Inhibin Secretion Research Articles

High and low dose of luzindole or 4-phenyl-2-propionamidotetralin (4-P-PDOT) reverse bovine granulosa cell response to melatonin.

Communication between oocytes and granulosa cells ultimately dictate follicle development or atresia. Melatonin is also involved in follicle development. This study aimed to investigate the effects of melatonin and its receptor antagonists on hormone secretion, as well as gene expression related to hormone synthesis, TGF-β superfamily, and follicle development in bovine granulosa cells, and assess the effects of melatonin in the presence of 4-P-PDOT and luzindole. Bovine ovaries were collected from a local abattoir and follicular fluid (follicle diameter 5-8 mm) was collected for granulosa cell isolation and culture. Granulosa cells and culture medium were collected 48 h after treatment with melatonin at high dose concentrations (10-5 M) and low dose concentrations (10-9 M) in the absence/presence of 4-P-PDOT and luzindole (10-5 M or 10-9 M). Furthermore, the expression level of genes related to hormonal synthesis (CYP11A1, CYP19A1, StAR, and RUNX2), TGF-β superfamily (BMP6, INHA, INHBA, INHBB, and TGFBR3), and development (EGFR, DNMT1A, and FSHR) were detected in each experimental group by real-time quantitative PCR. In addition, the level of hormones in culture medium were detected using ELISA. Both 10-5 M and 10-9 M melatonin doses promoted the secretion of inhibin A and progesterone without affecting the production of inhibin B and estradiol. In addition, both promoted the gene expression of INHA, StAR, RUNX2, TGFBR3, EGFR, and DNMT1A, and inhibited the expression of BMP6, INHBB, CYP11A1, CYP19A1, and FSHR. When combined with different doses of 4-P-PDOT and luzindole, they exhibited different effects on the secretion of inhibin B, estradiol, inhibin A, and progesterone, and the expression of CYP19A1, RUNX2, BMP6, INHBB, EGFR, and DNMT1A induced by melatonin. High and low dose melatonin receptor antagonists exhibited different effects in regulating hormone secretion and the expression of various genes in response to melatonin. Therefore, concentration effects must be considered when using luzindole or 4-P-PDOT.

Identification of a window of androgen sensitivity for somatic cell function in human fetal testis cultured ex vivo

BackgroundReduced androgen action during early fetal development has been suggested as the origin of reproductive disorders comprised within the testicular dysgenesis syndrome (TDS). This hypothesis has been supported by studies in rats demonstrating that normal male development and adult reproductive function depend on sufficient androgen exposure during a sensitive fetal period, called the masculinization programming window (MPW). The main aim of this study was therefore to examine the effects of manipulating androgen production during different timepoints during early human fetal testis development to identify the existence and timing of a possible window of androgen sensitivity resembling the MPW in rats.MethodsThe effects of experimentally reduced androgen exposure during different periods of human fetal testis development and function were examined using an established and validated human ex vivo tissue culture model. The androgen production was reduced by treatment with ketoconazole and validated by treatment with flutamide which blocks the androgen receptor. Testicular hormone production ex vivo was measured by liquid chromatography-tandem mass spectrometry or ELISA assays, and selected protein markers were assessed by immunohistochemistry.ResultsKetoconazole reduced androgen production in testes from gestational weeks (GW) 7–21, which were subsequently divided into four age groups: GW 7–10, 10–12, 12–16 and 16–21. Additionally, reduced secretion of testicular hormones INSL3, AMH and Inhibin B was observed, but only in the age groups GW 7–10 and 10–12, while a decrease in the total density of germ cells and OCT4+ gonocytes was found in the GW 7–10 age group. Flutamide treatment in specimens aged GW 7–12 did not alter androgen production, but the secretion of INSL3, AMH and Inhibin B was reduced, and a reduced number of pre-spermatogonia was observed.ConclusionsThis study showed that reduced androgen action during early development affects the function and density of several cell types in the human fetal testis, with similar effects observed after ketoconazole and flutamide treatment. The effects were only observed within the GW 7–14 period—thereby indicating the presence of a window of androgen sensitivity in the human fetal testis.

In vitro production of mouse morphological sperm in artificial testis bioengineered by 3D printing of extracellular matrix

Since autologous stem cell transplantation is prone to cancer recurrence, in vitro sperm production is regarded a safer approach to fertility preservation. In this study, the spermatogenesis process on testicular tissue extracellular matrix (T-ECM)-derived printing structure was evaluated. Ram testicular tissue was decellularized using a hypertonic solution containing triton and the extracted ECM was used as a bio-ink to print an artificial testis. Following cell adhesion and viability examination, pre-meiotic and post-meiotic cells in the study groups (as testicular suspension and co-culture with Sertoli cells) were confirmed by real-time PCR, flow-cytometry and immunocytochemistry methods. Morphology of differentiated cells was evaluated using transmission electron microscopy (TEM), toluidine blue, Giemsa, and hematoxylin and eosin (H&E) staining. The functionality of Leydig and Sertoli cells was determined by their ability for hormone secretion. The decellularization of testicular tissue fragments was successful and had efficiently removed the cellular debris and preserved the ECM compounds. High cell viability, colonization, and increased expression of pre-meiotic markers in cultured testicular cells on T-ECM-enriched scaffolds confirmed their proliferation. Furthermore, the inoculation of neonatal mouse testicular cells onto T-ECM-enriched scaffolds resulted in the generation of sperm. Morphology evaluation showed that the structure of these cells was quite similar to mature sperm with a specialized tail structure. The hormonal analysis also confirmed production and secretion of testosterone and inhibin B by Leydig and Sertoli cells. T-ECM printed artificial testis is a future milestone that promises for enhancing germ cell maintenance and differentiation, toxicology studies, and fertility restoration to pave the way for new human infertility treatments in the future.

Efficacy of Deep Breathing Exercise and Aerobic Exercise on Perimenopausal Women

Introduction: Perimenopause means “around the menopause”. The hormonal changes that occur during the perimenopausal transition are the gametogenic failure which is characterized by the reduced inhibin secretion which occurs in the early follicular phase, increased serum FSH and decreased fertility. The aim of this study is to deterine the effectiveness of deep breathing exercises and aerobic exercises on perimenopausal Women Methods: A total of 50participants will take part in the study. The participants will be chosen based on Inclusion criteria by a convenient sampling method. The participants will be divided into two groups: Group A: Control group (n=25) and Group B: Experimental group(n=25). The level of perimenopausal symptoms will assessed using menopause rating scale before the commencement of the intervention in both the groups. The Control group will receive Jacobson’s relaxation technique and the experimental group will receive deep breathing exercise and aerobic exercise. The treatment will be demonstrated to the participants through video demonstration. After the completion of the treatment protocol the perimenopausal symptoms will be evaluated again using the menopause rating scale in both the groups to assess the reduction of perimenopausal symptoms in both the groups and to find the effectiveness of deep breathing exercise and aerobic exercise in reduction perimenopausal symptoms. Results: The values were statistically analyzed using the Wilcoxon signed-rank test. There was a statistically significant improvement in the experimental group. The result was significant at p<.05. Conclusion: This study, therefore, displays that deep breathing exercise and aerobic exercise are effective in reducing the somatic, psychological, and urogenital symptoms in perimenopausal women. KEYWORDS: Deep breathing exercise, Aerobic exercise, and perimenopausal symptoms.

The effect of exosomes derived from human ovarian epithelial cancer cells on the secretion of AMH and Inhibin in granulosa cells

The effect of exosomes derived from human ovarian epithelial cancer cells on the secretion of AMH and Inhibin in granulosa cells

White Matter Stroke Induces a Unique Oligo-Astrocyte Niche That Inhibits Recovery.

Subcortical white matter stroke is a common stroke subtype. White matter stroke stimulates adjacent oligodendrocyte progenitor cells (OPCs) to divide and migrate to the lesion, but stroke OPCs have only a limited differentiation into mature oligodendrocytes. To understand the molecular systems that are active in OPC responses in white matter stroke, OPCs were virally labeled and laser-captured in the region of partial damage adjacent to the infarct in male mice. RNAseq indicates two distinct OPC transcriptomes associated with the proliferative and limited-regeneration phases of OPCs after stroke. Molecular pathways related to nuclear receptor activation, ECM turnover, and lipid biosynthesis are activated during proliferative OPC phases after stroke; inflammatory and growth factor signaling is activated in the later stage of limited OPC differentiation. Within ECM proteins, Matrilin-2 is induced early after stroke and then rapidly downregulated. Prediction of upstream regulators of the OPC stroke transcriptome identifies several candidate molecules, including Inhibin A-a negative regulator of Matrilin-2. Inhibin A is induced in reactive astrocytes after stroke, including in humans. In functional assays, Matrilin-2 induces OPC differentiation, and Inhibin A inhibits OPC Matrilin-2 expression and inhibits OPC differentiation. In vivo, Matrilin-2 promotes motor recovery after white matter stroke, and promotes OPC differentiation and ultrastructural evidence of remyelination. These studies show that white matter stroke induces an initial proliferative and reparative response in OPCs, but this is blocked by a local cellular niche where reactive astrocytes secrete Inhibin A, downregulating Matrilin-2 and blocking myelin repair and recovery.SIGNIFICANCE STATEMENT Stroke in the cerebral white matter of the brain is common. The biology of damage and recovery in this stroke subtype are not well defined. These studies use cell-specific RNA sequencing and gain-of-function studies to show that white matter stroke induces a glial signaling niche, present in both humans and mice, between reactive astrocytes and oligodendrocyte progenitor cells. Astrocyte secretion of Inhibin A and downregulation of oligodendrocyte precursor production of Matrilin-2 limit OPC differentiation, tissue repair, and recovery in this disease.

Effects of Insulin on Porcine Neonatal Sertoli Cell Responsiveness to FSH In Vitro.

There is ongoing debate as to whether the decline of sperm production in recent times may be related to a parallel increase in the rate of obesity and diabetes. Lower anti-Müllerian hormone (AMH) and inhibin B secretion have been observed in young hyperinsulinemic patients compared to healthy controls, suggesting a Sertoli cell (SC) dysfunction. The pathophysiological mechanisms underlying SC dysfunction in these patients are poorly understood. To the best of our knowledge, no evidence is available on the effects of insulin on SC function. Therefore, this study was undertaken to assess the effects of insulin on basal and follicle-stimulating hormone (FSH)-stimulated SC function in vitro. To accomplish this, we evaluated the expression of AMH, inhibin B and FSHR genes, the secretion of AMH and inhibin B and the phosphorylation of AKT473 and SC proliferation on neonatal porcine SC after incubation with FSH and/or insulin. We found that similar to FSH, the expression and secretion of AMH is suppressed by insulin. Co-incubation with FSH and insulin decreased AMH secretion significantly more than with FSH alone. Insulin had no effect on the expression and secretion of the inhibin B gene, but co-incubation with FSH and insulin had a lower effect on inhibin B secretion than that found with FSH alone. FSH and/or insulin increased AKT473 phosphorylation and SC proliferation. In conclusion, the results of this study showed that insulin modulates SC function. We hypothesize that hyperinsulinemia may therefore influence testicular function even before puberty begins. Therefore, particular care should be taken to avoid the onset of hyperinsulinemia in children to prevent a future deleterious effect on fertility.

Control of ovarian follicular development to the gonadotrophin-dependent phase: a 2006 perspective

In sheep, as in other mammals, ovarian follicular growth is regulated mainly by intraovarian growth factors during early development with pituitary hormones increasingly important during the final phases to ovulation. Most follicles are present as primordial structures and these express many hundreds of genes that fulfil an array of housekeeping and signalling functions. Once growth has been initiated, at least two oocyte-derived growth factors, namely growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), are critical for ongoing development to ovulation, most likely by regulating the proliferative and differentiative functions of adjacent follicular cells. In sheep, the granulosa cell populations double some 12–14 times and a well-defined thecal layer differentiates before antrum formation and the time taken to complete this process varies between 50–150 days with very little follicular atresia. During preantral growth, FSH and LH receptors coupled to the cyclic AMP second messenger system develop in granulosa and thecal cells respectively. From the late preantral stage, GDF9, BMP15 and perhaps other factors are thought to regulate gene expression in cumulus cells to enhance metabolic cooperativity with the oocyte and in mural granulosa cells to regulate their responses to pituitary hormones. In sheep, artral follicular development is characterized by a much faster rate of growth, additional increases in the numbers of granulosa (4–5 more doublings) and thecal cells, an increased level of steroid and inhibin secretion in response to FSH and LH, but also by most follicles undergoing atresia. The final number of follicles that go on to ovulate is dependent upon FSH as well as the intrafollicular concentrations of GDF9 and BMP15.

Correlation of oxidation-reduction potential with hormones, semen parameters and testicular volume.

Seminal oxidative stress (OS) is a major cause of male factor infertility and can be measured as oxidation-reduction potential (ORP). Studies showed significant negative relationships of ORP with sperm count, motility or DNA integrity. Since these parameters are also positively or negatively associated with reproductive hormones follicle-stimulating hormone (FSH), luteinising hormone (LH), testosterone, testicular volume and the occurrence of varicocele, it is important to understand the mechanistic relationship between ORP and hormonal and/or testicular parameters. Therefore, we studied the relationship between ORP levels, standard hormone profiles and testicular volume in infertile men with and without varicocele. Results show a highly significant negative relationship of ORP with testicular volume and significantly positive correlations with FSH and LH. Yet, when adding varicocele as covariate, the relationship with FSH/LH became nonsignificant. Contrary, the presence of varicocele had only a contributing influence on the association of ORP with the testis volume. No association was found with estradiol. We propose that since OS causes degeneration of Sertoli cell with testicular shrinkage, such negative effect would result in a negative feedback on the hypothalamus with less inhibin secretion. This may result in increased secretion of LH and FSH. Thus, systemic and/or local OS may be responsible for smaller testis volumes.

Cumulin and FSH Cooperate to Regulate Inhibin B and Activin B Production by Human Granulosa-Lutein Cells In Vitro.

The oocyte-secreted factors bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) interact functionally, and it is hypothesized that this interaction may be mediated by formation of a GDF9:BMP15 heterodimer termed cumulin. GDF9 and BMP15 regulate folliculogenesis and ovulation rate and have been shown to regulate inhibin and activin, local regulators of folliculogenesis. The objective of this study was to determine whether cumulin regulates granulosa cell inhibin and activin production and whether this requires cooperation with FSH. Human granulosa-lutein (hGL) cells collected from patients undergoing in vitro fertilization were cultured with or without FSH with various forms of recombinant cumulin (native and cysteine mutants, with or without the prodomains), and cysteine mutant GDF9 or BMP15. Messenger RNA expression of the subunits of inhibins/activins (INHA, INHBA, INHBB) and secretion of inhibin A, inhibin B, and activin B were measured. Mature forms and proforms of cumulin stimulated comparable INHBB mRNA expression and secretion of inhibin B and activin B, whereas GDF9 or BMP15 exhibited no effect. Cumulin, but not GDF9 or BMP15, interacted synergistically with FSH to increase INHBB mRNA and inhibin B expression. FSH markedly stimulated INHA, which encodes the α subunit of inhibin A/B, and suppressed activin B. Cumulin with or without FSH did not significantly alter inhibin A. Together these data demonstrate that cumulin, but not GDF9 or BMP15, exerts paracrine control of FSH-induced regulation of inhibin B and activin B. The prodomains of cumulin may have a minimal role in its actions on granulosa cells.

Stimulation with recombinant FSH acutely and significantly increases inhibin B and estradiol in women after GnRH antagonist treatment: a model to study FSH regulation of ovarian function

Obesity is associated with decreased pituitary output of LH and FSH, inadequate folliculogenesis, and reduced ovarian steroid and peptide hormone production. It remains unclear whether the underlying pathophysiology is related to abnormal hypothalamic-pituitary dynamics, abnormal ovarian environment, or both. The persistent deficit in FSH secretion, despite the lack of restraint and normal pituitary FSH reserve, remains unexplained. FSH pulsatility is challenging to evaluate, given its long half-life and low signal-to-noise ratio, and current analytic methods fail to adequately characterize the hormone secretion dynamics of FSH and its feedback. We hypothesized that both Inhibin B and Estradiol (E2) levels will be acutely increased following exogenous, pulsatile recombinant (r) FSH administration, after a GnRH antagonist blockade. This will establish a model to study ovarian, FSH-stimulated E2 and inhibin secretion, independent of the pituitary, to determine mechanisms underlying the FSH/ovarian dysregulation in obesity. A pilot, cohort study of 9 normal-weight, cycling women at the University of Colorado School of Medicine. Participants (mean age 26.3 ± 4.9 years; BMI 22.2 ± 1.3 kg/m2) underwent frequent blood sampling (q10 min) during a 26-hour overnight study visit. Following an initial baseline10-hour collection to assess unstimulated LH and FSH secretion, Cetrotide (EMD Serono) was administered subcutaneously twice: 3 mg at hour 10 and 0.25mg at hour 16. Frequent blood sampling resumed (hours 16-26) and rFSH (Gonal-F; 30 IU) was given intravenously every hour. LH, FSH and E2 were measured by immunoassays (Centaur XP, Siemens). Functional sensitivity is 0.1 mIU/mL and the intra- and inter-assay coefficients of variation were 3.8 and 4.1%, respectively. Inhibin B was analyzed by ELISA (Beckman Coulter). Functional sensitivity is 10 pg/mL and the intra- and inter-assay coefficients of variation were 1.8 and 4.9%, respectively. Statistical significance was determined by paired t test. Normal LH & FSH pulsatility was confirmed for the 10-hour baseline frequent sampling period for all subjects. Administration of Cetrotide reliably suppressed endogenous LH and FSH secretion. Repeated boluses of rFSH mimicked endogenous FSH pulses and significantly increased FSH levels over the course of the infusion (p<0.001). Starting from 6 hours post rFSH administration (after 180 IU) we observed a significant increase in both inhibin B (60%) and E2 (80%) production (p=0.01 and p<0.01 respectively). We herein report that exogenous FSH, administered in a pulsatile fashion after GnRH blockade, induced significant, acute increases in both E2 and Inhibin B, in normal weight women. This experimental paradigm can be used to investigate FSH feedback mechanisms in obesity or other environmental insults impacting ovarian function.

Sex-related differences in the associations between maternal dioxin-like compounds and reproductive and steroid hormones in cord blood: The Hokkaido study

BackgroundPrenatal exposure to dioxin-like compounds (DLCs) irreversibly affects fetal reproductive and steroid hormone synthesis. ObjectiveThis study aimed to assess the relationships between maternal DLCs and cord blood reproductive and steroid hormones. MethodsParticipants in this study were pregnant women who enrolled in the Sapporo Cohort of the Hokkaido Study between 2002 and 2005. We quantified 29 DLCs during the 2nd and 3rd trimesters in maternal blood. Additionally, we measured the concentrations of progesterone, estradiol (E2), testosterone (T), androstenedione, dehydroepiandrosterone (DHEA), cortisol, cortisone, sex hormone-binding globulin (SHBG), luteinizing hormone (LH), follicle-stimulating hormone (FSH), prolactin, inhibin B, and insulin-like factor-3 (INSL3) in cord blood samples. ResultsData from 183 mother-child pairs were analyzed. We observed sex-dependent associations of DLCs on T/E2 ratios, DHEA, cortisol, cortisone, adrenal androgen/glucocorticoid (AA/GC: sum of DHEA and androstenedione)/(sum of cortisol and cortisone) ratios and SHBG. An increase in maternal DLCs related to decreased T/E2 ratios and SHBG and inhibin B levels, and increased AA/GC ratios and FSH and DHEA levels in male cord blood samples. However, an increase in maternal mono-ortho polychlorinated biphenyls related to increased cortisol, cortisone, and SHBG levels, and decreased DHEA levels and AA/GC ratios in female cord blood samples. ConclusionsPrenatal exposure to DLCs alters steroidogenesis and suppresses the secretion of inhibin B in male cord blood. Relationships between maternal DLCs and cord blood hormones differ between boys and girls. Further studies are required to clarify whether the effects of in utero exposure to DLCs on adrenal hormones extend into infancy and puberty.

Male Central Hypogonadism in Paediatrics - the Relevance of Follicle-stimulating Hormone and Sertoli Cell Markers.

The definition of male hypogonadism, used in adult endocrinology, is not fully applicable to paediatrics. A clear understanding of the developmental physiology of the hypothalamic-pituitary-testicular axis is essential for the comprehension of the pathogenesis of hypogonadal states in boys and for the establishment of adequate definitions and classifications in paediatric ages. This is particularly true for central hypogonadism, usually called hypogonadotropic in adults. Because childhood is a period characterised by a physiological state of low gonadotropin and testosterone production, these markers of hypogonadism, typically used in adult endocrinology, are uninformative in the child. This review is focused on the physiological importance of prepubertal Sertoli cell markers – anti-Müllerian hormone (AMH) and inhibin B – and of the intratesticular actions of follicle-stimulating hormone (FSH) and testosterone during early infancy and the first stages of pubertal development. We discuss the role of FSH in regulating the proliferation of Sertoli cells – the main determinant of prepubertal testicular volume – and the secretion of AMH and inhibin B. We also address how intratesticular testosterone concentrations have different effects on the seminiferous tubule function in early infancy and during pubertal development.

Overexpression of C/EBPβ Affects The Cell Cycle Regulators and Spermatogenesis Related Genes Expression And Function of Bovine Sertoli Cells.

CCAAT/enhancer-binding protein beta (C/EBPβ), an important transcriptional factor, plays a pivotal role in the regulation of female germ cell development. However, the role of C/EBPβ on the development of male germ cells has not been reported. In this study, we constructed the recombinant adenovirus plasmids of bovine C/EBPβ gene and harvested the subsequent adenoviruses, and then assessed the mRNA levels of spermatogenesis-related genes (real-time PCR) and secretion of inhibin B after 48h of Ad-C/EBPβ recombinant adenovirus infection in bovine sertoli cells (SCs). We found that overexpression of exogenous C/EBPβ gene upregulated the mRNA expressions of spermatogenesis-related genes, including Pdgfa, Claudin, Caspase-3, Occludin, kit1 and Cyclin E, and decreased the mRNA levels of Cyclin D1. Meanwhile, overexpression of exogenous C/EBPβ gene significantly increases the amounts of secreted inhibin B. In conclusion, the results indicate that the C/EBPβ gene plays an important regulatory role in regulation of the cell cycle regulators and spermatogenesis-related genes expression and function of bovine SCs.

Effect of kisspeptin challenge on testosterone and inhibin secretion from invitro testicular tissue of adult male rhesus monkey (Macaca mulatta).

Kisspeptin expression has been found in gonads but a direct role of kisspeptin in reproduction is not known. The objective of this study was to find a dose and time related effect of kisspeptin on testicular hormones secretion of adult male rhesus monkey (n=5). Kisspeptin (1, 10, 100, 1000pm) was incubated to a culture of testes (100mg fragments) of male rhesus monkey and medium for hormone (testosterone and inhibin) measurement was collected after 30, 60 and 120min. 10 IU hCG (180min) and 50ng FSH (60 and 120min) were incubated to the culture for checking testicular cells ability to secrete hormones invitro. Kisspeptin did not significantly (P<0.05) increase the testosterone and inhibin levels at any dose. However, one way anova at pooled doses showed an increase in testosterone levels and paired t-test at pooled doses showed inhibin decrease after 120min of incubation suggesting an independent effect of time. hCG and FSH significantly (P<0.05) increased hormone concentration compared to the basal groups. We concluded that kisspeptin has no role in testicular regulation related to testosterone and inhibin release but kisspeptin may have other roles in testicular regulation.

An Extra Arm May Impair the Open Hand.

In the paper, “Inhibin Biosynthesis and Activity Are Limited by a Prodomain-Derived Peptide,” Walton et al (1) purport that processing of inhibin-α can result in a 43-amino acid pro-α peptide from an alternate cleavage site. This propeptide is projected to have an amphipathic α-helical structure that can wrap around the open hand or butterfly-shaped mature inhibin protein, thereby hindering inhibin association with β-glycan. The function of this proinhibin α-peptide is investigated as an inhibitor of mature inhibin action. The inhibins and activins are produced as prepro-proteins in which nonactive domains that are important to protein folding and dimerization of the molecules are cleaved, releasing the final active products. The β-subunits for inhibin and activin have only one classical cleavage site, but the α-inhibin subunit has another cleavage site between the pro-α-peptide and the N-peptide (Figure 1A). Uncleaved or incompletely cleaved inhibins have been described for many years as high-molecular-weight or big inhibins, with varying demonstrations of bioactivity (2,–4). A free α-subunit in association with the C portion, without the N portion, has also been described and is produced at high levels by a variety of normal and abnormal tissues (5, 6), but this is the first evaluation of a functional small prodomain cleavage peptide for inhibin. Figure 1. A, Inhibin precursor with the cleavage sites. Note the two cleavage sites on the α-subunit and the single cleavage site on the β-subunit. B, Inhibin A/B binding to β-glycan. C, Pro-α-peptide intercalating between inhibin ... The authors also noted some time ago that three hydrophobic amino acids in the prodomain of the inhibin α-subunit were critical to proper folding and secretion of mature inhibins (7). In that work they used mutagenesis evidence to propose a model of interaction of the N-terminal prodomain with a region near the C terminus of the proprotein to hold it in a coiled conformation that would allow the covalent bonds with the similarly held β-proprotein subunits and promote further processing. In that work they also proposed that the entire cleaved prodomain could remain associated with the active inhibin in a noncovalent way, such as the fact that the latency-associated peptide remains associated with TGFβ (8). In this work they have focused on the role of the small 43-amino acid propeptide. Not only do they demonstrate the necessity of cleavage of this promolecule from the N and C regions of inhibin for production of mature peptide to occur, but they also provide strong evidence for a role of this unassuming peptide in modulating the bioactivity of the mature inhibins. Yet it does not seem to interfere with activin activity. Thus, inhibin protein may come along with its own tiny inhibitor after processing, with the little associated prodomain peptide intercalating itself between the interface with inhibin A or B and the β-glycan receptor (Figure 1, B and C). Yet a function of β-glycan is to inhibit the activation of the activin receptor signaling cascade (9, 10). So the inhibitor is packaged with its own inhibitor, like a functional mini-me, providing direct modulation of inhibin function. This has profound implications biologically if this relationship is shown to occur in vivo. Activin action is already known to be tightly regulated on many levels, both directly and indirectly. From the expression of excess inhibin α-subunits (favoring inhibin production over activin), to follistatin or α-macroglobin binding, to production of inhibitory smads that can directly modulate the activin signaling cascade; the action of activin is tightly regulated in tissue-specific ways, and its effects are profoundly concentration and tissue specific (11, 12). Entry into this already complex system regulating activin, the action of a small amphipathic molecule that could be delivered to a specific site to block inhibin and thereby increase activin action and possibly result in increased unbound β-glycan may have great usefulness clinically and as a research tool across several overlapping fields of endocrinology.

Elevated non-esterified fatty acid concentrations during in vitro murine follicle growth alter follicular physiology and reduce oocyte developmental competence

To study how long-term elevated non-esterified fatty acid (NEFA) concentrations, typical in metabolic disorders such as obesity or type 2 diabetes, affect murine follicular development, follicle quality, and subsequent oocyte developmental competence in vitro. Experimental study. In vitro culture setting. Female and male 13-day old, B6CBAF1 mice of proven fertility were sacrificed for harvesting ovaries and epididymal sperm, respectively. Early secondary murine follicles were cultured in vitro in the presence of NEFAs until the antral stage (12 days). Treatments consisted of one or a mixture of NEFAs (stearic acid [SA], palmitic acid [PA], oleic acid [OA]) in physiological (basal) or pathological (high SA, high OA, high NEFA) concentrations. Follicular development; follicle and oocyte diameters; secretion of progesterone, estradiol, and inhibin B; and luteinized granulosa cell gene expression patterns were investigated. Oocytes from NEFA-exposed follicles were fertilized in vitro, and presumptive zygotes were cultured until the blastocyst stage. Exposure to high SA reduced follicle diameters and day-12 antrum formation. Elevated NEFA concentrations changed luteinized granulosa cell messenger-ribonucleic acid abundance of genes related to energy/fatty acid/steroid metabolism, apoptosis, and oxidative stress. High NEFA and high SA treatments increased progesterone synthesis, compared with high OA follicles. Oocyte developmental competence was substantially reduced in oocytes retrieved from high OA-, high SA-, and high NEFA-exposed follicles compared with basal-treated follicles. This study showed, for the first time, that lipolysis-linked, elevated NEFA concentrations can potentially impair fertility, by altering follicular physiology and reducing oocyte developmental competence.

Melatonin regulates the development and function of bovine Sertoli cells via its receptors MT1 and MT2

Melatonin and its receptors are found in the testis of many species, where they mediate testicular functions. The present study aimed to investigate the expression of melatonin receptors (MT1 and MT2) in bovine Sertoli cells (SCs), using reverse transcription polymerase chain reaction (RT-PCR) and western blot. In addition, we assessed the mRNA levels of spermatogenesis-related genes (real-time PCR) and secretion of inhibin B after treatment with various concentrations (0, 80, 160, and 320pg/mL) of melatonin at different time points (24, 48, or 72h). We found that bovine SCs express MT1 and MT2 receptors, which were regulated by melatonin in time- and dose-dependent manners after treatment with melatonin. Exogenous melatonin up-regulated the expression of spermatogenesis-related genes, including Cyclin D1, Cyclin E, Pdgfa, Dhh, Occludin, and Claudin, and decreased the mRNA levels of P21 and Kit1 in a time or dose-dependent manner. Meanwhile, melatonin supplementation significantly affected Inhba, Inhbb and Inha mRNA expression. These findings were consistent with inhibin B levels detected in the culture medium. In conclusion, exogenous melatonin acts via its receptors and appears to play regulatory roles in the development and function of bovine SCs.

Interleukin-6 disrupts blood-testis barrier through inhibiting protein degradation or activating phosphorylated ERK in Sertoli cells

It has been recently ascribed to several inflammatory cytokines (i.e. TGF-β3, TNF-α, and IL-1) a functional role in regulating Sertoli cell blood-testis barrier (BTB) dynamics. In the testis, IL-6 inhibits meiotic DNA synthesis during the seminiferous epithelium cycle, reduces sperm motility and influences the secretion of transferrin and inhibin B by Sertoli cells. Also, it has been shown that IL-6 affects tight junction permeability in Sertoli cells, but, little is known about its role in regulating the BTB. The aim of this study was to investigate the molecular mechanisms by which IL-6 affects BTB dynamics. We show that IL-6 perturbs the integrity of the BTB, and alters the normal localization and steady-state levels of BTB integral membrane proteins. We demonstrated that IL-6 regulates the BTB by inhibiting the degradation of BTB constitutive proteins and activating ERK-MAPK pathways. Our results provide mechanistic insight into the roles of IL-6 in regulating BTB dynamics.

Comment on "complete androgen insensitivity syndrome: optimizing diagnosis and management".

We read with interest the paper of Pizzo et al. [1], confirming that adolescence is a key period for the diagnosis of 46,XY disorders of sex development (DSD) [2]. However, in our opinion, some points should be better addressed. Among these, we have the following. The authors stated that the reported girl showed “normal intellectual function,” but this information had little relevance because mental function is not impaired in females with 46,XY DSD, reaching adolescence without any clinical suspicions. Indeed, this statement might be stigmatizing for these persons. The statement that this adolescent was affected by hypergonadotropic hypogonadism (HH) is correct, according to the high LH and FSH values shown in Table 1 [1]. Very low levels of both 17β-estradiol and testosterone were also shown [1]. This endocrine pattern is not typical of late adolescent and young adult females with complete androgen insensitivity syndrome (CAIS) and intact testes. On the contrary, these persons show high/normal levels of both 17β-estradiol (for a person with a 46,XY karyotype) and testosterone [3–5]. In addition, adolescent/young adult women with CAIS did not show HH: LH is in high normal range or slightly elevated (due to the androgen resistance at central level), but FSH is in normal range (due to unaffected inhibin secretion from Sertoli cells) [3–5]. In addition, SHBG was reported within adult male range by Pizzo et al. [1], suggesting a normal sensitivity to the low levels of androgens in this girl [6]. Indeed, SHBG is within normal female range in women with CAIS, due to peripheral androgen resistance [5]. All these findings show poor agreement with the affirmed diagnosis of CAIS [1]. The clinical phenotype of the reported girl did not match with the phenotype of adolescents with CAIS; in fact, the latter shows normal breast development related to the relatively high normal estrogen levels and unopposed androgen action [3, 4, 7] and does not show hypotrophic breasts [1]. It is quite surprising that hormonal replacement therapy was started before any diagnosis was established. Histological findings do not completely agree with the diagnosis of CAIS, in particular the absence of Leydig cells, which are abundant in adolescent females with CAIS and sometimes formed aggregates up to 2 mm in diameter [8, 9]. 46,XY karyotype, female phenotype, and absence of mullerian derivatives may be present in several 46,XY DSD; they should be excluded before diagnosis of CAIS by optimal clinical, endocrine, and genetic investigations [10]. For example, the testosterone/Δ4-androstenedione ratio in the adolescent reported by Pizzo et al. [1] is very low (0.13; normal values >0.8 [11]). Thus, diagnoses of 17β-hydroxysteroid-dehydrogenase deficiency type 3 or 46,XY gonadal dysgenesis [11, 12] must be considered in the diagnostic process. Risk of gonadal cancer largely varies among 46,XY DSD. For example, in CAIS is very low at least during the first two decades of life [13–15]. Thus, delayed gonadal removal can be recommended to permit both full sexual development [15] and better bone health [16]. If diagnosis of CAIS is certain, surgery can be postponed—at least until the legal age at which the propositus can participate in decision making [1, 15, 17–19]. We are concerned and in complete disagreement with the decision to perform gonadectomy without full disclosure and assent of the adolescent [17–19]. In conclusion, some findings are poorly consistent with a diagnosis of CAIS, which should be confirmed by molecular analysis of androgen receptor gene [2, 7]. In our experience, more than 25% of the females referred to our departments with clinical/endocrine diagnosis of CAIS did not have this diagnosis confirmed by genetic analysis [20]. Clinical approach should be changed according to the new guidelines for management of persons with 46,XY DSD and directly involving the girl in the decision process [2, 18, 21]. We also stress that multidisciplinary team evaluation in tertiary centers with documented experience in this field must be guaranteed to each person with 46,XY DSD for optimal holistic management [21], especially before performing irreversible surgical procedures.