Genetic disorders and spermatogenesis.

Male infertility affects one man in twenty and a genetic basis seems likely in at least 30% of those men. Genetic regulation of fertility involves the inter-related processes of testicular development, spermatogenesis (involving germ cell mitosis, meiosis and spermatid maturation), and their endocrine and paracrine regulation. In regard to spermatogenesis, particular attention has been given to the Yq11 region, where some spermatogenesis genes ('azoospermia factors') appear to be located. Several candidate genes have been identified but have not been shown to have a defined or essential role in spermatogenesis. Microdeletions of Yq11 are found in approximately 15% of azoospermic or severely oligospermic men. The complexity of the genetic control of male fertility is demonstrated by the evidence for genes involved in spermatogenesis and sexual differentiation on the X chromosome and autosomes. Better understanding of the genetic regulation of normal spermatogenesis will provide new probes for clinical studies; however, at present the majority of spermatogenic failure remains without an identified genetic linkage. The advent of intracytoplasmic sperm injection permits fertility in many previously sterile men and presents the possibility of their transmission of infertility; appropriate counselling is required.

Localization of follistatin in the rat testis.

The cellular localization of the activin-binding protein, follistatin, in the rat testis has been a matter of some controversy with different investigators claiming that Sertoli cells, Leydig cells or germ cells are the primary cell types containing this protein. The localization of mRNA encoding follistatin was re-examined using reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization as well as the distribution of follistatin by immunohistochemistry. The results demonstrate that mRNA encoding follistatin is located in many germ cells including type B spermatogonia, primary spermatocytes with the exception of the late leptotene and early zygotene stages, and spermatids at steps 1 to 11. It is also found in Sertoli cells and endothelial cells but not in Leydig cells. Immunohistochemistry, using two different antisera to follistatin, showed that this protein was localized to spermatogonia, primary spermatocytes at all stages except the zygotene stage, spermatids at all stages and to endothelial cells and Leydig cells in the intratubular regions. The failure to detect mRNA for follistatin in Leydig cells using RT-PCR and in situ hybridization suggests that the immunohistochemical localization in these cells reflects binding of follistatin produced elsewhere. The widespread localization of follistatin, taken together with its capacity to neutralize the actions of activin, may indicate that follistatin modulates a range of testicular actions of activin, many of which remain unknown.

Metabolic profile changes in the testes of mice with streptozotocin-induced type 1 diabetes mellitus

Contrary to the traditional view, recent studies suggest that diabetes mellitus has an adverse influence on male reproductive function. Our aim was to determine the effect of diabetes on the testicular environment by identifying and then assessing perturbations in small molecule metabolites. Testes were obtained from control and streptozotocin-induced diabetic C57BL/6 mice, 2, 4 and 8 weeks post-treatment. Diabetic status was confirmed by glycated haemoglobin, non-fasting blood glucose, physiological condition and body weight. A novel extraction procedure was utilized to obtain protein free, low-molecular weight, water soluble extracts which were then assessed using H-1 nuclear magnetic resonance spectroscopy. Principal component analysis of the derived profiles was used to classify any variations, and specific metabolites were identified based on their spectral pattern. Characteristic metabolite profiles were identified for control and type 1 diabetic animals with the most distinctive being from mice with the largest physical deterioration and loss of body weight. Eight streptozotocin-treated animals did not develop diabetes and displayed profiles similar to controls. Diabetic mice had decreases in creatine, choline and carnitine and increases in lactate, alanine and myo-inositol. Betaine levels were found to be increased in the majority of diabetic mice but decreased in a few animals with severe loss of body weight and physical condition. The association between perturbations in a number of small molecule metabolites known to be influential in sperm function, with diabetic status and physiological condition, adds further impetus to the proposal that diabetes influences important spermatogenic pathways and mechanisms in a subtle and previously unrecognized manner.

Fasciola hepatica: Comparative effects of host resistance and parasite intra-specific interactions on size and reproductive histology in flukes from rats infected with isolates differing in triclabendazole sensitivity

The efficacies of putative fasciolicides and vaccines against Fasciola hepatica are frequently monitored in clinical and field trials by determination of fluke egg output in host faeces and by worm counts in the host liver at autopsy. Less often used are parameters based on fluke size and histology, yet these can provide important indications of specific effects on the development of particular germ-line or somatic tissues, especially in relation to the timing and profligacy of egg production. In this study. F. hepatica metacercariae of two distinct isolates, the triclabendazole (TCBZ)-sensitive Cullompton isolate and the TCBZ-resistant Oberon isolate, were administered to rats as single-isolate or mixed-isolate infections. At autopsy 16 weeks later individual adult flukes were counted, measured and the reproductive organs were examined histologically. The degree of development of the testis tubules in each fluke was represented by a numerical score, based on the proportion of the histological section profiles occupied by testis tissue. The level of anti-F. hepatica antibody in the serum of each rat was determined by ELISA. It was found that Cullompton flukes were significantly larger than Oberon flukes, and that significantly more Cullompton metacercariae developed to adults than Oberon metacercariae. The Cullompton flukes showed histological evidence of aspermy and spermatogenic arrest, which was reflected in quantitatively reduced testicular development, as compared with the Oberon isolate. In Cullompton flukes, parthenogenetic egg development is implied. The size of Cullompton and Oberon flukes was significantly related to the number of adult flukes recovered, to the number of metacercariae administered, and to the percentage success of infection. The testis development score in both isolates was significantly related to the number of adult flukes recovered but not to the number of metacercariae administered, or to the percentage success of infection. Fluke size was positively related to testis score for both isolates, and a significant negative relationship was found between percentage success of infection and metacercarial dose. The results are interpreted in terms of differing interactions between various numbers of young flukes and host immunity during invasion of and migration in the hepatic parenchyma, and of fluke intra-specific (possibly pheromonal) stimulatory effects in the final stages of development, within the host bile ducts. No significant relationships were found between host antibody levels and fluke size or testis score. False positive serological reactions were found in some rats that had been infected, but found to harbour no flukes at autopsy. Clearly the act of eliminating the flukes involved generation of an immune response. (C) 2011 Elsevier B.V. All rights reserved.

Long Term Use of HU210 Adversely Affects Spermatogenesis in Rats by modulating the Endocannabinoid System

Recent societal acceptance of cannabinoids as recreational and therapeutic drugs has posed a potential hazard to male reproductive health. Mammals have a highly sophisticated endogenous cannabinoid (ECS) system that regulates male (and female) reproduction and exo-cannabinoids may influence it adversely. Therefore it is imperative to determine their effects on male reproduction so that men can make informed choices as to their use. Here, an animal model was used to administer HU210, a synthetic analogue of ?9-tetrahydrocannabinol (THC) and potent cannabinoid receptor (CB) agonist to determine its effects on reproductive organ weights, spermatogenesis, testicular histology and sperm motility. Its effects on the physiological endocannabinoid system were also investigated. Spermatogenesis was markedly impaired with reductions in total sperm count after 2 weeks of exposure. Spermatogenic efficiency was depleted, and Sertoli cell number decreased as exposure time increased with seminiferous tubules showing germ cell depletion developing into atrophy in some cases. Sperm motility was also adversely affected with marked reductions from 2 weeks on. HU210 also acted on the sperm’s endocannabinoid system. Long term use of exo-cannabinoids has adverse effects on both spermatogenesis and sperm function. These findings highlight the urgent need for studies evaluating the fertility potential of male recreational drug users.

The wbbD gene of E. coli strain VW187 (O7:K1) encodes a UDP-Gal:GlcNAc{alpha}-pyrophosphate-R {beta}1,3-galactosyltransferase involved in the biosynthesis of O7-specific lipopolysaccharide

In this work, we demonstrate that the wbbD gene of the O7 lipopolysaccharide (LPS) biosynthesis cluster in Escherichia coli strain VW187 (O7:K1) encodes a galactosyltransferase involved in the synthesis of the O7-polysaccharide repeating unit. The galactosyltransferase catalyzed the transfer of Gal from UDP-Gal to the GlcNAc residue of a GlcNAc-pyrophosphate-lipid acceptor. A mutant strain with a defective wbbD gene was unable to form O7 LPS and lacked this specific galactosyltransferase activity. The normal phenotype was restored by complementing the mutant with the cloned wbbD gene. To characterize the WbbD galactosyltransferase, we used a novel acceptor substrate containing GlcNAcalpha-pyrophosphate covalently bound to a hydrophobic phenoxyundecyl moiety (GlcNAc alpha-O-PO(3)-PO(3)-(CH(2))(11)-O-phenyl). The WbbD galactosyltransferase had optimal activity at pH 7 in the presence of 2.5 mM MnCl(2). Detergents in the assay did not increase glycosyl transfer. Digestion of enzyme product by highly purified bovine testicular beta-galactosidase demonstrated a beta-linkage. Cleavage of product by pyrophosphatase and phosphatase, followed by HPLC and NMR analyses, revealed a disaccharide with the structure Gal beta1-3GlcNAc. Our results conclusively demonstrate that WbbD is a UDP-Gal: GlcNAcalpha-pyrophosphate-R beta1,3-galactosyltransferase and suggest that the novel synthetic glycolipid acceptor may be generally applicable to characterize other bacterial glycosyltransferases.

The role of sexual steroids in the modulation of growth hormone (GH) secretion in humans

Sex steroids contribute to modulate GH secretion in man. However, both the exact locus and mechanism by which their actions are exerted still remain not clearly understood. We undertook a number of studies designed to ascertain: (1) whether or not sudden or chronic changes in circulating gonadal steroids may affect GH secretion in normal adults; and (2) the reason(s) for gender-related dimorphic pattern of GH release. The pituitary reserve of GH, as evaluated by means of a GHRH challenge, was similar in women with anorexia nervosa and in normally menstruating women. Estrogenic receptor blockade with tamoxifen (TMX) did not significantly change GHRH-induced GH response in these normal women. Therefore, acute or chronic hypoestrogenism apparently had no important effects at level of somatotrophs. In another group of normal women we tested the possibility that changes in circulating estrogens might induce changes in the hypothalamic-somatotroph rhythm (HSR). GHRH challenges were performed throughout a menstrual cycle, and again after having achieved functional ovarian blockade with a GnRH agonist treatment. Short-term ovarian blockade did not significantly affect the parameters of GH response to GHRH, although it was accompanied by an increase in the number of women ina refractory HSR phase at testing. This suggested a low potentiating effect on the basic pattern of somatostatin (SS) release occurring as a consequence of the decrease in circulating estrogens. In normal men, neither the GH response to GHRH nor the HSR were affected by functional testicular blockade (after GnRH agonist treatment). However, the administration of testosterone enanthate (250 mg) to another group of men increased both the GHRH-induced GH release and the number of subjects in a spontaneous secretory HSR phase at testing; these were reversed by estrogenic receptor blockade with TMS. In another group of normal men, the fraction of GH secreted in pulses (FGHP) during a nocturnal sampling period was significantly decreased by testicular blockade. Other parameters of GH secretion, such as the number of GH pulses and their mean amplitude (A), and the mean plasma GH concentration (MCGH), showed a slight, although not significant, decrease following the lack of androgens. The administration of testosterone enanthate (500 mg) reversed these parameters to values similar to those in the basal study. Interestingly, when tamoxifen was given after testosterone enanthate, A, MCGH and FGHP increased to values significantly higher than in any other experimental condition in that study. In all, these data suggest that 17ß-estradiol may participate in GH modulation by inhibiting the hypothalamic release of somatostatin, while testosterone stimulates it. The results obtained after estrogenic receptor blockade appear to indicate that the effect of testosterone in such a modulation is dependent on its aromatization to 17ß-estradiol. The differential levels of this steroid in both sexes might account for the sexual dimorphic pattern of GH secretion. From other data in the literature, obtained in rats, and our preliminary data in children with constitutional delay of growth and puberty, it is tempting to speculate that the effect of 17ß-estradiol may be exerted by modifying the functional activity of a-2 adrenergic pathways involved in the negative modulation of SS release.

KATNAL1 regulation of sertoli cell microtubule dynamics is essential for spermiogenesis and male fertility

Spermatogenesis is a complex process reliant upon interactions between germ cells (GC) and supporting somatic cells. Testicular Sertoli cells (SC) support GCs during maturation through physical attachment, the provision of nutrients, and protection from immunological attack. This role is facilitated by an active cytoskeleton of parallel microtubule arrays that permit transport of nutrients to GCs, as well as translocation of spermatids through the seminiferous epithelium during maturation. It is well established that chemical perturbation of SC microtubule remodelling leads to premature GC exfoliation demonstrating that microtubule remodelling is an essential component of male fertility, yet the genes responsible for this process remain unknown. Using a random ENU mutagenesis approach, we have identified a novel mouse line displaying male-specific infertility, due to a point mutation in the highly conserved ATPase domain of the novel KATANIN p60-related microtubule severing protein Katanin p60 subunit A-like1 (KATNAL1). We demonstrate that Katnal1 is expressed in testicular Sertoli cells (SC) from 15.5 days post-coitum (dpc) and that, consistent with chemical disruption models, loss of function of KATNAL1 leads to male-specific infertility through disruption of SC microtubule dynamics and premature exfoliation of spermatids from the seminiferous epithelium. The identification of KATNAL1 as an essential regulator of male fertility provides a significant novel entry point into advancing our understanding of how SC microtubule dynamics promotes male fertility. Such information will have resonance both for future treatment of male fertility and the development of non-hormonal male contraceptives.

Transforming Growth Factor-beta superfamily:evaluation as breast cancer biomarkers and preventive agents

The Transforming Growth Factor-beta (TGFbeta) superfamily of cytokines is comprised of a number of structurally-related, secreted polypeptides that regulate a multitude of cellular processes including proliferation, differentiation and neoplastic transformation. These growth regulatory molecules induce ligand-mediated hetero-oligomerization of distinct type II and type I serine/threonine kinase receptors that transmit signals predominantly through receptor-activated Smad proteins but also induce Smad-independent pathways. Ligands, receptors and intracellular mediators of signaling initiated by members of the TGFbeta family are expressed in the mammary gland and disruption of these pathways may contribute to the development and progression of human breast cancer. Since many facets of TGFbeta and breast cancer have been recently reviewed in several articles, except for discussion of recent developments on some aspects of TGFbeta, the major focus of this review will be on the role of activins, inhibins, BMPs, nodal and MIS-signaling in breast cancer with emphasis on their utility as potential diagnostic, prognostic and therapeutic targets.

Mullerian inhibiting substance inhibits breast cancer cell growth through an NFkappa B-mediated pathway

Müllerian inhibiting substance (MIS), a member of the transforming growth factor-beta superfamily, induces regression of the Müllerian duct in male embryos. In this report, we demonstrate MIS type II receptor expression in normal breast tissue and in human breast cancer cell lines, breast fibroadenoma, and ductal adenocarcinomas. MIS inhibited the growth of both estrogen receptor (ER)-positive T47D and ER-negative MDA-MB-231 breast cancer cell lines, suggesting a broader range of target tissues for MIS action. Inhibition of growth was manifested by an increase in the fraction of cells in the G(1) phase of the cell cycle and induction of apoptosis. Treatment of breast cancer cells with MIS activated the NFkappaB pathway and selectively up-regulated the immediate early gene IEX-1S, which, when overexpressed, inhibited breast cancer cell growth. Dominant negative IkappaBalpha expression ablated both MIS-mediated induction of IEX-1S and inhibition of growth, indicating that activation of the NFkappaB signaling pathway was required for these processes. These results identify the NFkappaB-mediated signaling pathway and a target gene for MIS action and suggest a putative role for the MIS ligand and its downstream interactors in the treatment of ER-positive as well as negative breast cancers.

Label-free based quantitative proteomics analysis of primary neonatal porcine Leydig cells exposed to the persistent contaminant 3-methylsulfonyl-DDE

Evidence that persistent environmental pollutants may target the male reproductive system is increasing. The male reproductive system is regulated by secretion of testosterone by testicular Leydig cells, and perturbation of Leydig cell function may have ultimate consequences. 3-Methylsulfonyl-DDE (3-MeSO₂-DDE) is a potent adrenal toxicants formed from the persistent insecticide DDT. Although studies have revealed the endocrine disruptive effect of 3-MeSO₂-DDE, the underlying mechanisms at cellular level in steroidogenic Leydig cells remains to be established. The current study addresses the effect of 3-MeSO₂-DDE on viability, hormone production and proteome response of primary neonatal porcine Leydig cells. The AlamarBlue™ assay was used to evaluate cell viability. Solid phase radioimmunoassay was used to measure concentration of hormones produced by both unstimulated and Luteinizing hormone (LH)-stimulated Leydig cells following 48h exposure. Protein samples from Leydig cells exposed to a non-cytotoxic concentration of 3-MeSO₂-DDE (10μM) were subjected to nano-LC-MS/MS and analyzed on a Q Exactive mass spectrometer and quantified using label-free quantitative algorithm. Gene Ontology (GO) and Ingenuity Pathway Analysis (IPA) were carried out for functional annotation and identification of protein interaction networks. 3-MeSO₂-DDE regulated Leydig cell steroidogenesis differentially depending on cell culture condition. Whereas its effect on testosterone secretion at basal condition was stimulatory, the effect on LH-stimulated cells was inhibitory. From triplicate experiments, a total of 6804 proteins were identified in which the abundance of 86 proteins in unstimulated Leydig cells and 145 proteins in LH-stimulated Leydig cells was found to be significantly regulated in response to 3-MeSO₂-DDE exposure. These proteins not only are the first reported in relation to 3-MeSO₂-DDE exposure, but also display small number of proteins shared between culture conditions, suggesting the action of 3-MeSO₂-DDE on several targeted pathways, including mitochondrial dysfunction, oxidative phosphorylation, EIF2-signaling, and glutathione-mediated detoxification. Further identification and characterization of these proteins and pathways may build our understanding to the molecular basis of 3-MeSO₂-DDE induced endocrine disruption in Leydig cells.