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.     

Decreased pulsatile LH secretion in heifers superovulated with eCG or FSH

This study was designed to test the hypothesis that treatment with super-ovulatory drugs suppresses endogenous pulsatile LH secretion. Heifers (n=5/group) were superovulated with eCG (2500 IU) or FSH (equivalent to 400 mg NIH-FSH-P1), starting on Day 10 of the estrous cycle, and were injected with prostaglandin F(2alpha) on Day 12 to induce luteolysis. Control cows were injected only with prostaglandin. Frequent blood samples were taken during luteolysis (6 to 14 h after PG administration) for assay of plasma LH, estradiol, progesterone, testosterone and androstenedione. The LH pulse frequency in eCG-treated cows was significantly lower than that in control cows (2.4 +/- 0.4 & 6.4 +/- 0.4 pulses/8 h, respectively; P<0.05), and plasma progesterone (3.4 +/- 0.4 vs 1.8 +/- 0.1 ng/ml, for treated and control heifers, respectively; P<0.05) and estradiol concentrations (25.9 +/- 4.3 & 4.3 +/- 0.4 pg/ml, for treated and control heifers, respectively; P<0.05) were higher compared with those of the controls. No LH pulses were detected in FSH-treated cows, and mean LH concentrations were significantly lower than those in the controls (0.3 +/- 0.1 & 0.8 +/- 0.1, respectively; P<0.05). This suppression of LH was associated with an increase in estradiol (9.5 +/- 1.4 pg/ml; P<0.05 compared with controls) but not in progesterone concentrations (2.1 +/- 0.2 ng/ml; P>0.05 compared to controls). Both superovulatory protocols increased the ovulation rate (21.6 +/- 3.9 and 23.0 +/- 4.2, for eCG and FSH groups, respectively; P>0.05). These data demonstrate that super-ovulatory treatments decrease LH pulse frequency during the follicular phase of the treatment cycle. This could be explained by increased steroid secretion in the eCG-trated heifers but not in FSH-treated animals.     

Development of preovulatory follicles expected to form short-lived corpora lutea in beef cows

Oestrus, expected to be followed by a short luteal phase, was induced in post-partum cows by weaning their calves at 35 days after parturition. Ovaries containing the first preovulatory follicles (Type F) formed after parturition were collected 3 h after the onset of oestrus. For comparison, preovulatory follicles (Type C) were collected 3 h after the onset of oestrus in normally cycling cows. The number of granulosa cells was determined and the concentrations of receptors for follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in granulosa cells and for LH in theca cells were measured. Concentrations of oestradiol-17 beta, testosterone, androstenedione and progesterone in follicular fluid were also measured. Type F follicles contained about twice the number of granulosa cells (based on DNA) as did Type C follicles (45.8 +/- 11.3 and 24.5 +/- 3.9 micrograms DNA/follicle, respectively; P less than 0.05) but these cells had fewer receptors for LH (0.13 +/- 0.02 vs 0.29 +/- 0.03 fmol/micrograms DNA; P less than 0.01) and FSH (0.61 +/- 0.08 vs 1.3 +/- 0.29 fmol/micrograms DNA; P less than 0.08) than did those from Type C follicles. Additionally, there were fewer receptors for LH in theca tissue from Type F than from Type C follicles (28.3 +/- 5.2 vs 51.3 +/- 6.1 fmol/follicle; P less than 0.01). Concentrations of oestradiol-17 beta (475.8 +/- 85.6 vs 112.9 +/- 40.0 ng/ml; P less than 0.01) and androstenedione (214.1 +/- 48.7 vs 24.7 +/- 7.7 ng/ml; P less than 0.01) in follicular fluid were higher in Type C than in Type F follicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Estradiol induces and progesterone inhibits the preovulatory surges of luteinizing hormone and follicle-stimulating hormone in heifers

Objectives were to determine: 1) whether estradiol, given via implants in amounts to stimulate a proestrus increase, induces preovulatory-like luteinizing hormone (LH) and follicle-stimulating hormone (FSH) surges; and 2) whether progesterone, given via infusion in amounts to simulate concentrations found in blood during the luteal phase of the estrous cycle, inhibits gonadotropin surges. All heifers were in the luteal phase of an estrous cycle when ovariectomized. Replacement therapy with estradiol and progesterone was started immediately after ovariectomy to mimic luteal phase concentrations of these steroids. Average estradiol (pg/ml) and progesterone (ng/ml) resulting from this replacement were 2.5 and 6.2 respectively; these values were similar (P greater than 0.05) to those on the day before ovariectomy (2.3 and 7.2, respectively). Nevertheless, basal concentrations of LH and FSH increased from 0.7 and 43 ng/ml before ovariectomy to 2.6 and 96 ng/ml, respectively, 24 h after ovariectomy. This may indicate that other ovarian factors are required to maintain low baselines of LH and FSH. Beginning 24 h after ovariectomy, replacement of steroids were adjusted as follows: 1) progesterone infusion was terminated and 2 additional estradiol implants were given every 12 h for 36 h (n = 5); 2) progesterone infusion was maintained and 2 additional estradiol implants were given every 12 h for 36 h (n = 3); or 3) progesterone infusion was terminated and 2 additional empty implants were given every 12 h for 36 h (n = 6). When estradiol implants were given every 12 h for 36 h, estradiol levels increased in plasma to 5 to 7 pg/ml, which resembles the increase in estradiol that occurs at proestrus. After ending progesterone infusion, levels of progesterone in plasma decreased to less than 1 ng/ml by 8 h. Preovulatory-like LH and FSH surges were induced only when progesterone infusion was stopped and additional estradiol implants were given. These surges were synchronous, occurring 61.8 +/- 0.4 h (mean +/- SE) after ending infusion of progesterone. We conclude that estradiol, at concentrations which simulate those found during proestrus, induces preovulatory-like LH and FSH surges in heifers and that progesterone, at concentrations found during the luteal phase of the estrous cycle, inhibits estradiol-induced gonadotropin surges. Furthermore, ovarian factors other than estradiol and progesterone may be required to maintain basal concentrations of LH and FSH in heifers.     

Ovarian function in ewes during the transition from breeding season to anoestrus

Transrectal ovarian ultrasonography was conducted in six Western white-faced ewes for 35 days from the last oestrus of the breeding season, to record the number and size of all ovarian follicles > or = 3 mm in diameter and luteal structures. Blood samples were collected once a day for estimation of serum concentrations of follicle-stimulating hormone (FSH), oestradiol and progesterone. Each ewe had five follicular waves (follicles growing from 3 to > or = 5 mm in diameter) over the scanning period. The duration of the growth phase of the largest ovarian follicles did not differ (P > 0.05) between waves, but follicular static and regressing phases decreased significantly (P < 0.05) after the decline in serum progesterone concentrations at the end of the last luteal phase of the breeding season. The intervals between the five follicular waves were: 9.2+/-0.4, 5.2+/-0.7, 8.3+/-0.8 and 5.8+/-0.7 days; the two shorter intervals differed (P < 0.05) from the two longer intervals. Using the cycle-detection program, rhythmic increases in serum FSH concentrations were detected in all ewes; the amplitude, duration and periodicity of FSH fluctuations did not vary (P > 0.05) throughout the period of study. The number of identified FSH peaks (7.8+/-0.5 peaks per ewe, per scanning period) was greater (P < 0.05) than the number of emerging follicular waves. Serum concentrations of oestradiol remained low (< or = 1 pg/ml) on most days, in five out of the six ewes studied, and sporadic elevations in oestradiol secretion above the non-detectable level were not associated with the emergence of follicular waves. The ovulation rate was lower than that seen during the middle portion of the breeding season (November-December) in white-faced ewes but the transitional ewes had larger corpora lutea (CL). Maximal serum concentrations of progesterone appeared to be lower and the plateau phase of progesterone secretion appeared to be shorter during the last luteal phase of the ovulatory season in comparison to the mid-breeding season of Western white-faced ewes. During the transition into anoestrus in ewes, the endogenous rhythm of FSH release is remarkably robust but the pattern of emergence of sequential follicular waves is dissociated from FSH and oestradiol secretion. Luteal progesterone secretion is suppressed because of fewer ovulations and diminished total luteal volume, but it may also result from diminished gonadotropic support. These season-related alterations in the normal pattern of ovine ovarian cycles appear to be due to reduction in ovarian responsiveness to gonadotropins and/or attenuation in secretion of luteinizing hormone (LH) occurring at the onset of the anovulatory season in ewes.     

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)     

Effects of luteinizing hormone on superovulatory and steroidogenic responses of rat ovaries to infusion with follicle-stimulating hormone

Immature female rats were infused s.c. continuously over a 60-h period with a partially purified porcine pituitary follicle-stimulating hormone (FSH) preparation having FSH activity 4.2 x NIH-FSH-S1 and luteinizing hormone (LH) activity 0.022 x NIH-LH-S1. High rates of superovulation were observed in rats receiving 1 U FSH/day, with 69 +/- 11 oocytes/rat recovered as cumulus-enclosed oocytes from oviducts on Day 1 (equivalent to the day of estrus). Addition of LH to the FSH, at dosages equivalent to 2.5-100 micrograms/day NIH-LH-S1 equivalents (2.5-100 mU) resulted in a dose-related inhibition of superovulation, reaching a nadir of 15 +/- 7 oocytes recovered from rats receiving 50 mU LH/day together with 1 U FSH/day. At the two highest LH doses, 50 and 100 mU/day, ovulation was advanced so that 12 +/- 3 and 15 +/- 4 oocytes, respectively, were recovered from oviducts of these rats flushed on the morning of Day 0, compared to none in rats infused with FSH alone. Ovarian steroid concentrations (ng/mg) observed on the morning of Day 0 in rats infused with FSH alone were progesterone, 0.50 +/- 0.13; testosterone, 0.16 +/- 0.08; androstenedione, 0.06; and estradiol, 0.23 +/- 0.05. On the morning of Day 1, ovarian progesterone concentrations in rats infused with FSH alone had risen to 3.30 +/- 0.33 ng/mg, whereas concentrations of testosterone, androstenedione, and estradiol, had fallen to essentially undetectable levels. Addition of LH to the FSH infusion resulted in dose-related increases, on Day 0, of all four steroids up to a dosage of 25 mU LH/day. At higher LH dosages, Day 0 ovarian concentrations of androgens and estradiol fell markedly, while progesterone concentrations continued to increase. Histological examination of ovaries revealed increases in the extent of luteinization of granulosa cells in follicles with retained oocytes on both Days 0 and 1 in rats infused with 25 and 50 mU LH/day together with 1 U FSH/day, compared to those observed in rats receiving FSH alone. These findings indicate that the elevated progesterone levels on Day 0 and inhibition of ovulation observed at these LH doses were due to premature luteinization of follicles, thus preventing ovulation. At lower LH doses, no sign (based on histologic or steroidogenic criteria) of premature luteinization was evident, suggesting that the decreased superovulation in these rats was due to decreased follicular maturation and/or increased atresia rather than to luteinization of follicles without ovulation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Temporal interrelationships among luteolysis, FSH and LH concentrations and follicle deviation in mares

The effect of altered LH concentrations on the deviation in growth rates between the 2 largest follicles was studied in pony mares. The progestational phase was shortened by administration of PGF2alpha on Day 10 (Day 0=ovulation; n=9) or lengthened by daily administration of 100 mg of progesterone on Days 10 to 30 (n=11; controls, n=10). All follicles > or = 5 mm were ablated on Day 10 in all groups to initiate a new follicular wave. The interovulatory interval was not altered by the PGF2alpha treatment despite a 4-day earlier decrease in progesterone concentrations. Time required for growth of the follicles of the new wave apparently delayed the interval to ovulation after luteolysis. The FSH concentrations of the first post-ablation FSH surge were not different among groups. A second FSH surge with an associated follicular wave began by Day 22 in 7 of 11 mares in the progesterone group and in 0 of 19 mares in the other groups, indicating reduced functional competence of the largest follicle. A prolonged elevation in LH concentrations began on the mean day of wave emergence (Day 11) in the prostaglandin group (19.2 +/- 2.2 vs 9.0 +/- 0.7 ng/mL in controls; P<0.05), an average of 4 d before an increase in the controls. Concentrations of LH in the progesterone group initially increased until Day 14 and then decreased so that by Day 18 the concentrations were lower (P<0.05) than in the control group (12.9 +/- 1.6 vs 20.2 +/- 2.6 ng/mL). Neither the early and prolonged increase nor the early decrease in LH concentrations altered the growth profile of the second-largest follicle, suggesting that LH was not involved in the initiation of deviation. However, the early decrease in LH concentrations in the progesterone group was followed by a smaller (P<0.05) diameter of the largest follicle by Day 20 (26.9 +/- 1.7 mm) than the controls (30.3 +/- 1.7 mm), suggesting that LH was necessary for continued growth of the largest follicle after deviation.     

Changes in gonadotrophin secretion and ovarian antral follicular activity in seasonally breeding sheep throughout the year

Overall, significantly more antral follicles greater than or equal to 1 mm diameter were present in Romney ewes during anoestrus than in the breeding season (anoestrus, 35 +/- 3 (mean +/- s.e.m.) follicles per ewe, 23 sheep; Day 9-10 of oestrous cycle, 24 +/- 1 follicles per ewe, 22 sheep; P less than 0.01), although the mean numbers of preovulatory-sized follicles (greater than or equal to 5 mm diam.) were similar (anoestrus, 1.3 +/- 0.2 per ewe; oestrous cycle, 1.0 +/- 0.1 per ewe). The ability of ovarian follicles to synthesize oestradiol did not differ between anoestrus and the breeding season as assessed from the levels of extant aromatase enzyme activity in granulosa cells and steroid concentrations in follicular fluid. Although the mean plasma concentration of LH did not differ between anoestrus and the luteal phase of the breeding season, the pattern of LH secretion differed markedly; on Day 9-10 of the oestrous cycle there were significantly more (P less than 0.001) high-amplitude LH peaks (i.e. greater than or equal to 1 ng/ml) in plasma and significantly fewer (P less than 0.001) low amplitude peaks (less than 1 ng/ml) than in anoestrous ewes. Moreover, the mean concentrations of FSH and prolactin were significantly lower during the luteal phase of the cycle than during anoestrus (FSH, P less than 0.05, prolactin, P less than 0.001). It is concluded that, in Romney ewes, the levels of antral follicular activity change throughout the year in synchrony with the circannual patterns of prolactin and day-length. Also, these data support the notion that anovulation during seasonal anoestrus is due to a reduced frequency of high-amplitude LH discharges from the pituitary gland.     

The effect of smoking on serum progesterone, estradiol, and luteinizing hormone levels over a menstrual cycle in normal women

Since smoking has been shown to affect serum progesterone and estradiol levels in postmenopausal women, we evaluated the levels of these hormones and luteinizing hormone (LH) over an entire menstrual cycle (17 points) in eight healthy nonsmokers and eight healthy smokers. The total length of the cycle and the lengths of the follicular and luteal phases did not differ between the groups. There was no difference in estradiol, progesterone, or LH levels during the periovulatory and luteal phases. Follicular-phase serum progesterone, which had a level 37% higher in smokers, showed a plateau in both groups (28.3 +/- 5.7 ng/dl versus 20.7 +/- 5.7; P less than 0.0001). Follicular-phase serum estradiol showed a rising curve in both groups. The mean value in smokers was slightly higher than that in nonsmokers (107 pg/ml versus 95; P approximately 0.05); during the early part of the follicular phase, prior to the rapid preovulatory increase, the difference was greater (23%) and of higher statistical significance (80 pg/ml versus 65; P less than 0.001). The follicular-phase LH levels of smokers were skewed downward from the levels in nonsmokers, presumably by negative feedback from the elevated estradiol and progesterone levels; the difference was significant (P less than 0.001). The elevations of serum progesterone and estradiol in smokers probably represent activation of adrenocortical secretion by smoking. The greater and more clear-cut rise of progesterone than of estradiol is probably due to the fact that essentially all of the follicular-phase serum progesterone is secreted by the adrenal, while only part of the follicular-phase serum estradiol comes from the adrenal (via androstenedione and estrone).     

Plasma estradiol FSH and LH concentration after dominant follicle aspiration in the cow

This work investigates the estrogenic role of the dominant follicle with regard to regulation of plasma FSH and LH concentration. Eight Holstein-Friesian cows were used for aspiration of the dominant follicle using ultrasound guidance during the early, mid and late stages of the luteal phase. Blood samples were collected at 15-min intervals from 4 h before until 7 h after aspiration. Plasma progesterone concentration increased from 0.7 to 7.2 ng mL-1 from early to mid luteal phase and then fell slightly to 5.9 ng mL-1 in the late luteal phase, but remained unaffected by follicle puncture. The follicular aspirate contained a thousandfold higher estradiol, than plasma concentration but its estradiol:progesterone ratio remained at around 2 at each stage of the luteal phase. Aspiration caused plasma estradiol concentration to fall from 1.4 to 0.7, 1.8 to 1.0 and 1.7 to 0.8 pg mL-1 in the early, mid and late stages of the luteal phase, respectively (P < 0.05). At the same time, mean plasma FSH concentration was increased from 1.1 to 1.8, 1.7 to 2.9 and 0.8 to 1.9 ng mL-1 (P < 0.05), respectively. The results suggest that estradiol secreted from dominant follicles selectively regulates gonadotropin secretion, since aspiration of the dominant follicle at any stage of the cycle affected circulating FSH but did not appear to influence the mean LH concentration.     

Effect of overexpression of inhibin alpha (1-32) fragment on bovine granulosa cell proliferation, apoptosis, steroidogenesis, and development of co-cultured oocytes

The objective was to determine the effects of an inhibin alpha (1-32) fragment gene on proliferation, apoptosis, and steroidogenesis of bovine granulosa cells (GC) isolated from medium and small follicles (diameter >4-8 and 1-4mm, respectively), and the effect of GC, previously transfected with pEGISI, on oocyte maturation and in vitro embryo development. To enhance expression of the inhibin alpha (1-32) fragment, GC were transfected with pEGISI. Transfection inhibited (P<0.05) GC proliferation (88.8+/-2.1%; mean+/-S.E.M.) compared to the control and EGFP groups (100% and 97.5+/-2.1%) from medium follicles, with no significant effect on GC from small follicles. Apoptosis was higher (P<0.01) in transfected GC than in controls. Transfection increased (P<0.05) estradiol synthesis from both medium and small follicles (0.57+/-0.13 and 0.86+/-0.13 pg/mL vs. 0.19+/-0.05 and 0.35+/-0.09 pg/mL in controls) after culturing for 48 h, with suppression (P<0.05) in transfected GC after 96 h. Transfection reduced (P<0.05) progesterone synthesis in GC from both medium and small follicles (24.5+/-3.4 and 75.4+/-4.6 ng/mL vs. 45.42+/-5.33 and 117.32+/-11.99 ng/mL in controls) after culture for 48 h, with no significant difference after 96 h. Maturation rate of oocytes co-cultured with transfected GC from medium follicles was decreased relative to control (61.5+/-6.8% vs. 71.2+/-5.7%, P<0.05), with no significant effect on embryo development. In conclusion, overexpression of inhibin alpha (1-32) fragment regulated GC development; effects on subsequent oocyte maturation were both time- and stage-dependent.     

Inhibition of thecal androstenedione production by exogenous progesterone in the cyclic hamster

Implants of progesterone on the day of dioestrus II in the hamster induced on the following day an increase in circulating levels of progesterone (6.0 +/- 0.7 ng/ml, N = 8; sesame oil controls, less than 0.5 ng/ml, N = 6) and a decline in serum levels of LH (5.3 +/- 0.4 ng/ml; controls 12 +/- 2 ng/ml) and oestradiol (10 +/- 2 pg/ml; controls 69 +/- 5 pg/ml). The production of androstenedione and oestradiol by antral follicles in vitro was reduced in progesterone-treated hamsters when compared with controls, but progesterone production was not affected. Aromatizing activities of antral follicles were the same in progesterone-treated and sesame oil-treated hamsters. Androstenedione production by theca was significantly less in progesterone-treated hamsters than in controls. On dioestrus II, LH replacement therapy (200 micrograms ovine LH by osmotic minipump inserted s.c.) prevented the decline in follicular androstenedione and oestradiol production induced by progesterone alone, and also prevented the decline in thecal androstenedione production in vitro. The results indicate that exogenous progesterone on dioestrus II lowers circulating levels of LH by the following day, inhibits thecal androstenedione production and thus reduces follicular oestradiol production without alteration in aromatizing ability.     

Progesterone and 17beta-estradiol, but not follicle stimulating hormone, alter the sex ratio of murine embryos fertilized in vitro

The objective was to determine the effects of adding progesterone, 17beta-estradiol (17beta-E2), and FSH during in vitro fertilization on development and sex ratio of murine embryos. Progesterone (33-330 pg/mL), 17beta-estradiol (17beta-E2); 10-70 pg/mL), and FSH (0.01-0.05 IU/mL), were added to human tubal fluid (HTF); this medium (with or without hormones) was used to pre-incubate sperm (2h) and to co-incubate sperm and oocytes (6h). Thereafter, the ova were washed and incubated in mM16 medium and embryo sex was determined (by PCR) on Day 4 (insemination=Day 0). There was no effect (P>0.05) of hormone treatments on rates of cleavage (6 h after cessation of co-incubation with sperm). The only significant effects of added hormones on development were a decrease in the rate of development to at least the morula stage in 165 pg/mL progesterone (0.46+/-0.03 vs. 0.54+/-0.05 in the control, mean+/-S.D.; P<0.05) and a decrease in the blastocyst rate in 0.03 IU/mL FSH (0.34+/-0.00 vs. 0.42+/-0.04 in the control, P<0.05). However, the ratio of male to female embryos was 1.61 and 2.90 following the addition of 99 pg/mL progesterone and 70 pg/mL 17beta-E2, respectively; both of these ratios were different (P<0.01) than in the control group (1.20). In contrast, the addition of FSH to the medium had no significant effect on this ratio (range, 0.78-1.02). We concluded that the addition of progesterone and estradiol to the media during in vitro fertilization did not enhance embryonic development, but significantly increased the proportion of male murine embryos.     

Transitory increases of luteinizing hormone and follicle-stimulating hormone following luteectomy in hemiovariectomized primates significantly increase progesterone secretion in vitro by the subsequent corpus luteum

Ten chronically hemiovariectomized cynomolgus and rhesus monkeys were luteectomized 5.5 +/- 0.3 days after the midcycle luteinizing hormone (LH) and follicle-stimulating hormone (FSH) surge in two consecutive cycles. The corpus luteum (CL) was removed, weighed, dispersed with collagenase and the luteal cells counted. Luteal cells (50,000/ml) were incubated in Ham's F10 medium for 3 h at 37 degrees C either in the presence or absence of 100 ng/ml human chorionic gonadotropin (hCG). Daily blood samples were taken from the monkeys throughout the study for determination of LH, FSH, estradiol (E2) and progesterone levels. Within 5 days following each luteectomy (LX), all monkeys responded with a significant increase in FSH and LH (P less than 0.05). Ovulatory LH/FSH surges occurred 14.4 +/- 0.5 days after the first LX. Hormonal profiles of serum progesterone prior to the first and second LX, CL weight and number of luteal cells/CL were similar (P greater than 0.05). However, luteal cells obtained at the second LX produced more progesterone (P less than 0.05) in vitro under basal and hCG-stimulated conditions than cells from the first LX. The areas under the LH and FSH curves following the first LX were highly correlated (P less than 0.05) with the in vitro progesterone production following the second LX. Thus, the monkeys with the largest areas under the LH and FSH curves subsequently had the highest in vitro progesterone production.     

Immunoreactive luteinizing hormone, estradiol, progesterone, testosterone, and androstenedione levels during the breeding season and anestrus in Siberian tigers

Seasonal analysis of 1239 captive births of Siberian tigers (Panthera tigris altaica) indicated a peak in April to June (P less than 0.001). Studies on seven animals in Minnesota indicated that behavioral heat cycles and ovarian follicular phase cycles began in late January and ceased in early June. Behavioral observation of 12 heat cycles in four tigers yielded an estrous length of 5.3 +/- 0.2 days and an interestrous interval of 25.0 +/- 1.3 days. Hormone assays on weekly blood samples (N = 180) from three female tigers indicated 16 cycles in two breeding seasons. Peak estradiol-17 beta levels were 46.7 +/- 6.0 pg/ml (N = 17) and interestrous concentrations were 8.7 +/- 0.66 pg/ml (N = 28) during the breeding season. Anestrous estradiol levels were 4.2 +/- 0.5 pg/ml (N = 70). The interestrous interval between estradiol peaks was 24.9 +/- 1.3 days (N = 9) with two outliers of 42 days. Serum progesterone concentrations from February to June were 1.2 +/- 0.15 ng/ml (N = 32), providing no evidence for ovulation or corpus luteum formation. Luteinizing hormone (LH) levels were 0.56 +/- 0.04 ng/ml (N = 180). Serum testosterone (r=0.71, P less than 0.001) and androstenedione levels (r=0.75, P less than 0.001) were correlated with estradiol during the breeding season. The duration of anestrus was 8 mo in two of these tigers. The interval was shortened in one tiger by exposure to a 16L:8D photoperiod. The Siberian tiger appears to be a polyestrous seasonal breeder and an induced ovulator whose breeding season may be synchronized by photoperiod.     

Steroid and gonadotropin hormone levels in young African women with filarial infection

Serum levels of dehydroepiandrosterone sulfate (DHEAS), testosterone (T), progesterone (P), estradiol (E2), prolactin (PRL), cortisol (F) and gonadotropins (FSH, LH) were analysed by radioimmunoassay for 125 schoolgirls aged 14-16, in a zone of endemic filariasis 3 days after menses. Two groups were identified: the infected group in which 38 subjects had circulating Loa loa and or Mansonella perstans microfilariae as determined by the Knott's concentration technique, and the non-infected group (87 subjects without microfilaremia). All results are expressed as the mean +/- SD. No significant difference was found between the two groups for age (14.47 +/- 1.37 yr vs 14.50 +/- 1.37 yr) or for body wt (46.10 +/- 8.45 kg vs 47.06 +/- 8.26 kg). There was a tendency to lower levels of DHEAS in the infected group by comparison with controls (54.92 +/- 37.34 micrograms/dl vs 66.80 +/- 47.18 micrograms/dl) while in the same infected group more subjects had higher levels of prolactin by comparison with the control group (10.85 +/- 14.16 ng/ml vs 9.80 +/- 5.56 ng/ml). Testosterone, progesterone, estradiol levels and the LH/FSH ratio were lower in the infected group than in the non-infected group (P: 0.25 +/- 0.12 ng/ml vs 0.33 +/- 0.20 ng/ml, P less than 0.025; T: 0.55 +/- 0.17 ng/ml vs 0.62 +/- 0.19 ng/ml, P less than 0.05; E2: 32.95 +/- 19.63 pg/ml vs 66.98 +/- 54.83 pg/ml, P less than 0.001; LH/FSH: 0.91 +/- 0.44 vs 1.30 +/- 0.84, P less than 0.005) respectively. No significant difference was found between the two groups for F; however FSH levels correlated negatively with F levels only in the microfilaremia group (r = -0.38, n = 38, P less than 0.05). Our results suggest that the presence of microfilaremia in our subjects may have contributed to reduced steroid levels, perhaps by involvement of the cyclic AMP kinase system. These observations may explain the delayed menarche and androgen secretion found during puberty in a similar population living in the same zone of endemic filariasis. Microfilaremia should therefore be considered an environmental factor which mediates endocrine disorders in subjects living in tropical filariasis areas.     

Relationship between serum luteinizing hormone and estradiol in prepubertal boars

Effects of estradiol on serum luteinizing hormone (LH) were studied in prepubertal boars. In Exp. 1, 15-wk-old boars were given (iv) 50 mug estradiol, 1 mg testosterone or 1.5 ml ethanol. Estradiol (P<0.05) decreased LH over a 2.5-hr period, but testosterone did not. In Exp. 2, an estradiol implant reduced LH sample variance (P<0.01) while LH (547 +/- 96 vs 655 +/- 43 pg/ml) and estradiol (14.2 +/- 3.3 vs 18.4 +/- 1.0 pg/ml; control vs implant) were unchanged in 12-wk-old boars. Pulsatile LH releases (4.3 +/- 1.1 vs 3.0 +/- 0.4 pulses/pig/8 hr; control vs treated) and pulse amplitude (272 +/- 34 vs 305 +/- 40 pg/ml) were not affected. The implant tended to decrease serum testosterone (4.86 +/- 0.75 vs 7.66 +/- 1.51 ng/ml; P<0.10). In Exp. 3, LH was higher after zero implants than after four implants (279 +/- 7 vs 227 +/- 9 pg/ml; P<0.01), and LH after two implants was also higher than after four implants (263 +/- 7 pg/ml; P<0.01) in 14-wk-old boars in a Latin square design. Peak LH after 40 mug gonadotropin releasing hormone (GnRH) was less after two and four implants (1,100 +/- 126 and 960 +/- 167 pg/ml, respectively; P<0.01) than after zero implants (1,742 +/- 126 pg/ml). Slope of the first 20 min of LH response to GnRH was greater after zero implants (45.3 pg/min; P<0.05) than after either two or four implants (20.6 and 16.9 pg/min, respectively). Implant treatment decreased serum testosterone (P<0.025) but increased estradiol (P<0.10). Small changes in serum estradiol resulted in changes in LH. These changes in sample variance and mean LH were recognized by boars as different from normal because serum testosterone decreased. Changes in LH may result from estradiol's negative effect on pituitary responsiveness to endogenous GnRH because response to exogenous GnRH was depressed by estradiol.     

Seasonal effects on attempts to synchronize estrus and ovulation by intravaginal application of progesterone-releasing device (PRID) in mares

To investigate seasonal effects on the efficacy of estrus synchronization in mares, we administered a progesterone-releasing device (PRID) intravaginally to eight Haflinger mares for 11 days. In January 3 of 8 mares responded to the treatment with estrus and ovulation, in March 7 with estrus and 6 of 7 mares with ovulation, in June 6 of 7 and in October 7 of 8 mares with estrus and ovulation. Follicle distribution patterns at PRID insertion were different between January/October, March/June and June/October (P<0.05). Number of follicles decreased during PRID treatment in January, March and June (difference of number of follicles at Day 12 minus number of follicles at Day 1: -4.2+/-2.7, -0.9+/-0.9 and -4.9+/-1.5 follicles), while it increased in October (3.9+/-1.2 follicles; P<0.05). Mean progesterone concentrations were lowest in January (0.3+/-0.1 ng mL(-1)) when compared with March (3.5+/-1.8 ng mL(-1); P=0.063), June (4.4+/-1.4 ng mL(-1); P<0.05) and October (2.2+/-0.9 ng mL(-1); P<0.05). At Day 2 of PRID treatment, mean progesterone concentrations significantly increased in all mares. Except from January, mean LH concentrations decreased within one day after PRID insertion and remained at low levels during treatments in January and March. Total secretion of LH during PRID-treatment was significantly lower in January and March when compared with June and October. In the 5 of 7 mares that ovulated during PRID treatment a distinct increase of plasma LH concentrations after ovulation was detected. Administration of the progesterone releasing intravaginal device PRID combined with the PGF2alpha analogue cloprostenol was able to induce estrus and ovulation in mares at different times of the year. However, efficacy of the treatment was not satisfactory concerning effectiveness in relation to season and synchrony of intervals from removal of PRID to ovulation in mares.     

Concentrations of steroids and gonadotropins in follicular fluid from normal heifers and heifers primed for superovulation

The concentrations of six steroids and of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were measured in follicular fluid from preovulatory and large atretic follicles of normal Holstein heifers and from preovulatory follicles of heifers treated with a hormonal regimen that induces superovulation. Follicular fluid from preovulatory follicles of normal animals obtained prior to the LH surge contained extremely high concentrations of estradiol (1.1 +/- 0.06 micrograms/ml), with estrone concentrations about 20-fold less. Androstenedione was the predominant aromatizable androgen (278 +/- 44 ng/ml; testosterone = 150 +/- 39 ng/ml). Pregnenolone (40 +/- 3 ng/ml) was consistently higher than progesterone (25 +/- 3 ng/ml). In fluid obtained at 15 and 24 h after the onset of estrus, estradiol concentrations had declined 6- and 12-fold, respectively; androgen concentrations had decreased 10- to 20-fold; and progesterone concentrations were increased, whereas pregnenolone concentrations had declined. Concentrations of LH and FSH in these follicles were similar to plasma levels of these hormones before and after the gonadotropin surges. The most striking difference between mean steroid levels in large atretic follicles (greater than 1 cm in diameter) and preovulatory follicles obtained before the LH surge was that estradiol concentrations were about 150 times lower in atretic follicles. Atretic follicles also had much lower concentrations of LH and slightly lower concentrations of FSH than preovulatory follicles. Hormone concentrations in follicles obtained at 12 h after the onset of estrus from heifers primed for superovulation were similar to those observed in normal preovulatory follicles at estrus + 15 h, except that estrogen concentrations were about 6-40 times lower and there was more variability among animals for both steroid and gonadotropin concentrations. Variability in the concentrations of reproductive hormones in fluid from heifers primed for superovulation suggests that the variations in numbers of normal embryos obtained with this treatment may be due, at least in part, to abnormal follicular steroidogenesis.