Role of FSH glycan structure in the regulation of Sertoli cell inhibin production.

Variations in follicle-stimulating hormone (FSH) carbohydrate composition and structure are associated with important structural and functional changes in Sertoli cells (SCs) during sexual maturation. The aim of the present study was to investigate the impact of FSH oligosaccharide structure and its interaction with gonadal factors on the regulation of monomeric and dimeric inhibin production at different maturation stages of the SC. Recombinant human FSH (rhFSH) glycosylation variants were isolated according to their sialylation degree (AC and BA) and complexity of oligosaccharides (CO and HY). Native rhFSH stimulated inhibin α-subunit (Pro-αC) but did not show any effect on inhibin B (INHB) production in immature SCs isolated from 8-day-old rats. Activin A stimulated INHB and had a synergistic effect on FSH to stimulate Pro-αC. The less acidic/sialylated rhFSH charge analogues, BA, were the only charge analogue mix that stimulated INHB as well as the most potent stimulus for Pro-αC production. Native rhFSH stimulated both Pro-αC and INHB in SCs at a more advanced maturation stage, isolated from 20-day-old rats. In these cells, all rhFSH glycosylation variants increased INHB and Pro-αC production, even in the presence of growth factors. The BA preparation exerted a more marked stimulatory effect on INHB and Pro-αC than the AC. Glycoforms bearing high mannose and hybrid-type oligosaccharides, HY, stimulated INHB and Pro-αC more effectively than those bearing complex oligosaccharides, CO, even in the presence of gonadal growth factors. These findings demonstrate the modulatory effect of FSH oligosaccharide structure on the regulation of inhibin production in the male gonad.

Hypogonadotropic Hypogonadism in Infants with Congenital Hypopituitarism: A Challenge to Diagnose at an Early Stage.

BACKGROUND: Combined pituitary hormone deficiency (CPHD) presents a wide spectrum of pituitary gland disorders. The postnatal gonadotropic surge provides a useful period to explore the gonadotropic axis for assessing the presence of congenital hypogonadotropic hypogonadism (CHH). AIM: To explore the functioning of the hypothalamic-pituitary-gonadal axis in the postnatal gonadotropic surge for an early diagnosis of CHH in newborns or infants suspected of having CPHD. SUBJECTS AND METHODS: A cohort of 27 boys under 6 months and 19 girls under 24 months of age with suspected hypopituitarism was studied. Serum concentrations of LH, FSH, testosterone, inhibin B, anti-Müllerian hormone (AMH) and estradiol were measured, and male external genitalia were characterized as normal or abnormal (micropenis, microorchidism and/or cryptorchidism). RESULTS: CPHD was confirmed in 36 out of 46 patients. Low LH and testosterone levels were found in 66% of the hypopituitary males, in significant association with the presence of abnormal external genitalia. This abnormality had a positive predictive value of 93% for CHH. No significant association was observed between serum FSH, AMH and inhibin B and the patient's external genitalia. CONCLUSION: In newborn or infant boys with CPHD, LH and testosterone concentrations measured throughout the postnatal gonadotropic surge, together with a detailed evaluation of the external genital phenotype, facilitate the diagnosis of CHH at an early stage.

Utilidad de la hormona anti-Mülleriana (AMH) y la inhibina B en el diagnóstico del hipogonadismo en el niño

Durante la infancia, el eje hipotálamo-hipófiso-testicular se encuentra parcialmente quiescente: bajan los niveles de gonadotrofinas y la secreción de testosterona disminuye siguiendo a la caída de la LH. Por el contrario, las células de Sertoli están activas, como lo demuestran los niveles séricos de hormona anti-mülleriana (AMH) e inhibina B. Por lo tanto, el hipogonadismo en la infancia puede ser puesto en evidencia, sin necesidad de pruebas de estímulo, si se evalúa la función de las células de Sertoli. La AMH sérica es alta desde la vida fetal hasta el inicio de la pubertad. La producción testicular de AMH aumenta en respuesta a la FSH pero es potentemente inhibida por los andrógenos. La inhibina B es alta en los primeros años de la vida, luego disminuye parcialmente aunque permanece claramente más alta que en las mujeres, y aumenta nuevamente en la pubertad. Las concentraciones séricas de AMH e inhibina B son indetectables en pacientes anórquidos. En el hipogonadismo primario que afecta a todo el testículo, establecido durante la vida fetal o la infancia, todos los marcadores testiculares están bajos. Cuando en el hipogonadismo están afectadas sólo las células de Leydig, la AMH y la inhibina B sérica son normales y/o altas, mientras que están bajas cuando se ven afectadas las células de Sertoli. La AMH y la inhibina B están bajas en varones con hipogonadismo central en edad prepuberal y continúan bajas en edad puberal. El tratamiento con FSH induce un aumento en los niveles séricos de los marcadores de la célula de Sertoli. En conclusión, la determinación de los niveles séricos de AMH e inhibina B es útil para evaluar la función testicular, sin necesidad de pruebas de estímulo, y orientar el diagnóstico etiológico en el hipogonadismo masculino en pediatría.

During childhood, the hypothalamic-pituitary-gonadal axis is partially quiescent: gonadotropin and testosterone levels decrease, but Sertoli cells remain active, as shown by serum anti-Müllerian hormone (AMH) and inhibin B levels. Therefore, hypogonadism may be diagnosed during childhood, without the need for stimulation tests, provided Sertoli cell function is assessed. Serum AMH levels are high from fetal life until the onset of puberty. Testicular AMH production increases in response to FSH but is potently inhibited by androgens. Serum inhibin B levels are high until the age of 3-4 years in boys; although they decrease thereafter, they remain clearly higher than in girls of the same age. During the early stage of puberty, serum inhibin B increases again to reach adult values. AMH and inhibin B are undetectable in the serum of anorchid patients. In boys with fetalonset primary hypogonadism affecting the whole testicular parenchyma, AMH and inhibin B are low in serum. Conversely, they are normal or high when only the interstitial tissue of the gonads is impaired. AMH and inhibin B are low in children with central hypogonadism and persist low during pubertal age. FSH treatment induces an increase in both Sertoli cell markers. In conclusion, the determination of serum AMH and inhibin B levels is useful for the assessment of testicular function, without the need for stimulation tests, in pediatric patients.

The glycan structure in recombinant human FSH affects endocrine activity and global gene expression in human granulosa cells.

The aim of this study was to analyse the biological response to different recombinant human FSH (rhFSH) glycosylation variants on the endocrine activity and gene expression at whole-genome scale in human granulosa-like tumor cell line, KGN. The effects of differences in rhFSH sialylation and oligosaccharide complexity were determined on steroid hormone and inhibin production. A microarray approach was used to explore gene expression patterns induced by rhFSH glycosylation variants. Set enrichment analysis revealed that hormone sialylation and oligosaccharide complexity in rhFSH differentially affected the expression of genes involved in essential biological processes and molecular functions of KGN cells. The relevance of rhFSH oligosaccharide structure on steroidogenesis was confirmed assessing gene expression by real time-PCR. The results demonstrate that FSH oligosaccharide structure affects expression of genes encoding proteins, growth factors and hormones essential for granulosa cells function.

Carbohydrate complexity and proportion of serum FSH isoforms reflect pituitary-ovarian activity in perimenopausal women and depot medroxyprogesterone acetate users.

BACKGROUND: FSH is synthesized and secreted in multiple glycosylation variants with different oligosaccharide structures; the endocrine milieu regulates the composition of FSH carbohydrate moiety. OBJECTIVES: To characterize serum FSH isoforms according to their sialic acid content and oligosaccharide complexity in regularly menstruating women and in depot medroxyprogesterone acetate (DMPA) users during the menopausal transition. Subjects and methods Ten regularly menstruating perimenopausal women aged 45-52, with mid-follicular phase FSH levels < or =10 IU/l and 10 regularly menstruating women, aged 20-39, were included. Blood samples were collected on the ninth day of the menstrual cycle. Twenty DMPA users were divided into two groups (n = 10) according to age: DMPA(1), age range 20-39 and DMPA(2), age range 45-52. Blood samples were collected 90 +/- 5 days after the last injection of DMPA. Oestradiol (E(2)), inhibin B (Inh B), Pro-alphaC levels and the relative abundance of FSH isoforms on the basis of charge (preparative isoelectric focusing) and carbohydrate complexity (Concanavalin A chromatography) were determined. RESULTS: Decreased Inh B and moderately elevated E(2) levels were observed in perimenopausal women associated with an increase in FSH sialylation and a decrease in its oligosaccharide complexity. DMPA induced changes in the hormonal profile and FSH molecular microheterogeneity; the secreted hormone was more heterogeneous and its oligosaccharides were less complex under this condition. CONCLUSION: Serum FSH glycoforms with increased sialylation and decreased oligosaccharide complexity reflect the decline of the gonadal activity induced either by age or by the use of a DMPA as a contraceptive.

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.

Effect of dihydrotestosterone on pituitary follicle-stimulating hormone isoforms in adult male rats treated with a gonadotropin-releasing hormone antagonist.

The effect of androgens on the oligosaccharide structure of follicle-stimulating hormone (FSH) isoforms was studied in adult male rats treated with a potent gonadotropin-releasing hormone (GnRH) antagonist. Animals were castrated (Cx), and 24 h later were treated with physiological doses of either testosterone propionate (T) or dihydrotestosterone propionate (DHT) (0.25 mg/rat in corn oil, daily, s.c.) for 7 days. The antiandrogen flutamide (F; 5 mg/day/rat, twice a day, s.c.) was administered to either Cx-T or Cx-DHT rats to block androgen action. The GnRH antagonist Org 30276 (Ant; 1 mg/kg/day, s.c.) was injected to both Cx- and Cx-DHT treated rats. FSH serum levels reached normal values in Cx rats treated with either T or DHT, whereas those treated with F retained Cx conditions. Both Cx-Ant and Cx-Ant-DHT treated animals presented normal serum FSH levels. Concanavalin A affinity chromatography was used to isolate pituitary FSH isoforms according to their carbohydrate inner structure. An increased proportion of FSH isoforms bearing complex-type oligosaccharides was observed in Cx rats treated with T or DHT, whereas the proportion of these isoforms was reduced in Cx and Cx-T-F or Cx-DHT-F rats. These results demonstrate that functional androgens are needed to complete the oligosaccharide processing of FSH. In addition, the proportion of the different pituitary FSH isoforms after DHT replacement was equivalent to that found when GnRH action was blocked, indicating that androgens are involved in the regulation of carbohydrate incorporation to the FSH molecule by acting mainly at the pituitary level, independently of GnRH action.

Inhibin a increases apoptosis in early ovarian antral follicles of diethylstilbestrol-treated rats.

The purpose of this study was to evaluate the role of inhibin A in follicular development and apoptosis-related mechanisms in preantral and early antral follicles from prepubertal diethylstilbestrol (DES)-treated rats. Granulosa cells isolated from the ovaries of 23- to 25-day-old rats were cultured in serum-free medium containing FSH (20 ng/ml), transforming growth factor beta (5 ng/ml), and estradiol (50 ng/ml) in the presence or absence of different concentrations of recombinant human inhibin A. (3)H-Thymidine incorporation was decreased in the presence of Inh, but no significant changes were observed in progesterone and estradiol levels in culture medium. An increase in low molecular weight DNA fragmentation indicative of apoptosis and an increase in the levels of Bax protein with no changes in Bcl-2 protein levels were evident in early antral follicles incubated for 24 h with Inh. For each animal, Inh (0.5 micro g/ovary) was injected intrabursally in one ovary, and the contralateral ovary served as a control. Ovarian histology revealed an inhibitory effect of Inh treatment on the follicular development induced by DES. At 24 h after Inh injection, the number of preantral follicles was increased compared with controls, whereas the number of early antral follicles was decreased. In addition, in vivo Inh treatment caused an increase in the percentage of apoptotic cells in preantral and early antral follicles. These results suggest that inhibin produced by the dominant follicle may act as a paracrine factor inhibiting the growth of neighboring follicles, thus participating in the mechanism of follicular selection.

Variations in soluble and particulate ABP of rat testis during sexual development

La proteína ligadora de andrógenos (ABP) fue determinada en las fracciones citosólica (cABP) y particulada (pABP), obtenidas por centrifugación diferencial de homogenatos testiculares de ratas. Se prepararon homogenatos en condiciones de estabilización para el ABP por el agregado de testosterona 350 nM al "buffer" de homogeneización. Se observó que tanto los niveles por mg/prot de cABP como de pABP eran máximos entre los 22 y 32 días de edad de la rata, disminuyendo a partir de allí durante la maduración sexual. Cuando los resultados se expresaron por órgano, ambas proteínas aumentaron con la edad del animal. La pABP pudo ser solubilizada en condiciones en que mantuvo su capacidad de unión a andrógenos, procediéndose entonces a su fotomarcación y posterior cromatografía en una columna de Sephadex G-0200, usando el ABP de citosol de epidídimo como control. Se observó que el ABP no sólo comparte con el cABP las mismas características físico-químicas para la unión de andrógenos, sino también el volumen de exclusión en la cromatografía mencionada. Dado que la pABP está solamente presente en la célula de Sertoli, probablemente represente ABP antes de ser secretado. Su localización subcelular sugiere un posible papel del ABP en la distribución de andrógenos testiculares

Acute effect of hOG on testicular and epididymal levels of androgen binding protein (ABP) in the immature rat

Se estudió en ratas inmaduras el efecto agudo de la hOG y la testosterona sobre los niveles testiculares (fracciones particulada y soluble) y epididimarios (fracción soluble) de la proteína ligadora de andrógenos (ABP). La administración de una única inyección de hOG (10 UI/rata) produjo una leve disminución de la proteína ligadora de andrógenos (ABP) en el testículo (fracciones particulada y soluble). Este efecto fue observado una y cuatro horas después del tratamiento. Simultáneamente se observó un aumento significativo en la concentración de ABP en el epidídimo (1 h: 169 ñ 5; 4 h: 182 + 5vs. control: 113 ñ13 fmol/mg proteína, media ñ ESM). El aumento en la concentración de testosterona en testículo y epidídimo sugeriría que el efecto observado podría estar mediado por la estimulación gonadatrófica de la síntese de andrógenos en la célula de Leydig. Para comprobar esta hipótesis, el efecto de la hOC se estudió en ratas tratadas previamente con aminoglutetimida para bloquear la esteroidogénesis. En este modelo experimental no se observó un aumento en la concentración de ABP epididimario por la acción de la gonadotrofina. finalmente, la administración de propionato de testosterona indujo un incremento en la concentración de ABP epididimario 4 h después del tratamiento (212 + 18vs. 127 ñ 28 fmol/mg proteína, media ñ ESM). Los resultados obtenidos sugieren que la elevación de los androgénos testiculares inducida por hOG produce un pasaje rápido de ABP desde el testículo hacia el epidídimo