Amplification of the proliferative response to alloantigen by a factor present in an extract of syngeneic thymic lymphoid cells.

A synergistic interaction in the proliferative response to alloantigen has been previously noted when intact thymus cells are cultured with post-thymic (peripheral) lymphoid cells. In the present study, a factor extracted from the thymus has been shown to similarly enhance the reactivity of syngeneic lymph node cells and thus to retain the amplifier activity of intact thymus cells. The factor has no effect on lymphoid cell proliferation in the absence of alloantigen. Cells with amplifier activity are found in highest concentration in the thymus but also may be detected in spleen cells that are nonadherent to nylon wool. The factor is shown in these experiments to be derived from thymic lymphoid cells and to act primarily upon post-thymic (peripheral) lymphoid cells. As such, this factor appears to be distinct from various other thymus factors that have been localized to thymic reticuloepithelial elements and that are thought to effect predominantly the differentiation of T-cell precursors.     

Prolactin effects on basal and gonadotrophin-stimulated progesterone accumulation by PMSG-primed mouse ovaries in vitro

To examine the effects of prolactin (Prl) and human chorionic gonadotrophin (hCG) on progesterone production by murine ovarian explants, immature female mice were injected with 4 IU pregnant mare's serum gonadotrophin (PMSG) to induce follicular maturation. After 24 or 40 h mice were killed, ovaries removed, cut into fragments and maintained as explants for 24 h in the presence or absence of ovine or human Prl (25-2500 ng/ml). None of these doses of Prl affected basal progesterone accumulation into media over 24 h. To determine if Prl could modify the capacity of ovarian explants to respond to gonadotrophin, ovaries were incubated with 25 IU/ml hCG for 3 h after an initial 24 h incubation period with or without Prl. Prl had no effect on basal progesterone accumulation but significantly enhanced hCG-stimulated progesterone accumulation during the 3 h incubation period. We conclude that Prl does not inhibit but may enhance progesterone secretion by pre-ovulatory follicles in the mouse.     

Characterization of epidermal growth factor-related proteins from human urinary chorionic gonadotrophin

A urinary chorionic gonadotrophin (hCG) preparation, mitogenic for ovarian carcinoma cells, was analysed by gel filtration through Sephadex G-100 Superfine. The resulting fractions were tested for hCG and for properties of the epidermal growth factor (EGF) by radioimmunoassays (RIA) in comparison with their ability to stimulate the growth of EFO-27nu ovarian carcinoma cells. The elution profile of the RIA activities for hCG corresponded to molecular weights of 12 and 71 kDa, whereas the mitogenic activity was found in peak fractions eluting at 7, 11 and 52 kDa, indicating the presence of mitogenic substances distinct from hCG or its beta-subunit. In comparison experiments, radiolabelled recombinant human EGF eluted at 7 kDa from the column. The profile of EGF immunoreactivity determined in the eluant fractions of hCG preparation A correlated with the mitogenic potential. Eluant fractions with growth-promoting activity competed with 125I-labelled EGF in binding to EFO-27nu cells; the inhibition of EGF binding was correlated with the mitogenic potential and the EGF immunoreactivity. We assume that the 7 kDa component of the gel filtration eluate corresponds to monomeric EGF; the high molecular weight mitogens may represent EGF precursor protein fragments of various molecular size classes.     

Effects of a sustained release formulation of the gonadotrophin-releasing hormone agonist histrelin on serum concentrations of gonadotrophins and oestradiol, and ovarian LH/human chorionic gonadotrophin receptor content in the rat.

Pituitary and ovarian function were studied during the loss and recovery of oestrous cyclical activity in rats following treatment with a sustained release formulation of the gonadotrophin-releasing hormone (GnRH) agonist [imidazole benzyl-D-His6,Pro9-ethylamide]-GnRH (histrelin). A single s.c. injection of microencapsulated histrelin (10-300 micrograms peptide/kg) induced a dose-dependent disruption of normal oestrous cyclical activity with a persistent dioestrous-like vaginal cytology. In preliminary studies, pituitary gland stimulation and desensitization were demonstrated when serum LH and FSH levels were greater 1 week after administration of 10 micrograms microencapsulated histrelin/kg compared with 300 micrograms microencapsulated histrelin/kg. Changes in pituitary and ovarian function were assessed over time following injection of microencapsulated histrelin (100 micrograms peptide/kg). LH secretion was maximal within 8 h and then gradually declined, remaining at dioestrous levels from days 7 to 28. Serum oestradiol concentrations remained low and rose above dioestrous levels only on day 28. In contrast, ovarian LH/human chorionic gonadotrophin (LH/hCG) receptor content fell within 8 h and, after a nadir on day 7, slowly returned to dioestrous levels by day 28. The increase in ovarian LH/hCG receptor content preceded any significant change in pituitary gonadotrophin secretion, indicating a differential pattern of recovery for pituitary and ovarian function. Subsequent studies tested the possibility that these temporal differences in pituitary and ovarian function may result from histrelin acting directly on these tissues. Treatment with histrelin microcapsules (300 micrograms peptide/kg) prevented any increase in LH secretion in response to a GnRH challenge 3 days later, indicating a direct action of histrelin on the pituitary gland.(ABSTRACT TRUNCATED AT 250 WORDS)     

An inverse relationship between inhibin and follicle-stimulating hormone during the period of ovulation induced by human chorionic gonadotrophin in dioestrous rats

To investigate the mechanism of the selective surge of FSH during the period of ovulation induced by human chorionic gonadotrophin (hCG) in dioestrous rats, inhibin activity in ovarian vein plasma was determined at varying time-intervals after treatment with hCG using the primary monolayer culture system of anterior pituitary cells. Inhibin activity in ovarian vein plasma had already decreased 6 h after injection of hCG, when concentrations of FSH in the plasma were still low in three of four animals. Inhibin activity further decreased 12-18 h after hCG, when a selective surge of FSH occurred. Inhibin activity increased to the level before hCG treatment 24 h after the treatment, when ovulation was completed and the FSH surge terminated. These results suggest that the selective surge of FSH occurs as a consequence of the decrease in inhibin secretion from the ovary, which is perhaps due to the ovulation dose of hCG altering the functional activity of the granulosa cells in the large Graafian follicles.     

Effect of gonadotrophins on progesterone secretion by cultured granulosa cells obtained from human preovulatory follicles

Granulosa cells were obtained from human preovulatory follicles in 31 women undergoing in vitro fertilization and embryo transfer due to tubal infertility. Follicular maturation was stimulated and synchronized by treatment with Clomiphene or human menopausal gonadotrophin (hMG), or both, plus human chorionic gonadotrophin (hCG). Follicles were aspirated by ultrasound guided puncture approximately 34-36 h after the hCG injection. The granulosa cells were washed and suspended in modified medium 199 containing 10% foetal bovine serum and cultured as monolayers for 6-8 days in the absence and presence of hormones and reactants. Progesterone formation was analyzed by RIA. In general, the cells underwent morphological luteinization and secreted high amount of progesterone. Under basal conditions the secretion of progesterone was highest during the first 2 days in culture and then gradually declined. Progesterone secretion was stimulated by human LH, hCG and the adenylate cyclase stimulator forskolin, with a maximal effect between days 2-6. The beta-adrenergic agonist isoproterenol in preliminary experiments potentiated the stimulatory effect of hCG but had no own stimulatory effect. No clear differences in progesterone secretion or responsiveness to in vitro stimulation relating to the various in vivo stimulation protocols were found.     

Contrasting effects of prolactin on luteal and follicular steroidogenesis

To determine whether prolactin affects both luteal and follicular production of testosterone and oestradiol, pseudopregnant rats, either intact or hypophysectomized on day 8, were injected daily between days 8 and 9 with 1.5 i.u. human chorionic gonadotrophin (hCG), 250 micrograms prolactin or a combination of both. Control rats were given vehicle. On day 9, blood was obtained from the ovarian vein and corpora lutea and follicles were isolated and incubated in vitro for 2 h. Administration of hCG to intact rats increased ovarian secretion of testosterone and oestradiol dramatically, but did not affect progesterone secretion. Hypophysectomy on day 8 of pseudopregnancy was followed by a drop in ovarian steroid secretion. Prolactin treatment of hypophysectomized rats markedly enhanced progesterone production but had no stimulatory effect on either testosterone or oestradiol. In contrast, hCG dramatically enhanced ovarian secretion of both testosterone and oestradiol without affecting progesterone secretion. Prolactin administered together with hCG antagonized the stimulation of both testosterone and oestradiol secretion by hCG, yet increased progesterone production. When the specific effects of hCG and prolactin administration on follicles and corpora lutea were studied separately, it was found that hCG treatment in vivo greatly stimulated testosterone and oestradiol production by both tissues in vitro. Since hCG only marginally affected aromatase activity in the follicle, had no effect on aromatase activity in luteal cells and did not increase progesterone synthesis, it appears that hCG acts to increase the formation of androgen substrate for oestradiol biosynthesis. Prolactin, administered with or without hCG, inhibited both basal and hCG-stimulated testosterone and oestradiol synthesis by the follicle. In sharp contrast to its inhibitory effect on follicular production of steroids, prolactin appears to be essential for LH stimulation of testosterone and oestradiol by the corpus luteum. In the absence of prolactin, luteal cells gradually ceased to respond to LH and decreased their output of testosterone and oestradiol. Prolactin administration to hypophysectomized rats did not affect luteal cell production of either steroid. However, corpora lutea of rats treated with prolactin responded to the hCG challenge with an increase in testosterone and oestradiol synthesis. In summary, results of this investigation demonstrate that prolactin affects follicular and luteal production of testosterone and oestradiol in opposite ways.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of human chorionic gonadotrophin on the release of cyclic AMP and progesterone from the sheep ovary at different stages of the oestrous cycle.

The rate of release of cyclic AMP by sheep ovaries containing a corpus luteum was determined at different stages of the cycle before and up to 60 min after an intra-arterial (ia) injection of 500 IU human chorionic gonadotrophin (hCG). The median cyclic AMP concentration in arterial plasma and of ovarian venous plasma following hCG stimulation was 93.2 and 98.0 pmol/ml, respectively. The ovaries of ewes examined at Days 1 and 2 of the cycle showed no response to hCG, whereas in 2 sheep at Day 3, hCG caused a slight response, and in 13 sheep examined between Days 5-18, hCG caused a marked increase in cAMP release. In 5 of the sheep in which both ovarian veins were cannulated, only the ovary with a corpus luteum responded to hCG with an increased secretion rate of cyclic AMP and progesterone. The results indicate a lack of responsiveness in the newly formed corpus luteum to hCG.     

Progestin receptor in the thymus of ovariectomized immature rats

By using the synthetic progestin promegestone (R5020), the location and characteristics of progestin receptors in the thymic cytosols from immature ovariectomized oestrogen-treated rats were determined. Tritiated promegestone bound to the cytosol with high affinity (dissociation constant (Kd) = 2.0 +/- 0.3 nmol/l; promegestone greater than progesterone greater than oestradiol greater than corticosterone testosterone) and low capacity (number of binding sites (Bmax) = 143.0 +/- 13.5 fmol/mg protein). These values were appropriate for progestin receptors. However, an extremely high dose of dexamethasone (10 mumol/l; 1000-fold excess over [3H]promegestone) slightly inhibited the specific binding. Progestin receptors were predominantly located in the reticuloepithelial (RE)-cell fraction, with few in the thymocyte T-cell fraction. The receptor level was raised (24.9 +/- 11.3 (S.E.M.) to 143.0 +/- 13.5 fmol/mg protein) with increased doses of oestrogen (0-30 micrograms) administered in vivo. Using sucrose density gradient ultracentrifugation it was found that the thymic progestin receptor had a sedimentation coefficient of 9S under low-salt conditions. These results clearly suggest that the thymus of the immature female rat contains a specific progestin receptor which is mainly located in the RE cells.     

Human chorionic gonadotrophin-induced suppression of serum inhibin involves reduction in ovarian inhibin alpha-subunit messenger RNA levels in the pregnant mare serum gonadotrophin-primed female rat

We have investigated the effect of administration of human chorionic gonadotrophin (hCG) to immature female rats pretreated with pregnant mare serum gonadotrophin (PMSG) on ovarian inhibin alpha-subunit mRNA, serum inhibin and circulating gonadotrophin levels. PMSG stimulation alone caused a 5-fold increase in relative inhibin alpha-subunit mRNA levels and a 12-fold increase in serum inhibin by 48 h. However, injection of hCG at 40 h suppressed PMSG-stimulated ovarian inhibin alpha-subunit mRNA and serum inhibin to levels at 48 h not statistically significantly different from controls. Serum follicle-stimulating hormone (FSH) fell after PMSG treatment, but rose after combined PMSG-hCG treatment. Serum luteinizing hormone (LH) was unchanged after PMSG alone but also rose after combined PMSG and hCG therapy. In conclusion, hCG suppresses ovarian inhibin synthesis in PMSG-stimulated immature female rats with preservation of the reciprocal relationship between inhibin and FSH. This immature female rat model therefore provides further insight into the effects of LH or hCG on granulosa cell inhibin production just prior to ovulation.     

Ovarian production of progesterone and 20 alpha-dihydroprogesterone in vitro following prostaglandin F2 alpha induced luteolysis in the superluteinized rat

Superluteinized rats were injected with the prostaglandin F2 alpha (PGF2 alpha) analogue cloprostenol to induce luteolysis. The treatment decreased progesterone production of ovarian homogenates from 8.9 +/- 0.5 to 4.0 +/- 0.7 nmol/ovary/10 min (mean +/- SEM) within 40 min. tochondrial fractions isolated from control and cloprostenol treated animals produced 4.7 +/- 0.4 and 2.8 +/- 0.3 nmol progesterone/ovary/10 min, respectively. Thus, the PGF2 alpha analogue treatment significantly reduced mitochondrial progesterone production. Addition of the 15 000 X g supernatant fraction did not influence the progesterone production rates of the mitochondrial fraction. The basal progesterone secretion from quartered ovaries decreased from 1.50 +/- 0.15 to 0.38 +/- 0.05 nmol/ovary during the initial 15 min of incubation following cloprostenol administration. hCG and N6,O2'-dibutyryladenosine 3':5'-cyclic monophosphate (DBC) stimulated the progesterone secretion from quartered ovaries, but the response was delayed in ovaries obtained from cloprostenol treated animals. Although the response was delayed, the progesterone secretion following cloprostenol treatment was re-activated with cAMP either directly or via hCG. The increment in progesterone secretion above unstimulated controls in response to DBC was not influenced by the cloprostenol treatment while the increment caused by hCG was decreased. Our data suggest that: 1) PGF2 alpha deactivates mitochondrial progesterone production, 2) this deactivation may be overcome by cAMP, and 3) PGF2 alpha decreases gonadotrophin responsive adenylyl cyclase.     

The relationship between hCG and relaxin secretion in normal pregnancies vs peri-implantation spontaneous abortions1

OBJECTIVE We determined the ovarian response to human chorionic gonadotrophin (hCG) in terms of relaxin and progesterone secretion during the peri-implantation period of normal and failing pregnancies. We wished to test the hypotheses that relaxin production in failing pregnancies is different from that in normal pregnancies, that relaxin is a reliable, quantitative indicator of the biological activity of endogenous hCG, and that relaxin is a useful predictor of peri-implantation spontaneous abortions. DESIGN Daily blood samples were collected in a prospective longitudinal study from insemination patients. PATIENTS Women undergoing artificial insemination in natural cycles with non-frozen donor semen at a University clinic. MEASUREMENTS Serum LH, hCG, relaxin and progesterone were measured and the relationship between hCG and the ovarian hormones was evaluated in the peri-implantation period of normal pregnancies and spontaneous abortions. RESULTS Nine of 23 conceptive cycles resulted in a spontaneous abortion between 16 and 70 days after the LH peak. In all normal and failing pregnancies there was a close qualitative relationship between hCG secretion and relaxin production. Six of nine failing pregnancies were associated with abnormally low hCG secretion. Six of the spontaneous abortions were associated with rates of relaxin secretion which were higher than the mean of 14 normal pregnancies. No such alterations in progesterone concentrations were observed. In cases where hCG was extremely low, the quantitative relationship between hCG and relaxin was different from that in cases of normal hCG concentrations. CONCLUSIONS There is a close temporal relationship between the secretion of trophoblastic hCG and ovarian secretion of relaxin in the peri-implantation period of normal and failing pregnancies. In failing pregnancies there is substantial variability in the quantitative relationship between relaxin and hCG, indicating that relaxin is not a reliable quantitative indicator of hCG bioactivity. Contrary to previous reports, relaxin concentrations in failing pregnancies tended to be higher than or equal to concentrations in normal pregnancies until the loss was imminent. Because of this relaxin is not a useful predictor of peri-implantation spontaneous abortions.     

The configuration of the alpha and beta subunit domains in single-chain bovine LH analogs influences the secretion and steroidogenic response

The gonadotrophins LH, FSH and human (h) CG are non-covalent heterodimers composed of a common alpha and the hormone-unique beta subunit. LH regulates the production of androgens and progestins in the follicle, and the levels of these steroids are critical for the ovarian function. Structural features of the gonadotrophins involved in the steroidogenic response of the ovary are not completely understood. As an approach to address how the topology of the ligand affects steroidogenesis we exploited the single-chain (SC) gonadotrophin methodology because manipulating the relative position of the tethered subunit domains in SC hCG analogs enabled to change in the conformation, secretion, receptor binding and adenylyl cyclase activity. We genetically engineered a SC bovine LH analog with a linker derived from the CTP domain of the hCGbeta subunit, NH2-alpha-CTP-LHbeta-COOH (denoted as alphaCTPLHbeta; AB configuration) and evaluated the secretion form transfected CHO cells and steroidogenesis in follicular derived cells in comparison to the variant NH2-LHbeta-CTP-alpha-COOH (LHbetaCTPalpha; BA configuration). The secretion of the analogs from CHO cells was quantitative, and that of alphaCTPLHbeta was more efficient than that of LHbetaCTPalpha The experiments suggested that both variants were N- and O- glycosylated, though the posttranslational modifications are likely to be non-identical in the AB and BA analogs. The analogs stimulated progesterone secretion by immortalized rat granulosa cells that express the rat LH receptor but the EC50 of alphaCTPLHbeta (AB orientation) was higher by 20 fold, as compared to LHbetaCTPalpha (BA). In primary cultures of bovine theca cells, alphaCTPLHbeta stimulated progesterone release with a reduced sensitivity (by at least 50 folds) and smaller magnitude over the basal levels (about 3 folds) relative to LHbetaCTPalpha. In contrast, the accumulation of androstenedione in the media of the same primary cultures appeared to be nearly identical. As a result, the androstenedione/progesterone ratio for the alphaCTPLHbeta analog was significantly increased relative to LHbetaCTPalpha (2-3 folds). This unequal response suggests a distinct regulation of progesterone and androstenedione biosynthesis. Our data demonstrate major differences in steroid balance following stimulation of the receptor with structural LH analogs and provide further insight into gonadotrophin regulation of ovarian steroid production.     

Identification of nuclear triiodothyronine receptors in the thymic epithelium.

Thymic epithelial cell physiology is known to be under neuroendocrine control. In particular, thyroid hormones modulate thymic hormone secretion by thymic epithelial cells in vivo and in vitro, thus suggesting the existence of specific receptors for those hormones in this component of the thymic microenvironment. Yet, thyroid hormone-binding sites have previously been detected only in crude thymus fractions and lymphocytes. We, thus, decided to search for T3 receptors in the thymic epithelium, by using an antinuclear T3 receptor monoclonal antibody. In situ immunohistochemical analysis of thymic frozen sections showed nuclear labeling of both lymphoid and nonlymphoid cells in the cortex and medulla. Moreover, in vitro studies using thymic epithelial cell lines and the so-called thymic nurse cells revealed a positive reaction in the chromatin, with nucleoli remaining negative. Immunoblot data clearly showed a single protein band of 57K reactive with the antinuclear T3 receptor antibody in murine thymus extracts as well as in the thymic epithelial cell lines. Lastly, in vitro treatment of these cells with T3 resulted in a transient, yet profound, down-modulation of the receptor. In conclusion, our findings provide molecular evidence that the action of thyroid hormones on thymic epithelium occurs via the typical 57K nuclear T3 receptors.     

Neuroendocrine control of thymus physiology

The thymus gland is a central lymphoid organ in which bone marrow-derived T cell precursors undergo differentiation, eventually leading to migration of positively selected thymocytes to the peripheral lymphoid organs. This differentiation occurs along with cell migration in the context of the thymic microenvironment, formed of epithelial cells, macrophages, dendritic cells, fibroblasts, and extracellular matrix components. Various interactions occurring between microenvironmental cells and differentiating thymocytes are under neuroendocrine control. In this review, we summarize data showing that thymus physiology is pleiotropically influenced by hormones and neuropeptides. These molecules modulate the expression of major histocompatibility complex gene products by microenvironmental cells and the extracellular matrix-mediated interactions, leading to enhanced thymocyte adhesion to thymic epithelial cells. Cytokine production and thymic endocrine function (herein exemplified by thymulin production) are also hormonally controlled, and, interestingly in this latter case, a bidirectional circuitry seems to exist since thymic-derived peptides also modulate hormonal production. In addition to their role in thymic cell proliferation and apoptosis, hormones and neuropeptides also modulate intrathymic T cell differentiation, influencing the generation of the T cell repertoire. Finally, neuroendocrine control of the thymus appears extremely complex, with possible influence of biological circuitry involving the intrathymic production of a variety of hormones and neuropeptides and the expression of their respective receptors by thymic cells.     

A factor from bursa of Fabricius inhibits in vitro the chorionic gonadotropin response of the chick testis

The effect of bursa of Fabricius on the endocrine function of the chick testis was studied using an isolated testis cell preparation. Testosterone secretion, both basal and under hCG stimulation, was measured in the incubation medium. The bursal extract inhibited the response of the testis cells to hCG detected as a reduced testosterone secretion. The normal basal secretion of the testis cells was not modified by the bursal extract. To analyze some characteristics of the bursal factor, fractions of approximately known molecular weight were obtained by filtering through Amicon membranes. The active factor was found in the fraction corresponding to 1000-10000 Da. Its activity disappeared after heating of trypsin incubation, suggesting the peptidic nature of the bursal factor. Tissue extracts from gut and spleen did not modify the hCG response of the testis cells. There was inhibition of the hCG response by thymus extracts of the newly hatched chicken. These results add new evidence of a modulatory effect of the immunogenic organs on the endocrine function of the testis in the newly hatched chicken.     

Content of chorionic gonadotropin in human fetal tissues.

As part of a study on the physiological role of hCG in the human fetus, the hCG concentrations in homogenates of various fetal tissues were measured using a hCG beta subunit RIA. The mean concentrations (picograms of hCG per mg wet tissue +/- SEM; n greater than 10, unless otherwise indicated) found in human fetuses of 12-20 weeks were: ovary, 46.9 +/- 4.3; testis, 8.2 +/- 1.7; kidney, 20.3 +/- 2.8; thymus, 11.5 +/- 1.2; adrenal, 2.6 +/- 0.4; lung, 3.4 +/- 0.7; liver, 1.8 +/- 0.2; spleen, 1.4 +/- 0.4 (n = 5); muscle, 2.4 +/- 0.8 (n = 6); and meconium, 356 +/- 104. That the immunoreactive material measured behaved like hCG was determined by RIA of the supernatants. Parallelism was demonstrated between dilution curves for the tissue homogenates and the hCG standard for all tissues except meconium. A rat Leydig cell in vitro bioassay was used to demonstrate that there was hCG biological activity in the supernatants in ovarian, thymic, and renal tissues. The mean ratios of biological to immunological activities were 5.3 in kidney (n = 4), 1.6 in thymus (n = 3), and 1.3 in ovary (n = 2). Blood content of the tissues was determined from measurements of hemoglobin levels and it was found that for the ovary, testis, kidney, and thymus, hCG concentrations were higher than could be explained by the presence of circulating hCG in the tissues. These results, together with our previous results of the binding and effects of hCG in the human fetal testis, support the fact that the fetal testis is a target organ for hCG in the stimulation of steroidogenesis. The presence of high levels of hCG in the ovary, thymus, kidney, and meconium poses questions for further study of the possible physiological role of hCG.     

Restoration of the gonadotrophin response to LH-RH and oestrogen administration in patients after molar abortion.

The relation of the hypothalamic-pituitary effect on gonadotrophin secretion and the concentration of serum hCG after molar abortion was investigated. Gonadotrophin release after a bolus injection of LH-RH or conjugated oestrogens was observed in 17 women until 5 months after molar abortion. Little if any response of gonadotrophin was observed at a hCG level of 100 mIU/ml or more, but the response of LH and FSH to LH-RH were restored to normal when the serum hCG level decreased to below 100 mIU/ml. A normal response of LH to conjugated oestrogens was observed at a serum hCG level of 20 mIU/ml or less. These findings suggest that a high level of hCG interferes with pituitary gonadotrophin secretion, that the threshold hCG levels for normal responses of gonadotrophins to LH-RH and oestrogen are 100 and 20 mIU/ml, respectively, and that secretion of gonadotrophins is restored even in the presence of a low hCG level.     

Effects of infantile thymectomy on ovarian functions and gonadotrophin-induced ovulation in prepubertal mice: role of thymulin

The effects of thymectomy performed on 10-day-old (Tx-10) mice on spontaneous puberty and the ovulatory response induced by gonadotrophin treatment were analysed, together with the effects of thymulin replacement from 10 days of age. Infantile thymectomy induced a delay of puberty, a decrease in serum 17beta-oestradiol concentration and a reduced total number of follicles. Injection of thymulin (12 ng/g body weight) to Tx-10 mice resulted in an earlier onset of puberty, a decrease in the weights of ovaries and uterus, and an increase in serum 17beta-oestradiol concentrations. In control and Tx-10 mice, treatment with pregnant mare serum gonadotrophin (PMSG) (5 IU) at 25 days of age resulted in ovulation and the numbers of ova shed by ovulating animals were similar. When the animals were injected with 1 IU PMSG ovulation did not occur. In Tx-10 mice, both 1 and 5 IU PMSG increased the number of follicles to values similar to those observed in the controls. In Tx-10 mice the sequential injection of PMSG (1 IU) and human chorionic gonadotrophin (hCG) (3 IU) resulted in ovulation, but the number of ova shed was lower than in controls. When these animals were injected daily with thymulin, an increase in the number of ova shed and serum 17beta-oestradiol concentrations was observed. The uterine weight of Tx-10 mice was always significantly reduced in response to gonadotrophin treatment. Thymulin injection in PMSG-hCG-treated Tx-10 mice provoked a significant increase in uterine weight. The results suggest that the presence of the thymus after the neonatal period is necessary to normal ovarian development and function. The increase in gonadotrophin-induced ovarian response produced by thymulin replacement indicates that this peptide has a role in this process as one of the connecting signals between thymus and ovaries.     

Effect of cytokines and growth factors on the secretion of inhibin A, activin A and follistatin by term placental villous trophoblasts in culture

OBJECTIVE: Maternal serum inhibin A and activin A are higher in pre-eclampsia than in normal pregnancy. The placenta is a source of these proteins in pregnancy. The aim of this study was to investigate the effect of growth factors and proinflammatory cytokines that are raised in pre-eclampsia on the secretion of dimeric inhibin A, activin A and follistatin by villous cytotrophoblasts in culture. DESIGN AND METHODS: Villous cytotrophoblasts were prepared from term placentae and cultured in serum-free media. Cells were treated with increasing concentrations of tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta, transforming growth factor (TGF)-beta1, granulocyte and monocyte colony-stimulating factor (GMCSF), inhibin A, activin A and follistatin for 2 days. Culture supernatants were assayed for human chorionic gonadotrophin (hCG), inhibin A, activin A and follistatin as appropriate. Experiments were repeated at least three times with each cytokine or growth factor and the data pooled. RESULTS: Cytotrophoblasts syncytialise and spontaneously secrete hCG, inhibin A and activin A in culture. Follistatin levels were <20 pg/ml in most experiments. Activin A secretion was increased in culture in a dose-dependent manner by IL-1beta (approximately 150%, P<0.05), TNF-alpha (approximately 35%, P=0.02) and GMCSF (approximately 100%, P<0.01). hCG secretion was inhibited in a dose-dependent manner by TNF-alpha (50%, P<0.05). Inhibin A was stimulated by IL-1beta ( approximately 30%, P=0.05). Inhibin A, activin A, follistatin or TGF-beta1 did not have a significant effect on any measured parameters. CONCLUSIONS: These data show that inflammatory cytokines increase the secretion of activin A by trophoblasts in culture. The presence of very low levels, or no follistatin (<20 pg/ml) in the culture media suggests 'free' activin A could have autocrine/paracrine effects on cytotrophoblasts. Inhibin A secretion was stimulated by IL-1beta. However, absence of an effect by the other cytokines investigated on inhibin A in this study suggests that the mechanism(s) involved in increasing maternal circulating levels of inhibin A and activin A in pre-eclampsia are controlled differentially.