Growth factors in ovarian function.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
J K Findlay
Publications and source records attributed to J K Findlay.
Effects of immunization against recombinant bovine inhibin alpha subunit on circulating concentrations of gonadotrophins in ewes.
Immunization of ewes against a pure recombinant preparation of the alpha subunit of bovine inhibin (alpha-bI) resulted in a three- to fourfold increase in ovulation rate, associated with antibodies in plasma recognizing pure native 31 kDa inhibin. The aim of this study was to examine the effects of this immunization on basal and GnRH-stimulated plasma concentrations of FSH and LH in ewes during the anoestrous and breeding seasons. The groups were untreated control ewes (n = 5), control ewes treated with keyhole limpet haemocyanin (KLH alone, n = 4), ewes treated with alpha-bI alone (n = 4) and alpha-bI-KLH conjugate-treated ewes (n = 3). There were no effects of immunization on basal FSH or LH in anoestrous ewes, despite the presence of antibodies recognizing 31 kDa inhibin. In the breeding season, immunization against alpha-bI resulted in increased basal (follicular phase, P less than 0.1; luteal phase P less than 0.05) and GnRH-stimulated (follicular phase only, P less than 0.001) release of FSH, but not LH. The data are compatible with the hypotheses that the increase in ovulation rate in immunized ewes is due to an increase in circulating FSH concentrations and that inhibin may only have a major peripheral influence on FSH in sheep during the breeding season.
Anestrus↗
Growth hormone and insulin-like growth factor-I accelerate PMSG-induced differentiation of granulosa cells.
To investigate the nature of the interactions between growth hormone (GH), insulin-like growth factor-I (IGF-I) and follicle stimulating hormone (FSH), their temporal and dose-related effects on steroidogenesis were studied in granulosa cells from stilboestrol-treated immature rats, stimulated in vitro with pregnant mare's serum gonadotropin (PMSG) or FSH. GH in the presence of PMSG enhanced aromatase activity and progesterone synthesis above that induced by maximally stimulating doses of PMSG alone, and accelerated PMSG-induced peak levels for both progesterone and aromatase activity. IGF-I also enhanced PMSG-induced aromatase activity and progesterone production, and accelerated their peak responses in a similar fashion to the effects observed for GH. The stimulatory actions of IGF-I could still be observed after the removal of FSH from the cultures, and appeared to be partly independent of cAMP. It is concluded that both GH and IGF-I act on FSH-induced granulosa cells to accelerate the differentiation of the follicular cell to a lutein cell.
Animals↗
Transforming growth factor beta enhances basal and FSH-stimulated inhibin production by rat granulosa cells in vitro.
Transforming growth factor beta (TGF beta) caused a dose-dependent increase in both basal and follicle-stimulating hormone (FSH)-stimulated inhibin production by rat granulosa cells in culture. The TGF beta dose-response curve in the absence of FSH was approximately parallel to that in the presence of either a minimally effective dose (1 ng/ml) or a maximally effective dose (30 ng/ml) of FSH, suggesting an additive effect of these two agents on inhibin production. There was also a suggestion of an increased sensitivity of granulosa cell inhibin production to FSH when the cells were coincubated with TGF beta. The time course study showed that similar to FSH, the stimulatory effect of TGF beta on basal and FSH-stimulated inhibin production was evident on day 1 and was maximal by day 4. In addition, epidermal growth factor (EGF) reduced FSH-stimulated inhibin production with an ID50 value of 1.3 ng/ml. Coincubation of cells with EGF and 1 ng TGF beta/ml enhanced greatly the inhibitory action of EGF on FSH-induced inhibin production (ID50 less than 0.1 ng/ml). It is concluded that: (1) TGF beta directly stimulates inhibin production by rat granulosa cells and the combined effect with FSH was largely additive, (2) the inhibitory effect of EGF on FSH-induced inhibin production was enhanced by TGF beta, (3) individual members of the TGF beta/inhibin gene family regulate ovarian function, not only by direct action on follicle cells but also indirectly by influencing the production rate of other members of that family.
Animals↗
Effect of purified 31K bovine inhibin on the specific binding of gonadotropin-releasing hormone to rat anterior pituitary cells in culture.
The mechanism by which inhibin decreases the responsiveness of the pituitary gonadotroph to GnRH in terms of secretion of gonadotropins is largely unknown. We studied the effect of pure 31K bovine inhibin on the specific binding of GnRH to rat anterior pituitary cells in culture using iodinated Buserelin as tracer. Results showed that treatment of cultured anterior pituitary cells from adult male rats with inhibin (0-30 U/ml) for 72 h decreased Buserelin binding in a dose-dependent manner. In the presence of a maximally inhibiting dose of manner. In the presence of a maximally inhibiting dose of inhibin, Buserelin binding decreased progressively with time, reaching a minimum of 42% of the control value after 3 days. Exposure of pituitary cells for 3 days to the inhibin-related peptides transforming growth factor-beta (up to 400 pM) and Müllerian inhibitory substance (up to 100 nM) did not decrease binding of Buserelin, suggesting that the effect was specific to inhibin. Inhibin did not compete with iodinated Buserelin for GnRH-binding sites when they were added to the assay tube simultaneously. In addition, treatment with inhibin halved the number, but did not change the affinity, of GnRH-binding sites and had no effect on either cell number of cell viability. It is concluded that the reduction by inhibin of rat gonadotroph responsiveness to GnRH may be partly related to a decrease in the number of GnRH receptors on the cell surface.
Animals↗
Comparison of inhibin immunological and in vitro biological activities in human serum.
A comparison of serum inhibin levels in men and women was undertaken using a sensitive sheep pituitary cell in vitro bioassay and a newly developed heterologous RIA. The RIA was based on an antiserum raised to bovine 31K inhibin using [125I]31K inhibin as tracer. Bovine inhibin alpha- and beta-subunits, bovine activin-A, transforming growth factor-beta, and Mullerian inhibitory substance did not cross-react in the RIA. In both assays, dilutions of serum gave response lines parallel to that of the partially purified human follicular fluid inhibin preparation used as standard. Negligible levels of both bio (B)- and immuno (I) activities were found in serum from women with premature ovarian failure or castrated men. In ovulation-induced cycles, serum B inhibin levels increased progressively from the early to the late follicular phase and remained at the late follicular phase level during the early and midluteal phases. Serum I inhibin levels also rose during the follicular phase, but declined during the early luteal phase before increasing again in the midluteal phase. As a consequence, inhibin B:I ratios varied during the treatment cycle, with high ratios in early follicular (2.86) and early luteal (2.25) phases and a low ratio in the midluteal phase (1.09). Similar changes in serum B:I ratios also occurred during the midcycle and midluteal phases of normal cycles. The B:I ratio was lower (0.35) in normal men. We conclude that the largely similar pattern of inhibin biological and immunological activities in serum obtained during a variety of physiological conditions support the validity of the RIA procedure, and the B:I ratio of serum inhibin varies during the follicular and luteal phases of the cycle and is low in men. Potential reasons for these changes in B:I ratio include the presence of interfering substances in either the bioassay or the RIA, the presence of inhibin isoforms, and/or modulation of secreted forms by sex steroids.
Adult↗
Serum inhibin concentrations rise throughout normal male and female puberty.
Using a newly developed, sensitive, and specific RIA, we measured the serum concentrations of inhibin, together with those of FSH, LH, and sex steroids, throughout puberty in 99 boys and 102 girls attending a suburban Melbourne school. Serum inhibin levels rose from a geometric mean level of 161 U/L (range, 87-310; 67% confidence interval) at stage I puberty in boys to 442 U/L (range, 300-626) at stage V, while corresponding values in girls were 97 U/L (range, 46-204) and 231 U/L (range, 187-372), respectively. Serum inhibin concentrations were strongly correlated with age and serum FSH, LH, testosterone, and estradiol; all hormones increased in parallel in both boys and girls. After adjustment for age, the partial correlation coefficients remained significant only for testosterone in the boys. We hypothesize that gonadal inhibin production is stimulated by rising gonadotropin levels during pubertal development.
Adolescent↗
Ovine follicular fluid inhibits thymidine incorporation by 3T3 fibroblasts in vitro.
The ability of antral follicular fluid obtained from sheep follicles to inhibit 3T3 fibroblasts maintained for 48 h in concentrations of fetal calf serum optimal for cell growth was examined. Addition of pooled follicular fluid to cultures resulted in a dose-dependent and reversible inhibition of [3H]thymidine incorporation. Serum from ovariectomized ewes, fetal calf serum, bovine inhibin, oestradiol-17 beta, testosterone, cortisol or progesterone were without effect over a range of doses. Treatment of pooled follicular fluid with charcoal-dextran did not reduce inhibitory activity which was only partly removed by heating at 85 degrees C. Fluid obtained from large follicles (greater than 5 mm) was more potent as an inhibitor than was fluid obtained from smaller (less than 5 mm) follicles. Gel chromatography of pooled fluid resolved two peaks of inhibitory activity associated with material of Mr approximately 180,000 and less than 10,000 respectively. No inhibitory activity was evident in fractions of serum from ovariectomized ewes chromatographed in an identical manner. These results indicate that ovine follicular fluid contains two components able to inhibit reversibly mitosis of 3T3 fibroblasts in vitro.
Animals↗
Interferon-alpha mimics effects of ovine trophoblast protein 1 on prostaglandin and protein secretion by ovine endometrial cells in vitro.
Ovine endometrial cells were isolated from ovariectomized oestrogen and progesterone-treated ewes and maintained in primary culture. In-vitro treatment with human interferon-alpha 2 (Roferon, Hoffman La Roche) (5, 50 IU/ml) or purified ovine trophoblast protein 1 (oTP-1, 30 ng/ml) significantly attenuated PGF2 alpha (25 +/- 17, 29 +/- 17, 28 +/- 9% +/- SEM of control [no in-vitro treatment = 100%] respectively, N = 4 ewes) and PGE (11 +/- 4, 16 +/- 4, 16 +/- 5% of control) release from the cultured cells. Fluorography of two dimensional polyacrylamide gel electrophoretic analyses of proteins secreted by the cells following 35S-methionine incorporation, revealed that synthesis and secretion of the same "pregnancy-related" proteins was stimulated by both interferon-alpha 2 and oTP-1. Thus, interferon-alpha 2 (which has sequence homology with oTP-1) acts on the ovine endometrium, eliciting similar biological responses to those of oTP-1.
Animals↗
Effects of bovine inhibin, transforming growth factor-beta and bovine Activin-A on granulosa cell differentiation.
To determine whether inhibin and its related peptides might act locally to control granulosa cell function and differentiation, the dose- and time-dependent effects of bovine inhibin, the homo-dimer of the beta-chain of bovine inhibin (Activin-A) and porcine TGF beta on rat granulosa cell aromatase activity and progesterone synthesis were investigated in vitro. TGF beta enhanced FSH-induced aromatase activity and progesterone synthesis, and accelerated the peak response for progesterone synthesis. Activin-A on the other hand, augmented FSH-induced aromatase activity while arresting progesterone synthesis, and anti-luteinization effect. By contrast, exogenous inhibin had no detectable effect on the steroidogenic potential of these cells. Thus TGF beta and Activin, unlike their similar effects on the release of FSH by the pituitary, appear to affect ovarian granulosa cell function in different fashion, under conditions where inhibin itself has no effect.
Activins↗
Progesterone in vitro stimulates secretion of a specific protein by ovine epithelial endometrial cells.
Cultured ovine epithelial endometrial cells from oestrogen-treated ovariectomized ewes were treated in vitro with combinations of oestradiol-17 beta (E), progesterone (P) and the P-receptor antagonist RU486 (each 10(-6) to 10(-9) M), in the presence of [35S]methionine. Neither DNA content of dishes nor total protein were increased in treatment compared with control dishes. Incorporation of [35S] into secreted protein was lower from cells treated in vitro with P or E + P (10(-9) M) than from those treated with E (10(-9) M, P less than 0.01). Incorporation of [35S] into cellular protein was decreased by P (10(-9) M, P less than 0.025). SDS-PAGE analysis of secreted proteins enabled measurement of levels of a 46K protein which is secreted maximally following E + P administration in vivo. In vitro, P either alone or with E (each 10(-7) M) increased the abundance of the 46K protein in cell secretions by a factor of 1.5 +/- 0.1 (N = 9) or 1.8 +/- 0.3 (N = 10) respectively (P less than 0.01) compared with controls. The administration of E (10(-7) M) or either or both steroids at 10(-9) M, was without effect. RU486 alone (10(-6) to 10(-8) M) was also without effect but in the presence of E + P or P, blocked the increase in the 46K protein, suggesting this effect is mediated via binding of P to its receptor.
Animals↗
Direct inhibition of rat granulosa cell inhibin production by epidermal growth factor in vitro.
The effect of epidermal growth factor (EGF) on inhibin production by rat granulosa cells has been investigated using a recently developed inhibin radioimmunoassay (RIA). Granulosa cells from intact immature diethylstilbestrol (DES)-treated rats were exposed to EGF (1-100 ng/ml) in the presence or absence of FSH for varying periods in vitro. An inhibitory effect of EGF on basal inhibin secretion was evident at day 2 of culture and was sustained over the subsequent 2 days. This action on basal inhibin secretion was dose-dependent, and maximal inhibition to 50% of control was observed at a dose of 100 ng EGF/ml at day 4. EGF also inhibited basal progesterone secretion in a similar manner. EGF caused a dose-dependent inhibition of FSH-stimulated inhibin secretion, with an ID50 (0.5 ng/ml, 0.08 nM) about one-eighth that in the absence of FSH. In addition, EGF also inhibited the stimulation of inhibin production by 8-Br-cAMP and prostaglandin E2. To exclude the possibility that EGF was toxic to the granulosa cells, several biochemical parameters related to cell growth were measured. EGF treatment did not alter cell number but slightly increased [3H]thymidine incorporation into cellular DNA. The effect of EGF on [35S]methionine incorporation into cellular protein was biphasic, being stimulatory at doses less than 10 ng/ml but inhibitory at 100 ng/ml. The present data have demonstrated a direct inhibitory effect of EGF on basal and FSH-stimulated inhibin production by granulosa cells suggesting an important regulatory role of this growth factor in the differentiation of ovarian function.
Animals↗
Intragonadal control mechanisms.
On the weight of the evidence presented above, it is concluded that regulation at a local, intragonadal level is an integral part of the overall regulation of gonadal function in both sexes. The interaction between cells within a gonad extends beyond the same cell type to include germ cell-somatic cell interactions as well. We believe this local interaction between cell types facilitates the differing requirements of the various developmental stages of germ cells within the gonad, which would not be possible by simply varying the afferent pituitary hormone supply. We re-emphasize that the local factors responsible for these interactions are acting in conjunction with the pituitary hormones, and, in some cases, may be their proximate regulators. A more controversial phenomenon is the possibility of an interaction between the gonads which does not involve the hypothalamic-pituitary axis. The little evidence which is available to support this hypothesis comes mainly from studies on ovarian function, particularly recruitment and selection of follicles. More research on this phenomenon is warranted. Not surprisingly there are many parallels between the testes and ovaries with respect to the nature and action of local regulators. For example, the intragonadal action of steroids, the local modulation of the response of target cells to FSH, the influence of macrophages on steroidogenesis and the presence of mitotic and meiotic regulators are common to both sexes. It would not be surprising if the chemical nature of these factors in the ovary and testes are similar. If the ever-increasing list of factors and activities being discovered in the gonads is any guide, the phenomena outlined in this review are just the beginning of an extensive list of cell-cell interactions occurring within and between the gonads. No doubt the gonads will share with other organs the same interactions between cells which are required for normal cellular function. The uniqueness of the gonads lies in their protection and production of germ cells. The challenge of the future for reproductive biologists will be to discover and describe the interactions within and between germ cells which are obligatory for normal reproductive function, and to apply that information to devising ways of overcoming infertility and regulating fertility.
Animals↗
The maternal ovary is not the source of circulating inhibin levels during human pregnancy.
The concentration of immunoreactive inhibin in serum was measured in three pregnant women with premature ovarian failure involved in a donor oocyte in-vitro fertilization programme. Inhibin was not detectable in peripheral serum prior to conception but rose within 2-4 weeks of embryo transfer, whereafter levels rose gradually during pregnancy (less than 20 weeks 1.22 U/ml (0.85-1.76) versus greater than 20 weeks 2.28 U/ml (1.42-3.67), P less than 0.01; geometric mean +/- 67% confidence interval) and were similar to those observed in 24 normal pregnant women. hCG rose in parallel with inhibin during early gestation, but declined after 3 months. FSH levels were elevated before conception and were suppressed during pregnancy. In conclusion (i) immunoreactive inhibin is detectable from early gestation in women with no endogenous ovarian function indicating that the maternal ovary does not contribute significantly to inhibin secretion during pregnancy; (ii) the trophoblast is the likely source of inhibin during pregnancy; (iii) the regulation of hCG and inhibin secretion differs throughout gestation; and (iv) inhibin may have a role in FSH regulation during pregnancy and/or a local role within the feto-placental unit.
Chorionic Gonadotropin↗
Influence of dose and route of administration of bovine follicular fluid and the suppressive effect of purified bovine inhibin (Mr 31,000) on plasma FSH concentrations in ovariectomized ewes.
Ovariectomized Merino ewes were used to develop an in-vivo bioassay for purified bovine inhibin of Mr 31,000. Various doses (0.25, 0.5, 1 or 2 ml) of bovine follicular fluid, given either by the intravenous (i.v.) or intracarotid route (i.c.) resulted in significant linear dose-related suppression of plasma FSH and interval to maximum suppression. Control ewes (1.0 ml steer plasma) showed no significant change in FSH over the same period. Doses of 470 and 2590 U of pure inhibin given i.v. caused a significant suppression of FSH in plasma in all ewes. The in-vivo potency estimate of the high dose (2760 U, 1420-4690 fiducial limits) agreed well with the in-vitro assay of potency. There were no significant changes observed in mean plasma LH after treatment with the higher dose of pure inhibin. There were no rebound effects of treatment with bovine follicular fluid or pure inhibin on FSH concentrations above that of controls. It is concluded that the form of bovine inhibin of Mr 31,000, which is believed to be the predominant circulating form, is biologically active when administered in vivo.
Animals↗
Isolation of inhibin from ovine follicular fluid.
Two forms of inhibin with molecular weights of 65,000 and 30,000 (65 and 30 kD) were isolated from ovine follicular fluid using a combination of gel permeation chromatography, reversed-phase high-performance liquid chromatography and preparative polyacrylamide gel electrophoresis. The 65 kD form was partially purified approximately 315-fold whilst the 30 kD form was isolated as two isoforms (29 and 30 kD) of similar biological activity and in greater than 95% purity (1210-fold purification and 4.2% recoveries). On reduction the 30 kD form resolved into four components of 36, 31, 20-21 and 16 kD of which the 20-21 and 16 kD components were similar to the corresponding inhibin subunits isolated from porcine and bovine follicular fluid. The 36 kD component was established as a non-reducible inhibin-like material, based on its binding to antiserum raised against bovine 58 kD inhibin. The nature of the remaining non-reducible 31 kD component is unknown. Two NH2-terminal amino acid sequences (first 13 amino acids) identified in purified 30 kD inhibin were identical to the corresponding subunit amino acid sequences of bovine 31 kD inhibin.
Animals↗
Regulation of the secretion of FSH in domestic ruminants.
Explore the source record for details and available documents.
Animals↗
Oocyte donation and in vitro fertilization for hypergonadotropic hypogonadism: clinical state of the art.
Explore the source record for details and available documents.
Adult↗