Hormonal Regulation of Follicle-Stimulating Hormone Glycosylation in Males.

The Follicle-Stimulating Hormone plays an important role in the regulation of gametogenesis. It is synthesized and secreted as a family of glycoforms with differing oligosaccharide structure, biological action, and half-life. The presence of these oligosaccharides is absolutely necessary for the full expression of hormone bioactivity at the level of the target cell. The endocrine milieu modulates the glycosylation of this hormone. During male sexual development a progressive increase in FSH sialylation and in the proportion of glycoforms bearing complex oligosaccharides are the main features in this physiological condition. In late puberty, FSH oligosaccharides are largely processed in the medial- and trans-Golgi cisternae of the gonadotrope and remain without changes throughout adult life. In experimental models, the absence of gonads severely affects FSH sialylation; androgen administration is able to restore the characteristics observed under physiological conditions. The expression of ST6 beta-galactoside alpha-2,6-sialyltransferase 1 is hormonally regulated in the male rat; it decreases after short periods of castration but increases markedly at longer periods of androgen deprivation. Although ST3 beta-galactoside alpha-2,3-sialyltransferase 3 is expressed in the male rat pituitary it is not influenced by changes in the endocrine milieu. The oligosaccharide structure of FSH has an impact on the Sertoli cell endocrine activity. In more advanced stages of Sertoli cell maturation, both sialylation and complexity of the oligosaccharides are involved in the regulation of inhibin B production; moreover, FSH glycoforms bearing incomplete oligosaccharides may enhance the stimulatory effect exerted by gonadal growth factors. In this review, we discuss available information on variation of FSH glycosylation and its hormonal regulation under different physiological and experimental conditions, as well as the effect on Sertoli cell endocrine activity.

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.

Spreading the clinical window for diagnosing fetal-onset hypogonadism in boys.

In early fetal development, the testis secretes - independent of pituitary gonadotropins - androgens and anti-Müllerian hormone (AMH) that are essential for male sex differentiation. In the second half of fetal life, the hypothalamic-pituitary axis gains control of testicular hormone secretion. Follicle-stimulating hormone (FSH) controls Sertoli cell proliferation, responsible for testis volume increase and AMH and inhibin B secretion, whereas luteinizing hormone (LH) regulates Leydig cell androgen and INSL3 secretion, involved in the growth and trophism of male external genitalia and in testis descent. This differential regulation of testicular function between early and late fetal periods underlies the distinct clinical presentations of fetal-onset hypogonadism in the newborn male: primary hypogonadism results in ambiguous or female genitalia when early fetal-onset, whereas it becomes clinically undistinguishable from central hypogonadism when established later in fetal life. The assessment of the hypothalamic-pituitary-gonadal axis in male has classically relied on the measurement of gonadotropin and testosterone levels in serum. These hormone levels normally decline 3-6 months after birth, thus constraining the clinical evaluation window for diagnosing male hypogonadism. The advent of new markers of gonadal function has spread this clinical window beyond the first 6 months of life. In this review, we discuss the advantages and limitations of old and new markers used for the functional assessment of the hypothalamic-pituitary-testicular axis in boys suspected of fetal-onset hypogonadism.

Testicular function during adolescence in boys with type 1 diabetes mellitus (T1D): absence of hypogonadism and differences in endocrine profile at the beginning and end of puberty.

AIM: Conflicting results regarding testicular function in adults with type 1 diabetes (T1D) have been reported, but little is known about Leydig and Sertoli cell function during puberty in boys treated with multiple daily insulin doses. Our aim was to assess testicular function in boys with T1D. METHODS: Pubertal boys with T1D (n = 71) and healthy control boys (Control group; n = 104) who were 10-18 years were studied. Both groups were matched by pubertal stage, age, and BMI. Total testosterone (TT), calculated free testosterone (cfT), SHBG, inhibin B, AMH, and gonadotropin levels were determined. RESULTS: At the beginning of puberty, the T1D group had higher levels of SHBG (p = 0.003) and similar androgen levels than the Control group. At the end of puberty, higher TT, and cfT were observed in T1D compared to the Control group (p < 0.01 and p < 0.001, respectively). Gonadotropins and AMH were similar in both groups. Regression analysis showed that T1D was a significant factor, even after adjusting for Tanner stage and BMI-SDS, affecting TT, cFT, and SHBG levels. BMI-SDS was a significant factor affecting TT and SHBG levels. Higher HbA1c had a negative effect on total testosterone and cFT and a positive effect on SHBG levels in T1D boys. CONCLUSION: Adolescents with T1D do not exhibit hypogonadism, as shown by normal gonadotropin, testosterone, inhibin B, and AMH levels. However, in T1D boys, HbA1c and BMI-SDS had a negative association with testosterone levels. Elevated testosterone levels are observed during late puberty, which were not present earlier.

Male Central Precocious Puberty: Serum Profile of Anti-Müllerian Hormone and Inhibin B before, during, and after Treatment with GnRH Analogue.

We aimed to describe the functional changes of Sertoli cells, based on the measurement of serum anti-Müllerian hormone (AMH) and inhibin B during treatment with GnRHa and after its withdrawal in boys with central precocious puberty. Six boys aged 0.8 to 5.5 yr were included. AMH was low at diagnosis in patients >1 yr but within the normal range in younger patients. AMH increased to normal prepubertal levels during treatment. After GnRHa withdrawal, AMH declined concomitantly with the rise in serum testosterone. At diagnosis, inhibin B was elevated and decreased throughout therapy, remaining in the upper normal prepubertal range. In patients with testicular volume above 4 mL AMH remained higher in spite of suppressed FSH. After treatment withdrawal, inhibin B rose towards normal pubertal levels. In conclusion, AMH did not decrease in patients <1 yr reflecting the lack of androgen receptor expression in Sertoli cells in early infancy. Serum inhibin B might result from the contribution of two sources: the mass of Sertoli cells and the stimulation exerted by FSH. Sertoli cell markers might provide additional tools for the diagnosis and treatment followup of boys with central precocious puberty.

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.

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.

Sertoli cell markers in the diagnosis of paediatric male hypogonadism.

During childhood, the pituitary-testicular axis is partially dormant: testosterone secretion decreases following a drop in luteinising hormone levels; follicle-stimulating hormone (FSH) levels also go down. Conversely, Sertoli cells are most active, as revealed by the circulating levels of anti-Müllerian hormone (AMH) and inhibin B. Therefore, hypogonadism can best be evidenced, without stimulation tests, if Sertoli cell function is assessed. Serum AMH is high from fetal life until mid-puberty. Testicular AMH production increases in response to FSH and is potently inhibited by androgens. Inhibin B is high in the first years of life, then decreases partially while remaining clearly higher than in females, and increases again at puberty. Serum AMH and inhibin B are undetectable in anorchid patients. In primary or central hypogonadism affecting the whole gonad established in fetal life or childhood, all testicular markers are low. Conversely, when hypogonadism only affects Leydig cells, serum AMH and inhibin B are normal. In males of pubertal age with central hypogonadism, AMH and inhibin B are low. Treatment with FSH provokes an increase in serum levels of both Sertoli cell markers, whereas human chorionic gonadotrophin (hCG) administration increases testosterone levels. In conclusion, measurement of serum AMH and inhibin B is helpful in assessing testicular function, without need for stimulation tests, and orientates the aetiological diagnosis of paediatric male hypogonadism.

Gonadotrophin secretion pattern in anorchid boys from birth to pubertal age: pathophysiological aspects and diagnostic usefulness.

CONTEXT: The biphasic ontogeny of serum gonadotrophins observed in normal children also exists in girls with gonadal dysgenesis, although with higher levels. However, limited data exist in prepubertal boys with anorchia. OBJECTIVE: To investigate whether the existence of testicular tissue is required for gonadotrophin downregulation in boys. Secondarily, we analysed the prevalence of high gonadotrophins and its diagnostic value to assess the presence or absence of testes in childhood. STUDY DESIGN: In a retrospective, semi-longitudinal study, we compared serum gonadotrophin levels in 35 boys with anorchia aged 0-18 years, in 29 bilaterally cryptorchid boys with abdominal testes and in 236 normal boys. RESULTS: In anorchid boys, follicle stimulating hormone (FSH) and luteinizing hormone (LH) were abnormally high in the first months after birth, then decreased progressively. LH decreased more readily than FSH and dropped to normal values in up to 70% of anorchid patients before the usual age of pubertal onset, when both gonadotrophins increased again to very high levels. In cryptorchid boys, FSH was elevated in a significantly (P < 0·0001) lower proportion of cases. Below the age of 6 years, FSH below 2 IU/l ruled out anorchia and LH above 5 IU/l confirmed anorchia with high accuracy. Between 6 and 11 years, FSH or LH levels above 5 IU/l were highly specific for the absence of testes. CONCLUSIONS: The U-shaped pattern of serum gonadotrophins observed in normal males from birth to puberty was also found in anorchid boys, but with gonadotrophin levels considerably elevated. Serum gonadotrophin levels may normalize in anorchid boys during late childhood only to rise again at puberty. The presence of testicular tissue results in restrain of gonadotrophin secretion in most patients, even if the testes are cryptorchid.

Prolactin (PRL) induction of cyclooxygenase 2 (COX2) expression and prostaglandin (PG) production in hamster Leydig cells.

Serum prolactin (PRL) variations play a crucial role in the photoperiodic-induced testicular regression-recrudescence transition in hamsters. We have previously shown that cyclooxygenase 2 (COX2), a key enzyme in the biosynthesis of prostaglandins (PGs), is expressed mostly in Leydig cells of reproductively active hamsters with considerable circulating and pituitary levels of PRL. In this study, we describe a stimulatory effect of PRL on COX2/PGs in hamster Leydig cells, which is mediated by IL-1ß and prevented by P38-MAPK and JAK2 inhibitors. Furthermore, by preparative isoelectric focusing (IEF), we isolated PRL charge analogues from pituitaries of active [isoelectric points (pI): 5.16, 4.61, and 4.34] and regressed (pI: 5.44) hamsters. More acidic PRL charge analogues strongly induced COX2 expression, while less acidic ones had no effect. Our studies suggest that PRL induces COX2/PGs in hamster Leydig cells through IL-1ß and activation of P38-MAPK and JAK2. PRL microheterogeneity detected in active/inactive hamsters may be responsible for the photoperiodic variations of COX2 expression in Leydig cells.

Are Klinefelter boys hypogonadal?

UNLABELLED: Male hypogonadism implies decreased function of one or more testicular cell population, i.e. germ, Leydig and/or Sertoli cells. In the normal prepubertal boy, Sertoli cells are very active, as indicated by high anti-Müllerian hormone (AMH) and inhibin B secretion, whereas the functional activity of Leydig cells is minimal, as evidenced by low testosterone production, and germ cells do not undergo the full spermatogenic process. Klinefelter syndrome is the most frequent cause of hypogonadism in the adult male. In this review, we discuss whether the gonadal failure is already established during infancy and childhood. In Klinefelter syndrome, there is increased germ cells degeneration from mid-foetal life - resulting in a decreased number at birth - which persists during infancy and childhood and becomes dramatic during puberty. Controversial results exist in the literature regarding Leydig cell function in Klinefelter boys: while some authors have found normal to low testosterone levels in infancy and childhood, others have reported normal to high values. Sertoli cell products AMH and inhibin B are normal in prepubertal boys and only decline during mid- to late puberty. CONCLUSION: Klinefelter syndrome is a primary hypogonadism affecting all testicular cell populations. Germ cells are affected from foetal life, and a severe depletion occurs at puberty. Leydig cell function may be normal or mildly affected in foetal and early postnatal life. Sertoli cell function is not impaired until mid- to late puberty, as reflected by normal AMH and inhibin B in Klinefelter boys.

Regulation of inhibin/activin expression in rat early antral follicles.

The aim of the present study was to determine the endocrine activity of cultured early antral follicles (EAF) isolated from prepubertal diethylstilbestrol-treated rats. The effect of steroidogenic substrates and FSH on steroid, inhibin A and B, Pro-alphaC and activin A production was evaluated. Androsterone was the predominant steroid produced by EAF. The addition of androstenedione, androstenedione+FSH and progesterone stimulated oestradiol production, whereas 25-hydroxycholesterol (25-OH-Chol) increased progesterone production. Inhibin A, B, Pro-alphaC, and activin A were produced under basal conditions. The predominance of inhibin B over inhibin A was not affected by the addition of androstenedione or progesterone. Inhibin A and activin A production was stimulated by FSH. 25-OH-Chol increased Inha, Inhba and Inhbb mRNA expression and the production of the three molecular forms of inhibins but decreased activin A production. These results show that FSH and the steroid follicular microenvironment differentially modulate the gene expression of inhibin/activin subunits, their assembly and secretion.

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.

Seminiferous tubule function in delayed-onset X-linked adrenal hypoplasia congenita associated with incomplete hypogonadotrophic hypogonadism.

OBJECTIVE: X-linked adrenal hypoplasia congenita (AHC, OMIM 300200) due to mutations in the DAX-1 gene is frequently associated to hypogonadotrophic hypogonadism (HHG, OMIM 238320). Clinical variants with delayed-onset have been recognized. The objective of this study is to assess Sertoli cell function throughout pubertal development in patients with childhood-onset AHC due to stop mutations in the DAX-1 gene. DESIGN: Observational follow-up study of gonadotrophin pulsatility pattern, and serum levels of antimüllerian hormone and inhibin B through pubertal development in these patients. PATIENTS: Three patients belonging to two families with AHC were included in this study. MEASUREMENTS: The gonadotrophic pattern, serum inhibin B and antimüllerian hormone were determined in relation to clinical Tanner stage of pubertal development. RESULTS: One patient showed a marked elevation in serum FSH concomitantly with low inhibin B and antimüllerian hormone levels, indicating a primary testicular dysfunction. The other two patients showed a gonadotrophic pattern of HHG, and their serum levels of inhibin B and antimüllerian hormone also reflected a moderate primary testicular dysfunction. The three patients were azoospermic. CONCLUSIONS: These cases give further insight into the clinical spectrum of phenotypes of the hypothalamic-pituitary-gonadal axis in patients with variants in hypogonadism associated with childhood-onset X-linked AHC due to DAX-1 mutations.

Establishment of testicular endocrine function impairment during childhood and puberty in boys with Klinefelter syndrome.

OBJECTIVE: To precisely characterize the chronology of testicular endocrine function impairment during childhood and adolescence in patients with Klinefelter syndrome. Design Retrospective chart review. Patients A total of 29 boys with Klinefelter syndrome with up to 12.3 years follow-up. MEASUREMENTS: Clinical features and serum hormone levels were analysed during follow-up. RESULTS: Of the 29 patients, 16 were prepubertal and 13 had already entered puberty at their first visit. Fifteen patients were followed up through late puberty. Before puberty, LH, FSH, testosterone, anti-Müllerian hormone (AMH) and inhibin B were within the expected range in almost all cases. However, levels of the inhibin alpha-subunit precursor Pro-alphaC were in the lowest levels of the normal range in most cases. During puberty, FSH levels increased earlier and more markedly than LH. Inhibin B and AMH declined to abnormally low or undetectable levels in advanced pubertal stages. Although testosterone and Pro-alphaC levels were within the reference ranges in most cases, they were abnormally low for the observed LH values. CONCLUSIONS: In Klinefelter syndrome, a mild Leydig cell dysfunction is present from early childhood in most cases and persists throughout puberty. Sertoli cell function is normal until mid puberty, when a dramatic impairment is observed.

Time course of the serum gonadotropin surge, inhibins, and anti-Müllerian hormone in normal newborn males during the first month of life.

CONTEXT: Newborns with ambiguous genitalia or males with nonpalpable gonads usually require an early assessment of the presence and functional state of testicular tissue. OBJECTIVE: Our objective was to characterize the precise ontogeny of the serum patterns of gonadotropins, testosterone, anti-Müllerian hormone (AMH), and inhibins in normal newborn boys. DESIGN: We conducted a cross-sectional and longitudinal study. SUBJECTS: Serum samples were obtained in 57 boys and 13 girls on d 2 of life. A second sample was obtained on d 7, 10, 15, 20, and 30 (boys) and on d 30 (girls). MAIN OUTCOME MEASURES: Serum levels of gonadotropins, testosterone, AMH, and inhibins were measured. RESULTS: In males, LH and FSH were undetectable or very low on d 2. By d 7, LH increased to 3.94 +/- 3.19 IU/liter (mean +/- sd) and FSH to 2.04 +/- 1.67 IU/liter. LH/FSH ratios were 0.40 +/- 0.11 (d 2) and 2.02 +/- 0.20 (d 30). AMH rose from 371 +/- 168 pmol/liter (d 2) to 699 +/- 245 pmol/liter (d 30), and inhibin B rose from 214 +/- 86 ng/liter (d 2) to 361 +/- 93 ng/liter (d 30). The inhibin alpha-subunit precursor (pro-alphaC) remained stable during the first month of life. Testosterone levels were 66 +/- 42 ng/dl (d 2), 82 +/- 24 ng/dl (d 20), and 210 +/- 130 ng/dl (d 30). A sexual dimorphism was observed in AMH and inhibin B (lower in girls on d 2 and 30), in LH/FSH ratio (lower in girls on d 30) and in testosterone (lower in girls on d 30). CONCLUSIONS: Sertoli cell markers AMH and inhibin B are the earliest useful markers indicating the existence of normal testicular tissue.

Controversial role of inhibin alpha-subunit gene in the aetiology of premature ovarian failure.

BACKGROUND: Premature ovarian failure (POF) is characterized by hypergonadotropic amenorrhoea before the age of 40. Inhibin alpha-subunit (INHalpha) gene is proposed as a candidate gene due to its role in negative feedback control of FSH. METHODS: Polymorphism -16C>T of INHalpha gene was studied in 61 POF patients and 82 controls above 40 years old (C > 40). Substitution 769G>A was studied in 59 POF patients, 76 C > 40 and 73 controls below 40 years old (C < 40). RESULTS: No significant difference in risk of POF development for -16T allele was found when comparing idiopathic POF (I-POF) with C > 40 (Odds ratio = 1.46; 95% confidence interval = 0.63-3.19). Implication of -16C>T polymorphism in serum inhibin levels was analysed in 46 controls, and no significant differences (P > 0.05) were found between CC and CT + TT genotype groups when comparing either mid-follicular phase Pro-alphaC and inhibin B values or mid-luteal phase Pro-alphaC and inhibin A values. Heterozygosity for substitution 769G>A was found in 1 of 59 POF woman, 2 of 76 C > 40 and 6 of 73 C < 40. Presence of this substitution in a relevant number of control subjects is herein described for the first time. CONCLUSION: Our results indicate that -16C>T and 769G>A variants in INHalpha gene may not be associated to POF disease.

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.