Direct enhancement of gonadotropin-stimulated ovarian estrogen biosynthesis by estrogen and clomiphene citrate.

The biosynthesis of ovarian aromatases and hence estrogen production are under the control of the gonadotropins, FSH and LH. Using a primary culture of rat granulosa cells, we now report that estrogens (diethylstilbestrol and 17 beta-estradiol) augment the stimulation of aromatase activity by FSH and LH. Moreover, clomiphene citrate, a drug widely used to induce ovulation in anovulatory women, also enhances gonadotropin-stimulated aromatase activity. These in vitro findings suggest that estrogens within the microenvironment of the ovarian follicles may exert a local autoregulatory effect on their own production via an ultra-short loop, positive feedback mechanism. In addition, the clinical efficacy of clomiphene citrate may derive partially from its direct augmentation of the gonadotropin-stimulated estrogen production at the ovarian level.

Estrogen augmentation of gonadotropin-stimulated progestin biosynthesis in cultured rat granulosa cells.

The influence of estrogens on gonadotropin-stimulated production of progesterone and 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OH-P) was examined in primary cultures of rat granulosa cells. Granulosa cells were cultured for 3 days with increasing concentrations of FSH in the presence or absence of either diethylstilbestrol (DES) or estradiol. FSH treatment increased progestin production in a dose-dependent manner, whereas treatment with estrogens alone were ineffective. In contrast, concomitant addition of either DES or estradiol augmented FSH-stimulated production of progesterone and 20 alpha-OH-P. Increasing concentrations of estradiol (10(-10) - 10(-7) M) augmented the stimulatory effect of FSH (30 ng/ml) on progesterone production in a dose-dependent manner with ED50 values of approximately 3 X 10(-9) M. The facilitatory action of estradiol was time-related, becoming significant after 36 h of treatment. Granulosa cells were also cultured for 2 days with FSH to induce functional LH receptors. The FSH-primed cells were treated for an additional 3 days with increasing concentrations of LH (0.3-30 ng/ml) in the absence or presence of DES (10(-7) M). LH stimulated progesterone and 20 alpha-OH-P production in a dose-dependent manner, whereas concomitant addition of DES further enhanced LH-induced progestin biosynthesis. (Bu)2cAMP also increased progesterone and 20 alpha-OH-P production by the granulosa cells; however, concurrent addition of DES did not augment the actions of (Bu)2cAMP. The effect of estrogens on gonadotropin-stimulated cAMP accumulation was also examined. FSH treatment dose-dependently increased cAMP accumulation, whereas concomitant treatment with estradiol further increased the FSH action. Similarly, LH treatment also stimulated cAMP accumulation in FSH-primed cells, whereas concurrent addition of DES further augmented LH action. Thus, the stimulatory effect of estrogens upon gonadotropin-stimulated progestin production may be related to the augmentation of cAMP biosynthesis. The present observations suggest that intraovarian estrogens may act locally to enhance the sensitivity of granulosa cells to FSH and LH, thereby increasing the biosynthesis of progestins and cAMP by the granulosa cells.

Mechanism of the direct action of gonadotropin-releasing hormone and its antagonist on androgen biosynthesis by cultured rat testicular cells.

The direct effects of GnRH and its agonistic and antagonistic analogs upon testicular androgen biosynthesis were studied in primary cultures of testicular cells obtained from adult hypophysectomized rats. Treatment of cultured cells with hCG (10 ng/ml) substantially increased testosterone production, while concomitant addition of GnRH or its agonist [des-Gly10, D-Ser(TBu)6,Pro9NHEt-GnRH] decreased hCG-stimulated testosterone production in a dose-related manner with ED50 values of 1.2 X 10(-9) and 4.5 X 10(-11) M, respectively. Treatment with 10(-6) M of either a GnRH partial peptide or a cyclic GnRH analog did not affect hCG action; however, the addition of a GnRH antagonist ([Ac-D-Phe1,D-p-Cl-Phe2,D-Trp3,6]GnRH) together with hCG and GnRH blocked the GnRH-induced decrease in testosterone production, with a half-maximal inhibitory dose ratio (antagonist to GnRH) of 0.15. The stimulatory effect of hCG became apparent by 8 h of incubation; no hCG effect was seen at this time in the presence of GnRH. Treatment with hCG increased cAMP accumulation, but GnRH administration did not affect hCG-induced cAMP accumulation. In contrast, treatment with GnRH depressed testosterone production induced by cholera toxin or (Bu)2cAMP. The inhibitory effect of GnRH on testosterone production (93% inhibition) was associated with decreases in hCG-induced 17 alpha-hydroxyprogesterone (39%) and delta 4-androstenedione (82%), but was not accompanied by a decrease in progesterone production. In cells incubated with cyanoketone and spironolactone to prevent pregnenolone metabolism, hCG stimulated pregnenolone biosynthesis, while concomitant GnRH treatment did not affect hCG action. In contrast, GnRH decreased hCG-induced testosterone production in cells treated with 10(-5) M progesterone. Similarly, GnRH decreased hCG-induced testosterone and androstenedione production in cells incubated with 10(-5) M 17 alpha-hydroxyprogesterone. The present results demonstrate that GnRH and its analogs exert direct actions on testicular cells through stereospecific recognition sites. The inhibitory effect of GnRH on testicular androgen production occurs at sites distal to the formation of cAMP and pregnenolone and may be due to decreases in the activity of the enzymes 17 alpha-hydroxylase and 17-20 desmolase.

Disparate effects of triphenylethylene antiestrogens on estrogen and progestin biosyntheses by cultured rat granulosa cells.

The influence of triphenylethylene antiestrogens on FSH-stimulated production of estrogen, progesterone, and 20 alpha-hydroxy-4-pregnen-3-one (20 alpha-OH-P) was examined in cultured rat granulosa cells. The cells were cultured with increasing concentrations of FSH, with or without diethylstilbestrol (DES) or various antiestrogens. After 3 days, medium progesterone and 20 alpha-OH-P contents were determined. Cells were reincubated for an additional 8 h with delta 4-androstene-3,17-dione, and estrogen formation was measured. Steroid production was negligible by cultures treated with DES or antiestrogens alone. FSH treatment increased estrogen and progestin production, while the addition of DES (10(-7) M) further enhanced FSH-stimulated steroidogenesis. Likewise, treatment with 10(-6) M of the antiestrogens tamoxifen (Tam), nafoxidine (Naf), or clomiphene citrate (CC) also enhanced FSH-stimulated aromatase activity. In contrast, the antiestrogens each inhibited FSH stimulation of progesterone and 20 alpha-OH-P production (greater than 73% inhibition at 300 ng/ml FSH). Increasing concentrations (3 X 10(-8)-10(-6) M) of the antiestrogens augmented the stimulatory effect of FSH (10 ng/ml) on estrogen production in a dose-related manner (CC greater than Tam greater than Naf). Similar doses of these antiestrogens inhibited the stimulatory effect of FSH (300 ng/ml) on progesterone and 20 alpha-OH-P production (Tam greater than CC greater than Naf). The observed inhibition of progestin production is associated with decreases in FSH-stimulated pregnenolone biosynthesis in antiestrogen-treated cells incubated with 10(-6) M cyanoketone. Furthermore, the antiestrogens inhibited the binding of [3H]estradiol to ovarian estrogen receptors with binding affinity constants of 364, 437, and 2144 nM for CC, Tam, and Naf, respectively. Thus, antiestrogens exert disparate actions on granulosa cell estrogen and progestin biosyntheses. Like estrogens, CC, Tam, and Naf enhance FSH-stimulated aromatase activity with potencies comparable with their abilities to interact with ovarian estrogen receptors. However, unlike estrogens, the antiestrogens inhibit FSH-stimulated progestin biosynthesis, partially via suppression of pregnenolone biosynthesis. The present granulosa cell culture system provides a valuable model for elucidating the disparate actions of estrogens and antiestrogens on ovarian steroidogenesis.

Expression of androgen receptor mRNA in the brain of Gekko gecko: implications for understanding the role of androgens in controlling auditory and vocal processes.

The neuroanatomical distribution of androgen receptor (AR) mRNA-containing cells in the brain of a vocal lizard, Gekko gecko, was mapped using in situ hybridization. Particular attention was given to auditory and vocal nuclei. Within the auditory system, the cochlear nuclei, the central nucleus of the torus semicircularis, the nucleus medialis, and the medial region of the dorsal ventricular ridge contained moderate numbers of labeled neurons. Neurons labeled with the AR probe were located in many nuclei related to vocalization. Within the hindbrain, the mesencephalic nucleus of the trigeminal nerve, the vagal part of the nucleus ambiguus, and the dosal motor nucleus of the vagus nerve contained many neurons that exhibited strong expression of AR mRNA. Neurons located in the peripheral nucleus of the torus in the mesencephalon exhibited moderate levels of hybridization. Intense AR mRNA expression was also observed in neurons within two other areas that may be involved in vocalization, the medial preoptic area and the hypoglossal nucleus. The strongest mRNA signals identified in this study were found in cells of the pallium, hypothalamus, and inferior nucleus of the raphe. The expression patterns of AR mRNA in the auditory and vocal control nuclei of G. gecko suggest that neurons involved in acoustic communication in this species, and perhaps related species, are susceptible to regulation by androgens during the breeding season. The significance of these results for understanding the evolution of reptilian vocal communication is discussed.

Norepinephrine regulates human chorionic gonadotrophin production by first trimester trophoblast tissue in vitro.

The effect of norepinephrine (NE) upon human chorionic gonadotrophin (hCG) production by 6-8 week gestation placental explants has been investigated. NE (5 micrograms/ml) enhanced hCG secretion significantly from the second day of treatment. The stimulatory effect of NE on hCG secretion could be abolished by the alpha 1-receptor specific antagonist prazosin (10(-4) M) and partly diminished by the beta 1-receptor specific antagonist atenolol (10(-4) M), but was not influenced by the alpha 2-receptor specific antagonist yohimbine (10(-4) M). The involvement of the alpha-receptor in the regulation of hCG secretion was further confirmed by addition of the alpha-receptor agonist clonidine (10(-6) M) which had a similar stimulatory effect on hCG release but the effect was antagonized by both prazosin and yohimbine. Further study showed that NE induced a significant increase in cyclic adenosine monophosphate (cAMP) production by trophoblast tissue. Cyclic AMP secretion in the NE-treated group was fivefold higher than that of the control group. Both the protein kinase C (PKC) specific activator 1-deoyl-2-acetyl-sn-glycerol (OAG) and the PKC non-specific activator phorbol-12-myristate-13-acetate (PMA) had a stimulatory effect on hCG secretion, while the PKC inhibitor, 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (H7) diminished the hCG secretion stimulated by NE. The effect of NE was blocked by the voltage-dependent calcium channel blocker nifedipine but not by the voltage-independent calcium channel blocker gadolinium chloride (GdCl3). On the other hand, anti-gonadotrophin releasing hormone (GnRH) IgG and the GnRH antagonist (D-Phe2, D-Trp6)-GnRH did not influence the stimulatory effect of NE on hCG release. The results indicate that NE regulates hCG production in human first trimester trophoblast tissue. The effect of NE was mainly mediated by alpha 1 and partly by beta 1 receptors. Cyclic AMP, the PKC signal transduction pathway and the voltage-dependent calcium channels were involved in NE action.

A study of the effect of B-EP and naloxone on the function of the hypothalamo-pituitary-testicular axis of the rat.

To investigate whether endogenous opioid peptides (EOP) play an important role in intragonadal regulation of testicular function and regulation of the hypothalamic-pituitary-gonadal axis of the male rat, the authors employed two principal methods: culture of testicular Leydig cells and Sertoli cells, and in vitro perifusion of hypothalamo-pituitary Leydig cells of the adult rat. The results demonstrated that incubation of Leydig cells with B-endorphin (B-EP 10(-9) = 10(-6) mol/L) or naloxone (NAL 10(-5) = 10(-8) mol/L) manifested no significant changes of non-stimulated or hCG-stimulated testosterone secretion both in 20 and 60 day-old rats. Similar results were obtained when the cells were treated with B-EP (10(-10) = 10(-7) mol/L) for 48 h during culture. Pretreatment of incubated Leydig cells with B-EP in similar concentrations for 48 h showed no effect on the response to hCG stimulation. In addition, treatment with B-EP in vitro for 24 or 72 h manifested no effects on estradiol production by aromatization of cultured Sertoli cells. Neither NAL 10(-5) given in vitro nor NAL (5 mg/body weight) injected subcutaneously 1 h before decapitation affected LH and testosterone release from the perifused hypothalamo-pituitary Leydig cells system. These results could not support the hypothesis that B-EP is a local regulator of testicular function. The physiological significance of EOP in regulating the function of gonadal axis of adult male rat remains to be investigated further.

Effects of gossypol on rat Sertoli and Leydig cells in primary culture and established cell lines.

In order to evaluate the direct effect of gossypol on testicular cells, we used primary cultures of rat Leydig and Sertoli cells. No alteration in Leydig cell survival, morphology, or testosterone production was seen during three days of culture with up to 3 microgram/ml gossypol. With higher concentrations (3 to 7 microgram/ml) of gossypol, there was a reduction in cell survival but no change in androgen secretion. In contrast, there was a marked change in Sertoli cell morphology after five days of gossypol treatment. Large vacuoles and electron dense granules appeared in the cytoplasm, but these effects were reversed within six days of removing gossypol from the medium. There was a significant decrease in androgen binding protein (ABP) secretion by Sertoli cells in the presence of gossypol. We also tested the effect of gossypol on the growth of three established cell lines. Two Sertoli-derived cell lines, TM4 and TR-ST, were more sensitive than a Leydig-derived cell line (TM3). These results suggest that, of the somatic cell types in the testis, the Sertoli cells are most sensitive to gossypol.

Stimulatory effect of norepinephrine on progesterone production by human first trimester placenta explants in vitro.

Norepinephrine (NE) was investigated for its effect on progesterone secretion from 6-8 week gestation human trophoblast tissue cultured in serum-free medium. Norepinephrine (5 micrograms/ml) enhanced progesterone secretion significantly on the third day of treatment. The stimulatory effect of NE on progesterone production was abolished by the alpha 1-receptor specific antagonist prazosin (10(-4) M) (P < 0.05), but was not influenced by the alpha 2-receptor specific antagonist yohimbine (10(-4) M) and the beta 1-receptor specific antagonist atenolol (10(-4) M). On the other hand, the alpha-receptor agonist clonidine (10(-6) M) had a similar stimulatory effect on progesterone release but the effect was antagonized by both the alpha 1-antagonist prazosin and the alpha 2-antagonist yohimbine. Further study showed that NE induced a significant increase in cyclic adenosine monophosphate (cAMP) production by trophoblast tissue. Cyclic AMP secretion in the NE treated group was fivefold higher than that of the control group. The effect of NE was blocked by the voltage-dependent calcium channel blocker nifedipine (100 microM) but not by the voltage-independent calcium channel blocker gadolinium chloride (GdCl3) (10 microM). In addition, anti-gonadotropin releasing hormone (GnRH) IgG (5 micrograms/ml) and GnRH antagonist, (D-Phe2, D-Trp6)-GnRH (10(-6) M) did not influence the stimulatory effect of NE on progesterone release. The results indicate that NE regulates progesterone production in human first trimester trophoblast tissue. The effect of NE was mediated by the alpha 1 receptors. Cyclic AMP and voltage-dependent calcium channel were involved in its action.

Effects of multiglycosides of Tripterygium wilfordii (GTW) on rat fertility and Leydig and Sertoli cells.

Primary cultures of rat Leydig and Sertoli cells were used to evaluate the direct effects of GTW on testicular cells and to compare these to the effects of gossypol acetate. Both GTW and gossypol acetate can affect the survival of Leydig and Sertoli cells. But Sertoli cells are much more sensitive than Leydig cells, either to gossypol acetate or GTW. Leydig and Sertoli cells all died when they were exposed to gossypol acetate or GTW at a dose of 3.0 micrograms/ml or 30 micrograms/ml, respectively, for 24 hours. The cell survival-time course demonstrated that the cell numbers were decreased after 2 hours, and especially so after 8 hours. No significant changes were observed in testosterone production in Leydig cells after 24 hours of exposure to 1.0-20 micrograms/ml GTW. The forward motility of epididymal spermatozoa was completely lost and fertility of rat was significantly inhibited after the treatment of GTW in vivo. It is concluded that GTW does affect the fertility of rat and viability of cultured rat Leydig and Sertoli cells.

Changes in the serum concentrations of gonadotrophins and oestradiol in peripubertal Chinese Beijing black gilts.

Four gifts of the Chinese Beijing Black breed, all from the same litter, had their blood serum concentrations of luteinizing hormone (LH), follicle stimulating hormone (FSH) and oestrogens (E2) measured by radioimmunoassay at intervals of 20 days between the ages of 5 and 185 days. The concentrations of LH declined sharply between days 5 to 25 and remained low until rising again from day 85 and reaching a peak at day 105 before declining again and remaining constant. The concentrations of FSH increased from day 85, peaking at day 105, followed by a slow decline to day 185. Serum E2 concentrations were high at day 5 then decreased dramatically, but increased considerably at 185 days. These results are similar to those reported from European breeds.

Study on reproductive endocrinology of human placenta (II)--Hormone secreting activity of cytotrophoblast cells.

The capability of cytotrophoblast cells to produce hCG, progesterone, estrogen, cGnRH and beta-endorphin in vitro has been demonstrated in serum-free culture medium. Before experiment, a 24-h preculture was carried out in order to remove the endogenous hormones of the tissue. During a period of 8 days' culture, the cytotrophoblast cells could constantly produce a small amount of hCG. The production of progesterone rose rapidly and became doubled within six days. The estrogen secretion showed a similar pattern in the presence of androstenedione, a precursor of estrogen, indicating the elevation of aromatase activity in the cells. The elevation of the enzyme activity has been further demonstrated not to be induced by androstenedione. In both cytotrophoblast and syncytiotrophoblast cell cultures, cGnRH was only detected in the culture of cytotrophoblast cells, with a value up to 4 pg/10(5) cells/24 h. However, beta-endorphin was identified both in cytotrophoblast and syncytiotrophoblast cells. Its content increased significantly in the medium of cytotrophoblast cell culture from the 4th to 6th days, but declined in the medium of syncytiotrophoblast cell culture. The results demonstrate clearly that the cytotrophoblast cells are the sole origin of GnRH in human placenta and are also able to synthesize beta-endorphin and steroid hormones. The findings indicate that there is no such a sharp functional demarcation existing between these two kinds of trophoblast cells as suggested before. The data are of significance for a better understanding of the mechanism of hormonal regulation in placenta.

Study on reproductive endocrinology of human placenta--culture of highly purified cytotrophoblast cell in serum-free hormone supplemented medium.

A new method of long-term culture of cytotrophoblast cells in serum-free medium has been developed. Cytotrophoblast cells were isolated with cold trypsin and purified by unit gravity sedimentation through BSA density gradients. The cells were cultured in the FD medium with supplement of EGF, insulin, transferrin and sodium selenite. They could survive over three weeks. The results showed that both EGF and insulin stimulated hCG and progesterone secretion and that sodium selenite elevated hCG output but not progesterone secretion. Transferrin produced synergistic effect with EGF and insulin on hCG and progesterone secretion but it was ineffective when used alone. This study demonstrates that the four growth factors mentioned above are essential for the survival of cytotrophoblast cells in vitro. It is therefore suggested that EGF, insulin and selenium may possibly be involved in the regulation of hCG and progesterone secretion in the human placenta.

Study on reproductive endocrinology of human placenta (III)--Hormonal regulation of progesterone production by trophoblast tissue of first trimester.

The effect of hormones on progesterone secretion by 6-8 week human trophoblast tissue cultured in serum-free medium has been investigated. GnRH at low concentration (10(-10)-10(-8) mol/L) stimulated progesterone secretion, while high dose (10(-6)-10(-5) mol/L) produced inhibitory effect. The progesterone secretion could be significantly decreased by addition of anti-hCG antiserum or monoclonal anti-hCG IgG in a dose- and time-dependent manner. Various concentrations of TRH, PGE2, PGF2 alpha, testosterone and estradiol were found to be ineffective. These data indicate clearly that progesterone production by human trophoblast tissue at early gestation stage is under the modulation of GnRH and hCG.

[GnRH receptor and the regulation of its gene expression].

The function of GnRH(gonadotropin-releasing hormone, LHRH) is mediated by GnRH receptor (GnRHR). The study on GnRHR is one of the highlights in neuroendocrinology and reproductive biology. The latest study of GnRHR, including molecular structure of GnRHR, regulation of GnRHR gene expression, distribution of GnRHR, the regulation of GnRHR by peptide and steroid hormone, as well as signal transduction mediated by GnRHR, are reviewed in the present article. Studies on GnRHR will make great contribution to the understanding of the regulation of reproduction and its action for therapy of malignant tumor.

The effects of growth factors on human normal placental cytotrophoblast cell proliferation.

The effects of growth factors were investigated on the proliferation of a normal placental cytotrophoblast cell line (NPC). Epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha) and insulin-like growth factor-I (IGF-I) stimulated NPC cell proliferation. In contrast, TGFbeta1 was found to be a negative regulator, inhibiting EGF-induced cell proliferation. When EGF/TGF alpha receptor was analysed by radio-ligand binding, two binding sites of different affinities were revealed in the proliferating NPC cells but only the low affinity binding site was detected in the non-proliferating cytotrophoblast cells in primary cultures. The results suggest that EGF stimulates cytotrophoblast proliferation through high affinity binding sites.

[Effect of prolactin on production of acid phosphatase in rat prostatic cells and its mechanism].

The present study revealed the effect of prolactin (PRL) and sex steroid hormones on production of prostatic acid phosphatase (PAP) of the immature rat prostatic epithelial cells in serum-free medium. The results showed that PRL significantly stimulated synthesis and secretion of PAP, but failed to synergize with dihydrotestosterone or estradiol. Dihydrotestosterone or estradiol alone did not promote PAP production. Studies on the mechanism of PRL action indicated that adenyl cyclase was not activated in the action of PRL: Synergistic effect between PRL and prostaglandin E2 and F2 alpha was revealed in the stimulation of PAP production, and the effect of PRL could be blocked by indomethacin (a prostaglandin synthesis inhibitor). In conclusion, PRL directly stimulated PAP production of the cultured rat prostatic epithelial cells in serum-free medium and prostaglandin was involved in PRL action.

Establishment and characterization of a cytotrophoblast cell line from normal placenta of human origin.

A cell line has been established from human placentae at the first trimester of normal pregnancy. The cell line was obtained by culture of purified cytotrophoblast cells in serum-free medium supplemented with epidermal growth factor, insulin, dexamethasone and 0.1% bovine serum albumin. The cells can be subcultured for >30 passages in one to three splits. All the cells were mononuclear epithelial-like cells positive to cytokeratin 18, gonadotrophin-releasing hormone (GnRH), neuropeptide Y, neurotensin, leucine-enkephalin, dopamine and 5-hydroxytryptamine immunocytochemical staining. The cells secreted GnRH, progesterone and oestradiol (in the presence of testosterone) but little human chorionic gonadotrophin and no beta-endorphin. The cell line showed human karyotypes and had a population doubling time of 48 h in serum-free medium. However, the cells would stop growing in the medium containing fetal bovine serum. A normal cytotrophoblast cell line established in serum-free medium will be particularly useful in the study of cytotrophoblast cell proliferation and differentiation.

Neuropeptides and neurotransmitters in human placental villi.

The human placenta contains many kinds of bioactive substances which are more or less similar to those from the hypothalamic-pituitary-gonadal axis. Most of the studies were carried out mainly with term placenta. The present study, therefore, was attempted to identify, quantify and characterize these substances in the human placenta at the early pregnancy. Using the RIA, immunohistochemistry, HPLC, tissue culture and intrauterine injection methods, we have found that: (1) many kinds of neuropeptides and neurotransmitters are present in the placental villi; (2) LH-RH, NT and SRIF positive immunoreactive granules are localized in the cytotrophoblast and those of beta-EP, 5-HT positive granules in the syncytiotrophoblast; (3) synthetic LH-RH and dynorphin (Dyn) stimulate the hCG secretion of the early placental villi in vitro, and (4) the antisera of LH-RH, NT, Dyn and NE antagonist-alpha-MPT significantly reduced the number of blastocyst implantations in the early pregnant rat. These results indicate that in the human placenta there possibly exists a self-regulation mechanism for the synthesis and secretion of placental hormones and neurotransmitters. Therefore, the human placenta can be regarded as a neuroendocrine organ.