Elevated expression of inhibin α gene in sterile allotriploid crucian carp.

Inhibin and Activin, belong to the transforming growth factor ß superfamily (TGF-ß), which associate with the regulation of the reproductive process by the modulation of the hypothalamic-pituitary-gonad (HPG) axis. In this study, we reported the molecular cloning and tissue expression of inhibin α in allotriploid crucian carp and its parent- diploid red crucian carp. The full-length cDNA of inhibin α were respectively 1632 bp and 1642 bp in allotriploids and diploids, which both consisted of a 1044 bp open reading frame (ORF) encoding 347 amino acids. Real-time quantitative PCR (RT-qPCR) showed that allotriploids and diploids had significant expression of inhibin α in testis and ovary, and the expression of inhibin α in the gonads of allotriploids was higher than that of diploids. The immunohistochemistry indicated that the ovarian development of allotriploids was abnormal, and the expression of Inhibin α in the ovary of allotriploids was higher than that of diploids. Results of co-immunoprecitation (co-IP) demonstrated that the Inhibin α and Activin ßA, Inhibin α and Activin ßB can form dimers. These findings suggested that the elevated expression of inhibin α and the competitive binding of Inhibin α subunit with Activin ß subunits in allotriploids may be releted to the sterility of allotriploids. Furthermore, these results will facilitate the investigation of reproduction characteristics in allotriploids and provide theoretical basis for the study of polyploid breeding in the future.

Targeting NAD-dependent dehydrogenases in drug discovery against infectious diseases and cancer.

Dehydrogenases are oxidoreductase enzymes that play a variety of fundamental functions in the living organisms and have primary roles in pathogen survival and infection processes as well as in cancer development. We review here a sub-set of NAD-dependent dehydrogenases involved in human diseases and the recent advancements in drug development targeting pathogen-associated NAD-dependent dehydrogenases. We focus also on the molecular aspects of the inhibition process listing the structures of the most relevant molecules targeting this enzyme family. Our aim is to review the most impacting findings regarding the discovery of novel inhibitory compounds targeting the selected NAD-dependent dehydrogenases involved in cancer and infectious diseases.

Polymorphisms of four candidate genes and their correlations with growth traits in blue fox (Alopex lagopus).

To improve the accuracy and genetic progress of blue fox breeding, the relationships between genetic polymorphisms and growth and reproductive traits of the blue fox were investigated. MC4R, MC3R, INHA and INHBA were selected as candidate genes for molecular evolution and statistical analyses. Single-factor variance analyses showed that the MC4R (g.267C > T, g.423C > T, and g.731C > A) and MC3R (g.677C > T) genotypes had significant impacts on body weight, chest circumference, abdominal perimeter and body mass index (BMI) (P < 0.05) in blue fox. The MC4R and MC3R combined genotypes had significant effects on the body weight and abdominal circumference. The different genotypes of INHA g.75G > A had significant effects on female fecundity, whereas the different genotypes of INHBA g.404G > T and g.467G > T and the INHA and INHBA combined genotypes had significant effects on male fecundity. The proteins encoded by the open reading frames (ORFs) of different polymorphic loci were predicted and analysed. The aims of this study were to identify genetic markers related to growth and reproduction in the blue fox and to provide an efficient, economical and accurate theoretical approach for auxiliary fox breeding.

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.

Activins in reproductive biology and beyond.

BACKGROUND: Activins are members of the pleiotrophic family of the transforming growth factor-beta (TGF-ß) superfamily of cytokines, initially isolated for their capacity to induce the release of FSH from pituitary extracts. Subsequent research has demonstrated that activins are involved in multiple biological functions including the control of inflammation, fibrosis, developmental biology and tumourigenesis. This review summarizes the current knowledge on the roles of activin in reproductive and developmental biology. It also discusses interesting advances in the field of modulating the bioactivity of activins as a therapeutic target, which would undoubtedly be beneficial for patients with reproductive pathology. METHODS: A comprehensive literature search was carried out using PUBMED and Google Scholar databases to identify studies in the English language which have contributed to the advancement of the field of activin biology, since its initial isolation in 1987 until July 2015. 'Activin', 'testis', 'ovary', 'embryonic development' and 'therapeutic targets' were used as the keywords in combination with other search phrases relevant to the topic of activin biology. RESULTS: Activins, which are dimers of inhibin ß subunits, act via a classical TGF-ß signalling pathway. The bioactivity of activin is regulated by two endogenous inhibitors, inhibin and follistatin. Activin is a major regulator of testicular and ovarian development. In the ovary, activin A promotes oocyte maturation and regulates granulosa cell steroidogenesis. It is also essential in endometrial repair following menstruation, decidualization and maintaining pregnancy. Dysregulation of the activin-follistatin-inhibin system leads to disorders of female reproduction and pregnancy, including polycystic ovary syndrome, ectopic pregnancy, miscarriage, fetal growth restriction, gestational diabetes, pre-eclampsia and pre-term birth. Moreover, a rise in serum activin A, accompanied by elevated FSH, is characteristic of female reproductive aging. In the male, activin A is an autocrine and paracrine modulator of germ cell development and Sertoli cell proliferation. Disruption of normal activin signalling is characteristic of many tumours affecting reproductive organs, including endometrial carcinoma, cervical cancer, testicular and ovarian cancer as well as prostate cancer. While activin A and B aid the progression of many tumours of the reproductive organs, activin C acts as a tumour suppressor. Activins are important in embryonic induction, morphogenesis of branched glandular organs, development of limbs and nervous system, craniofacial and dental development and morphogenesis of the Wolffian duct. CONCLUSIONS: The field of activin biology has advanced considerably since its initial discovery as an FSH stimulating agent. Now, activin is well known as a growth factor and cytokine that regulates many aspects of reproductive biology, developmental biology and also inflammation and immunological mechanisms. Current research provides evidence for novel roles of activins in maintaining the structure and function of reproductive and other organ systems. The fact that activin A is elevated both locally as well as systemically in major disorders of the reproductive system makes it an important biomarker. Given the established role of activin A as a pro-inflammatory and pro-fibrotic agent, studies of its involvement in disorders of reproduction resulting from these processes should be examined. Follistatin, as a key regulator of the biological actions of activin, should be evaluated as a therapeutic agent in conditions where activin A overexpression is established as a contributing factor.

Computational designing of a poly-epitope fecundity vaccine for multiple species of livestock.

Inhibin and follistatin are known to reduce fecundity by inhibiting the actions of activin and FSH. Thus, the immunoneutralization of these hormones is a rational proposal for augmenting reproductive performance. The present study describes a comprehensive computational methodology comprising of a consensus approach of several B- and Th-cell epitope prediction tools for the identification of epitopic regions within the structure of these hormones that can be incorporated into a poly-epitope fecundity vaccine. The proposed peptide (RGD-WSPAALRLLQRPPEEPA-KK-YSFPISSILE) should be effective in multiple animal species, generating good immunological memory.

Anti-Müllerian hormone (AMH), inhibin-α, growth differentiation factor 9 (GDF9), and bone morphogenic protein-15 (BMP15) mRNA and protein are influenced by photoperiod-induced ovarian regression and recrudescence in Siberian hamster ovaries.

Exposure of Siberian hamsters to short photoperiod (SD) inhibits ovarian function, including folliculogenesis, whereas function is restored with their transfer to long photoperiods (LD). To investigate the mechanism of photo-stimulated recrudescence, we assessed key folliculogenic factors-anti-Müllerian hormone (AMH), inhibin-α, growth differentiation factor-9 (GDF9), and bone morphogenic protein-15 (BMP15)-across the estrus cycle and in photo-regressed and recrudescing ovaries. Adult hamsters were exposed to either LD or SD for 14 weeks, which respectively represent functional and regressed ovaries. Select regressed hamsters were transferred back to LD for 2 (post-transfer week 2; PTw2) or 8 weeks (PTw8). Ovaries were collected and fixed in formalin for immunohistochemistry or frozen in liquid nitrogen for real-time PCR. AMH, inhibin-α, GDF9, and BMP15 mRNA and protein were detected in all stages of the estrus cycle. Fourteen weeks of SD exposure increased (P < 0.05) ovarian AMH, GDF9, and BMP15, but not inhibin-α mRNA levels as compared to LD. Transfer of regressed hamsters to stimulatory long photoperiod for 8 weeks returned AMH and GDF9 mRNA levels to LD-treated levels, and further increased mRNA levels for inhibin-α and BMP15. Immunostaining for AMH, inhibin-α, GDF9, and BMP15 proteins was most intense in preantral/antral follicles and oocytes. The overall immunostaining extent for AMH and inhibin-α generally mirrored the mRNA data, though no changes were observed for GDF9 or BMP15 immunostaining. Shifts in mRNA and protein levels across photoperiod conditions suggest possible syncretic roles for these folliculogenic factors in photo-stimulated recrudescence via potential regulation of follicle recruitment, preservation, and development.

Effect of a DNA vaccine harboring two copies of inhibin α (1-32) fragments on immune response, hormone concentrations and reproductive performance in rats.

The objective was to investigate the effects of a novel DNA vaccine (pcISI) harboring two copies of inhibin α (1-32) fragments on immune response, hormone concentrations and reproductive performance in rats. Female Wistar rats (n=18 per group) were immunized (twice, 4 wk apart) with 10, 50, or 100 µg (T1, T2 and T3, respectively), of the pcISI plasmid. At 4 wk after the second immunization, plasma antibody titers were higher (P<0.05) in T3 than in either T1 or T2 (0.341±0.123, 0.236±0.068, and 0.251±0.077, respectively, mean±SD). Concurrrently, plasma concentrations of FSH and estradiol were highest (P<0.05) in T3, and were higher (P<0.05) in T1 and T2 than in control groups. For antibody-positive rats, there was a correlation (P<0.01) between antibody titer and FSH concentrations after two pcISI immunizations. The number of mature follicles in the T3 group (46.00±4.65) was higher (P<0.05) than in two control groups (29.25±3.72 and 27.92±3.48), and also higher (P<0.05) than in T1 and T2 (37.17±4.99 and 38.75±7.09). Antibody-positive rats had more mature ovarian follicles than negative rats (46.75±4.23 vs. 35.60±3.38, P<0.05). Moreover, litter size and number of placentas were increased (P<0.05) in the pcISI immunization groups, except for the T1 group, compared to the control groups. In conclusion, the pcISI DNA vaccine successfully induced a humoral immune response, improved reproductive hormone concentrations, stimulated follicular development, and increased number of placentas and litter size. Furthermore, 100 µg yielded the best immune response.

Bisphenol AF may cause testosterone reduction by directly affecting testis function in adult male rats.

Although in vitro studies have indicated that Bisphenol AF (BPAF) might be a more dangerous endocrine disruptor than Bisphenol A (BPA), no information on reproductive toxicity in animals is available. In this study, the effects of BPAF exposure on the testis and the related mechanisms of toxicity were investigated. Sprague-Dawley (SD) male rats were exposed to BPAF (0, 2, 10, 50 and 200 mg/kg/d) for 14 days. Total cholesterol levels in serum were decreased in rats given a dose of 50 and 200 mg/kg/d. BPAF concentration in the testes increased with increasing doses of BPAF. Reduced serum testosterone and increased luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels were observed in rats in the higher dose groups. Furthermore, BPAF exposure resulted in a dramatic decline in genes and protein involved in cholesterol biosynthesis, transport and steroid biosynthesis. Similarly, the testicular mRNA levels of inhibin B, estrogen receptor (ERα) and luteinizing hormone receptor (LHR) also decreased in rats given a dosage of 200 mg/kg/d BPAF. Together, these data demonstrate that BPAF-induced inhibition of testosterone production primarily resulted from the alteration of genes and proteins in the testosterone biosynthesis pathway.

New targets for old hormones: inhibins clinical role revisited.

Inhibins are gonadal peptide hormones belonging to the transforming growth factor-ß (TGF-ß) superfamily that regulate the pituitary follicle stimulating hormone (FSH) secretion by negative feedback mechanisms. It is evident that the understanding of inhibins function in the hypothalamic-pituitary-gonadal axis will provide insights into physiology and pathology of the gonadal function. In recent years, a great deal of attention has been focussed on clinical relevance of measuring circulating inhibins in normal and disease state. The past few years also have witnessed the emergence and discovery of extra pituitary action of inhibins that might provide further insights into the underlying diseases like cancer especially in the reproductive axis and various other new endocrine target organs. In this review after systematic analysis of literature, we discuss briefly the known and recent advances in function of these hormones highlighting also its structure, production and mechanisms of signal transduction. Also this review discusses about the physiological relevance of inhibin association in the normal function to the development of reproductive cancers. Finally, we describe evidence from various emerging studies that inhibins make an important contribution to other physiological functions apart from reproduction which reveals new endocrine target organs of inhibins. The emerging view is inhibin participates in multiple ways to regulate the function in different cell types and still complete repertoire of its actions is under investigation.

Peripheral concentrations of inhibin A, ovarian steroids, and gonadotropins associated with follicular development throughout the estrous cycle of the sow.

We investigated changes in peripheral concentrations of inhibin A, total inhibin, steroids, and gonadotropins throughout the intact estrous cycle of the sow in relation to ovarian changes determined by daily transrectal ultrasonography. All visible follicles of 3 mm or more in diameter were classified as small (> or =3 and <6 mm) or large (> or =6 mm). Follicular recruitment was identified in two periods of the cycle: one from the late luteal to the follicular phase, characterized by an increase in the number of small follicles followed by the appearance of large follicles; and another during the early luteal phase, consisting only of increased numbers of small follicles. Plasma concentrations of inhibin A increased (P<0.05), coinciding with the two periods of follicle emergence. Estradiol (E(2)) levels increased (P<0.05) during the follicular phase, but not during the early luteal phase. An inverse relationship (P<0.01) between the patterns of inhibin and FSH concentrations was noted around the two periods of follicle emergence, but there was no relationship (P> or =0.1) between the patterns of plasma E(2) and FSH during the early luteal phase. In conclusion, measurement of plasma inhibin A levels combined with ultrasonographic examination of the ovaries revealed two periods of synchronous follicular growth during the sow's estrous cycle. The results strongly suggest that inhibin A functions as a negative feedback regulator of FSH secretion throughout the estrous cycle, whereas E(2) appears to influence FSH secretion only during the follicular phase.

Characterization of cAMP/PKA/CREB signaling cascade in the bonnet monkey corpus luteum: expressions of inhibin-alpha and StAR during different functional status.

Luteinizing hormone mediates its nuclear action primarily by activating cAMP/Protein kinase A (PKA) pathway leading to phosphorylation of cAMP response element binding (CREB) family of transcription factors. Earlier studies have documented altered cAMP responsiveness of luteal cells during maturation, and in the rhesus monkey, extinction of CREB expression following luteinization and ovulation. In the course of studies aimed at characterizing LH-cAMP signaling pathway, we serendipitously discovered that CREB is after all present in the monkey corpus luteum (CL). The present experiments were carried out to examine the PKA activity, CREB expression and RT-PCR expression of inhibin-alpha (Inh-alpha) subunit and steroidogenic acute regulatory protein (StAR) in CL obtained from a variety of model systems. PKA activity in the CL was maintained throughout the luteal phase. Messenger RNA expression by RT-PCR and Northern analyses and protein levels employing antibodies specific to total- and phospho-forms demonstrated presence of CREB in the CL. Additionally, immuno-histo/cytochemical analyses, Electrophoretic mobility shift assays and chromatin immunoprecipitation assays for Inh-alpha and StAR genes further confirmed the presence of CREB in the CL. The present study, contrary to an earlier report, demonstrates the presence of CREB (both transcript and protein) in the monkey CL. Also, analysis of expression of Inh-alpha and StAR genes (considered to be cAMP responsive), during different functional status of CL suggests that LH regulates their expression perhaps by cAMP/PKA/CREB pathway.

N-linked oligosaccharides direct the differential assembly and secretion of inhibin alpha- and betaA-subunit dimers.

The biosynthetic pathway governing inhibin heterodimer (alpha/beta) and activin homodimer (beta/beta) assembly and secretion from ovarian granulosa cells is not fully understood. Here, we examined the role of inhibin subunit glycosylation in the assembly and secretion of mature inhibin A and activin A. Inhibition of subunit glycosylation by tunicamycin treatment of alpha- and beta(A)-expressing CHO cell lines reduced inhibin but not activin secretion. Dimeric inhibin A is preferentially secreted from parental isogenic wild-type (wt) cell lines (alpha(wt)beta(wt)). Mutation of a single glycosylation site at asparagine 268 (alpha(Delta268)beta(wt)) reduces inhibin secretion by 78% and permits beta/beta assembly and secretion. Conversely, gain of a glycosylation (GOG) site in the analogous region of the beta(A)-subunit (alpha(wt)beta(GOG327)) enhances inhibin A secretion. The present study demonstrates that N-linked glycan sites direct heterodimer vs. homodimer assembly, and prevention of glycosylation abrogates inhibin secretion. These data support a definitive role for site-specific N-glycosylation in governing inhibin/activin dimer assembly and secretion.

Benchmarking sets for molecular docking.

Ligand enrichment among top-ranking hits is a key metric of molecular docking. To avoid bias, decoys should resemble ligands physically, so that enrichment is not simply a separation of gross features, yet be chemically distinct from them, so that they are unlikely to be binders. We have assembled a directory of useful decoys (DUD), with 2950 ligands for 40 different targets. Every ligand has 36 decoy molecules that are physically similar but topologically distinct, leading to a database of 98,266 compounds. For most targets, enrichment was at least half a log better with uncorrected databases such as the MDDR than with DUD, evidence of bias in the former. These calculations also allowed 40x40 cross-docking, where the enrichments of each ligand set could be compared for all 40 targets, enabling a specificity metric for the docking screens. DUD is freely available online as a benchmarking set for docking at http://blaster.docking.org/dud/.

Analysis of the function of activin betaC subunit using recombinant protein.

Activins, TGF-beta superfamily members, have multiple functions in a variety of cells and tissues. Additional activin beta subunit genes, betaC and betaE, have been identified in humans and rodents. To explore the role of activin betaC subunit, we generated recombinant human activin C using Chinese hamster ovary cells. Recombinant activin C from the conditioned medium was purified by consecutive hydrophobic, size-exclusion, and high performance liquid chromatography. SDS-PAGE and Western blot analysis of the purified protein revealed that activin C formed disulfide bridges. However, activin C had no effect on the proliferation of cultured liver cells. Furthermore, there were no significant differences in erythroid differentiation and follicle stimulating hormone secretion in vitro. It was also shown that immunoreactive bands indicated the hetrodimer of activin betaC, and inhibin alpha subunits were detected in the conditioned medium from the activin C-producing cells, which were stably transfected with inhibin alpha subunit cDNA. This suggests that activin betaC subunit may have been present and that it may exert its effect as inhibin C.

Structural basis for a functional antagonist in the transforming growth factor beta superfamily.

Within the transforming growth factor beta superfamily, the agonist-antagonist relationship between activin and inhibin is unique and critical to integrated reproductive function. Activin acts in the pituitary to stimulate follicle-stimulating hormone, and is antagonized by endocrine acting, gonadally derived inhibin. We have undertaken a mutational analysis of the activin betaA subunit to determine the precise structural aspects that contribute to inhibin antagonism of activin. By substituting specific amino acid residues in the activin betaA subunit with similarly aligned amino acids from the alpha subunit, we have pinpointed the residues required for activin receptor binding and activity, as well as for inhibin antagonism of activin through its receptors. Additionally, we have identified an activin mutant with a higher affinity for the activin type I receptor that provides structural evidence for the evolution of ligand-receptor interactions within the transforming growth factor beta superfamily.

Significance of inhibin in reproductive pathophysiology and current clinical applications.

The human reproductive process is regulated by complex mechanisms that involve many organs, including the brain, gonads and endocrine system. It has been more than 70 years since the name 'inhibin' was used to describe a substance produced in the gonads that negatively regulates pituitary secretion. Inhibin B controls FSH secretion via a negative feedback mechanism. It is a glycoprotein hormone secreted by the Sertoli cells of the testis and granulosa and theca cells of the ovary. Serum inhibin B concentrations are positively related to testicular volume and sperm counts. Current understanding of inhibin physiology and pathology in the human suggests that inhibin B may be of importance as a marker of Sertoli cell function in men with infertility and as a prognostic indicator in women undergoing ovulation induction therapy. Inhibin concentrations are elevated in patients with granulosa cell tumours and in post-menopausal women with mucinous ovarian cancers. Immunoreactivity against the inhibin alpha-subunit was identified in all cases of adrenal cortical adenoma and carcinoma, and levels are suppressed in the malignant prostate disease. This article discusses the structure, regulation and clinical use of inhibin and other related substances.

Inhibin B as a potential biomarker of testicular toxicity.

Inhibin B is a glycoprotein produced predominantly by Sertoli cells which regulates pituitary FSH release by a negative feedback loop. The regulation of inhibin B is complex with changes in the pattern of secretion occurring during development, and many factors such as FSH, testosterone, Sertoli cell proliferation and germ cell complement likely to contribute to overall production. Systemic inhibin B concentrations seem to reflect the extreme ends of spermatogenic status with high levels of inhibin B observed in normal, fertile individuals and lower levels of inhibin B in individuals with severe damage to the testis as a result of germ cell depletion. Inhibin B has proved valuable in epidemiological studies exploring male infertility with data showing that inhibin B combined with FSH measurements has a higher positive predictive value for detecting male infertility than either alone. Inhibin B is proposed as a potential biomarker of testicular toxicity in rodent toxicity studies to compliment traditional endpoints. In pharmaceutical development, inhibin B might allow better linkage between animal study results and subsequent monitoring of testicular function in clinical trials. An international, intercompany project has been initiated to evaluate the overall suitability and limitations of inhibin B as a biomarker of testicular toxicity.