Effect of sialylation and complexity of FSH oligosaccharides on inhibin production by granulosa cells.

Granulosa cell (GC) inhibin A and B production is regulated by FSH and gonadal factors. This gonadotrophin is released as a mixture of glycoforms, which induce different biological responses in vivo and in vitro. Our aim was to determine the effect of recombinant human FSH (rhFSH) glycosylation variants on inhibin A and B production by rat GCs. Preparative isoelectro focusing was used to isolate more acidic/sialylated (pH <4.00) and less acidic/sialylated (pH >5.00) rhFSH charge analogues. Concanavalin A was used to isolate unbound and firmly bound rhFSH glycoforms on the basis of their oligosaccharide complexity. GCs, obtained from oestrogen-primed immature rats, were cultured with either native rhFSH or its glycosylation variants. Inhibin A and B were determined using specific ELISAs. Results were expressed as mean±s.e.m. Under basal conditions, inhibin A was the predominant dimer produced (inhibin A: 673±55; inhibin B: 80±4  pg/ml). More acidic/sialylated charge analogues stimulated inhibin B production when compared to inhibin A at all doses studied; by contrast, less acidic/sialylated charge analogues stimulated inhibin A production and elicited no effect on inhibin B. Glycoforms bearing complex oligosaccharides showed a potent stimulatory effect on inhibin B when compared to inhibin A production (i.e. dose 1  ng/ml: 4.9±0.5 vs 0.9±0.1-fold stimulation, P<0.001). Glycoforms bearing hybrid-type oligosaccharides favoured inhibin A production (i.e. dose 4  ng/ml 2.9±0.1 vs 1.6±0.1-fold stimulation, P<0.05). These results show that the sialylation degree as well as the complexity of oligosaccharides present in the rhFSH molecule may be considered additional factors that differentially regulate dimeric inhibin production by rat GCs.

Transforming growth factor beta1 regulates follistatin mRNA expression during in vitro bovine granulosa cell differentiation.

In order to test the hypothesis that transforming growth factor beta (TGF-beta) acts by FS regulation on bovine granulosa cells in in vitro differentiation, we analyzed the effect of TGF-beta1 on follistatin mRNA expression in three differentiation states of bovine granulosa cells. We showed a positive regulation of FS mRNA after TGF-beta1 (1 ng/ml) treatment of freshly isolated granulosa cells from small-medium antral follicles (2-8 mm). This effect was abolished by the addition of exogenous follistatin (100 ng/ml), suggesting that this effect could be mediated by activin. Although these cells showed a similar effect on FS mRNA expression after treatment with activin-A, a soluble form of activin receptor type IIA was unable to inactivate the TGF-beta effect. When we tested the TGF-beta effect on FS mRNA in different granulosa cell states, TGF-beta1 regulation was associated with progesterone production only in freshly isolated cells. The amount of total activin-A produced by first passage cells (dedifferentiated cells), was ten times smaller than the one measured in a conditioned medium from freshly isolated cells (mature cells). The TGF-beta1-dependent FS mRNA expression persisted in first passage cells without changes with FS addition. On the other hand, the BGC-1 granulosa cell line (immature cells) produced large amounts of activin-A regulated by TGF-beta1 and an invariable steady state of FS mRNAs. In summary, our results showed that FS mRNA expression is regulated by TGF-beta1 independently of activin effects in differentiated granulosa cells.

Low risk of impaired testicular Sertoli and Leydig cell functions in boys with isolated hypospadias.

CONTEXT: Isolated hypospadias may result from impaired testicular function or androgen end-organ defects or, alternatively, from hormone-independent abnormalities of morphogenetic events responsible for urethral seam. OBJECTIVE: The objective was to evaluate the relative prevalence of hormone-dependent etiologies in boys with isolated hypospadias. DESIGN, PATIENTS, AND MAIN OUTCOME MEASURES: We studied endocrine testicular capacity in 61 patients with isolated hypospadias and 28 with hypospadias associated with micropenis, cryptorchidism, or ambiguous genitalia. Serum anti-Müllerian hormone and inhibin B were used as Sertoli cell markers. A human chorionic gonadotropin test was performed to evaluate Leydig cell function. RESULTS: Testicular dysfunction was observed in 57.1% and androgen end-organ defects in 7.2% of patients with hypospadias associated with cryptorchidism, micropenis, or ambiguous genitalia. In the remaining 35.7%, the disorder was idiopathic. The presence of ambiguous genitalia predicted the existence of testicular or end-organ dysfunction with 81.8% specificity. Isolated hypospadias was associated in 14.8% of patients with testicular dysfunction and in 6.5% of cases with end-organ defects; in 78.7% of cases, the condition was idiopathic. The occurrence of isolated hypospadias ruled out the existence of testicular or end-organ disorders with 80.0% sensitivity. Altogether our data indicate that the risk for the existence of an underlying testicular or end-organ dysfunction is low in patients with isolated hypospadias (odds ratio, 0.13; 95% confidence interval, 0.05-0.36; P < 0.001). CONCLUSIONS: Boys with isolated hypospadias are more likely to have normal endocrine testicular and androgen end-organ functions, suggesting that transient disruption of morphogenetic events in early fetal life may be the predominant underlying cause.

Altered serum profile of inhibin B, Pro-alphaC and anti-Müllerian hormone in prepubertal and pubertal boys with varicocele.

OBJECTIVE: Anti-Müllerian hormone (AMH) and inhibin B are reliable markers of Sertoli cell function. The aim of the present study was to assess the functional state of Sertoli cells in order to detect early changes in the testicular function of prepubertal and pubertal patients with untreated grade II or III varicocele. DESIGN AND PATIENTS: Seven prepubertal and 55 pubertal boys with untreated grade II or III varicocele were studied. Seven prepubertal and 43 pubertal normal boys were considered as controls. MEASUREMENTS: Serum levels of gonadotrophins, testosterone, inhibin B and Pro-alphaC and AMH were determined by time-resolved immunofluorometric assays, radioimmunoassay (RIA) and specific enzyme-linked immunosorbent assays (ELISAs), respectively. RESULTS: Inhibin B and Pro-alphaC serum levels were higher in prepubertal patients with varicocele than in controls (P < 0.001). No further increment in inhibin B and Pro-alphaC levels was observed in pubertal patients with varicocele. Higher levels of AMH were found in patients in Tanner stages I, III, IV and V when compared to normal boys by Tanner stage (P < 0.05, P < 0.01, P < 0.01, P < 0.001, respectively). The direct correlation found in normal boys between inhibin B levels and LH, testosterone and testicular volume was not observed in patients with varicocele. CONCLUSIONS: The altered serum profile of gonadal hormones observed in untreated prepubertal and pubertal patients with varicocele may indicate an early abnormal regulation of the seminiferous epithelium function.