The electrophysiological effects of tetraphenylphosphonium on vascular smooth muscle

OBJECTIVE: To investigate whether luteal secretion of inhibin-a is altered in the perimenopausal transition and to evaluate whether luteal inhibin secretion is correlated with other markers of ovarian reserve such as FSH and inhibin-b. DESIGN: Prospective study. SETTING: Reproductive Endocrinology Laboratories at The Ohio State University. PATIENT(S): Twenty-five women 39-52 years of age with regular menstrual cycles. INTERVENTION(S): Daily urine samples were monitored (LH predictor kit) to identify the day of ovulation. Blood samples obtained on days 6 and 8 after the LH surge and on day 3 of the subsequent follicular phase were assayed for FSH, E2, progesterone. inhibin-a, and inhibin-b. MAIN OUTCOME MEASURE(S): Serum levels of inhibin-a, inhibin-b, FSH, E2, and progesterone. RESULT(S): Luteal phase inhibin-a and follicular phase inhibin-b were correlated inversely with age in perimenopausal women. In addition, luteal phase inhibin-a and follicular phase inhibin-b levels were correlated inversely with follicular phase FSH levels. CONCLUSION(S): Both luteal phase inhibin-a and follicular phase inhibin-b levels are correlated inversely with age during the fifth decade of life. These findings suggest that corpus luteum function is altered during the perimenopausal transition. Moreover, these direct measures of ovarian function may be more sensitive indicators of "ovarian reserve" than indirect indicators such as pituitary FSH secretion.     

Recombinant human inhibin-A administered early in the menstrual cycle alters concurrent pituitary and follicular, plus subsequent luteal, function in rhesus monkeys

Inhibin, a suppressor of pituitary FSH secretion in nonprimate species, may also act in the ovary to regulate follicular development. To examine whether inhibin has similar actions in primates, female rhesus monkeys (n = 3/treatment), exhibiting regular menstrual cycles, received sc injections of either vehicle or 60 micrograms/kg recombinant human inhibin-A at 0800 and 1600 h for 5 days beginning at menses. The vehicle-treated monkeys displayed menstrual cycles of normal length, with the follicular (11.3 +/- 2.5 days, mean +/- SE) and luteal (16.3 +/- 2.5 days) phases demarcated by midcycle peaks in serum estradiol (E) and bioactive LH. After the first inhibin injection, levels of immunoreactive inhibin A peaked at 10 ng/mL within 1 h and returned to baseline (< 0.1 ng/mL) before the second injection 8 h later. Although serum E and LH did not change, bioactive FSH decreased (to 66% of pretreatment levels, P < 0.05) within 8 h. Within 1 day, circulating bioactive FSH was less (P < 0.05) in inhibin-treated monkeys, compared with controls. By 2-3 days, serum E levels were also markedly (P < 0.05) reduced in inhibin-treated animals, whereas bioactive LH rose 3-fold (P < 0.05). After inhibin treatment, the midcycle rises in serum E and LH were delayed; hence, the follicular phase was prolonged (15.0 +/- 2.6 days, P < 0.05), compared with controls. Although the patterns and levels of serum LH circulating during the subsequent luteal phase seemed comparable in both groups, mean progesterone levels were suppressed to 2-3 ng/mL (P < 0.05) during the midluteal phase in inhibin-treated monkeys. However, the length of the luteal phase in inhibin-treated cycles (13.0 +/- 2.6 days) was not significantly altered. We conclude that exogenous inhibin rapidly diminishes pituitary FSH secretion in female monkeys during the early follicular phase of the menstrual cycle. This action, and/or other actions directly on the ovary, leads to subsequent effects on follicular steroidogenesis and pituitary LH secretion that culminate in an aberrant ovarian cycle characterized by an insufficient luteal phase. The study identifies, for the first time, possible activities and roles of inhibin during the ovarian cycle in primates.     

A feature of hematological findings in myelodysplastic syndromes

Ovine FSH (40: g per bird daily for 10 days) increased ovarian weight, follicular size, phosphatase activities, and RNA and protein levels in tree pie (Dendrocitta vagabunda), but exogenous ovine LH (40 micrograms per bird daily for 10 days) with the same dose and duration caused depletion of ovarian cholesterol and ascorbic acid concentrations with a rise in sialic acid and glycogen levels of the ovary. In contrast, prolactin (LTH: 5 I.U. per bird daily for 10 days) administration showed reverse biochemical changes to those of FSH. The findings suggest that FSH induces mainly ovarian follicular growth and LH stimulates ovarian steroidogenesis, but LTH is antigonadal in this wild avian species.     

The follicle-stimulating hormone (FSH) threshold/window concept examined by different interventions with exogenous FSH during the follicular phase of the normal menstrual cycle: duration, rather than magnitude, of FSH increase affects follicle development

According to the threshold concept, FSH concentrations need to surpass a distinct level to stimulate ovarian follicle growth. The window concept stresses the significance of a limited duration of elevated FSH levels above the threshold for single dominant follicle selection. The aim of this study was to investigate effects on follicle growth of increased FSH levels, differing in duration and magnitude of elevation, during the follicular phase. Twenty-three normo-ovulatory (cycle length, 26-31 days), young (age, 20-31 yr) women volunteered for this study. In all subjects a series of daily transvaginal sonography scans of the ovaries and blood sampling [for FSH and estradiol (E2) determinations] were performed during two consecutive cycles. The first study cycle (control cycle) started 10 days after urinary assessment of the LH surge in the preceding cycle (DayLH) and was concluded on the day of ovulation assessed by transvaginal sonography scans. The second series of daily monitoring (intervention cycle) started 10 days after DayLH in the control cycle. After randomization, subjects received either 375 IU urinary FSH, s.c., as a single injection on Day(LH+14) (group A; n = 11) or 75 IU daily from Day(LH+19) until Day(LH+23) (group B; n = 12). In group A, FSH levels increased on the day after injection to a median concentration of 10.1 IU/L, which was 1.9 times higher (P < 0.01) than levels on matching days during the control cycle. Concentrations returned to basal levels 3 days after injection. In group B, a moderate elevation of FSH concentrations (15% increase; P < 0.05) was observed compared to levels during the control cycle. In group A, E2 concentrations increased (P = 0.03) 1 day after FSH injection and returned to baseline levels within 2 days. In group B, E2 levels started to increase after the first injection of FSH and remained significantly higher (P < 0.01) during the following 5 days compared to those on matching days in the control cycle. Compared to matching days in the control cycle an increased number of follicles 8-10 mm in size was found in group A (P < 0.01) during the period from Day(LH+14) until Day(LH+19), without an increase in follicles 10 mm or larger thereafter. In contrast, in group B, the numbers of both 8- to 10-mm and 10-mm or larger follicles were higher during the period from Day(LH+19) until Day(LH+24) in group B (P = 0.02 and P < 0.01, respectively). Results from the present study suggest that a brief, but distinct, elevation of FSH levels above the threshold in the early follicular phase does not affect dominant follicle development, although the number of small antral follicles did increase. In contrast, a moderate, but continued, elevation of FSH levels during the mid to late follicular phase (effectively preventing decremental FSH concentrations) does interfere with single dominant follicle selection and induces ongoing growth of multiple follicles. These findings substantiate the FSH window concept and support the idea of enhanced sensitivity of more mature follicles for stimulation by FSH. These results may provide the basis for further investigation regarding ovulation induction treatment regimens with reduced complication rates due to overstimulation.     

Expression of messenger ribonucleic acid for inhibin subunits and ovarian secretion of inhibin and estradiol at various stages of the sheep estrous cycle

The relationship between expression of inhibin mRNA and ovarian secretion of estradiol (E2) and immunoactive inhibin was investigated at midluteal phase and throughout the follicular phase of the sheep estrous cycle. At laparotomy, timed samples of ovarian blood were collected and ovaries were removed from 39 Scottish Blackface ewes (ovulation rate 1.3 +/- 0.1) on Day 10 of the luteal phase or 24, 48, 60, 72, or 84 h after injection of cloprostenol (PG; 100 micrograms) on Days 10-12. Ovaries were removed and fixed for in situ hybridization using 35S-labeled antisense riboprobes transcribed from inhibin alpha, beta A, and beta B cDNAs. LH, E2, and inhibin concentrations were determined by RIA. On the basis of peripheral LH levels and the presence of estrogen-active follicles (E-A; > or = 3 mm in diameter secreting > 1 ng/min E2) or recent ovulations, animals were grouped as follows: presurge (24 or 48 h post-PG; LH < 5 ng/ml; n = 7), midsurge (with E-A; LH > 5 ng/ml; n = 6), late surge (large follicle not E-A; LH > 5 ng/ml; n = 4), postsurge (large follicle not E-A; LH < 5 ng/ml; n = 7), and postovulation (n = 10). As expected, E2 secretion by the "active" ovary (containing preovulatory follicle) tended to increase with follicular development such that secretion was maximal at midsurge and then declined. E2 secretion by the "inactive" ovary was low at all stages. Immunoactive inhibin, in contrast, was secreted in substantial quantities by both ovaries, although secretion from active ovaries was higher at all stages (p < 0.05). Effects of stage on secretion were not significant, but immunoactive inhibin secretion from active ovaries was high in postsurge animals when E2 secretion was very low. Hybridization for inhibin mRNA was specific for granulosa cells of antral follicles. While most sheep in the luteal (4 of 5), presurge (2 of 3), and midsurge groups (5 of 5) had at least one inhibin-positive large follicle (expressing both alpha- and beta-subunit mRNA), none were present between the LH surge and ovulation (late and postsurge groups). Inhibin mRNA was undetectable in midcycle CL, but 4 of 10 recent ovulations hybridized weakly with the alpha probe and one very weakly with the beta A probe. The mean number of inhibin-positive large follicles per animal (in those having at least one) was 1.3 +/- 0.15 (n = 15 ewes).(ABSTRACT TRUNCATED AT 400 WORDS)     

Hormone-induced proliferation and differentiation of granulosa cells: a coordinated balance of the cell cycle regulators cyclin D2 and p27Kip1

The proliferation and terminal differentiation of granulosa cells are critical for normal follicular growth, ovulation, and luteinization. Therefore, the in situ localization and hormonal regulation of cell cycle activators (cyclin D1, D2, and D3) and cell cycle inhibitors (p27Kip1 and p21Cip1) were analyzed in ovaries of mice and rats at defined stages of follicular growth and differentiation. Cyclin D2 mRNA was specifically localized to granulosa cells of growing follicles, while cyclin D1 and cyclin D3 were restricted to theca cells. In hypophysectomized (H) rats, cyclin D2 mRNA and protein were increased in granulosa cells by treatment with estradiol or FSH and were increased maximally by treatment with both hormones. In serum-free cultures of rat granulosa cells, cyclin D2 mRNA was rapidly elevated in response to FSH, forskolin, and estradiol, indicating that estradiol as well as cAMP can act directly and independently to increase cyclin D2 expression. The levels of p27Kip1 protein were not increased in response to estradiol or FSH. In contrast, when ovulatory doses of human CG (LH) were administered to hormonally primed H rats to stimulate luteinization, cyclin D2 mRNA and protein were rapidly decreased and undetectable within 4 h, specifically in granulosa cells of large follicles. Also in response to LH, the expression of the cell cycle inhibitor p27Kip1 was induced between 12 and 24 h (p21Cip1 was induced within 4 h) and remained elevated specifically in luteal tissue. A critical role for cyclin D2 in the hormone-dependent phase of follicular growth is illustrated by the ovarian follicles of cyclin D2-/- mice, which do not undergo rapid growth in response to hormones, but do express markers of FSH/LH action, cell cycle exit, and terminal differentiation. Collectively, these data indicate that FSH and estradiol regulate granulosa cell proliferation during the development of preovulatory follicles by increasing levels of cyclin D2 relative to p27Kip1 and that LH terminates follicular growth by down-regulating cyclin D2 concurrent with up-regulation of p27Kip1 and p21Cip1.     

Immunocytochemical localization of vasoactive intestinal peptide and neuropeptide Y in the bovine ovary

The distribution of the neuropeptides vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY) was studied immunocytochemically in bovine ovaries from 3 mo of gestation up to and including puberty, and from adult cows at three stages of the estrous cycle. The appearance of VIP and NPY immunoreactivity of 4.5-6 mo of gestation coincided with the onset of follicular development. In contrast to NPY, VIP was first found in the cortex. Both VIP and NPY immunoreactivity increased with age. From 9 mo of gestation onwards, VIP and NPY were found around blood vessels and non-vascular smooth muscle cells, in the stroma near preantral follicles, and in the theca externa of antral follicles. In addition, VIP-positive cells were observed exclusively in the granulosa layer of the preovulatory follicle at the time of the LH surge. The distribution of VIP- and NPY-immunoreactive fibers in the ovary may point to an effect of these neuropeptides on various physiological processes, including follicle development and ovarian blood flow. In addition, the presence of VIP-positive cells in the granulosa layer of the preovulatory follicle is indicative of a role for VIP in ovulation.     

Changes in gonadotrophin response to gonadotrophin releasing hormone in normal women following bilateral ovariectomy

OBJECTIVE: Pituitary responsiveness to GnRH varies throughout the normal menstrual cycle. We have investigated whether there are differences in the ovarian mechanisms which regulate gonadotrophin secretion between the follicular and the luteal phase of the cycle. DESIGN: Normally ovulating women were studied during the first week following hysterectomy plus bilateral ovariectomy performed either in the mid- to late follicular phase (follicle size 16 mm) or in the early to midluteal phase (5 days post LH peak). The response of LH to a single dose of 10 micrograms GnRH was investigated 2 hours before the operation and every 12 hours after the operation until postoperative day 4 and every 24 hours until day 8. PATIENTS: Fourteen normally cycling premenopausal women with normal FSH (< 10 IU/l). Seven women were ovariectomized in the follicular and 7 in the luteal phase. MEASUREMENTS: Pituitary response to GnRH was calculated as the net increase in FSH (delta FSH) and LH (delta LH) at 30 minutes above the basal value. RESULTS: Basal levels of FSH and LH before the operation were significantly lower in the luteal than the follicular phase (P < 0.05), while those of oestradiol (E2) were similar. Also, similar were delta LH and delta FSH values. Serum progesterone and immunoreactive inhibin (Ir-inhibin) concentrations before the operation were higher in the luteal than the follicular phase (P < 0.05). Following the operation, serum E2, progesterone and Ir-inhibin values declined dramatically, while basal FSH and LH as well as delta FSH values showed a gradual and significant increase. The percentage increase in FSH and LH values (mean +/- SEM) on day 8 after the operation was similar in the follicular (453 +/- 99% and 118 +/- 35% respectively) and the luteal phase (480 +/- 71% and 192 +/- 45% respectively). In contrast to delta FSH, delta LH values after a temporal increase 12 hours from the operation, remained stable in the follicular phase and declined significantly in the luteal phase up to day 4. CONCLUSIONS: Basal gonadotrophin secretion during the normal menstrual cycle is predominantly under a negative ovarian effect. It is suggested that in contrast to FSH, the secretion of LH in response to GnRH is controlled by different ovarian mechanisms during the two phases of the menstrual cycle.     

Plasma hormones in clomiphene citrate therapy

Daily plasma follicle-stimulating hormone (FSH), luteinizing hormone (LH), estrone (E1), estradiol-17beta (E2), progesterone (P), androstenedione (A), and testosterone (T) were measured in six clomiphene citrate (Clomid) treated cycles. Three patients ovulated and 1 of them conceived during the study cycle. Three other patients failed to ovulate in spite of some evidence of ovarian response to clomiphene treatment in 2 of them. Plasma gonadotropin levels, of LH in particular, rose during the clomiphene therapy and reached a peak during Day 5 to Day 7 of therapy. Levels of plasma estrogens, both E1 and E2, gradually rose, reflecting follicular maturation in the ovary. When E2 reached a critical level as in the normal ovulatory cycle, it triggered an LH surge which consequently initiated ovulation. When the E2 level was inadequate or excessive, ovulation failed in spite of an LH surge. Following ovulation, plasma P rose and fell in a manner similar to the normal ovulatory cycle, with occasional values that exceeded the normal range. Levels of androgens, both A and T, rose during clomiphene therapy in some cases and T seemed to fluctuate in correlation with LH level. The possible local inhibitory influence of high E2 and T levels on follicular maturation in the ovary during clomiphene therapy is suspected in some cycles in which ovarian response was evident, but ovulation failed to occur.     

Long-term ovarian function in sheep after ovariectomy and transplantation of autografts stored at -196 C

We have previously demonstrated that ovarian function and fertility can be preserved in sheep after castration by autotransplantation of cryopreserved strips of ovarian cortex. In the current experiments we have investigated the long term survival of such grafts by detailed measurements of ovarian function for a period of nearly 2 yr after autotransplantation. After ovariectomy and transplantation of frozen/thawed grafts, the concentrations of FSH and LH rose to castrate levels for about 14 weeks before falling gradually to reach near-normal levels at about 60 weeks. In the breeding season from October 1994 to March 1995, all ewes had 5-10 estrous cycles that were similar in length to those in the 4 control ewes. Luteal function as indicated by the progesterone concentration was identical before and 11 months after transplantation. In contrast, the basal concentrations of FSH and LH were persistently raised throughout the luteal phase, but showed a normal decline during the follicular phase. The concentration of inhibin A in ovarian venous plasma measured at the end of the experiment 22 months after transplantation was significantly lower than that in control ewes (mean +/- SE, 409 +/- 118 vs. 1914 +/- 555 pg/ml; P < 0.004). Transplantation of frozen/thawed ovarian tissue to SCID mice demonstrated that about 28% of primordial follicles survived the procedure. All of the ovaries transplanted into sheep contained large antral follicles and/or cysts, but very few primordial oocytes when recovered at autopsy after 22 months. These results demonstrate that despite a drastic reduction in the total number of primordial follicles, cyclical ovarian function is preserved in sheep after autotransplantation of frozen/thawed ovarian tissue and provide experimental confirmation that such a technique could provide a means of preserving fertility in women undergoing chemo- or radiotherapy for malignant disease.     

The regulation mechanism of the female menstrual cycle

The hormonal patterns during menstrual cycle, which consist of cyclic alterations in gonadotropins, estradiol, and progesterone, are controlled by hypothalamic-pituitary-ovarian feedback mechanism. GnRH produced in hypothalamus acts on the pituitary cells to secrete FSH and LH, which stimulate the follicular development. The developed follicles secrete estradiol, progesterone, inhibin, activin, and follistatin. Estradiol and progesterone, at different concentrations and/or ratios, either positively or negatively control the feedback of hypothalamic-pituitary axis in regulating the secretion of GnRH, FSH and LH. Inhibin and follistatin selectively suppress, whereas activin enhances the secretion of FSH in the pituitary. Recently, various additional factors produced by the ovary have been identified to contribute to the follicular development by paracrine and/or autocrine regulation as well as to feedback on hypothalamic-pituitary unit.     

Effect of 48 h treatment with 17 beta-oestradiol or progesterone on follicular wave emergence and synchrony of ovulation in Bos indicus cows when administered at the end of a period of progesterone treatment

The objective of this study was to determine the effect of treatment with additional progesterone (P4) or 17 beta-oestradiol (E2) at the end of a period of P4 treatment on ovarian follicular development, ovulation time, and plasma gonadotrophin and steroid hormone concentrations of Bos indicus cows. Initially, two injections of PGF2 alpha were given 14 days apart, and at the time of the second injection (Day 0) all cows received a single P4-releasing controlled internal drug release (CIDR) device that was removed 10 days later. Control cows (Group 1, n = 8) received no other treatment. On Day 8, cows in Group 2 (n = 8) and Group 3 (n = 8) received either a s.c. implant containing E2, or a second CIDR device, respectively. All CIDR devices and E2 implants were removed at a similar time on Day 10. Treatment with E2 or P4 delayed mean (+/- SD) time of ovulation (113.1 +/- 25.6 h, 153.4 +/- 44.5 h and 150.8 +/- 25.1 h for Groups 1, 2 and 3, respectively; P < 0.05) and the mean time (+/- SD) of the luteinising hormone (LH) peak (87.4 +/- 24.5 h, 124.3 +/- 45.0 h and 122.3 +/- 25.04 h for Groups 1, 2 and 3, respectively; P < 0.05). Both treatments delayed the mean (+/- SD) day of emergence of the ovulatory follicle (7.7 +/- 3.6 days, 11.3 +/- 1.7 days and 11.1 +/- 1.5 days for Groups 1, 2 and 3, respectively; P < 0.05), and reduced the variability in the day of emergence of the ovulatory follicle (P < 0.05) compared with the control cows. Variability in age and duration of dominance of the ovulatory follicle was greater in control animals compared with treated animals (P < 0.05). Treatment with E2 on Days 9 and 10 did not alter mean concentrations of gonadotrophins in the cows in Group 2 compared with control cows (P > 0.05), whereas treatment of cows with an additional CIDR device resulted in greater mean concentrations of FSH and lesser concentrations of LH on Day 9 (P < 0.05) compared with cows in Groups 1 and 2. By Day 10 mean concentrations of gonadotrophins were similar among cows in all three groups. Concentrations of E2 were less in cows in Group 3 compared with cows in Groups 1 and 2 from Day 9 to Day 11 (P < 0.05). We conclude that treatment with either E2 or P4 can influence the pattern of ovarian follicular development and ovulation in cattle; however, the mechanism of action of the two treatments may differ. Atretogenic treatments for ovarian follicles applied at the end of a period of progesterone treatment did not improve synchrony of ovulation.     

Optimizing sampling strategies for estimating quality-adjusted life years

Ovarian organ culture was used to study the influence of various gonadotropin hormones (100 ng FSH, 100 ng LH, 100 ng LH added together with 100 ng FSH; 1 i.u. hCG or 10 i.u. PMSG) on growth and development of follicles as well as on steroid secretion by ovaries of postnatal, 15-day-old mice. Ovaries were aseptically removed and single organs were placed on a piece of lens paper which was supported by a stainless steel grid in the small organ culture dish. The cultures were maintained in medium M199 supplemented with 5% of calf serum in a CO2 incubator at 37 degrees C. The morphological changes and steroid secretion measured by appropriate RIAs were studied. The stages of follicular development and the incidence of particular types of follicles were scored. Progesterone and estradiol were detected in the medium by radioimmunoassay. Differences between control and gonadotropin stimulated ovaries were found in the number of the ovarian follicles in more advanced maturation stages. There was increased number of multilaminar and antral follicles in FSH and FSH plus LH treated cells. The adding of gonadotropin hormones to the culture medium stimulated significantly progesterone secretion. The most significant effect was observed in media of cultures treated with LH and hCG. As to estradiol secretion the highest stimulatory effect was seen in cultures supplemented with FSH alone, together with LH and with PMSG. The organ culture technique applied in the current study could be a suitable model of studying the interaction of various factors as well as its effect on ovarian differentiation and on selection of dominant follicles. This system allows maintaining the structural integrity of the whole ovary, thus in the physiological functional status of the organ.     

Follicular fluid hormone concentrations after ovarian stimulation using gonadotropin preparations with different FSH/LH ratios. I. Comparison of an FSH-dominant and a purified FSH preparation

OBJECTIVE: A small amount of LH is necessary for 17beta-estradiol production in the ovarian follicle. Human menopausal gonadotropin (hMG) contains equal amounts of FSH and LH activity, whereas recombinant FSH is a gonadotropin preparation without LH. The aim of the present randomized study was to investigate whether ovarian stimulation treatment with recombinant FSH or hMG resulted in different steroidal composition of follicular fluid. METHODS: Antral fluid from mature follicles was collected in in vitro fertilization cycles and concentrations of testosterone, androstenedione, estrone, estradiol, progesterone, FSH, and LH were determined. Seven patients (27 samples) were treated with hMG, 6 patients (22 samples) with recombinant FSH. RESULTS: Androgen, estrogen, progesterone, and FSH concentrations in follicular fluid tended to be lower in the group treated with recombinant FSH, but the variation was large and differences were statistically not significant. CONCLUSION: Treatment with a gonadotropin preparation containing no LH resulted in adequate androgen and estrogen levels in antral fluid of the ovarian follicle in women with normal endocrine profiles, even during pituitary suppression by a GnRH agonist. Apparently, the amount of endogenous LH was sufficient for steroid production within the follicle.     

How does daily treatment with human chorionic gonadotropin induce superovulation in the cyclic hamster?

Daily s.c. injection of 2.0 IU hCG per day, begun on Day 1 of the cycle (estrus), results in hamsters ovulating 20.7 +/- 0.7 eggs instead of the normal number of 13.3 +/- 0.5 (SEM). This is associated with a reduced rate of follicular atresia so that more of the 10 developing follicles per ovary (large preantral stages) normally recruited on Day 1 of the cycle mature and go on to ovulate. The hCG-treated follicles were larger than control follicles, but contained similar amounts of DNA/follicle; increased size of the antral cavity accounted for their greater size. Moreover, DNA synthesis was significantly reduced in the hCG follicles on Days 2 and 4. Thecal vascularity as judged by the number of red blood cells retained in the theca or microsphere uptake by follicles indicates that on Day 2, thecal blood flow was significantly lower in the hCG-treated animals than in controls. On the other hand, after hCG treatment begun on Day 1, serum levels and in vitro incubation of individual follicles revealed that on Day 2 and beyond, androstenedione (A) and estradiol (E2) levels were elevated. After hCG treatment, the elevated serum E2 correlated with reduced serum LH on Days 3 and 4 whereas FSH was unaffected. To study in vitro steroid accumulation, the 10 largest follicles (the developing follicles) were dissected from alternate left and right ovaries from control and hCG-treated animals and incubated individually, and their histology was then compared with the steroid profiles. Accumulation of A and E2 was significantly greater in the hCG-treated follicles than in controls in a 1-h basal incubation and after the addition of 50 ng LH. Progesterone accumulation usually did not differ between the control and hCG-treated follicles. Early stage 1 atretic follicles (judged by histology) were still capable of producing A and E2 in vitro, comparable to control follicles; but, as atresia progressed, the follicles synthesized only progesterone. This is consistent with the temporal pattern previously observed in a model of induced follicular atresia in the hamster [Greenwald, Biol Reprod 1989; 40:175-181]. It is concluded that superovulation resulting from hCG injections is due to thecal production of androgens from follicles normally destined for atresia. For the untreated cyclic hamster, the critical time for thecal androgen production is the first 2 days of the cycle. The aromatizable androgens are then converted into estrogens, which in turn may maintain the microenvironment of the antral cavity, which is essential for viability of the granulosa cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Endocrine activity of induced persistent follicles in sheep

This experiment was designed to examine gonadotropin requirements for the induction and maintenance of persistent ovarian follicles in sheep. At the time of prostaglandin (PG) treatment on the tenth day of an induced estrous cycle, 8 ewes (with one ovary autotransplanted to the neck) received an injection of a GnRH antagonist ([Ac-d-Nal1, d-4-C-1-Phe2, d-Trp3, d-Arg6, d-Ala10] GnRH.HOAc; 50 microg/kg s.c.), and continuous hourly injections of exogenous ovine LH (equivalent to 1.25 microg NIH-oLH-S26) began simultaneously with this first antagonist injection (time zero). Antagonist was given three times at 3-day intervals. On Day 6, LH injections were stopped in 4 ewes (group 2) but continued in 4 other ewes (group 1) until the end of the 10-day experiment. Ovarian vein blood was sampled daily every 15 min for a 2-h period around two injections of exogenous LH (this sampling included group 2 after Day 6). Additional jugular and ovarian vein blood samples were collected every 8 h throughout the experiment. Daily ultrasound examination revealed the presence of at least one large follicle (range 4- to 7.5-mm diameter) from Day 3 to Day 10 in all ewes, but no new growing follicles (> 2 mm) were detected for at least 6 days. After Day 2, secretion of estradiol was positively correlated with that of inhibin (r = 0.83, p < 0.001), whereas FSH concentrations were inversely related to inhibin (r = -0.71, p < 0.001) and estradiol (r = -0.81, p < 0.001). In the absence of an LH surge, estradiol and androstenedione secretion (range 5-20 ng steroid/min) was maintained from Day 1 to Day 8 in group 1; but in group 2, secretion decreased abruptly when the LH injections stopped. Thus, continued low-amplitude, high-frequency LH pulses were required to maintain estradiol secretion when concentrations of FSH were < 0.5 ng/ml. However, estradiol and androstenedione secretion decreased (and FSH concentrations increased) between Days 8 and 10 in the ewes that received continued LH injections (group 1), showing that atresia in estrogenic follicles was not due to a lack of gonadotropin availability but to changes within the follicle. For the first 3 days after administration of PG, androstenedione secretion was greater than that of estradiol (p < 0.05), but from Day 4 to 6 the secretion rates were similar (p < 0.1), suggesting that aromatase may be limiting in the first 3 days whereas provision of androstenedione precursors was altered as the follicle persisted. In group 2 on Days 7 and 8 when hourly LH injections had stopped, neither androstenedione nor estradiol secretion increased after one test injection of LH; in contrast, androstenedione but not estradiol secretion increased after a second LH test injection 1 h later, suggesting that secretion of androstenedione is controlled by repeated exposure to LH. In conclusion, persistent estrogenic follicles were produced in the follicular phase in sheep by treatment with a combination of GnRH antagonist and hourly pulses of LH. Secretion of estradiol was dependent on continued hourly LH pulses of approximately 1 ng/ml and the follicles remained estrogenic for 8 days, after which time the ability to secrete estradiol and androstenedione declined even with continued LH injections.     

An inactivating mutation of the luteinizing hormone receptor causes amenorrhea in a 46,XX female

Hypergonadotropic hypogonadism is characterized by decreased gonadal function due to the inability of the gonads to respond to pituitary gonadotropins. Hypergonadotropic hypogonadism in females has many causes, among which are ovarian dysgenesis and abnormalities of the ovarian receptors for the pituitary gonadotropins. We evaluated a woman who presented with amenorrhea due to hypergonadotropic hypogonadism, but who had structurally normal ovaries. She is a sister of two previously identified 46,XY male pseudohermaphrodites with Leydig cell hypoplasia. Injection of hCG did not cause any change in plasma levels of estradiol or progesterone, suggesting complete ovarian resistance to LH. Analysis of the DNA sequence of the LH receptor gene revealed that the patient is homozygous for the same single base change as her two brothers. This mutation causes substitution of an alanine residue by a proline at position 593. In vitro analysis of the mutant LH receptor in cultured human embryonic kidney 293 cells documented that the receptor is unable to stimulate adenylyl cyclase in response to hCG. Plasma levels of estradiol and progesterone were low, whereas LH and FSH levels were increased. On histological analysis of the ovary, follicles were seen at all developmental stages. Nonetheless, primary amenorrhea had been present for 5 yr, and repeated measurements of plasma estradiol and progesterone indicate that ovulation does not occur. These results document the existence of inherited LH resistance as a cause of primary amenorrhea in women. The combined clinical and molecular observations are consistent with previous experimental data suggesting that in humans, LH is necessary for ovulation but follicular maturation can occur in the presence of FSH alone.     

The presence of luteinized unruptured follicle syndrome and altered folliculogenesis in rats with surgically induced endometriosis

OBJECTIVE: Our purpose was to determine in the rat model whether endometriosis could influence ovarian function by altering oocyte release or folliculogenesis. STUDY DESIGN: We histologically examined the ovaries of reproductively cycling rats with (n = 16) and without (n = 10) surgically induced endometriosis. The rats in these two groups were further subdivided into unilaterally ovariectomized or ovarian-intact groups. Serial sections of ovaries were examined, and follicular development and frequency of luteinized unruptured follicles were determined. RESULTS: A significant tenfold increase in the number of luteinized unruptured follicles was observed in the ovaries from rats with endometriosis (2.7 per rat) compared with unoperated and sham-operated control groups (overall mean 0.26 per rat, p < 0.05). Additionally, ovaries from unilateral ovariectomized animals with endometriosis contained four times as many luteinized unruptured follicles (four per rat) as did the ovaries from bilaterally ovarian-intact rats with endometriosis (1.40 per rat, p < 0.01). Fewer follicles were present in rats with endometriosis (180 follicles per ovary) than in control rats (231 follicles per ovary, p < 0.05). CONCLUSION: In the rat model the presence of ectopic endometrium is associated with an increased frequency of luteinized unruptured follicles and altered follicular development.     

In vitro transfer rate of 14C from acetate-1-14C into ovarian steroids in the rat ovary during the estrous cycle and effects of LH and FSH

Fluctuations of ovarian biosynthetic activity and effects of exogenous LH and FSH on it during the estrous cycle were investigated by measuring in vitro transfer rates of 14C from 14C-1-acetate into progesterone (P), 20 alpha-hydroxy-pregn-4-en-3-one (20 alpha-OH-P) and estrogen (estradiol and estrone, E) in the ovarian homogenates from rats autopsied at 2 hour intervals. The transfer rate of 14C from 14C-1-acetate into P was lowest in the afternoon of estrus and increased from the morning of diestrus 1, making its peaks during the afternoon of diestrus 2 and in the midnight of proestrus. The transfer of 14C into 20 alpha-OH-P was high on the days of diestrus 2 and proestrus with its peak in the afternoon of the latter day. The maximum transfer of 14C into E in the afternoon of proestrus and a high rate in the morning of estrus with relatively low one in the midnight were observed. Exogenously injected LH (150 mug) or FSH (150 mug) was either stimulatory or inhiibitory to the transfer rates of 14C from 14C-1-acetate into ovarian steroids. During day time of diestrus 2 and midnight between proestrus and estrus, the transfer of 14C into P and 20 alpha-OH-P increased by LH, and during day time of proestrus and from the afternoon of estrus to the morning of diestrus 1 decreased. The transfer of 14C into E increased by LH from the afternoon of diestrus 2 to the morning of proestrus, and decreased during the afternoon of proestrus and from the afternoon of estrus to the morning of diestrus 2. Administration of FSH was also stimulatory or inhibitory. The 14C transfer into P and 20 alpha-OH-P increased by FSH from the afternoon of estrus to the morning of proestrus, but in the afternoon of proestrus they decreased. Transfer of 14C into E increased by FSH significantly on the days of diestrus 2 and proestrus, and slightly on the day of estrus, while it decreased in the afternoon of diestrus 1.     

Patterns of ovarian growth and development in cattle with a growth hormone receptor deficiency

Nutritionally induced changes in growth hormone (GH) and IGF-I are associated with decreased ovarian function and may partially explain infertility and anestrus in undernourished cattle. The reproductive importance of GH and IGF-I was tested in cattle with a GH receptor deficiency (GHRD) that have reduced blood IGF-I. Blood was collected daily for plasma, and ovaries were examined daily by ultrasonography for 3 wk during an estrous cycle (estrus = d 0) in GHRD (n = 8) and control (n = 8) cattle. On d 18, blood samples were collected every 10 min for 6 h to measure LH. The GHRD cattle had fewer small antral ovarian follicles (2 to 5 mm, P < .01). After estrous cycle d 5, the first-wave dominant follicle stopped growing in GHRD but continued growing in controls (P < .001). Size of the CL was equivalent for GHRD and controls until d 5, after which CL development slowed in GHRD (P < .01). Likewise, plasma progesterone concentrations were less in GHRD (P < .001). During the luteal phase, GHRD cattle failed to develop follicles greater than 10 mm in diameter (endocrine status x day, P < .05). Size and rate of growth of preovulatory follicles, plasma estradiol, plasma FSH, and plasma LH (d 18 bleed) were similar in GHRD and controls. In conclusion, an important role for GH, GH receptor, and IGF-I in ovarian function was supported because GHRD cattle had distinctly different patterns of ovarian development compared with control cattle.