Elevated peripheral concentrations of LH block ovulation and increase thecal and serum progesterone in the hamster

Insertion of osmotic minipumps containing 1 mg ovine LH on Day 1 (oestrus) elevated circulating serum concentrations of LH, progesterone and androstenedione when compared with values at pro-oestrus. Ovulation was blocked for at least 2 days at which time there were twice the normal numbers of preovulatory follicles. Follicular and thecal progesterone production in vitro was elevated when compared with that in pro-oestrous controls. Follicular and thecal androstenedione production in vitro was lower than in controls even though serum concentrations of androstenedione were elevated; the higher androstenedione values may be due to the increase in number of preovulatory follicles when compared with pro-oestrous controls. Follicles from LH-treated hamsters aromatized androstenedione to oestradiol and follicular production of oestradiol was similar to that in pro-oestrous follicles despite low follicular androstenedione production in the LH-treated group. Treatment with 20 i.u. hCG on Days 4 or 6 after insertion of an LH osmotic minipump on Day 1 induced ovulation of approximately 30 ova, indicating that the blockade of ovulation was not due to atresia of the preovulatory follicles. Serum progesterone concentrations on Days 2, 4 and 6 in LH-treated hamsters were greater than 17 nmol/l, suggesting that the blockade of ovulation might have been due to prevention of the LH surge by high serum progesterone concentrations.     

Exogenous progesterone reduces follicular prostaglandin E and 6-keto prostaglandin F-1 alpha in the cyclic hamster

In cyclic hamsters, exogenous progesterone (100 micrograms) administered s.c. at 09:00 h on the day of dioestrus II reduced prostaglandin (PG) E and 6-keto PGF-1 alpha but not PGF concentrations in preovulatory follicles measured at 09:00 h of pro-oestrus. The injection of 10 micrograms ovine LH (NIADDK-oLH-25) concurrently with 100 micrograms progesterone on dioestrus II prevented the decline in follicular PGE and 6-keto PGF-1 alpha values. Administration of LH alone did not significantly alter follicular PG concentrations. Inhibition of follicular PGE accumulation by progesterone was due to a decline in granulosa PGE concentration and not thecal PGE. Progesterone administration also reduced follicular oestradiol concentrations. Administration of oestradiol-17-cyclopentanepropionate (ECP) (10 micrograms) with progesterone did not prevent the decline in follicular PGE and 6-keto PGF-1 alpha but did increase follicular PGF concentrations. However, ECP given alone on dioestrus II reduced follicular PGE and increased PGF concentrations in preovulatory follicles on pro-oestrus. It is concluded that exogenous progesterone administered on dioestrus II inhibits granulosa PGE and 6-keto PGF-1 alpha accumulation in preovulatory follicles, probably by reducing serum LH concentrations, and that the granulosa cells, which are LH-dependent, are a major source of follicular PGE.     

Relationship between the preovulatory luteinizing hormone (LH) surge and androstenedione synthesis of preantral follicles in the cyclic hamster: detection by in vitro responses to LH

Preantral follicles of cyclic hamsters were isolated on proestrus, estrus and diestrus I, incubated for 3 h in 1 ml TC-199 containing 1 microgram ovine luteinizing hormone (LH) (NIH-S22), and the concentrations of progesterone (P), androstenedione (A) and estradiol (E2) determined by radioimmunoassay. At 0900-1000 h on proestrus (pre-LH surge) preantral follicles produced 2.4 +/- 0.3 ng A/follicle per 3 h, less than 100 pg E2/follicle and less than 250 pg P/follicle. At the peak of the LH surge (1500-1600 h) preantral follicles produced 1.8 +/- 0.2 ng P and 1.9 +/- 0.1 A and less than 100 pg E2/follicle. After the LH surge (1900-2000 h proestrus and 0900-1000 h estrus) preantral follicles were unable to produce A and E2 but produced 4.0 +/- 1.0 and 5.0 +/- 1.1 ng P/follicle, respectively. By 1500-1600 h estrus, the follicles produced 8.1 +/- 3.1 ng P/follicle but synthesized A (1.6 +/- 0.2 ng/follicle) and E2 (362 +/- 98 pg/follicle). On diestrus 1 (0900-1000 h), the large preantral-early antral follicles produced 1.9 +/- 0.3 ng A, 2.4 +/- 0.4 ng E2 and 0.7 +/- 0.2 ng P/follicle. Thus, there was a shift in steroidogenesis by preantral follicles from A to P coincident with the LH surge; then, a shift from P to A to E2 after the LH surge. The LH/follicle-stimulating hormone (FSH) surges were blocked by administration of 6.5 mg phenobarbital (PB)/100 g BW at 1300 h proestrus. On Day 1 of delay (0900-1000 h) these follicles produced large quantities of A (2.2 +/- 0.2 ng/follicle) and small amounts of E2 (273 +/- 27 pg/follicle) but not P (less than 250 pg/follicle).(ABSTRACT TRUNCATED AT 250 WORDS)     

Alteration of follicular steroid secretion and thecal morphology after in-vitro exposure of individual pig follicles to follicle regulatory protein

Medium-sized (4-6 mm) pig follicles were incubated for 10 h and then examined via light microscopy. Treatment with pig FSH resulted in significantly increased concentrations of oestradiol, testosterone, androstenedione and progesterone in the medium. Follicle regulatory protein (FRP) alone (1 micrograms/ml) decreased follicular secretion of oestradiol (56%) and progesterone (53%) but stimulated the secretion of testosterone (226%) and androstenedione (139%). In the presence of 1 ng FSH/ml, the inhibitory effect of FRP on oestradiol secretion was enhanced (74%), progesterone values were unaffected and secretion of testosterone and androstenedione were reduced by 66% and 53%, respectively. All effects of FRP were fully overcome by 1 micrograms FSH/ml. The incidence of atresia, as defined by granulosa cell pycnosis, was similar in all treatment groups (1-3 of 10 follicles per group). The remaining follicles had intact granulosa cells. However, follicles treated with FRP (1 micrograms/ml) + FSH (1 ng/ml) had pycnotic nuclei in the theca interna cells, in the presence of an intact stratum granulosum. External exposure of follicles to FRP may not reflect physiological conditions since, in vivo, thecal pycnosis is never observed before granulosa cell pycnosis. However, the present results indicate that FRP is potentially capable of altering both follicular morphology and steroidogenesis. We suggest that FSH and FRP interact to affect follicular development.     

Hormonal requirements for the growth and differentiation of hamster preantral follicles in long-term culture

Preantral follicles from pro-oestrous and oestrous hamsters were isolated enzymically (Stages 1-5) and by microdissection (Stage 6) and cultured for up to 168 h in the absence or presence of 100 ng ovine FSH or LH separately or combined or 1 or 10 micrograms progesterone or estradiol-17 beta in serum-free defined medium and exposed to 1 muCi [3H]thymidine for 24 h before termination. In the presence of insulin and hydrocortisone but not gonadotrophins, the morphology of follicles from pro-oestrous animals at Stages 1-4 (1-4 layers granulosa cells; no theca) were unaffected for up to 48 h whereas for Stages 5 (5-6 layers granulosa cells and developing theca) and 6 (7-8 layers granulosa cells and theca), atresia was prominent by 24 h. FSH significantly reduced the percentage of atretic follicles in Stages 1-5 throughout the culture period; but was effective only up to 96 h for Stage-6 follicles. LH was also effective, albeit to a lesser extent. FSH increased follicular labelling indexes during every 24-h labelling period and, during a pulse-chase period, follicular DNA content and granulosa cell numbers. FSH, but not LH, induced differentiation by 96 h of preantral follicles at Stage 6 into small antral stages (Stages 7-8). FSH and LH together induced almost the same effect as FSH alone. However, neither progesterone nor oestradiol had any significant long-term effects on DNA synthesis and oestradiol induced atresia beyond 24 h. Both FSH and LH induced follicular maturation in vitro as evident from increases in progesterone, androstenedione and oestradiol production. Follicles (Stages 1-4) collected from oestrous hamsters responded to FSH to a lesser extent than did those from pro-oestrous animals, possibly because of in-vivo exposure to periovulatory changes in gonadotrophins; however, an antrum formed in Stage-6 follicles by 72 h.     

Effects of season and phase of the estrous cycle on steroidogenesis and LH-FSH sensitivity of large ovine follicles perfused in vitro

The aims of this study were to compare stero?dogenesis (progesterone, androstenedione and estradiol production) and response to LH and FSH challenge by whole perifused follicles 4 to 5.5 mm in diameter, obtained at different periods of the breeding season (onset, middle, end), during anestrus and the luteal phase. We have observed that all follicles do not have the same stero?dogenetic potential and do not respond with the same intensity to LH and FSH. At the middle of the breeding season, LH and FSH supplementation was ineffective in increasing progesterone secretion by follicles (0.19+/-0.05 vs. 0.20+/-0.03 ng/mL). In contrast, gonadotrophin challenge elicited significant (P<0.05) increases in androstenedione (0.94+/-0.34 vs. 0.35+/-0.09 ng/mL) and estradiol (120+/-11 vs. 49+/-10 pg/mL) production immediately after its administration. At the onset of the breeding season, steroidogenesis was identical under both basal and gonadotrophin-stimulated conditions unlike that in middle of the breeding season. However follicles were more sensitive to the gonadotrophin challenge in terms of estradiol production than those collected at the middle of the breeding season (220+/-45 vs. 120+/-11 pg/mL). Follicles obtained at the end of the breeding season featured higher progesterone (2.61+/-0.81 vs. 0.19+/-0.05 ng/mL; P<0.05) and lower estradiol production (10+/-3 vs. 49+/-10 pg/mL; P<0.05) that was not influenced by LH and FSH. Basal androstenedione secretion was comparable to that observed at the middle of the breeding season (0.42+/-0.10 vs. 0.35+/-0.09 ng/mL), but the response to stimulation was significantly higher (1.82+/-0.61 vs. 0.94+/-0.34 ng/mL; P<0.05). In anoestrus and the luteal phase, follicles presented higher progesterone and androstenedione and lower estradiol concentrations (P<0.05) compared with those obtained during the follicular phase at the middle of the breeding season. In the luteal phase, follicles remained capable of responding to LH-FSH challenge by increasing estradiol secretion (9+/-1 before and 21+/-6 pg/mL after LH-FSH; P<0.05). In contrast, in the luteal phase, estradiol production was not increased by LH-FSH challenge (7+/-2 vs. 12+/-4 pg/mL).     

Differential regulation of pig theca cell steroidogenesis by LH, insulin-like growth factor I and granulosa cells in serum-free culture

The regulation of pig theca cell steroidogenesis was studied by the development of a physiological serum-free culture system, which was subsequently extended to investigate potential theca-granulosa cell interactions. Theca cells were isolated from antral follicles 6-9 mm in diameter and the effects of plating density (50-150x10(3) viable cells per well), LH (0.01-1.0 ng ml(-1)), Long R3 insulin-like growth factor I (IGF-I) (10, 100 ng ml(-1)) and insulin (1, 10 ng ml(-1)) on the number of cells and steroidogenesis were examined. The purity of the theca cell preparation was verified biochemically and histologically. Co-cultures contained 50x10(3) viable cells per well in granulosa to theca cell ratio of 4:1. Wells containing granulosa cells only were supplemented with 'physiological' doses of androstenedione or 100 ng ml(-1). Oestradiol production by co-cultures was compared with the sum of the oestradiol synthesized by granulosa and theca cells cultured separately. Oestradiol and androstenedione production continued throughout culture. High plating density decreased steroid production (P < 0.01). LH increased androstenedione (P < 0.001) and oestradiol (P < 0.05) synthesis and the sensitivity of the cells increased with time in culture. Oestradiol production was increased by 10 ng IGF-I ml(-1) (P < 0.001) but androstenedione required 100 ng ml(-1) (P < 0.001). Co-cultures produced more oestradiol than the sum of oestradiol synthesized by theca and granulosa cells cultured separately (P < 0. 001), irrespective of the androstenedione dose. This serum-free culture system for pig theca cells maintained in vivo steroidogenesis and gonadotrophin responsiveness. Thecal androstenedione and oestradiol production were differentially regulated and were primarily stimulated by LH and IGF-I, respectively. Theca-granulosa cell interactions stimulated oestradiol synthesis and this interaction was mediated by factors additional to the provision of thecal androgen substrate to granulosa cells.     

Steroid production by isolated theca and granulosa cells after initiation of atresia in the hamster

Hypophysectomized PMSG-primed hamsters were injected with PMSG antiserum and the theca and granulosa cells of the resulting atretic follicles were incubated in vitro. In the absence of added hormone, 17 alpha-hydroxyprogesterone and oestradiol production was not detectable in granulosa cells collected and incubated at 0, 12 and 24 h after antiserum. Progesterone production was not detected in control incubations at 0 h but was measurable with cells collected at 12 h after PMSG antiserum. When incubated with androstenedione or pregnenolone (10 ng/ml for each) 17 alpha-hydroxyprogesterone and progesterone production by granulosa cells were significantly increased at 0, 12 and 24 h after antiserum. Granulosa cells were capable of aromatizing androstenedione to oestradiol at all times examined. At 0 and 12 h after antiserum to PMSG, isolated thecal shells produced androstenedione. LH stimulation caused increased androstenedione production in all thecae at 0 h, in 50% of the thecae at 12 h and in none at 24 h after antiserum. Thecal shells produced 17 alpha-hydroxyprogesterone in response to LH at 0, 12 and 24 h after antiserum, and produced progesterone at all times examined. Thecae also responded to LH with increased progesterone production up to 72 h after antiserum. These experiments demonstrate that one important steroidogenic event in atresia may be the loss of activity of C 17,20 lyase in the theca leading to loss of substrate (androstenedione) for granulosa cell aromatization, although aromatase activity is present until at least 24 h after the induction of atresia.     

In vitro steroidogenesis by dissociated rat follicles, primary to antral, before and after injection of equine chorionic gonadotropin.

Prepubertal female rats were injected s.c. with 5.0 IU eCG, and ovaries were collected 24 and 48 h post-eCG, on Day 25, as well as from an untreated group also on Day 25. Large antral follicles were manually dissected, and the ovarian remnants were incubated with collagenase overnight to liberate preantral follicles from adhering stromal cells. The viability of the follicles was established by normal histology and lack of pyknotic granulosa cells (GCs) and by their ability to secrete steroids. After a 1-h baseline incubation, either 10 ng LH or 100 ng FSH was added for an additional hour, and the media-before and after gonadotropin administration-were used to measure progesterone, androstenedione, and estradiol by RIA. A distinct hierarchy existed in steroid synthesis, with the maximal production by the largest (700 microm) antral follicles. The major steroid that had accumulated after addition of LH at 48 h post-eCG was androstenedione (1099 pg/follicle per hour), followed by equal amounts of progesterone (155 pg/follicle per hour) and estradiol (191 pg/follicle per hour). There was a precipitous drop in steroid production by 550-microm and 400-microm antral follicles, especially in estradiol for the latter-sized follicles (0.08 pg/follicle per hour). Preantral follicles also produced progesterone and androstenedione after addition of LH. For example, follicles 222 microm in diameter with 4-5 layers of GCs and well-developed theca responded to LH at 48 h post-eCG by accumulating androstenedione (37 pg/follicle per hour) and progesterone (6 pg/follicle per hour) but negligible estradiol. The smallest follicles secreting steroids, 110-148 microm in diameter, had 2-4 layers of GCs. However, primary follicles (1 layer of GCs and no theca) did not synthesize appreciable amounts of any steroid. Although small preantral follicles were consistently stimulated by LH, FSH was ineffective. This result differs from findings in the hamster showing that intact preantral follicles with 1-4 layers of GCs and no theca respond to FSH by secreting progesterone in vitro (Roy and Greenwald, Biol Reprod 1987; 31:39-46). The technique developed to collect intact rat follicles should be useful for numerous investigations.     

Effect of LH on steroidogenesis by hamster follicles isolated at defined stages of development

Multiple follicular growth was stimulated in groups of immature female hamsters by the administration of PMSG. The ovaries were removed 72-78 or 96-102 h later. The animals at 96-102 h were subdivided according to whether the concentration of plasma progesterone was within the range of the concentrations found at the previous period (0.45-13.18 nmol/l; Group I) or above (Group II). Thecal and granulosa cells from batches of isolated follicles (591-740 micron) were cultured together in the presence or absence of LH and the products of steroidogenesis were measured in the culture fluid. The results showed that there was a significant increase in the production of progesterone and 17 alpha-hydroxyprogesterone in follicles from Groups I and II at 96-102 h. In the presence of LH there was a significant increase in the yield of progesterone and 17 alpha-hydroxyprogesterone over the control values at all time periods. There were, however, reductions in the yields of 17 alpha-hydroxyprogesterone and androstenedione in the presence of LH at 96-102 h compared with 72-78 h and in Group II compared with Group I, although the concentrations of 17 alpha-hydroxyprogesterone were approximately 40-fold those of androstenedione. In addition, the production of oestradiol decreased significantly in follicles from both groups at 96-102 h and was reduced further in the presence of LH. It is concluded that the major rate-limiting step of steroidogenesis in hamster follicles undergoing the oestrogen-progestagen shift involves the side-chain cleavage of 17 alpha-hydroxyprogesterone.     

Interleukin-1 alpha increases thecal progesterone production of preovulatory follicles in cyclic hamsters

Adult cyclic hamsters were used to study the effects of interleukin-1 alpha (IL-1 alpha) on in vitro steroidogenesis in preovulatory follicles. IL-1 alpha increased progesterone secretion by preovulatory follicles during a 24-h incubation in RPMI-1640 medium containing hCG (100 mIU/ml) (progesterone levels: 17.5 +/- 2.2 vs. 10.6 +/- 1.9 ng/follicle/ml, p less than 0.05). IL-1 alpha alone had no effect on follicular steroidogenesis. The source of increased progesterone secretion was the thecae (9.8 +/- 1.0 vs. 5.8 +/- 0.4 ng/2 thecae/ml, p less than 0.01) and not the granulosa cells (6.6 +/- 0.2 vs. 6.8 +/- 0.5 ng/20,000 viable granulosa cells/ml). IL-1 alpha also stimulated production of testosterone in thecae of preovulatory follicles. The follicular progesterone increase was dependent on the time of incubation and dose of IL-1 alpha. IL-1 alpha at 5-50 U/ml maximally stimulated progesterone production in the preovulatory follicles, and no significant effect of IL-1 alpha was observed until the 12th hour of incubation. The effects of IL-1 alpha on in vitro steroidogenesis in preantral follicles, experimentally induced atretic preovulatory follicles, and newly formed corpora lutea were examined. IL-1 alpha in the presence of hCG also significantly increased progesterone secretion by atretic preovulatory follicles. In the incubation of preantral follicles or newly formed corpora lutea, however, IL-1 alpha did not alter steroidogenesis. These results indicate that IL-1 alpha stimulates progesterone secretion by preovulatory follicles and that the target tissue for this effect is the thecal layer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of pentobarbitone sodium and bromocriptine on follicular oestradiol production in the rat

Injection of an ovulation-blocking dose of pentobarbitone sodium given in the early afternoon of pro-oestrus in rats decreased follicular oestradiol production in vitro the next day (2.42 +/- 0.11 ng/4 h/follicle in pro-oestrous rats, 0.49 +/- 0.04 ng/4 h/follicle in pentobarbitone-treated rats). Pentobarbitone, given 1 day earlier (at dioestrus II), prevented the increase in oestradiol production that normally occurs between di-oestrus II and pro-oestrus. Injection of a subovulatory amount of hCG (0.5 i.u.) given after pentobarbitone injection inhibited the decrease in follicular oestradiol production induced by pentobarbitone. The pentobarbitone-induced decrease in oestradiol production was also prevented by bromocriptine (1 mg) given at di-oestrus II (15:00 h) and pro-oestrus (09:00 h). Bromocriptine is an effective inhibitor of prolactin secretion and this suggests therefore that the decrease in follicular oestradiol production after pentobarbitone is due to the preovulatory surge of prolactin. However, pretreatment with bromocriptine also inhibited the effect of pentobarbitone on oestradiol production when pentobarbitone was given at di-oestrus II. Moreover, when ergocornine (another inhibitor of prolactin secretion) was used instead of pentobarbitone to block ovulation, follicular oestradiol production was also decreased the next day. In contrast to bromocriptine, ergocornine was not able to prevent the pentobarbitone-induced decrease in follicular oestradiol production. These results indicate that the decrease in follicular oestradiol production after pentobarbitone injection is due to inhibition of the serum concentrations of LH rather than the preovulatory surge of prolactin. How bromocriptine (but not ergocornine) prevents the pentobarbitone-induced decrease in oestradiol production is not clear.     

Improvement of follicular development rather than gonadotrophin secretion by thyroxine treatment in infertile immature hypothyroid rdw rats

Despite extensive study of reproductive abnormalities in female hypothyroid animals, little is known of folliculogenesis and gonadotrophin secretion in spontaneously hypothyroid animals, especially in response to exogenous hormone treatment. In this study, follicular development and plasma hormone concentrations in the presence or absence of thyroxine and eCG treatment were investigated in infertile immature spontaneously hypothyroid rdw rats. Administration of thyroxine once a day from day 21 to day 29 after birth resulted in increases in body weight (P < 0.001) and ovary mass on day 30 (P < 0.01). Similar populations of both healthy and atretic antral follicles ranging from 101 to 400 micrometer in diameter were observed in control rdw and normal rats. In rdw rats, thyroxine treatment markedly increased the number of healthy antral uniovular follicles 101-400 or > 550 micrometer in diameter in the absence or presence of eCG, respectively. Combined treatment of thyroxine and eCG in rdw rats also markedly increased the number of healthy antral biovular follicles. Thyroxine treatment did not affect the population of atretic antral follicles, but resulted in decrease in the number of atretic large antral follicles (> 400 microm) in the presence of eCG. Plasma oestradiol concentrations in rdw rats given both thyroxine and eCG were significantly higher than they were in rdw rats given eCG alone (P < 0.001). There were no significant differences in plasma FSH concentrations on day 28 between rdw (10.7 +/- 1.6 ng ml(-1)) and normal rats (12.0 +/- 1.4 ng ml(-1); P > 0. 05). Although there were no significant differences in plasma LH concentrations between control rdw (1.9 +/- 0.1 ng ml(-1)) and normal rats on day 30 (1.8 +/- 0.1 ng ml(-1); P > 0.05), eCG treatment increased plasma LH to a peak concentration 52 h after injection in normal (24.9 +/- 2.4 ng ml(-1)) but not in rdw rats treated with thyroxine (4.8 +/- 0.3 ng ml(-1); P < 0.05). In conclusion, the results of the present study indicate that thyroxine treatment improves follicular development but does not rescue the defect of the preovulatory surge of LH in eCG-primed rdw rats.     

Relationship between concentrations of cortisol in ovarian follicular fluid and various biochemical markers of follicular differentiation in cyclic and anovulatory cattle

Concentrations of cortisol were determined in pooled fluid of small (less than 10 mm) and large (greater than or equal to 10 mm) follicles of cyclic cattle (Exp. 1), and in fluid of the largest follicle of 17 post-partum anovulatory cows (Exp. 2). In Exp. 1, concentrations of cortisol in small follicles were greater (P less than 0.05) than in large follicles (14.7 versus 13.2 ng/ml), and varied significantly with stages of the cycle; small and large follicles had the highest cortisol concentration during the early luteal phase of the cycle. Large follicles had 2-fold greater concentrations of oestradiol than did small follicles, whereas small follicles had 2-fold greater concentrations of androstenedione than did large follicles. Across pools of follicular fluid, cortisol concentrations were correlated only to androstenedione concentrations (r = 0.65, P = 0.07). In Exp. 2, concentrations of cortisol did not significantly differ between oestrogen-active (oestradiol greater than progesterone in follicular fluid) and oestrogen-inactive (progesterone greater than oestradiol) follicles, although oestrogen-active follicles had a 24-fold greater concentration of oestradiol than did oestrogen-inactive follicles. Cortisol concentrations were correlated to hCG binding capacity of thecal cells (r = -0.35, P = 0.08) and to follicular diameter (r = 0.45, P less than 0.05). These results suggest that normally fluctuating concentrations of cortisol in follicular fluid of cattle play little or no active role in follicular differentiation in vivo.     

Changes in oestrogen biosynthesis in preovulatory rat follicles after blockage of ovulation with pentobarbitone sodium

Follicles isolated 1 and 2 days after pentobarbitone sodium injection at pro-oestrus were incubated with C-21 steroids or aromatizable C-19 steroids. Addition of testosterone or androstenedione (50 ng/ml) increased oestradiol production by ovulation-blocked follicles, while addition of progesterone or 17 alpha-hydroxyprogesterone was ineffective. LH-stimulated oestradiol production was lower in follicles isolated 1 and 2 days after pentobarbitone sodium injection, but progesterone production was elevated compared to pro-oestrous follicles. Total steroidogenesis, measured by pregnenolone production in the presence of inhibitors of pregnenolone conversion, did not differ on the 3 days. The activity of C17-20 lyase, measured in follicular homogenates, decreased between pro-oestrus and the next day. Aromatase and 17 alpha-hydroxylase activities also decreased, but the activity of these enzymes was always considerably higher than that of C17-20 lyase. It is concluded that the decrease in follicular oestradiol production after injection of pentobarbitone sodium was due primarily to a decrease in the activity of the enzyme system responsible for the conversion of 17 alpha-hydroxyprogesterone to androstenedione, thereby limiting the amount of substrate available for aromatization to oestrogen.     

Hormone concentrations before and after semen-induced ovulation in the bactrian camel (Camelus bactrianus)

During the follicular phase of bactrian camels, basal concentrations of LH were 2.7 +/- 1.2 ng/ml. By 4 h after insemination peak values of 6.9 +/- 1.0 ng/ml occurred. In addition, a smaller LH peak (5.4 +/- 2.5 ng/ml) appeared 1 day before regression of the follicle began in unmated camels. During the follicular phase peripheral plasma progesterone values were low (0.36 +/- 0.28 ng/ml), but values increased to reach 1.73 +/- 0.74 ng/ml at 3 days and 2.4 +/- 0.86 ng/ml at 7 days after ovulation. Plasma oestradiol-17 beta concentrations were 26.8 +/- 9.0 pg/ml during the follicular phase and 30.8 +/- 5.1 pg/ml when the follicle was maximum size. Values fell after ovulation but rose to 29.8 +/- 6.5 pg/ml 3 days later.     

Endocrine and ovarian responses associated with the first-wave dominant follicle in cattle.

To examine endocrine and biochemical differences between dominant and subordinate follicles and how the dominant follicle affects the hypothalamic-pituitary-ovarian axis in Holstein cows, the ovary bearing the dominant follicle was unilaterally removed on Day 5 (n = 8), 8 (n = 8), or 12 (n = 8) of synchronized estrous cycles. Follicular development was followed daily by ultrasonography from the day of detected estrus (Day 0) until 5 days after ovariectomy. Aromatase activity and steroid concentrations in first-wave dominant and subordinate follicles were measured. Intact dominant and subordinate follicles were cultured in 4 ml Minimum Essential Medium supplemented with 100 microCi 3H-leucine to evaluate de novo protein synthesis. Five days after unilateral ovariectomy, cows were resynchronized and the experiment was repeated. Follicular growth was characterized by the development of single large dominant follicles, which was associated with suppression of other follicles. Concentrations of estradiol-17 beta (E2) in follicular fluid and aromatase activity of follicular walls were higher in dominant follicles (438.9 +/- 45.5 ng/ml; 875.4 +/- 68.2 pg E2/follicle) compared to subordinate follicles (40.6 +/- 69.4 ng/ml; 99.4 +/- 104.2 pg E2/follicle). Aromatase activity in first-wave dominant follicles was higher at Days 5 (1147.1 +/- 118.1 pg E2/follicle) and 8 (1028.2 +/- 118.1 pg E2/follicle) compared to Day 12 (450.7 +/- 118.1 pg E2/follicle). Concentrations of E2 and androstenedione in first-wave dominant follicles were higher at Day 5 (983.2 +/- 78.2 and 89.5 +/- 15.7 ng/ml) compared to Days 8 (225.1 +/- 78.6 and 5.9 +/- 14.8 ng/ml) and 12 (108.5 +/- 78.6 and 13.0 +/- 14.8 ng/ml). Concentrations of progesterone in subordinate follicles increased linearly between Days 5 and 12 of the estrous cycle. Plasma concentrations of FSH increased from 17.9 +/- 1.4 to 32.5 +/- 1.4 ng/ml between 0 and 32 h following unilateral removal of the ovary with the first-wave dominant follicle. Increases in plasma FSH were associated with increased numbers of class 1 (3-4 mm) follicles in cows that were ovariectomized at Day 5 or 8 of the cycle. Unilateral ovariectomy had no effects on plasma concentrations of LH when a CL was present on the remaining ovary. First-wave dominant follicles incorporated more 3H-leucine into macromolecules and secreted high (90,000-120,000) and low (20,000-23,000) molecular weight proteins that were not as evident for subordinate follicles at Days 8 and 12.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of the peripubertal pattern of LH and FSH secretion on in-vitro oestradiol-17 beta secretion and follicular growth within juvenile rat ovaries

Juvenile rat ovaries were placed in perfusion culture and exposed to (1) no gonadotrophin, (2) tonic NIH-FSH (200 ng RP-1 equiv./ml) or (3) NIH-FSH + NIH-LH pulses (2/h, amplitude = 80 ng RP-1 equiv./ml). After 3 h of perifusion, the ovaries were prepared for histological analysis and the perifusate assayed for oestradiol-17 beta. Since the NIH-FSH preparation is contaminated with LH, a second experiment was conducted using recombinant bovine LH and FSH. Ovaries were perifused for 3 h with (1) no hormones, (2) recombinant FSH (200 ng/ml) or (3) recombinant FSH plus 25 ng recombinant LH/ml. NIH-FSH alone increased the number of mid-size antral follicles (P less than 0.05) and decreased the number of small antral follicles (P less than 0.05). Pulsatile LH in the presence of FSH increased the number of mid-size antral follicles without reducing the number of small antral follicles. Studies with recombinant FSH and LH demonstrated that both FSH and LH are necessary to stimulate follicles to grow, indicating that the growth-promoting property of the NIH-FSH is due to the presence of both FSH and LH. Regardless of whether NIH or recombinant gonadotrophins were used, follicular growth was induced without increasing oestradiol secretion. These results demonstrate that enhanced oestradiol secretion is not essential for the induction of follicular growth, while both LH and FSH are necessary to stimulate small antral follicles to grow into mid-size antral follicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Epinephrine intracerebroventricular stimulation modifies the LH effect on ovarian progesterone and androstenedione release

This study investigates the interaction between the effect of epinephrine intracerebroventricular (icv) injection and LH on the progesterone concentration in ovarian vein blood (Po) in vivo, and also, on the release of ovarian progesterone and androstenedione in vitro, in rats on dioestrus day 2. When 2 mg ovine LH were injected in vein (i.v.), Po increased reaching 120+/-12.2 and 151+/-17.5 ng ml(-1) at 22 and 25 min, respectively. Another group of rats was injected intracerebroventricular with 5 microgram epinephrine at time zero, and with 2 mg ovine LH i.v. 3 min later. This time Po decreased during the first 3 min, then increased, reaching 64+/-7.1 ng ml(-1) at 25 min, lower than the Po obtained 22 min after LH i.v. injection only (P<0.01). Moreover, rats were injected i.v. with 2 mg ovine LH at time zero, and 7 min later with epinephrine intracerebroventricular. Po increased during the first 7 min, decreased until the 13th minute and reached 70+/-8.9 ng ml(-1) at 25 min, lower than the Po obtained 25 min after LH i.v. injection only (P<0.01). In other experience, rats with one (either right or left) superior ovarian nerve transected (SON-t), were injected intracerebroventricular with epinephrine. Five minutes later, the ovaries were removed and incubated in vitro with LH. Both ovaries (right or left) in which the SON was intact at time of epinephrine i. c.v. injection, showed a reduction of progesterone and androstenedione released in vitro (P<0.05). These results suggest that, on dioestrus day 2, the central adrenergic stimulus competes with LH in the release of ovarian progesterone. Also, the neural input that arrives at the ovary through the SON would antagonize the ovarian progesterone and androstenedione response to LH.     

A comparison of hormone levels in follicle-lutein-cysts and in normal bovine ovarian follicles

Insulin-like growth factors 1 and 2 (IGF-1 and 2), oxytocin, progesterone, estradiol and ubiquitin were measured in bovine follicle-lutein-cysts and in follicular fluid after the classification of ovarian follicles by size (Class I = <4 mm; Class II = 5-8 mm; Class III = 9-12 mm; Class IV = preovulatory; Class V = cystic). It was found that IGF-1 concentrations increased during growth from 280 ng/ml in small follicles to 489 ng/ml in preovulatory follicles; IGF-2 appeared to remain constant in follicular fluid and in cysts (275 ng/ml). Oxytocin values were low in Class I, II and III follicles (30 pg/ml) but increased in preovulatory and cystic follicles (75 pg/ml). Estradiol increased significantly only in preovulatory follicles. Ubiquitin, a protein reflecting cellular replicative activity, could be found in bovine follicular fluid in high concentrations: 1.6 mug/ml in Class I,II and III follicles with the highest amounts in preovulatory follicles (2.3 mug/ml). In contrast with normal follicles, cysts were found to have a minimal concentration of ubiquitin (0.3 mug/ml). Progesterone levels were 5 times higher in cysts (325 ng/ml) and IGF-1 concentrations were markedly higher in cystic follicles (881 ng/ml) than in the other follicles. Simultaneously, maximum gene expression for IGF-1 was found in granulosa/lutein cells of cystic follicles (Class V), suggesting de novo synthesis of IGF-1. Between the different follicle classes progesterone, oxytocin and IGF-1 concentrations correlated positively (r=0.82). Hormonal levels in follicle-lutein-cysts indicated an arrested stage of insufficient luteinization as a possible result from the premature release of LH or from the release of amounts of LH inadequate to cause ovulation.