Effects of follicle regulatory protein on thecal aromatase and 3 beta-hydroxysteroid dehydrogenase activity in medium- and large-sized pig follicles

Theca was excised from large (greater than 8 mm) and medium-sized (3-6 mm) pig follicles and prepared as monolayer cultures in serum-free media. After 24 h cells were treated with (1) M199 (control), (2) 5 i.u. hCG, (3) 100 micrograms or 100 ng FRP or (4) hCG (5 i.u.) + FRP (100 micrograms or 100 ng). At 3, 6, 12, 24 and 48 h after treatment, progesterone, oestradiol, androstenedione and testosterone were measured in media. Formation of progesterone by microsomal fractions incubated (37 degrees C) with 1 microM-pregnenolone + 5-microM-NAD+ for 1 h was used as a measure of 3 beta-HSD activity. Aromatase activity was determined by incubating cells with [3H]testosterone for 3 h (37 degrees C) and measuring 3H2O release. In theca from large follicles, hCG enhanced 3 beta-HSD activity after 48 h (P less than 0.05) and secretion of progesterone after 36 h. FRP alone inhibited 3 beta-HSD activity at 36 and 72 h, but had little effect on progesterone secretion. FRP inhibited (P less than 0.05) the hCG-induced increase in 3 beta-HSD activity at 36, 48 and 72 h. HCG enhanced aromatase activity after 48 h while FRP prevented (P less than 0.05) the hCG-induced increase in aromatase activity at 48 and 72 h. Secretion of oestradiol was enhanced (P less than 0.05) at 48 h but inhibited at 72 h by hCG. FRP alone had little effect on secretion of oestradiol but hCG + FRP was inhibitory at 72 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thecal cell 3-beta hydroxysteroid dehydrogenase activity: modulation by human chorionic gonadotropin, progesterone, estradiol-17 beta and dihydrotestosterone

Thecal cell steroidogenesis plays a major role in folliculogenesis within the porcine ovary. Accordingly, the effects of physiological concentrations of steroids on 3 beta-hydroxysteroid dehydrogenase activity (3 beta-HSD) were determined. Theca was excised from large porcine follicles and prepared in a monolayer culture in 1 ml of serum-free media. Cells were treated 24 h after culture as follows: (1) control, (2) hCG (5 IU); (3) progesterone (P, 3 micrograms); estradiol-17 beta (E, 4 micrograms); 5 beta-dihydrotestosterone (DHT, 1 microgram); (4) hCG + P or E or DHT. At 3, 6, 12, 24 and 48 h after treatment, media were assessed for P levels. For 3 beta-HSD activity, P formation by microsomal fractions incubated with 1 microM pregnenolone + 5 microM NAD+ for 1 h (37 degrees C) was monitored. Thecal cell P secretion increased from 27 to 72 h. hCG significantly (P less than 0.05) increased P levels after 36 h compared to controls. E or E + hCG decreased P levels at 36, 48, and 72 h and DHT prevented the hCG-induced increase in P secretion. 3 beta-HSD activity in thecal microsomes increased significantly from 27 to 72 h. hCG had little effect on 3 beta-HSD activity compared with controls from 27 to 36 h, but significantly (P less than 0.05) decreased 3 beta-HSD activity at 48 and 72 h. However, P or P + hCG significantly (P less than 0.05) decreased 3 beta-HSD activity at all times. In addition, E or E + hCG significantly (P less than 0.05) decreased 3 beta-HSD activity at 48 and 72 h. DHT prevented the hCG-induced decrease in 3 beta-HSD activity. In conclusion, porcine thecal secretion of P and microsomal 3 beta-HSD activity increased during 72 h of culture. Paradoxically, the addition of hCG to cultures enhanced media P concentrations but inhibited 3 beta-HSD activity. Further, the addition of E to cultures decreased media concentrations of P while P or E decreased 3 beta-HSD activity. Therefore, paracrine/autocrine effects of locally produced steroids may play a role in modulating thecal cell steroidogenesis.     

Effect of human parathyroid hormone on the cAMP production and the endocrine functions of trophoblast cells from first trimester placenta.

Our previous study on teratocarcinoma cells suggested the role of human parathyroid hormone (hPTH) in early development of the placenta. The purpose of this study was to evaluate the possible role of hPTH on the functions of first trimester trophoblast cells. Adenylate cyclase activity in crude membranes from first trimester human placental villous tissue is stimulated 2-fold by hPTH (1-34) (10(-6) mol.l-1) from 265 +/- 32 to 532 +/- 80 pmol of cAMP/mg protein/15 min. A similar stimulation of adenylate cyclase is observed in human term placental villous tissue but not in 3 different choriocarcinoma cell lines. In order to evaluate the possible role of hPTH on the functions of first trimester human trophoblast cells, these cells were isolated by dispase and cultured (2 x 10(5) cells per plate) in DMEM supplemented with 20% fetal calf serum with or without 100 ng/ml of epidermal growth factor (EGF), for 4 d. On d 2 of culture, hPTH (10(-7) mol.l-1) stimulates cAMP production of these cells from 0.52 +/- 0.2 to 2.58 +/- 0.57 pmol.h-1 per 10(6) cells (mean +/- SEM). As compared to control (30 ng/ml), the output of hCG is increased by 1.5- (NS), 2- (P less than 0.01) and 3- (P less than 0.01) fold by EGF, hPTH, and hPTH added with EGF, respectively. Dibutyryl cAMP (10(-3) mol.l-1) increased hCG secretion by 3-fold (P less than 0.05). EGF and hPTH added separately or together significantly stimulated (P less than 0.01) the secretion of free alpha subunit 2-fold from 35 ng/ml to 70 ng/ml. In contrast, hPTH and EGF added separately did not change the secretion of free beta hCG. However, added together, they significantly increased (P less than 0.01) the secretion of free beta hCG after 48 h of culture, maximal stimulation (2.5 fold) being observed at d 4 of culture. In conclusion, human trophoblast cells are target cells for hPTH. hPTH acts in association with EGF in promoting expression of endocrine activity of these cells, such as hCG secretion. Trophoblast cells provide a model for the study of the cooperative effect between a peptide hormone and a growth factor in the regulation of endocrine function.     

Ovarian steroid synthesis in tissue culture after administration of hormones and epidermal growth factor

The features of steroidogenesis of immature mouse ovaries in culture under the influence of follicle-stimulating hormones (FSH), human chorionic gonadotropin (hCG), epidermal growth factor (EGF) and insulin have been investigated during the period of reinitiation of meiosis in the oocytes. Secretion of progesterone is stimulated after addition of FSH, hCG and of insulin and EGF combination to the medium. EGF increases FSH-stimulated progesterone secretion and inhibits estradiol secretion. The ratios progesterone/estradiol and testosterone/estradiol increase, when EGF is added to the culture medium. It is analogous to the action of hCG. It is suggested that EGF may be an intrafollicular EGF regulator of luteinizing hormone action on the sex and somatic cells of the mammalian ovaries.     

Effect of FSH and HCG on progesterone secretion by cultured oocyte-cumulus complexes and granulosa cells from porcine antral follicles: Influence of follicular development

Oocyte-cumulus complexes and granulosa cells were harvested from small (1–2 mm), medium (3–5 mm), and large (6–12 mm) porcine antral follicles and cultured for 2 and 3 days. The effects of various doses of purified hCG and human FSH on progesterone secretion and monolayer formation were examined. After a 2-day culture period it was found that FSH was more effective in stimulation of progesterone secretion by cultured oocyte-cumulus complexes than in granulosa cells harvested from small follicles (P < 0.01), whereas hCG was more effective in stimulating progesterone secretion in granulosa cells than in oocytecumulus complexes harvested from large follicles. In contrast, after a 3-day culture period, granulosa cells secreted more progesterone compared to oocytecumulus complexes under control conditions or in the presence of hCG or FSH. After 3 days both FSH and hCG stimulated progesterone secretion by oocytecumulus complexes and granulosa cells; however, the hormone effect was greater upon granulosa cells than oocyte-cumulus complexes. After 3 days of culture in the case of both follicular cell types, there was a greater response to FSH in the case of cells harvested from small compared to large follicles. The reverse was true in the case of hCG responsiveness. Monolayer formation ability of oocyte-cumulus complexes was greater in the case of complexes harvested from small and medium than complexes harvested from large follicles. Addition of hCG to the cultures led to a dose-dependent decrease in monolayer formation by oocyte-cumulus complexes harvested from all sizes of follicles.     

Effects of progesterone receptor blockers on human granulosa-luteal cell culture secretion of progesterone, estradiol, and relaxin

We have developed culture methods for human luteinizing granulosa cells (GLC) that support the timely and dynamic secretion of estrogen (estradiol-17beta; E(2)), progesterone (P(4)), and relaxin (Rlx) in patterns that mimic serum hormone concentrations during the luteal phase of the menstrual cycle. Additional hCG, to simulate rescue of the corpus luteum, prevented the normal decline in GLC hormone production. To test the importance of the P(4) receptor in P(4) production, GLC were treated in vitro with two P(4) receptor antagonists. Human GLC received one of two hCG support protocols: a Baseline group simulating the normal luteal phase or a Rescue group simulating early pregnancy. Baseline and Rescue groups were treated with either RU-486 or HRP2000 either early or late in the cell culture period. The effects of treatments or control on ovarian steroid and peptide hormone production were determined (significant difference was P < 0.05). In the Rescue group, late treatment resulted in an immediate and dramatic decline in E(2), P(4), and Rlx secretion to nearly nondetectable levels within 1 day after treatment, and hormones remained depressed for the remaining 10 days of culture. In contrast, early treatment resulted in a decline in steroid hormone secretion that returned to control levels within 5 days of cessation of treatment, and Rlx secretion was delayed for approximately 5 days more than in controls. The data support the hypothesis that P(4) may be a required autocrine factor, not only for its own production but also for the maintenance of full endocrine function of the corpus luteum.     

Response of porcine theca and granulosa cells to GH during short-term in vitro culture

In Experiment 1, the influence of exogenous GH on steroid secretion by granulosa and theca interna cells recovered from small (1-3 mm), medium (4-6 mm) and large (8-12 mm) follicles was tested. In the second experiment, theca cells (Tc) and granulosa cells (Gc) obtained from large follicles were cultured separately or in two types, Tc/Gc co-culture, where both types of cells were mixed in one well or Gc and Tc were separated by cell culture membrane inserts. In the third experiment, the influence of GH on the morphology of Gc and Tc cells and activity of Delta(5),3beta-hydroxysteroid dehydrogenase (3beta-HSD) was studied. Cells were grown in the control medium (M199+5% of calf serum) or supplemented with 100 ng/ml GH. Testosterone (10(-7) M) was added as the aromatase substrate to granulosa cells cultures. The media were assayed after 48 h of culture for progesterone and oestradiol by RIA. GH added to the culture media had no effect on oestradiol and progesterone secretion by granulosa cells isolated from small and medium follicles while it stimulated both oestradiol and progesterone secretion by Gc isolated from large preovulatory follicles. A stimulatory effect on oestradiol secretion by Tc isolated from all size follicles was observed. GH did not stimulate progesterone secretion by Tc isolated from small follicles but stimulated progesterone secretion by Tc isolated from medium and large preovulatory follicles. Both co-culture systems exhibited synergistic effect on oestradiol secretion. The stimulatory effect on progesterone secretion under the influence of GH was observed in Gc cultured alone and Tc cultured alone. In contrast, the secretion of progesterone was attenuated in both co-culture systems and the addition of GH further augmented this attenuation. A statistically significant increase in oestradiol secretion was observed in all culture conditions. The addition of GH to the culture medium stimulated the activity of 3beta-HSD compared with the control culture from both types of cells. In conclusion, the present studies indicate that there are direct and follicular development stage dependent actions of GH on steroidogenesis of porcine follicular cells.     

表皮生长因子，生长抑素对人早孕胎盘hCG分泌及基因表达的影响

观察了表皮生长因子（ＥＧＦ）,生长抑素（ＳＳ）对体外培养的人早孕绒毛膜促性腺激素（ｈＣＧ）分泌及ｈＣＧβ－ｍＲＮＡ含量的影响。发现ＥＧＦ可明显刺激绒毛分泌ｈＣＧ,显著增加ｈＣＧβ－ｍＲＮＡ含量,生长抑素虽然对绒毛ｈＣＧ分泌及ｈＣＧ　β－ｍＲＮＡ含量无明显影响,但可抑制ＥＧＦ,ＧｎＲＨ刺激的ｈＣＧ分泌及ｈＣＧβ－ｍＲＮＡ水平。提示ＥＧＦ,ＳＳ在妊娠早期参与了ｈＣＧ分泌的调节。     

Contrasting effects of oestradiol-17 beta and human chorionic gonadotrophin on steroidogenesis in the rabbit corpus luteum

On Day 10 of pseudopregnancy, rabbits were given an i.v. injection of hCG (10-20 i.u.) that was sufficient to cause new ovulations and the loss of follicular oestradiol secretion. There was an immediate 3-4-fold rise in serum progesterone which returned to near prestimulation values (approximately 27 ng/ml) within 12 h in the presence of an implant containing oestradiol-17 beta. In the absence of oestradiol, serum progesterone continued to decline to reach low values (approximately 4 ng/ml) within 24 h and the original corpora lutea subsequently regressed. The administration of oestradiol 24 h after injection of hCG, when progesterone secretion was low, arrested any further decline in progesterone and then restored serum progesterone to normal values. This steroidogenic effect of oestradiol in vivo was a function of enhanced luteal steroidogenesis; corpora lutea removed and incubated for 12 h produced progesterone at high, linear rates, whereas the corpora lutea from animals that did not receive oestradiol produced low or insignificant quantities of progesterone in vitro. We conclude that hCG at these doses is compatible with continued responsiveness of the corpora lutea to oestrogen and that hCG produces its luteolytic effect primarily by ovulating follicles, thus stopping the secretion of the luteotrophic hormone, oestradiol.     

Progesterone production by luteal cells isolated from cynomolgus monkeys: effects of gonadotropin and prolactin during acute incubation and cell culture

Corpus luteum function in cynomolgus monkeys (Macaca fascicularis) during the menstrual cycle and immediately following parturition was evaluated through in vitro studies on progesterone production by dispersed luteal cells in the presence and absence of human chorionic gonadotropin (hCG) or human prolactin (hPRL). Luteal cells isolated between days 17-20 of the menstrual cycle secreted progesterone (P) during short-term incubation (21.6 +/- 1.2 ngP/ml/5 X 10(4) cells/3 hr, X +/- S.E., n = 7) and responded to the addition of 1-100 ng hCG with a significant (p less than 0.05) increase in P secretion. Cells removed the day of delivery secreted large, but variable (27.9-222 ng/ml, n = 4) amounts of P during short-term incubation. Moreover, hCG (100 ng/ml) stimulation of P production by cells at delivery (176 +/- 19% of control) was less than that of cells from the cycle of (336 +/- 65%). The presence of hPRL (2.5-5000 ng/ml) failed to influence P secretion by luteal cells during short-term incubation in the presence or absence of hCG. P production by luteal cells obtained following delivery declined markedly during 8 days of culture in Ham's F10 medium: 10% fetal calf serum. Continual exposure to 100 ng/ml of hCG or hPRL failed to influence P secretion through Day 2 of culture. Thereafter hCG progressively enhanced (p less than 0.05) P secretion to 613% of control levels at Day 8 of culture. In contrast, hPRL significantly increased P secretion (163% of control levels, p less than 0.05) between Day 2-4 of culture, but the stimulatory effect diminished thereafter. The data indicate that dispersed luteal cells from the cynomolgus monkey provide a suitable model for in vitro studies on the primate corpus luteum during the menstrual cycle, pregnancy, and the puerperium, including further investigation of the possible roles of gonadotropin and PRL in the regulation of luteal function in primates.     

Influences of epidermal growth factor and insulin-like growth factor-I on bovine blastocyst development in vitro

Experiments were carried out to investigate putative beneficial effects of adding epidermal growth factor (EGF) or insulin-like growth factor-I (IGF-I) for bovine embryo culture in chemically defined media. Presumptive zygotes (18 h post-insemination) were randomly assigned to culture treatments. In experiment 1, treatments involved additions of recombinant human EGF to provide concentrations of 0 ng (control), 1, 5, and 25 ng/ml. No differences were seen in numbers of 4-cell stage embryos between groups. A concentration of 5 ng/ml EGF but not 1 or 25 ng/ml during embryo culture improved percentages of 4-cell stage embryos reaching blastocysts compared to the control (P<0.05). Numbers of inner cell mass (ICM) cells and trophoblast cells of day 8 blastocysts were similar for the control and 5 ng/ml EGF-treated groups. In experiment 2, culture with recombinant human IGF-I in concentrations of 0 ng (control), 2, 10, and 50 ng/ml resulted in no differences in numbers of 4-cell stage embryos between groups. When compared to controls, IGF-I treatments at 10 and 50 ng/ml improved proportions of 4-cell stage embryos that reached blastocysts (P<0.05). In experiment 3, numbers of ICM cells of day 8 blastocysts were significantly higher after being cultured with 50 ng/ml of IGF-I compared to those of the controls (P<0.05). No additive effect of combining EGF (5 ng/ml) and IGF-I (50 ng/ml) was seen when results were compared to those following supplementation of the media with either EGF or IGF-I alone. In conclusion, both EGF and IGF-I could independently enhance bovine preimplantational development in chemically defined media and IGF-I but not EGF may play a mitogenic role during early bovine development.     

Oestradiol treatment increases the sensitivity of MCF-7 cells for the growth stimulatory effect of IGF-I

MCF-7 cells were grown in serum free medium (Dulbecco MEM without phenol red, supplemented with Costar SF-1 without insulin). Insulin was added as required and gave dose dependent growth stimulation at concentrations between 5 and 10,000 nM. Identical growth response curves were obtained for thymidine uptake and cell number. Oestradiol and insulin-like growth factor I (IGF-I) added individually both gave a dose dependent stimulation of cell growth in serum free medium containing 50 nM insulin. The growth stimulatory effect of oestradiol was to a large extent inhibited with suramine, a general inhibitor of growth factors, indicating that the effect of oestradiol was mediated through stimulating autocrine secretion of a growth factor.

To investigate a possible link between the effects of oestradiol and IGF-I, a specific IGF-I receptor antibody (IR-3), 10 μg/ml was used. These experiments were carried out with 2.5 nM insulin in the medium, a concentration at which insulin had no growth stimulatory effect. Stimulation was carried out for 18 h before assay of thymidine uptake. The effect of oestradiol was not significantly reduced by IR-3, indicating that IGF-I was not an autocrine mediator of oestradiol stimulation of cell growth under these conditions, whereas IR-3 extensively reduced growth stimulation by IGF-I. On long term stimulation (5 days) oestradiol had a marked stimulatory effect on cell number and IR-3 almost totally abrogated this effect. When oestradiol (1 nM) and IFG-I (2.5 nM) were added together, the combined effect on thymidine incorporation and cell number was significantly greater than additive. This synergistic effect on the IGF-I growth response was totally abolished by the IGF-I receptor antibody. The results suggest a cooperative interaction of oestradiol and IGF-I. It is concluded that growth stimulation of MCF-7 cells by long term treatment with oestradiol may be mediated through autocrine secretion of IGF-I.

The effect of short term stimulation of thymidine incorporation suggest that the growth response of oestradiol is more complex, and indicate that a cooperative interaction with IGF-I is involved, which is unrelated to stimulated autocrine secretion.     

In-vitro and in-vivo responsiveness of the corpus luteum of the mare to gonadotrophin stimulation

Dispersed horse luteal cells were used to evaluate the ability of horse LH, hCG and PMSG to stimulate progesterone secretion in vitro. Morphological characterization of these cells before gonadotrophin stimulation indicated the presence of two populations of cells based on cell diameters. In luteal cells incubated as suspended cells, horse LH and hCG stimulated (P less than or equal to 0.05) progesterone production at all levels of treatment. Stimulation of progesterone secretion by hCG was greater (P less than or equal to 0.05) than by horse LH over the range of concentrations utilized. When mares (N = 7) received an intramuscular injection of 1000 i.u. hCG on Days 3, 4 and 5 after the end of oestrus, there was an increase (P less than or equal to 0.05), in peripheral progesterone concentrations beginning on Day 7 and continuing until Day 14 compared with controls (N = 7). Peripheral progesterone concentrations continued to be elevated in hCG-treated mares for Days 15-30 after oestrus in those mares that conceived. Although treatment with hCG increased progesterone concentrations, it had no influence on anterior pituitary release of LH as measured by frequency and amplitude of LH discharge. We conclude that the mare corpus luteum is responsive to gonadotrophins in vitro and that exogenous hCG can enhance serum progesterone concentrations throughout the oestrous cycle and early pregnancy.     

Stimulation of progesterone secretion by cultured human granulosa cells with melatonin and catecholamines

Granulosa cells, aspirated from the follicles of patients undergoing treatment for in-vitro fertilization, were cultured in serum-supplemented medium. Adrenaline and noradrenaline stimulated a dose-related increase in progesterone secretion with a maximum stimulation at 10(-5) M, a response that was prevented by the beta-antagonist, propranolol. Adrenaline and hCG showed similar characteristics in their stimulation of progesterone secretion but there was no further increase in progesterone when the 2 compounds were added together. Melatonin stimulated progesterone secretion and, like adrenaline, this stimulation was prevented by propranolol. The ability of both adrenaline and melatonin to increase progesterone secretion was dependent on the degree of follicular development, as determined by peripheral oestradiol concentrations, on the day of laparoscopy. These results suggest that adrenaline and melatonin may have a physiological role in modulating luteal function and that melatonin may act by a beta-adrenergic-related mechanism.     

Gonadotropin-releasing hormone effects on placental hormones during gestation: II. Progesterone, estrone, estradiol and estriol

The release of progesterone (P), estrone (E1), estradiol (E2) and estriol (E3) from human placental tissue in vitro was found to be related to the gestational age of the placenta. The basal release of P, E1 and E2 on Day 1 of culture was highest from placentas of early gestation (9-13 wk). The release of P then declined, reaching a nadir by 15 wk, and continued at that level. The release of E1 and E2, reached a nadir at 17 weeks, and then again increased by term. In contrast, the basal release of E3 increased with increasing gestational age of the placenta. Thus, it appears that differing factors may influence placental P, E1, E2 and E3 production. In addition, the effect of synthetic gonadotropin-releasing hormone (GnRH) on these hormonal releases was studied. The stimulation of P by GnRH was greatest in placentas of 16 and 17 wk of gestation after extended culture when the basal release of P had declined. As much as a 240-fold increase was observed on the eighth day of culture. A large stimulation of P (32-fold) was also observed in the term placental cultures. A stimulation of E1 and E2 by GnRH was observed during the initial days of culture and in mid-gestational placental cultures (16-17 wk). A stimulation of E2 only was also observed at 13-15 wk and at term. A stimulation of E3 was observed in certain individual placentas. A correlation of the P and human chorionic gonadotropin (hCG) response to GnRH stimulation was noted, as well as an inverse relation of estrogens and hCG stimulation by GnRH. These data demonstrate that steroidogenic competence of the placenta differs with gestational age and that GnRH can influence steroid release. The degree and pattern of response to GnRH varied with the gestational age of the placenta and its endocrine milieu.     

Progesterone pretreatment has a direct effect on GnRH-induced preovulatory follicles to determine their ability to develop into normal corpora lutea in anoestrous ewes

In two experiments carried out during seasonal anoestrus, Romney Marsh ewes were treated with small-dose (250 ng) multiple injections of GnRH at 2-h intervals with and without progesterone pretreatment. In Exp. 1, 8/8 progesterone-primed ewes ovulated and produced functionally normal corpora lutea compared with 2/9 non-primed ewes. Follicles were recovered from similarly treated animals 18 or 28 h after the start of GnRH treatment (at least 14 h before the estimated time of the LH peak) and assessed in terms of diameter, granulosa cell number, oestradiol, testosterone and progesterone concentrations in the follicular fluid, oestradiol production in vitro and binding of 125I-labelled hCG to granulosa and theca. There were no significant differences in any of these measures in 'ovulatory' follicles recovered from the progesterone-pretreated compared to non-pretreated animals. In Exp. 2, follicles were removed from similar treatment groups just before and 2 h after the start of the LH surge. Unlike 'ovulatory' follicles recovered from the non-pretreated ewes, those recovered from progesterone-pretreated ewes responded to the LH surge by significantly increasing oestradiol secretion (P less than 0.01) and binding of 125I-labelled hCG (P less than 0.05) to granulosa cells. Overall there was also more (P less than 0.05) hCG binding to granulosa and theca cells from progesterone-pretreated animals. Non-ovulatory follicles recovered from progesterone-primed ewes had more (P less than 0.05) binding of 125I-labelled hCG to theca and a higher testosterone concentration in follicular fluid (P less than 0.05) than did those from non-primed ewes. These results suggest that inadequate luteal function after repeated injections of GnRH may be due to a poor response to the LH surge indicative of a deficiency in the final maturational stages of the follicle.     

Interactions of prostaglandins with subpopulations of ovine luteal cells. I. Stimulatory effects of prostaglandins E1, E2 and I2

Preparations of small and large steroidogenic cells from enzymatically dispersed ovine corpora lutea were utilized to study the in vitro effects of luteinizing hormone (LH) and prostaglandins (PG) E1, E2 and I2. Cells were allowed to attach to culture dishes overnight and were incubated with either LH (100 ng/ml), PGE1, PGE2, or PGI2 (250 ng/ml each). The secretion of progesterone by large cells was stimulated by all prostaglandins tested (P less than 0.05) while the moderate stimulation observed after LH treatment was attributable to contamination of the large cell population with small cells. Prostaglandins E1 and E2 had no effect on progesterone secretion by small cells, while LH was stimulatory at all times (0.5 to 4 hr) and PGI2 was stimulatory by 4 hr. Additional studies were conducted to determine if the effects of PGE2 upon steroidogenesis in large cells were correlated with stimulated activity of adenylate cyclase. In both plated and suspended cells PGE2 caused an increase (P less than 0.05) in the rate of progesterone secretion but had no effect upon the activity of adenylate cyclase or cAMP concentrations within cells or in the incubation media. Exposure of luteal cells to forskolin, a nonhormonal stimulator of adenylate cyclase, resulted in marked increases in all parameters of cyclase activity but had no effect on progesterone secretion. These data suggest that the actions of prostaglandins E1, E2 and I2 are directed primarily toward the large cells of the ovine corpus luteum and cast doubt upon the role of adenylate cyclase as the sole intermediary in regulation of progesterone secretion in this cell type.     

Effects of human chorionic gonadotrophin on contractile activity of steroid-primed pig myometrium in vitro

We examined the effects of (a) oestrogen and progesterone on concentrations of luteinizing hormone/human chorionic gonadotrophin (LH/hCG) receptors in uterine smooth muscle in vivo and (b) hCG on spontaneous myometrial contractions in vitro. Ovariectomized gilts received 2 ml corn oil (control; n = 5), 2 mg oestradiol benzoate (n = 6) or 20 mg progesterone (n = 5) for 5 days. Gilts were hysterectomized 8 h after the last injection and longitudinal sections of myometrium were incubated in modified Krebs' solution with 0 or 10 i.u. of hCG (n = 10/gilt) for 4 h at 37 degrees C in 95% O2:5% CO2. After incubation, myometrial sections were placed in a tissue chamber perfused with Krebs' solution and mechanical activity was recorded for 30 min. Cell membrane fractions were prepared from myometrial tissue not used for in-vitro studies and analysed for LH/hCG receptors. Treatment with oestradiol benzoate increased (P less than 0.01) the number of LH/hCG-binding sites compared with gilts receiving corn oil or progesterone. Incubation of myometrium with hCG reduced (P less than 0.01) the frequency and amplitude of spontaneous uterine contractions in gilts treated with oestradiol benzoate. In contrast, hCG had no effect (P greater than 0.05) on the pattern of myometrial contractions in gilts given corn oil or progesterone. These results indicate that oestradiol promotes the synthesis of LH/hCG receptors in pig myometrium and incubation of oestrogen-primed tissue with hCG has a quiescent effect on myometrial contractility.     

Gonadotropins increase concentrations of immunoreactive insulin-like growth factor-I in porcine follicular fluid in vivo

To evaluate the regulation of ovarian insulin-like growth factor-I (IGF-I) during follicular growth in vivo, we measured the concentration of this peptide in follicular fluid (FFL) of immature gilts during the induction of follicular development by pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). FFL concentrations of immunoreactive (i) IGF-I were compared with those of intrafollicular steroids and with concentrations of iIGF-I, estradiol (E2), and porcine growth hormone (GH) in serum. PMSG, administered at Time 0, induced a significant (p less than 0.01), time-dependent increase in intrafollicular iIGF-I that peaked 72 h after administration of the hormone, before the administration of hCG. During the first 72 h, the changes in ovarian iIGF-I paralleled those for progesterone and E2. After the administration of hCG at 72 h, FFL levels of E2 fell, those of iIGF-I remained constant, and progesterone rose. Serum E2 concentrations paralleled those in FFL. Since serum GH and IGF-I levels rise during spontaneous puberty in some species, these levels were also monitored. However, a significant treatment effect on serum GH and iIGF-I was not demonstrated. In summary, ovarian concentrations of iIGF-I are increased by gonadotropic hormones in vivo. The absence of concomitant changes in circulating levels of iIGF-I and GH suggests that the gonadotropin effects are exerted directly on the ovary. These results, together with more abundant data regarding secretion and action of IGF-I in cultured granulosa cells, suggest that IGF-I may function in an autocrine or paracrine fashion to amplify the actions of gonadotropins at an ovarian level.     

Glucocorticoid influence on porcine granulosa cell IGF-I and steroid hormone production in vitro

The effect of cortisol on granulosa cell (GC) insulin-like growth factor I (IGF-I) synthesis, and IGF-mediated steroid production was examined at various stages of follicle maturation. Granulosa cells were recovered from gilts on Days 14, 18, and 20 of the estrous cycle, while luteinizing GC were recovered on Day 21, just prior to ovulation. The cells were cultured in serum-free medium with increasing concentrations of cortisol (0, 1, 10, and 100 microg/mL) for 5 d with or without IGF-I stimulation (10 ng/mL). During culture all cells were supplemented with FSH and androstenedione (A4). Cellular IGF-I, progesterone (P4) and estradiol-17beta (E2) production was determined by specific radioimmunoassays (RIA), and cell proliferation was assessed. Granulosa cell IGF-I and steroid hormone synthesis increased (P<0.05) with follicle maturation. Direct exposure to high cortisol concentrations, however, altered both IGF-I synthesis and action. Cortisol treatment lowered (P<0.05) IGF-I production by GC recovered on Days 18, 20, and 21. Furthermore, it reduced (P<0.05) IGF-stimulated P4 synthesis at all stages and decreased (P<0.05) IGF-stimulated E2 synthesis by cells recovered on Day 14. In contrast, cortisol enhanced (P<0.05) FSH-stimulated P4 production by GC collected on Days 14 and 18. The opposing effects on FSH and IGF-I action indicate that cortisol did not promote an overall suppressive effect on cell function, nor did it impair cell proliferation. Hence, these results demonstrate that elevated cortisol concentrations can disrupt both IGF-I synthesis and IGF-mediated actions by porcine GC under in vitro conditions, and that specific disruptions are dependent on the stage of follicle maturation.