Differential changes in inhibin A, activin A, and total alpha-subunit levels in granulosa and thecal layers of developing preovulatory follicles in the chicken

Accumulating evidence implicates inhibins and activins as endocrine and local regulators of follicular development in mammals, and it was recently confirmed that inhibin/activin alpha and betaA genes are also expressed in the avian ovary. To investigate the potential involvement of these proteins in the chicken ovary, thecal and granulosa layers of the four largest follicles (F1-F4) and the most recent postovulatory follicle were collected from hens (10/group) killed 4, 12, and 20 h before the expected time of F1 ovulation. Inhibin A and activin A concentrations of tissue extracts (expressed per mg DNA) were measured using validated two-site enzyme-linked immunosorbent assays; total immunoreactive inhibin alpha-subunit (ir-alpha) was also measured by heterologous RIA (Monash assay). Inhibin A and ir-alpha were largely confined to the granulosa layer, whereas activin A was much more abundant in the thecal layer. Granulosa inhibin A contents were similar in F4 and F3, but increased approximately 40-fold from F3-F1 (P < 0.0001). As such, the F1 granulosa layer was by far the richest source of inhibin A in the chicken ovary, but contained very little activin A. Total ir-alpha in granulosa was much more abundant than inhibin A and increased only 3-fold from F4-F1 (P < 0.001). Activin A in both granulosa and theca showed little variation between F1 and F4 follicles (by ANOVA, P > 0.05). The inhibin A content of F1 granulosa was maximal 12 h before ovulation and had fallen approximately 6-fold (P < 0.0001) within 8 h, suggesting an inhibitory effect of the preovulatory LH surge on the F1 capacity to synthesize inhibin A. Inhibin A, activin A, and ir-alpha were all less in the postovulatory follicle compared with F1 before ovulation (P < 0.0001). In conclusion, application of the present two-site enzyme-linked immunosorbent assays to the chicken ovary revealed 1) divergent tissue distribution of inhibin A and activin A within preovulatory follicles, and 2) differential regulation of granulosa cell production of inhibin A and activin A dimers during preovulatory follicular development. These findings of dynamic changes in inhibin A, activin A, and total ir-alpha support the hypothesis that these proteins subserve regulatory roles during preovulatory follicular development in the hen.     

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)     

Activin A and inhibin B in extra-embryonic coelomic and amniotic fluids, and maternal serum in early pregnancy

Activin A and inhibin B levels were measured, using a two-site enzyme immunoassay, in extra-embryonic coelomic fluid, amniotic fluid and maternal serum samples retrieved from 23 healthy pregnant women, at 8 (n=8), 9 (n=8), and 10 (n=7) weeks of gestation. Dimeric activin A and inhibin B were measurable in all samples. Median (+/-SEM) activin A concentrations in coelomic fluid (0.98+/-0.34 ng/ml) were significantly higher than in maternal serum (0.68+/-0.05 ng/ml) and in amniotic fluid (0.09+/-0.04 ng/ml) (P<0.05). Maternal serum activin A levels were significantly higher than amniotic fluid concentrations. Median (+/-SEM) inhibin B concentrations in coelomic fluid (24.32+/-6.02 pg/ml) were significantly higher than in maternal serum (5.94+/-0.97 pg/ml) and in amniotic fluid (6.31+/-1.53 pg/ml) (P<0.05), while no significant difference between maternal serum levels and amniotic fluid concentrations was found. No significant difference in activin A and inhibin B levels in extra-coelomic fluid, amniotic fluid, and maternal serum throughout the 3 weeks of pregnancy was found. The present study showed that coelomic fluid is an important reservoir of activin A and inhibin B, supporting the hypothesis that the extra-embryonic coelom may have a secretory role during the first 11 weeks of gestation.     

Expression of inhibin alpha and inhibin/activin beta A subunits in the granulosa layer of the large preovulatory follicles of the hen

The expression of the mRNA for the inhibin/activin subunits (alpha and beta A) in the granulosa layer of the five largest preovulatory follicles of the hen was investigated. Total RNA from the granulosa layer of the F5 (the fifth largest) to F1 (the largest) follicles was extracted and analyzed by Northern blot analysis using homologous chicken inhibin alpha and beta A subunit cDNA probes. RNA loading was quantified by a cDNA probe of bovine 18S rRNA. Results showed that for the chicken inhibin alpha subunit mRNA signals (n = 3), the mean relative intensity for the F1, F2, F3, and F4 follicles was 0.50 +/- 0.10 ( +/- SEM,), 0.52 +/- 0.08, 0.59 +/- 0.06, and 0.81 +/- 0.04, respectively, compared to a mean relative intensity of 1.00 (p < 0.05) for the F5 follicle. For the beta A subunit mRNA signals (n = 3), the mean relative intensity for the F5, F4, F3, and F2 follicles was 0.25 +/- 0.06, 0.28 +/- 0.15, 0.40 +/- 0.17, and 0.48 +/- 0.10 (p < 0.05) for the F1 follicle. The inhibin alpha subunit was also estimated to be more abundantly expressed among follicles in the granulosa layer than was the beta A subunit. Our data indicate that the expression of inhibin alpha and beta A subunits is differentially regulated in the hen granulosa layer during follicular development. Expression of the alpha subunit is reduced with follicular development whereas inhibin beta A subunit expression is dramatically enhanced. In addition, the granulosa layer of the large preovulatory follicles may produce more inhibin alpha subunit than beta A subunit, and the F1 follicle may be the primary source of the beta A subunit for dimeric inhibin and/or activin in the hen.     

Recent development of nucleic acid drug

The dynamics of ovarian follicular development and the pattern of pituitary and ovarian hormone concentration were investigated during the luteal phase in ewes with autotransplanted ovaries. The follicles were measured by ultrasound and samples of ovarian and jugular venous blood were collected at intervals of 12 h. Blood samples were collected before and after a GnRH challenge (250 ng GnRH, i.v.) to allow the determination of basal and LH-stimulated concentration of ovarian steroids. Throughout the luteal phase, large antral follicles developed in three waves, each of which was preceded by a rise in the concentration of FSH (P < 0.05). The concentrations of oestradiol and androstenedione in the unstimulated and LH-stimulated samples were similar (P > 0.05) during the first 3 days of the luteal phase but differed thereafter, with the LH-stimulated being significantly higher than the basal concentrations (P < 0.05). In the first wave of follicular development the changes in follicular size were accompanied by an increase in the concentration of ovarian steroids and inhibin A. During the second follicular wave, although changes in follicle diameter were similar to the first wave (P > 0.05), the basal concentration of ovarian steroids and inhibin A remained unchanged throughout the period of emergence and demise of the large follicles. These results confirm that the development of large antral follicles during the luteal phase of the sheep occurs in successive waves that are associated with fluctuations in FSH secretion. However while the results strongly suggest that fluctuations in both inhibin A and oestradiol secretion control FSH during the first follicular wave, the cause of the FSH fluctuations associated with waves two and three is unclear. Final resolution of this issue may need to await the development of a specific assay for dimeric inhibin B.     

Paradoxical action of activin A on folliculogenesis in immature and adult mice

Activin A is a gonadal protein originally isolated from follicular fluid and is recognized as a local regulator of granulosa cell differentiation. Whether activin A promotes folliculogenesis, however, still remains unclarified. The present study was designed to elucidate the effect of activin A on follicular growth in in vitro follicle culture systems. Preantral follicles, 100-120 microm in diameter, were mechanically isolated from BDF1 hybrid immature mice (11 days old) and adult mice (8 weeks old), then cultured for 4 days in a serum-free medium supplemented with activin A (100 ng/ml), FSH (100 mIU/ml), and a combination of both. Follicular diameter was measured daily, and the amount of estradiol and inhibin released at day 4 was determined by RIA. Preantral follicles collected from immature mice showed a significant increase in diameter when cultured with activin A or both activin A and FSH. FSH alone showed no significant effect on the diameter of follicles from immature mice. In contrast, the diameter of preantral follicles from adult mice significantly increased in response to FSH. Activin A did not stimulate growth of follicles from adult mice, and more interestingly, blocked the effect of FSH. The inhibitory action of activin A was in part restored by co-culture with follistatin (100 ng/ml). These results indicate that activin A is folliculogenetic in the prepubertal mouse, but not in adults.     

Inhibin B in men with normal and disturbed spermatogenesis

Inhibin, a dimeric gonadal glycoprotein, inhibits the production and/or secretion of follicle stimulating hormone (FSH). The major species currently recognized are inhibin A (alphabeta A subunit) and inhibin B (alphabeta B subunit). In men, inhibin B seems to be the physiologically important form of inhibin. Therefore we measured serum inhibin B using a new two-site immunoenzymatic assay in 14 men (mean +/- SEM age, 34.5 +/- 0.7 years) with sperm counts >20 x 10(6)/ ml, in 35 men (mean +/- SEM age, 36.4 +/- 1.3 years) with oligozoospermia (sperm count <20 x 10(6)/ml) and in men with azoospermia (three orchidectomized men, three men with Klinefelter's syndrome, 10 men with Kallmann's syndrome). We compared inhibin B concentrations with serum FSH and sperm concentrations. In men with normal sperm concentrations (44.7 +/- 6.4 x 10(6)/ml), the concentration of inhibin was 223 +/- 18 pg/ml and of FSH 5.0 +/- 0.7 IU/l; in patients with low sperm concentrations (3.7 +/- 0.8 x 10(6)/ml), the concentration of inhibin B was 107 +/- 12 pg/ml and of FSH 12.2 +/- 1.5 IU/l. In all patients, except those with hypogonadotrophic hypogonadism, the relationship between inhibin B and FSH concentrations was inverse (r = -0.69, P < 0.0001). In all patients the sperm concentration was positively correlated with inhibin B concentrations (r = 0.70, P < 0.0001) and negatively correlated with FSH concentrations (r = -0.37, P < 0.01). We conclude that inhibin B may be a marker of exocrine testicular function and could offer improved diagnosis and treatment modalities for male infertility.     

Follicle stimulating hormone-granulosa cell axis involvement in the antifolliculotrophic effect of low dose mifepristone (RU486)

This study was designed to assess the involvement of follicle stimulating hormone (FSH)-granulosa and luteinizing hormone (LH)-theca axes in the antifolliculotrophic effect of mifepristone. Plasma gonadotrophins, including plasma LH bioactivity and pulsatility, oestradiol, testosterone and inhibin concentrations, and follicular growth were monitored in volunteer women treated with placebo or mifepristone in two consecutive cycles. Mifepristone was given either as a single dose of 5 mg (n = 7) when the leading follicle had reached a diameter between 12 and 14 mm, or as a multiple dose of 5 mg/day for 3 days, beginning when the leading follicle had reached a diameter between 14 and 16 mm (n = 5) or between 6 and 11 mm (n = 5). Following the single dose of mifepristone, follicular growth and the accompanying increase in plasma oestradiol were arrested at 12 and 36 h respectively without changes in gonadotrophin or testosterone serum concentrations. The 3 day regimen arrested follicular growth and oestradiol rise and decreased plasma inhibin concentrations when follicles were larger than 12 mm at the onset of treatment. These results indicate that the antifolliculotrophic action of mifepristone is associated with a selective compromise of the FSH-granulosa axis of dominant follicles that have passed a critical stage of growth.     

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.     

Selection, dominance and atresia of follicles during the oestrous cycle of heifers

This study examined the correlation between measurement of follicle growth by ultrasound, and measurement of intrafollicular ratios of oestradiol and progesterone concentrations and the serum concentrations of FSH during selection, dominance and atresia or ovulation of dominant follicles in heifers. Heifers were ovariectomized on days 0 (before LH surge), 1 (after LH surge, preovulation), 1 (postovulation), 3, 6 and 12 of the oestrous cycle. Blood samples were collected at 4-6 h intervals. After ovariectomy all follicles > or = 5 mm were measured and follicular fluid was aspirated. Follicles were classified by size according to ultrasound (F1, largest; F2, second largest; F3, all remaining follicles > or = 5 mm) and by the ratio of oestradiol:progesterone concentrations. During the follicular phase, a single dominant oestrogen-active follicle increased in diameter while serum concentrations of LH increased and FSH decreased (P < 0.05). On day 1 (after LH surge, preovulation), serum LH and FSH decreased to pre-surge concentrations (P < 0.0001), while follicle size and intrafollicular progesterone concentration increased and oestradiol concentration decreased (P < 0.05). A dominant nonovulatory follicle, classified as oestrogen-active on days 1, 3 and 6 and oestrogen-inactive on day 12, increased in size from day 1 to day 7 and lost dominance during days 10-12, coincident with the growth of multiple oestrogen-active follicles. The serum FSH concentration increased transiently (P < 0.05) before each new wave of dominant follicular growth. The overall correlation of ultrasound measurements of follicle diameter with measures of follicle size after ovariectomy was high.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential responses of granulosa cells from small and large follicles to follicle stimulating hormone (FSH) during the menstrual cycle and acyclicity: effects of tumour necrosis factor-alpha

This study determined effects of follicle stimulating hormone (FSH) alone and in combination with tumour necrosis factor (TNF), on granulosa cells from small (5-10 mm diameter) and large (>10-25 mm) follicles during follicular and luteal phases of the cycle and during periods of acyclicity. Granulosa cells were collected from ovaries of premenopausal women undergoing oophorectomy. The cells were cultured with human FSH (2 ng/ml) and testosterone (1 microM) in the presence or absence of human TNF-alpha (20 ng/ml). Media were removed at 48 and 96 h after culture and progesterone, oestradiol and cAMP in media were measured by radioimmunoassays. FSH stimulated the accumulation of oestradiol from granulosa cells of small follicles during the follicular and luteal phases but not during acyclicity; and TNF reduced oestradiol accumulation in the presence of FSH. Interestingly, in granulosa cells from small follicles, progesterone and cAMP secretion increased in response to FSH and neither was affected by TNF. Thus, TNF specifically inhibited the conversion of testosterone to oestradiol in granulosa cells from small follicles. FSH stimulated oestradiol production by granulosa cells of large follicles obtained only during the follicular phase of the cycle and TNF inhibited the FSH-induced oestradiol secretion. Granulosa cells obtained from large follicles during the luteal phase and during acyclicity did not accumulate oestradiol in response to FSH. However, FSH increased progesterone and cAMP secretion by granulosa cells obtained from large follicles during the follicular and luteal phases. During the luteal phase alone, TNF in combination with FSH increased progesterone accumulation above that of FSH alone. FSH did not increase progesterone, oestradiol or cAMP secretion by granulosa cells obtained from large follicles during acyclicity. Thus, FSH increases progesterone, oestradiol and cAMP secretion by granulosa cells of small follicles during the follicular and luteal phases and TNF appears to inhibit FSH-induced oestradiol secretion specifically in those cells. In large follicles, FSH-stimulated granulosa cell secretion of oestradiol is limited to the follicular phase and this effect can be inhibited by TNF. In addition, when granulosa cells of large follicles do not increase oestradiol secretion in response to FSH, TNF stimulates progesterone secretion.     

High levels of immunoreactive endothelin-1 in human follicular fluids

Follicular fluids were obtained from 180 follicles of 15 women undergoing follicular aspiration for in-vitro fertilization. Follicular development was induced by a combination of buserelin acetate and human menopausal gonadotrophin. Endothelin-1 (ET-1) concentrations in human follicular fluids were determined by specific radioimmunoassay. ET-1-like immunoreactivity (ET-1-LI) ranged from 338 to 928 pg/ml. ET-1-LI concentrations in follicular fluids obtained from immature (< 15 mm) follicles were significantly higher than those from mature (15-25 mm) and post-mature (> or = 25 mm) follicles. No correlation was found between the concentration of ET-1-LI, on the one hand, and that of oestradiol, progesterone, testosterone, prolactin, luteinizing hormone, insulin-like growth factor-I, prostaglandin E2 or platelet activating factor on the other, in follicular fluids. However, a significant positive correlation was observed between ET-1-LI concentration and follicle stimulating hormone and IGF-II concentrations, respectively. These data suggest that the high concentration of ET-1 found in follicular fluids may play some physiological role in follicular development.     

Distribution of histamine in laying hen ovary

In a laying hen, histamine was found to be present in all compartments of the ovary, i.e. stroma with follicles < 1 mm, small white (1-4 mm), large white (4-8 mm), atretic white, yellow preovulatory (8-35 mm) and postovulatory follicles. Stroma containing non-yolky follicles exhibited the highest histamine concentration (6080 +/- 331 ng/g wet wt. tissue) which differed significantly (P < 0.01) from histamine levels observed in all examined classes of ovarian follicles. High histamine concentration was found in small, large and atretic white follicles as well as in older postovulatory follicles whereas low levels of histamine contained yellow preovulatory and younger postovulatory follicles. Population of yolky white follicles presented significant (P < 0.01) differences in histamine level among small (4280 +/- 333), atretic (2940 +/- 193) and large (2010 +/- 110 ng/g) follicles. Within hierarchy of yellow preovulatory (F7-F1) follicles initial decrease in histamine concentration, from 859.3 +/- 51.5 ng/g in F7 follicle to 363.9 +/- 28.3 ng/g in F4 follicle, was followed by the increase as follicle matured, reaching the highest level in F1 follicle (711.4 +/- 35.9 ng/g). In postovulatory (P1-P5) follicles histamine concentration gradually increased as they were getting older, from 604.3 +/- 49.3 ng/g in P1 follicle to 2253 +/- 197 ng/g in P5 follicle. Determination of histamine in relation to ovulation revealed significant (P < 0.01) difference both in histamine concentration and content between the largest preovulatory F1 follicle and the largest postovulatory P1 follicle, being 0.5 h before and 0.5 h after ovulation, respectively. It is suggested that in chicken, ovarian histamine may play a role in the follicular development and/or the ovulatory process.     

Gonadotropic control of secretion of dimeric inhibins and activin A by human granulosa-luteal cells in vitro

PURPOSE: It is well established that human granulosa cells and luteal cells express inhibin/activin subunit protein and secrete immunoreactive inhibin. The gonadotropic control of secretion of different molecular forms of inhibin and activin A by granulosa-luteal cells (G-LCs) was investigated using recently developed specific enzyme immunoassays (EIAs). METHODS: Granulosa-luteal cells obtained at IVF egg pickup were cultured in a serum-free medium at 37 degrees C in a water-saturated incubator with 5% CO2 for up to 5 days. Experiments with varying concentrations of human FSH, hLH, and hCG were carried out. RESULTS: FSH raised the secretion of inhibin A and pro-alpha C-containing inhibins after 24 and 48 hr in culture. Inhibin B was raised after 24 hr and activin A was raised after 48 hr of FSH treatment. LH treatment for 24 hr stimulated inhibin A, inhibin B, pro-alpha C, and activin A. hCG stimulated G-LC secretion of inhibin A after 48 hr and pro-alpha C after 24 hr. Paradoxically, inhibin B secretion was inhibited by 1 and 10 ng/ml hCG after 48 hr. Activin A was stimulated by hCG after 24 and 48 hr of incubation. G-LC secretion of estradiol and progesterone was also stimulated significantly by LH and hCG. CONCLUSIONS: Secretion of dimeric inhibins and activin A is controlled differentially by gonadotropins.     

Healthy women and patients with endometriosis show high concentrations of inhibin A, inhibin B, and activin A in peritoneal fluid throughout the menstrual cycle

Inhibin A, inhibin B, and activin A are growth factors which play local autocrine/paracrine roles in reproductive tissues. Since peritoneal fluid hormone content may reflect in part ovarian and endometrial secretory activities, the present study aimed to evaluate: (i) whether inhibin alpha-, activin betaA- and betaB-subunits, and activin receptor type II and type IIB mRNA are expressed in peritoneal tissues; (ii) expression and secretion of inhibin A and B, and activin A in cultured endometriotic cells; and (iii) concentrations of inhibin A and B, and activin A in serum and in peritoneal fluid in healthy women and in patients with endometriosis throughout the menstrual cycle. A group of women (n = 72) was recruited at laparoscopy for infertility investigation and divided into two groups: (i) control healthy women (n = 35), (ii) women with endometriosis (n = 37). Both groups were subdivided according to the follicular and luteal phase of the menstrual cycle. At the time of laparoscopy, specimens of peritoneal tissues were collected from three healthy women, while endometriotic tissue samples were collected and cultured from three women with endometriosis. Peritoneal tissues and cultured endometriotic cells expressed inhibin alpha-, activin betaA-, and betaB-subunits, and activin receptors mRNAs; in addition, inhibin-related proteins were measurable in culture medium. In healthy women, inhibin A and B, and activin A concentrations in peritoneal fluid were significantly higher than in serum (P < 0.001), at both phases of the menstrual cycle. Peritoneal inhibin A and B, and activin A concentrations were not significantly different between healthy women and patients with endometriosis, either when evaluated according to the degree of the disease and/or to the phase of the menstrual cycle. In conclusion, the findings that high concentrations are present in peritoneal fluid and that menstrual cycle-related changes occur suggest that reproductive organs may contribute to inhibin-related proteins in peritoneal fluid.     

Sequential step-up and step-down dose regimen: an alternative method for ovulation induction with follicle-stimulating hormone in polycystic ovarian syndrome

This study was designed to compare both the effectiveness and safety of two low-dose gonadotrophin regimens (step-up versus sequential step-up and step-down) for ovulation induction in polycystic ovarian syndrome (PCOS) patients. In all, 56 infertile clomiphene citrate-resistant PCOS patients were included in this prospective randomized study. A total of 38 cycles were conducted with a classic step-up protocol, whereas for 35 cycles the follicle-stimulating hormone (FSH) threshold dose was reduced by half when the leading follicle reached 14 mm in diameter (sequential protocol). Serum oestradiol, progesterone and luteinizing hormone concentrations and follicular growth rate were evaluated during the cycle. At the time of human chorionic gonadotrophin administration, cycles treated with sequential protocol exhibited significantly lower oestradiol concentrations [434 +/- 45 versus 593 +/- 67 pg/ml (mean +/- SEM)] and the number of medium-sized (14-15 mm) follicles was significantly reduced (0.3 +/- 0.1 versus 0.8 +/- 0.2) compared with cycles treated with the classic step-up protocol. Moreover, in these cycles serum luteal oestradiol concentrations were decreased significantly (350 +/- 77 versus 657 +/- 104 pg/ ml) compared with the classic step-up protocol. A sequential step-up and step-down protocol seems to be a safe and effective regimen for ovulation induction in PCOS patients. Decreasing the FSH dose following step-up follicular selection may be an alternative method to avoid multifollicular development.     

Important microsatellite markers in the investigation of replication errors (RER) in colorectal carcinomas

PURPOSE: Our purpose was to assess the value of monitoring serum P and inhibin A to determine how values might improve the clinical monitoring of natural cycle in vitro fertilization (IVF)-embryo transfer (ET) patients. METHODS: All patients (n = 26) who underwent natural-cycle IVF-ET (n = 35) were analyzed. Groups were evaluated according to patients who had a spontaneous luteinizing hormone (LH) surge (group I) and women receiving human chorionic gonadotropin (hCG) who underwent subsequent oocyte aspiration (group II). Group II was further evaluated according to women who did (n = 10) and did not (n = 7) have an ET. All cycles were evaluated with serial transvaginal ultrasonography and serum estradiol, progesterone, and inhibin A. When follicle maturity was achieved, hCG, 10,000 IU, was administered intramuscularly if a LH surge was not detected. Transvaginal ultrasound-guided aspiration was performed 34-36 hr after hCG administration followed by a 48-hr transcervical ET. RESULTS: No differences were seen in cycles the day prior to (d-1) and the day of a spontaneous LH surge, (n = 18) or hCG (d-0)(n = 17) in group I or group II with respect to lead follicular diameter (d-1, 15.3 +/- 0.6 vs. 14.2 +/- 0.9 mm; d-0, 17.4 +/- 0.8 vs. 17.8 +/- 0.6 mm) and serum estradiol (d-1, 148 +/- 15 vs. 150 +/- 15 pg/ml; d-0, 218 +/- 15 vs. 199 +/- 16 pg/ml), respectively. However, serum progesterone was significantly elevated in group I compared with group II on d-1 (0.82 +/- 0.6 vs. 0.48 +/- 0.04 ng/ml; P < 0.05) and d-0 (1.1 +/- 0.12 vs. 0.63 +/- 0.08 ng/ml; P < 0.05). Inhibin A was significantly greater on d-1 in group I (24 +/- 2.5 vs. 15 +/- 2.2 pg/ml; P < 0.05). In group II, cycles that resulted in an ET (n = 10) compared with group II cycles that did not (n = 7) revealed a significant difference in serum progesterone (0.51 +/- 0.05 vs. 0.7 +/- 0.07 ng/ml; P < 0.05) and inhibin A (15 +/- 2.5 vs. 37.3 +/- 5 pg/ml; P < 0.05) the day of hCG. CONCLUSIONS: The possible application of serum progesterone and inhibin A in managing natural-cycle IVF-ET is suggested. These assays may predict women who should be set up for egg retrieval, while cancelling others in spite of the absence of an LH surge.     

Active transforming growth factor-beta in wound repair: determination using a new assay

The increase in serum FSH that accompanies female reproductive aging occurs before changes in estradiol (E2). A decrease in negative feedback from inhibin A (a product of the dominant follicle and corpus luteum) and/or inhibin B (secreted by developing follicles) may explain the rise in FSH with age. To test the hypothesis that decreases in inhibin A or inhibin B occur at an age at which the first increase in follicular phase FSH is evident, daily blood samples were obtained across the menstrual cycle from younger (<35 yr; n = 23) and older (35-46 yr; n = 21) cycling women. These cross-sectional studies were complemented by longitudinal data in 3 women studied at a 10-yr interval. In the early follicular phase, mean inhibin B was lower in older cycling women (88 +/- 7 vs. 112 +/- 10 pg/mL; P < 0.05) and FSH was higher (13.0 +/- 0.5 vs. 11.2 +/- 0.7 IU/L in older vs. younger, respectively; P < 0.04). In the mid- and late follicular phases, inhibin B was also lower in the older women (117 +/- 9 vs. 146 +/- 10 and 85 +/- 8 vs. 117 +/- 11 pg/mL; P < 0.04), whereas E2 was higher (105 +/- 14 vs. 68 +/- 5 and 240 +/- 27 vs. 163 +/- 9 pg/mL; P < 0.02), and no differences in FSH were observed in the two groups at these times. In women studied longitudinally, FSH and inhibin B varied inversely in the follicular phase. In the early luteal phase, mean inhibin B was lower in the older group (64 +/- 6 vs. 94 +/- 12 pg/mL; P < 0.03), and FSH was higher (12.5 +/- 1.0 vs. 9.7 +/- 0.6 IU/L; P < 0.03). In the mid- and late luteal phases, inhibin B was also lower in older subjects (21 +/- 2 vs. 33 +/- 5 and 22 +/- 2 vs. 36 +/- 6 pg/mL; P < 0.02). No difference in inhibin A, E2, or progesterone was observed across the luteal phase, between the two groups. However, in all subjects studied longitudinally, increased age was associated with a decrease in inhibin A, inhibin B, and progesterone in the absence of changes in E2. Our conclusions were: 1) reproductive aging is accompanied by decreases in both inhibin B and inhibin A; 2) the decrease in inhibin B precedes the decrease in inhibin A and occurs in concert with an increase in E2, suggesting that inhibin B negative feedback is the most important factor controlling the earliest increase in FSH with aging; 3) these studies suggest that the decrease in inhibin B is the earliest marker of the decline in follicle number across reproductive aging.     

Local regulation of primate granulosa cell aromatase activity

Granulosa cells produce inhibin and activin, proteins implicated in the local regulation of preovulatory follicular development. To assess interactions among FSH, LH, inhibin and activin on primate granulosa cell aromatase activity, we studied primary granulosa cell cultures from the ovaries of the common marmoset (Callithrix jacchus), a monkey with an ovarian cycle similar in length to the human cycle. The distinctive action of activin was augmentation of gonadotropin-responsive aromatase activity throughout antral follicular development. FSH-stimulated aromatase activity in granulosa cells from immature follicles was augmented many fold by picomolar amounts of activin. In cell cultures from preovulatory follicles, the presence of activin stimulated basal aromatase activity in the absence of gonadotropin, as well as augmenting the action of LH. Thus, locally produced activin has the potential to modulate aromatase activity in developing ovarian follicles. By contrast, inhibin or inhibin alpha-subunit purified from bovine follicular fluid had minimal effects on aromatase activity. The only significant effect was slight suppression of FSH-inducible aromatase activity in granulosa cells from immature follicles at an inhibin concentration of 100 ng/ml. The finding that inhibin has a negligible effect on aromatase activity in granulosa cells from mature follicles suggests that it is unlikely to exert a physiologically significant influence on aromatase activity in vivo. However, evidence from other studies suggests that inhibin might affect aromatization indirectly through acting locally to modulate thecal androgen (aromatase substate) production. Therefore, both inhibin and activin have the potential to contribute at different levels to paracrine and autocrine regulation of follicular oestrogen synthesis.     

A bolus of recombinant human follicle stimulating hormone at midcycle induces periovulatory events following multiple follicular development in macaques

The efficacy of follicle stimulating hormone (FSH) as an alternative to luteinizing hormone (LH)/human chorionic gonadotrophin (HCG) for the initiation of periovulatory events in primate follicles is unknown. A single bolus of 2500 IU recombinant (r)-hFSH was compared to 1000 IU r-HCG for its ability to promote oocyte nuclear maturation and fertilization, granulosa cell luteinization and corpus luteum function following r-hFSH (60 IU/day) induction of multiple follicular development in rhesus monkeys. Following the r-hFSH bolus, bioactive luteinizing hormone concentrations were <3 ng/ml. Peak concentrations of serum FSH (1455+/-314 mIU/ml; mean+/-SEM) were attained 2-8 h after r-hFSH, and declined by 96 h. Bioactive HCG concentrations peaked between 2-8 h after r-HCG and remained > or = 100 ng/ml for >48 h, while immunoreactive FSH concentrations were at baseline. The proportion of oocytes resuming meiosis and undergoing in-vitro fertilization (IVF) were comparable for r-hFSH (89%; 47+/-19%) and r-HCG (88%; 50+/-17%). In-vitro progesterone production and expression of progesterone receptors in granulosa cells did not differ between groups. Peak concentrations of serum progesterone in the luteal phase were similar, but were lower 6-9 days post-FSH relative to HCG. Thus, a bolus of r-hFSH was equivalent to r-HCG for the reinitiation of oocyte meiosis, fertilization and granulosa cell luteinization, but a midcycle FSH surge did not sustain normal luteal function in primates.