The psychological impact of gestational trophoblastic disease: a prospective observational multicentre cohort study.

OBJECTIVE: To evaluate the short-term psychological consequences of gestational trophoblastic disease (GTD). DESIGN: A prospective observational multicentre cohort study. SETTING: Nationwide in the Netherlands. POPULATION: GTD patients. METHODS: Online questionnaires directly after diagnosis. MAIN OUTCOME MEASURES: Hospital Anxiety and Depression Scale (HADS), Distress Thermometer (DT), Impact of Event Scale (IES) and Reproductive Concerns Scale (RCS). RESULTS: Sixty GTD patients were included between 2017 and 2020. Anxious feelings (47%) were more commonly expressed than depressive feelings (27%). Patients experienced moderate to severe adaptation problems in 88%. Patients who already had children were less concerned about their reproductivity than were patients without children (mean score 10.4 versus 15.0, P = 0.031), and patients with children experienced lower distress levels (IES mean score 25.7 versus 34.7, P = 0.020). In addition, patients with previous pregnancy loss scored lower for distress compared with patients without pregnancy loss (IES mean score 21.1 versus 34.2, P = 0.002). DISCUSSION: We recommend that physicians monitor physical complaints and the course of psychological wellbeing over time in order to provide personalised supportive care in time for patients who have high levels of distress at baseline. CONCLUSIONS: GTD patients experience increased levels of distress, anxiety and depression, suggesting the diagnosis has a substantial effect on the psychological wellbeing of patients. The impact of GTD diagnosis on intrusion and avoidance seems to be ameliorated in patients who have children or who have experienced previous pregnancy loss. TWEETABLE ABSTRACT: Patients with gestational trophoblastic disease (GTD) experience short-term psychological consequences such as distress, anxiety and depression, suggesting that the diagnosis GTD has a substantial effect on the psychological wellbeing of patients. Various patient characteristics affect the impact of GTD diagnosis.

Progesterone-induced cyclin G1 inhibits the proliferation of endometrial epithelial cell and its possible molecular mechanism.

Under normal conditions, progesterone inhi-bits the estrogen-induced proliferation of endometrial epithelium. Our previous studies have shown that cyclin G1 was progesterone-dependent in mouse endometrial epithelium at peri-implantation, and exogenous cyclin G1 suppressed the proliferation of endometrial cancer cells. The objectives of this study are to determine whether cyclin G1, as a negative regulator of the cell cycle, is involved in the antiproliferative action of progesterone on endometrial epithelial cells, and to explore the possible molecular mechanism of cyclin G1 inhibition. The siRNA-mediated elimination of cyclin G1 attenuated the antiproliferative action of progesterone on endometrial epithelial cells. Immunoprecipitation showed that progesterone-induced cyclin G1 could interact with PP2A to mediate its phosphatase activity. The block of PP2A activity also attenuated the antiproliferative action of progesterone on endometrial epithelial cells and increased the phosphorylated Rb. In conclusion, progesterone-induced cyclin G1 mediates the inhibitory effect of progesterone on endometrial epithelial cell proliferation possibly through the recruitment of PP2A to dephosphorylate Rb.

Molecular analysis of human endometrium: short-term tibolone signaling differs significantly from estrogen and estrogen + progestagen signaling.

Tibolone, a tissue-selective compound with a combination of estrogenic, progestagenic, and androgenic properties, is used as an alternative for estrogen or estrogen plus progesterone hormone therapy for the treatment of symptoms associated with menopause and osteoporosis. The current study compares the endometrial gene expression profiles after short-term (21 days) treatment with tibolone to the profiles after treatment with estradiol-only (E(2)) and E(2) + medroxyprogesterone acetate (E(2) + MPA) in healthy postmenopausal women undergoing hysterectomy for endometrial prolapse. The impact of E(2) treatment on endometrial gene expression (799 genes) was much higher than the effect of tibolone (173 genes) or E(2) + MPA treatment (174 genes). Furthermore, endometrial gene expression profiles after tibolone treatment show a weak similarity to the profiles after E(2) treatment (overlap 72 genes) and even less profile similarity to E(2) + MPA treatment (overlap 17 genes). Interestingly, 95 tibolone-specific genes were identified. Translation of profile similarity into biological processes and pathways showed that ER-mediated downstream processes, such as cell cycle and cell proliferation, are not affected by E2 + MPA, slightly by tibolone, but are significantly affected by E(2). In conclusion, tibolone treatment results in a tibolone-specific gene expression profile in the human endometrium, which shares only limited resemblance to E(2) and even less resemblance to E2 + MPA induced profiles.

Progesterone-induced miR-133a inhibits the proliferation of endometrial epithelial cells.

AIM: This study aimed to understand the role of miR-133a in progesterone actions, explore the regulative mechanism of the progesterone receptor, and investigate the effects of miR-133a on the progesterone-inhibited proliferation of mouse endometrial epithelial cells. METHODS: The expression of miR-133a induced by progesterone was detected by quantitative real-time PCR both in vivo and in vitro. Ishikawa subcell lines stably transfected with progesterone receptor subtypes were used to determine the receptor mechanism of progesterone inducing miR-133a. Specific miR-133a mimics or inhibitors were transfected into mouse uteri and primary cultured endometrial epithelial cells to overexpress or downregulate the miR-133a. The roles of miR-133a in the cell cycle and proliferation of endometrial epithelial cells were analysed by flow cytometry and Edu incorporation analysis. The protein levels of cyclinD2 in uterine tissue sections and primary cultured endometrial epithelial cells were determined by immunohistochemistry and Western blot analysis. RESULTS: Progesterone could induce miR-133a expression in a PRB-dependent manner in endometrial epithelial cells. miR-133a inhibited endometrial epithelial cell proliferation by arresting cell cycle at the G1 -S transition. Moreover, miR-133a acted as an inhibitor in downregulating cyclinD2 in endometrial epithelial cells. CONCLUSION: We showed for the first time that progesterone-induced miR-133a inhibited the proliferation of endometrial epithelial cells by downregulating cyclinD2. Our research indicated an important mechanism for progesterone inhibiting the proliferation of endometrial epithelial cells by inducing special miRNAs to inhibit positive regulatory proteins in the cell cycle.

The hormone replacement therapy drug tibolone acts very similar to medroxyprogesterone acetate in an estrogen-and progesterone-responsive endometrial cancer cell line.

Tibolone, a steroidogenic compound with both estrogenic and progestagenic properties, is used as an alternative for estrogen or estrogen plus progesterone hormone therapy for the treatment of symptoms associated with menopause and osteoporosis. We have evaluated whether the effect of tibolone on a human endometrial cell line is similar to, or comparable with, the effect of estradiol (E(2)), medroxyprogesterone acetate (MPA) or E(2) + MPA treatment. Using stable transfection techniques, the estrogen receptor (ER) expressing human endometrial cancer cell line, ECC1, was altered to also express both progesterone receptors (PRs). These cells were then used to assess growth regulation and expression profiling (Affymetrix U133plus2) under the influence of E(2) (1 nM), MPA (1 nM), E(2) + MPA or tibolone (100 nM). Growth assessment and comparison of profiles indicate that tibolone behaves predominantly like MPA. Furthermore, regulation of prereplication complex genes, such as the minichromosome maintenance genes, could be involved in the observed strong inhibition of growth by tibolone as well as MPA. In addition, in total, 15 genes were found to be specific for tibolone treatment. These genes were predominantly involved in regulation of the cell cycle and differentiation.

Characterization of a zinc-finger protein and its association with apoptosis in prostate cancer cells.

BACKGROUND: The transition from androgen-dependent to androgen-independent prostate cancer is not fully understood but appears to involve multiple genetic changes. We have identified a gene, GC79, that is more highly expressed in androgen-dependent LNCaP-FGC human prostate cancer cells than in androgen-independent LNCaP-LNO human prostate cancer cells. Physiologic levels (0.1 nM:) of androgens repress expression of GC79 messenger RNA (mRNA) in LNCaP-FGC cells. To determine the role of GC79, we cloned its complementary DNA (cDNA) and functionally characterized its product. METHODS: The differentially expressed GC79 gene was cloned from human prostate cDNA libraries, sequenced, and transfected into mammalian cells to study its function. Expression of GC79 was analyzed in various adult and fetal human tissues and in prostate glands of castrated rats. The association of GC79 expression and apoptosis was investigated in COS-1 and LNCaP cells transfected with GC79 cDNA. All statistical tests are two-sided. RESULTS: Sequence analysis indicates that GC79 encodes a large, complex, multitype zinc-finger protein, containing nine C(2)H(2)-type zinc-finger domains, a cysteine-rich region, and a GATA C(4)-type zinc-finger domain. Castration-induced androgen withdrawal increased the expression of GC79 mRNA in the regressing rat ventral prostate, suggesting that the expression of GC79 mRNA is associated with the process of apoptotic cell death in the rat ventral prostate. Transfection and induction of GC79 cDNA in both COS-1 and LNCaP prostate cancer cells led to an apoptotic index that was eightfold higher (P:<.001, two-sided Student's t test) than that observed in uninduced transfected cells. CONCLUSIONS: We have cloned an androgen-repressible gene, GC79, that is potentially involved in apoptosis. This finding may have implications for the development of androgen-independent prostate cancer and, ultimately, for the treatment of prostate cancer.

Differentially expressed genes in androgen-dependent and -independent prostate carcinomas.

Differential gene expression between androgen-dependent (LNCaP-FGC) and androgen-independent (LNCaP-LNO) prostate cancer cells has been investigated using RNA arbitrarily primed and differential display PCR of mRNA. Four differentially expressed cDNA transcripts were identified, of which differential expression was confirmed by Northern blot analysis. Sequence analysis revealed two unknown (JC19 and GC79) and two known genes [B-cell translocation gene 1 and UDP-glucuronosyltransferase 2B15 (UGT2B15)]. JC19, GC79, and B-cell translocation gene 1 were more highly expressed in LNCaP-FGC cells compared with LNCaP-LNO cells, whereas UGT2B15 was only expressed in LNCaP-LNO cells. Androgens and 1,25-dihydroxyvitamin D3 were able to down-regulate UGT2B15 mRNA in LNCaP-LNO cells. For GC79 mRNA, down-regulation was only observed with androgens in LNCaP-FGC cells. Expression of JC19 mRNA was studied using a panel of human prostate cancer xenografts. In androgen-dependent xenografts, expression of JC19 mRNA was much higher compared with androgen-independent xenografts, in which significant expression was hardly detectable. The mRNA expression pattern in the xenografts is in good agreement with that observed in the cell culture system. In conclusion, the differential display technique used in the present study allows analysis of gene expression in vitro and in vivo and can be used for the identification of important genes involved in androgen-independent prostate cancer development.

Tamoxifen treatment for breast cancer enforces a distinct gene-expression profile on the human endometrium: an exploratory study.

Tamoxifen treatment for breast cancer increases proliferation of the endometrium, resulting in an enhanced prevalence of endometrial pathologies, including endometrial cancer. An exploratory study was performed to begin to understand the molecular mechanism of tamoxifen action in the endometrium. Gene-expression profiles were generated of endometrial samples of tamoxifen users and compared with matched controls. The pathological classification of samples from both groups included atrophic/inactive endometrium and endometrial polyps. Unsupervised clustering revealed that samples of tamoxifen users were, irrespective of pathological classification, fairly similar and consequently form a subgroup distinct from the matched controls. Using SAM analysis (a statistical method to select genes differentially expressed between groups), 256 differentially expressed genes were selected between the tamoxifen and control groups. Upon comparing these genes with oestrogen-regulated genes, identified under similar circumstances, 95% of the differentially expressed genes turned out to be tamoxifen-specific. Finally, construction of a gene-expression network of the differentially expressed genes revealed that 69 genes centred around five well-known genes: TP53, RELA, MYC, epidermal growth factor receptor and beta-catenin. This could indicate that these well-known genes, and the pathways in which they function, are important for tamoxifen-controlled proliferation of the endometrium.

Molecular portrait of the progestagenic and estrogenic actions of tibolone: behavior of cellular networks in response to tibolone.

Tibolone is a synthetic steroid with estrogenic effects on brain, vagina, and bone without stimulating the endometrium. During tibolone treatment, it is thought that the progestagenic properties of tibolone stimulate cell differentiation, which effectively counterbalances the growth-stimulating effects of the estrogenic properties of tibolone. The objective of this study was to characterize the expression profile that reflects the endometrial responses to the separated estrogenic (growth-inducing) and progestagenic (growth-inhibiting) actions of tibolone, thus gaining insight into the counteracting effect of these properties of tibolone on the endometrium. The estrogenic action of tibolone was studied in the estrogen-responsive ECC1 cell line (expressing estrogen receptor alpha), and the progestagenic action was studied in the progesterone-responsive cell line Ishikawa PRAB-36 (expressing PRA and PRB). The data showed that the progestagenic and estrogenic effects of tibolone produce different expression profiles with a narrow overlap in genes; however, both properties modulate the same biological processes. The final genetic network analysis indicated that the estrogenic effect of tibolone is potentially counterbalanced by the progestagenic metabolite of tibolone via differential regulation of similar cellular processes. For example, both progestagenic and estrogenic properties stimulate proliferation, but they exert the opposite effect on apoptosis. The apoptosis network was stimulated by the progestagenic properties of tibolone; in contrast, the estrogenic effect of tibolone suppressed the apoptosis network. The current results indicate that this differential regulation is realized through modulation of a different group of genes and rarely via contraregulation of the same set of genes.

Characterization of an early growth response gene, which encodes a zinc finger transcription factor, potentially involved in cell cycle regulation.

Using differential display polymerase chain reaction, early growth response gene alpha (EGR alpha) was first isolated as a 291-base pair 3'-cDNA clone, which was highly expressed in the androgen-independent prostate carcinoma cell lines PC3 and DU145, as compared with the androgen-responsive prostate carcinoma cell line LNCaP. Full length cloning of the EGR alpha coding region revealed that EGR alpha was a new member of an important subfamily of nuclear zinc finger transcription factors (others members e.g. Sp1, EGR-2, and Wilms' tumor gene). Moreover, it was observed that EGR alpha, as with most Sp1 subfamily members, was conserved between mammalian species ranging from human to rabbit. Two hormones important for prostate development and differentiation were found to be potent regulators of EGR alpha mRNA expression. Androgens were observed to induce a down-regulation of EGR alpha mRNA expression (70% in 72 h), while epidermal growth factor induced a rapid transient up-regulation (6-fold in 100 min). The up-regulation was controlled at the transcriptional level and effectively blocked by staurosporine (which suggests the involvement of the protein kinase C pathway). Functional analysis demonstrated that EGR alpha could bind to, and stimulate transcription from, a basic transcription element (BTE) consensus sequence on DNA (BTE is a transcription-modulating sequence in the promoter region of some cytochrome P450 family members). Furthermore, in stage-synchronized prostate cells, EGR alpha mRNA was highly expressed in the early G1 phase of the cell cycle, similar to c-fos mRNA expression. These results indicated that the zinc finger transcription factor EGR alpha seems to play a role in cell cycle regulation.

Calcium regulation of androgen receptor expression in the human prostate cancer cell line LNCaP.

Elevation of intracellular calcium levels in the presence of normal androgen levels has been implicated in apoptotic prostate cell death. Since the androgen receptor (AR) plays a critical role in the regulation of growth and differentiation of the prostate, it was of interest to determine whether Ca2+ would affect the expression of androgen receptor messenger RNA (mRNA) and protein, thus affecting the ability of androgens to control prostate function. AR-positive human prostate cancer cells, LNCaP, were incubated with either the calcium ionophore A23187 or the intracellular endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin. Subsequently, AR mRNA and protein levels were assessed by Northern and Western blot analysis. Both A23187 and thapsigargin were found to down-regulate steady state AR mRNA levels in a time- and dose-dependent manner. AR mRNA began to decrease after 6-8 h of incubation with 10(-6) M A23187 or 10(-7) M thapsigargin, reaching a nadir at 16 and 10 h of incubation, respectively. In contrast, control mRNA (glyceraldehyde 3-phosphate dehydrogenase) did not change significantly during the treatments with either A23187 or thapsigargin. AR protein levels were found to be decreased after 12 h of incubation with either 10(-6) M A23187 or 10(-7) M thapsigargin. The decrease in AR mRNA and protein seemed to precede apoptosis, since neither A23187 (24 h) nor thapsigargin (30 h) was found to alter cell morphology within the treatment time. Cycloheximide and actinomycin D were unable to change the calcium-mediated decrease in AR mRNA, ruling out the necessity for de novo protein synthesis or a change in mRNA stability. Moreover, the decrease in AR mRNA induced by calcium does not seem to involve protein kinase C- or calmodulin-dependent pathways, since inhibitors of these cellular components had no effect. Nuclear run-on assays demonstrated little or no effects of either A23187 or thapsigargin treatment on AR gene transcription (8 h and 10 h). In conclusion, these studies show that intracellular calcium seems to be a potent regulator of AR gene expression in LNCaP cells.

Activin is produced by rat Sertoli cells in vitro and can act as an autocrine regulator of Sertoli cell function.

The production of activin by Sertoli cells isolated from 21-day-old rats was studied using the mesoderm-inducing activity of activin on Xenopus laevis animal cap explants, immunoprecipitation and Western blotting. Furthermore, the effects of recombinant bovine activin-A on rat Sertoli cell aromatase activity and FSH and androgen receptor gene expression were examined. Animal cap explants from Xenopus laevis blastulas elongated after culture in conditioned medium of Sertoli cells cultured with or without ovine FSH or conditioned medium of the mouse Sertoli cell-derived TM4 cell line. Animal cap explants cultured in control medium remained spherical. This elongation was also found in the more than 10-kilodalton fraction of the conditioned medium and after heating for 10 min at 95 C, indicating that heat-stable activin-like bioactivity is present in the culture medium. Immunoprecipitation of [35S]methionine-labeled proteins and Western blotting of Sertoli cell-conditioned medium with polyclonal antisera against the inhibition beta-subunits indicated the presence of 24- to 25-kilodalton activin-like immunoreactive material. Sertoli cell aromatase activity was dose-dependently stimulated by ovine FSH after 72 h of culture. Recombinant bovine activin-A partly inhibited this stimulation in a dose-dependent way. This inhibition was also found after 24 h of culture. Furthermore, basal and FSH-stimulated androgen receptor mRNA expression in Sertoli cells and binding of the synthetic androgen R1881 to Sertoli cells were decreased after 24 h of culture in the presence of recombinant bovine activin-A. In the same experiments, FSH receptor mRNA expression was not significantly affected. These results indicate that activin can act as an autocrine regulator of Sertoli cell function.

Transient down-regulation of androgen receptor messenger ribonucleic acid (mRNA) expression in Sertoli cells by follicle-stimulating hormone is followed by up-regulation of androgen receptor mRNA and protein.

Cooperative actions of FSH and androgens on initiation, maintenance, and restoration of spermatogenesis have been described. In the present experiments the regulatory effects of FSH on androgen receptor (AR) gene expression in Sertoli cells were studied. In immature rats injection of FSH (1 microgram/g BW, ip) resulted in a rapid down-regulation of testicular AR mRNA expression (4 h), followed by recovery to the control level (10 h). Using cultured immature Sertoli cells, a similar transient effect on AR mRNA expression was observed after the addition of FSH (500 ng/ml) or (Bu)2cAMP (0.5 mM). Cycloheximide treatment of the cells did not prevent the rapid FSH-induced down-regulation of AR mRNA expression, indicating that de novo protein synthesis is not required for this effect. Furthermore, using a transcriptional run-on assay, no marked decrease in the rate of AR gene transcription was found upon treatment of the cultured Sertoli cells with FSH for 2 or 4 h. This demonstrates that the short term effect of FSH or AR mRNA expression reflects a change in mRNA stability. The AR protein level was not markedly affected by the transient decrease in AR mRNA expression. When immature Sertoli cells were incubated with FSH for longer time periods (24-72 h), both AR mRNA and protein expression were increased. In Sertoli cells isolated from 15-day-old rats, this increase was higher (mRNA, 2- to 3-fold; protein, 2-fold) than in Sertoli cells isolated from 25-day-old animals. The results indicate that FSH plays a complex role in the regulation of AR expression in immature rat Sertoli cells.

Progestogenic effects of tibolone on human endometrial cancer cells.

Tibolone, a synthetic steroid acting in a tissue-specific manner and used in hormone replacement therapy, is converted into three active metabolites: a Delta(4) isomer (exerting progestogenic and androgenic effects) and two hydroxy metabolites, 3 alpha-hydroxytibolone (3 alpha-OH-tibolone) and 3beta-OH-tibolone (exerting estrogenic effects). In the present study an endometrial carcinoma cell line (Ishikawa PRAB-36) was used to investigate the progestogenic properties of tibolone and its metabolites. This cell line contains progesterone receptors A and B, but lacks estrogen and androgen receptors. When tibolone was added to the cells, complete conversion into the progestogenic/androgenic Delta(4) isomer was observed within 6 d. Furthermore, when cells were cultured with tibolone or when the Delta(4) isomer or the established progestagen medroxyprogesterone acetate was added to the medium, marked inhibition of growth was observed. Interestingly, 3 beta-OH-tibolone also induces some inhibition of growth. These growth inhibitions were not observed in progesterone receptor-negative parental Ishikawa cells, and progestagen-induced growth inhibition of PRAB-36 cells could readily be reversed using the antiprogestagen Org-31489. Upon measuring the expression of two progesterone-regulated genes (fibronectin and IGF-binding protein-3), tibolone, the Delta(4) isomer and medroxyprogesterone acetate showed similar gene expression regulation. These results indicate that tibolone, the Delta(4) metabolite, and to some extent 3 beta-OH-tibolone exert progestogenic effects. Tibolone and most likely 3 beta-OH-tibolone are converted into the Delta(4) metabolite.

A novel gene on human chromosome 2p24 is differentially expressed between androgen-dependent and androgen-independent prostate cancer cells.

Identification of genes involved in the transition from androgen-dependent to androgen-independent prostate cancer is important to extend our current knowledge of the disease. Using differential display RT-PCR analysis between androgen-dependent and androgen-independent prostate cancer cells, we have identified a novel gene, designated GC109. GC109 harbours a putative Cys-His cluster, a nuclear localisation signal, a leucine zipper and a ret finger protein (rfp)-like domain. GC109 mRNA expression in normal human tissues was found not to be restricted to the prostate. However, using a variety of 15 human cancer cell lines, GC109 mRNA was preferentially expressed in androgen-dependent LNCaP-FGC, compared with androgen-independent LNCaP-LNO, DU145 and PC3 human prostate cancer cells. Finally, the GC109 gene was mapped on human chromosome 2p24. Based on its protein domain structure and chromosomal localisation, we hypothesise that GC109 may be involved in chromosomal rearrangements in prostate cancer.

Immunization of carp (Cyprinus carpio) with a Vibrio anguillarum bacterin: indications for a common mucosal immune system.

Uptake and transport of formalin-killed Vibrio anguillarum bacteria were studied in the second gut segment of carp and the resulting reaction of the immune system was investigated. Within a few hours after anal administration antigenic determinants of bacteria were present in intraepithelial macrophages of the second gut segment. In gut and skin mucus and bile, immunoglobulins (Ig's) were detected, but the amount was much lower than found in serum. In mucus of the second gut segment, 4 times more Ig (per mg protein) was found than in the first segment. Upon oral or anal immunization, slightly enhanced antigen-specific Ig titers could be detected in skin mucus and bile, but only after a booster along the same route. The existence of a common mucosal immune system is discussed, with special reference to the significance of the second gut segment. After anal intubation an increase of antigen-specific Ig could also be observed in serum. Following a booster, an enhanced cal memory. After anal boosting equal levels of serum antibody were reached compared with two consecutive intramuscular injections. However, no significant antibody increase occurred in serum after oral immunization, not even when bacteria were administered daily with the food.

Expression of inhibin subunit mRNAs and inhibin levels in the testes of rats with stage-synchronized spermatogenesis.

Inhibin alpha- and beta B-subunit mRNA expression, and levels of bioactive and immunoreactive inhibin were studied in rat testes, synchronized for the stage of the cycle of the seminiferous epithelium by treating vitamin A-deficient rats with vitamin A. Measurement of inhibin subunit mRNA expression and inhibin levels was started directly after the start of vitamin A treatment, and continued for 65 days. Inhibin subunit mRNA expression, and testicular bioactive and immunoreactive inhibin levels increased after the start of vitamin A treatment, reaching maximum values after 9 days, when B spermatogonia and preleptotene spermatocytes had appeared in the stage-synchronized testes. The ratio between beta B- and alpha-subunit mRNA expression was high at that time-point, whereas the ratio between bioactive and immunoreactive inhibin remained low. These data suggest a relatively high production of activin at that moment, and this may play a role in the development of B spermatogonia into preleptotene spermatocytes during the initiation of spermatogenesis. Stage-dependency was demonstrated for inhibin subunit mRNA expression, and for the levels of bioactive and immunoreactive inhibin, in rats with complete spermatogenesis. Inhibin alpha-subunit mRNA expression was relatively high at stages V and XIII of the spermatogenic cycle, whereas beta B-subunit mRNA expression was high at stage XIII but not at stage V. This resulted in a high beta B/alpha subunit mRNA ratio at stage XIII. Since it has been shown that expression of the activin receptor is high at stages XIII-I, locally formed activin might play a role in the regulation of meiosis. Bioactive and immunoreactive inhibin were highly correlated during the cycle, with maximum levels at stages XIV-I. It was concluded that the production of inhibin, and possibly activin, is dependent on the stage of the cycle of the seminiferous epithelium; these growth factors might play a paracrine role in the differentiation of spermatogenic cells.

Regulation of inhibin beta B-subunit mRNA expression in rat Sertoli cells: consequences for the production of bioactive and immunoreactive inhibin.

In Sertoli cells from 21-day-old rats, the expression of the mRNA encoding the alpha-subunit of inhibin, and the production of immunoreactive inhibin are stimulated by follicle-stimulating hormone (FSH). In contrast, the amount of beta B-subunit mRNA is not increased after FSH treatment of the cells, and the ratio between bioactive and immunoactive inhibin decreases after stimulation with FSH. These data suggest that the beta B-subunit is the limiting factor in the production of bioactive inhibin. The aim of the present experiments was to investigate the effect of changes in the amount of beta B-subunit mRNA on the production of bioactive and immunoreactive inhibin. During early postnatal testicular development, the relative amounts of the 4.2 kb and 3.5 kb mRNAs encoding the beta B-subunit of inhibin changed markedly. The meaning of this changing ratio between beta B-subunit mRNAs is not clear, since both mRNAs are actively translated, as demonstrated by polysomal analysis. The total amount of beta B-subunit mRNA correlated with the in vitro production of bioactive inhibin as published earlier. Prolonged stimulation of cultured Sertoli cells from 14-day-old rats with 4 beta-phorbol 12-myristate 13-acetate (PMA) caused a decreased expression of the beta B-subunit mRNAs, presumably by down-regulation of protein kinase C. A similar effect was obtained after addition of the calcium ionophore A23187. Concomitantly, a decreased production of bioactive inhibin was observed. Furthermore, Western blotting revealed that secretion of the 32 kDa inhibin alpha beta-dimer was decreased, whereas secretion of the combination of the C-terminal part with the pro-region of the alpha-subunit was increased. It is concluded that the level of the beta B-subunit of inhibin is rate-limiting for the production of bioactive inhibin in cultured Sertoli cells, and that its expression can be influenced by modulation of protein kinase C, and/or intracellular calcium levels.

Follicle-stimulating hormone regulates androgen receptor mRNA in Sertoli cells.

Follicle-stimulating hormone (FSH) and testosterone stimulate the production of a variety of proteins by immature Sertoli cells. A highly purified Sertoli cell preparation was incubated for 3 days with FSH and testosterone. Both androgen receptor protein and mRNA concentrations were markedly increased by FSH. Testosterone also increased the androgen receptor protein concentration, but did not increase the expression of the androgen receptor mRNA. It is concluded that FSH plays a role in the responsiveness of Sertoli cells to testosterone.

Follitropin receptor down-regulation involves a cAMP-dependent post-transcriptional decrease of receptor mRNA expression.

The regulation by FSH (follitropin; follicle-stimulating hormone) of FSH receptor mRNA and protein (FSH binding) was studied using cultured Sertoli cells isolated from 21-day-old rats. FSH induced a dose-dependent and almost complete down-regulation of receptor mRNA at 4 h after addition of the hormone. At subsequent time points (16 h and later) the FSH receptor mRNA levels had returned close to control values. The effect of FSH was mimicked by dibutyryl cyclic AMP (dbcAMP) and forskolin, and the phosphodiesterase inhibitor methyl-isobutylxanthine (MIX) prolonged the FSH action. These findings indicate that the effect of FSH on its receptor mRNA was mediated by cAMP. A down-regulatory effect of FSH and dbcAMP on FSH receptor mRNA was also observed in the presence of the protein synthesis inhibitor cycloheximide, suggesting a direct effect of FSH/dbcAMP on the expression of the FSH receptor gene. Transcriptional run-on experiments revealed that FSH did not inhibit initiation of the FSH receptor gene; hence a post-transcriptional mechanism is involved. Binding of 125I-FSH to the cultured Sertoli cells was rapidly (4 h) decreased when the cells were incubated with FSH or FSH in combination with MIX. This effect can be explained by ligand-induced receptor sequestration. In contrast, incubation of Sertoli cells with dbcAMP had no effect on binding of 125I-FSH after 4 h, but resulted in a 60% loss of FSH binding sites after 24 h, probably caused by decreased mRNA expression. In conclusion, FSH receptor down-regulation in Sertoli cells is effected not only by the well-documented ligand-induced loss of receptors from the plasma membrane, but also involves a cAMP-mediated decrease of FSH receptor mRNA through a post-transcriptional mechanism.