol or progesterone secretion at other time points. FSH caused cell reaggregation at 48 h. In conclusion, this serum-free culture system is appropriate for the study of mechanisms of rabbit granulosa cell differentiation. FSH induced cytodifferentiation and reaggregation of granulosa cells. Androstenedione appeared to act synergistically with FSH to promote steroidogenesis. Fibronectin sustained progesterone secretion during differentiation.     

Leptin controls rabbit ovarian function in vivo and in vitro: Possible interrelationships with ghrelin

The aim of these in vivo and in vitro studies was to examine the role of leptin in the control of plasma hormone concentrations, reproduction, and secretory activity of ovarian granulosa cells.In in vivo experiments, 15 female European domestic rabbit (Oryctolagus cuniculus) were treated with leptin (5 μg animal⁻¹ d⁻¹ for 1 wk before induction of ovulation with 25 IU equine chorionic gonadotropin and 0.25 IU human chorionic gonadotropin), and 15 females constituted the control group (treated with phosphate-buffered saline). Plasma concentrations of progesterone (P₄), testosterone (T), estradiol (E₂), estrone sulfate (ES), and insulin-like growth factor I (IGF-I) were determined at the estimated day of ovulation by radioimmunoassay (RIA), and number, viability, and body weight of newborns were recorded at parturition. In in vitro experiments, granulosa cells were isolated from periovulatory ovarian follicles of five control and five females treated with ghrelin (10 μg animal⁻¹ d⁻¹ for 1 wk before induced ovulation). Isolated cells were cultured for 2 d with and without leptin (0, 1, 10, or 100 ng/mL medium). Secretion of P₄, T, E₂, IGF-I, and prostaglandin F (PGF) was assessed in culture medium by RIA.In in vivo experiments, leptin administrations reduced plasma P₄, T, E₂, ES, and IGF-I levels. Leptin treatments did not affect ovarian weight or total number and body mass of newborns, but the proportion of pregnant females and number of live newborns were significantly higher in leptin-treated females than that in control females. In in vitro experiments, leptin significantly decreased (at 1 and 10 ng/mL) or increased (at 100 ng/mL) P₄ secretion, promoted E₂ and IGF-I (both at 100 ng/mL) secretion, and reduced T (at 1 and 10 ng/mL) and PGF (at 10 ng/mL) secretion. Granulosa cells from ghrelin-treated animals secreted less P₄, T, E₂, and PGF, but not IGF-I, than that secreted by granulosa cells from control animals. Furthermore, pretreatment of animals with ghrelin suppressed or even reversed subsequent leptin effects on P₄, T, E₂, IGF-I, and PGF secretion by cultured granulosa cells.These observations (1) show for the first time that leptin can increase the number of live newborns in rabbits, (2) confirm previous data on the ability of leptin to control ovarian secretory activity both directly and via upstream mechanisms, (3) demonstrate the involvement of ghrelin in the control of rabbit ovarian secretory functions, and (4) suggest an antagonistic interrelationship between leptin and ghrelin in the rabbit.     

Relaxin, oxytocin, and prostaglandin effects on progesterone secretion from bovine luteal cells during different stages of gestation

To determine the effects of relaxin, oxytocin, and prostaglandin F2 alpha on progesterone secretion, bovine luteal cells from different stages of gestation were dispersed in Medium 199 with 200 units/ml penicillin, 1.0% kanamycin, 0.5% bovine serum albumin, and 400 units/ml collagenase. Cells (10(5) were cultured in 400 microliters of Dulbecco's modified Eagle's medium and Ham's F-12 medium containing fetal bovine serum and antibiotics, in Falcon multiwell plates, in a humidified environment of 95% O2 and 5% CO2 at 37 degrees C. Cells were cultured for 24 hr without treatment and thereafter with medium-hormone replacement every 24 hr. Progesterone was quantified from unextracted media by radioimmunoassay. Basal progesterone secretion after 24 hr was 1.81 +/- 0.14, 1.76 +/- 0.17, 0.54 +/- 0.49, and 0.57 +/- 0.21 pg/ml per viable luteal cell from 145-, 165-, 185-, and 240-day-old corpora lutea, respectively. Basal progesterone secretion increased (P less than 0.05) with time in culture. Relaxin induced a dose-dependent (greater than 100 ng/ml) increase in progesterone release, compared with the controls. Oxytocin and prostaglandin F2 alpha induced greater release (P less than 0.05) of progesterone than relaxin at all stages of gestation, but progesterone release was dependent on the stage of gestation and the duration in culture. Luteinizing hormone (100 ng/ml) stimulated whereas 17 beta-estradiol (50 ng/ml) inhibited progesterone secretion by luteal cells at all stages of gestation examined. Relaxin obliterated the prostaglandin- and oxytocin-induced progesterone secretion by bovine luteal cells from 145 to 214 days of gestation. Thus, relaxin, cloprostenol, and oxytocin regulate progesterone production by cultured bovine luteal cells, but hormone secretion was dependent on the stage of gestation.     

Evidence for multiple uterine modulators of rabbit luteal function: the effect of hysterectomy during pseudopregnancy in the estrogen-treated rabbit

Previous studies show that hysterectomy on Day 1 of pseudopregnancy prolongs serum progesterone secretion in estrogen-treated pseudopregnant rabbits. These studies were undertaken to determine the day of pseudopregnancy when uterine factors are released to alter luteal function. When hysterectomies were performed on either Day 5, 8, 10, or 13 of pseudopregnancy, serum progesterone concentrations were greater than 10 ng/ml between Days 18 and 27 of pseudopregnancy compared to levels of approximately 4 ng/ml in sham-hysterectomized rabbits on these same days. In contrast, serum progesterone levels were not elevated when hysterectomies were performed on Day 11 of pseudopregnancy and were only partially maintained when hysterectomies were performed on Day 12 of pseudopregnancy. Twice daily injections of prolactin (1.5 mg, s.c.) between Days 1 and 33 of pseudopregnancy were unable to mimic the effect of estradiol in the hysterectomized rabbit. Twice daily injections of indomethacin (8 mg/kg, s.c.) between Days 6 and 23 of pseudopregnancy lowered uterine and luteal prostaglandin F2 alpha levels approximately 10-fold on Day 24 of pseudopregnancy but did not maintain progesterone secretion. Serum cholesterol levels were not altered by hysterectomy on any day and were thus not related to the maintenance of progesterone production. These results suggest that the uterus produces both inhibitory and stimulatory factors that effect luteal progesterone secretion. First, an inhibitor is released between Days 10 and 11 of pseudopregnancy in estrogen-treated rabbits that prevents the rabbit corpus luteum from responding to estradiol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Progesterone and androgen secretion by isolated cultured bovine corpus luteum cells: effect of LH, hCG, PRL, estradiol 17beta and testosterone

Primary cell cultures of bovine corpora lutea were used in order to examine their morphology and secretion of progesterone and androgen in vitro. The cells were grown as monolayers up to 6 days at 37 degrees C medium 199 supplemented with 10% calf serum. The concentration of progesterone and androgen was measured using appropriate radioimmunoassays [1,3] respectively. Luteal cells were cultured with addition of the following amounts of hormones: 100 ng LH, 10 i.u. hCG, 100 ng PRL, 150 ng Estradiol 17 beta and 150 ng Testosterone/ml of culture medium. The luteal cells also created considerable amounts of androgens. It was found that only estradiol added to the culture medium caused an increase in the level of testosterone. Progesterone secretion following the addition of hormones increased under the influence of LH, T, and E2 in statistically significant manner while hCG and PRL had no statistically significant effects.     

Effect of IGF-I on pig oocyte maturation,fertilization, and early embryonic development in vitro,and on granulosa and cumulus cell biosynthetic activity

Porcine granulosa cells have been shown previously to both secrete and respond to insulin-like growth factor-I (IGF-I), suggesting an autocrine function of this peptide in the follicle. The present work was undertaken to determine possible effects of IGF-I on in vitro maturation, in vitro fertilization, and early embryonic development in culture. Granulosa and cumulus cell proliferation and differentiation based on ³H-thymidine uptake and progesterone production, respectively, were also assessed. The results showed that the cleavage rate of oocytes was markedly stimulated in a dose-dependent manner by the addition of IGF-I to the oocyte maturation medium (P < 0.05). Embryo development beyond the 8-cell stage was improved by IGF-I, reaching a maximum of 22% at 200 ng/ml IGF-I. Treatment with IGF-I after fertilization increased the percentage of total oocyte cleavage (P < 0.05) to approximately 52%, 43%, and 57% at, respectively, 25, 50, and 100 ng/ml IGF-I. ³H-thymidine incorporation by granulosa cells was significantly increased in cultures treated with FSH (3-fold) or IGF-I (6-fold) compared to the control. For the cumulus cells, FSH caused a similar increase (3-fold) in ³H-thymidine incorporation while IGF-I stimulated a 15-fold increase. Progesterone production by the granulosa cells was increased to the same extent by treatment with FSH or IGF-I (4.7 and 5.1-fold, respectively). However, for the cumulus cells, while FSH caused a marked 16-fold increase in progesterone production, IGF-I caused only a marginal increase of 2.5-fold. These results indicate a beneficial effect of IGF-I on in vitro porcine oocyte maturation and pre-implantation embryo development, suggesting a physiological role for IGF-I in vivo. The in vivo effect of IGF-I may be indirect via autocrine stimulation of cumulus and/or granulosa cells resulting in enhanced oocyte maturation and fertilization. © 1994 Wiley-Liss, Inc.