Goat mammary epithelial cells provide a better expression system for production of recombinant human bone morphogenetic protein 2 compared to Chinese hamster ovarian cells.

Bone Morphogenetic Protein 2 (BMP2), a member of the Transforming Growth Factor-ß (TGF-ß) super family of proteins and is instrumental in the repair of fractures. The synthesis of BMP2 involves extensive post-translational processing and several studies have demonstrated the abysmally low production of rhBMP2 in eukaryotic systems, which may be due to the short half-life of the bioactive protein. Consequently, production costs of rhBMP2 are quite high, limiting its availability to the general populace. Therefore, there is an urgent need to identify better in-vitro systems for large scale production of rhBMP2. In the present study, we have carried out a comparative analysis of rhBMP2 production by the conventionally used Chinese Hamster ovarian cells (CHO) and goat mammary epithelial cells (GMEC), upon transfection with appropriate construct. Udder gland cells are highly secretory, and we reasoned that such cells may serve as a better in-vitro model for large scale production of rhBMP2. Our results indicated that the synthesis and secretion of bioactive rhBMP2 by goat mammary epithelial cells was significantly higher as compared to that by CHO-K1 cells. Our results provide strong evidence that GMECs may serve as a better alternative to other mammalian cells used for therapeutic protein production.

Follicle-stimulating hormone-mediated decline in miR-92a-3p expression in pubertal mice Sertoli cells is crucial for germ cell differentiation and fertility.

Sertoli cells (Sc) are the sole target of follicle-stimulating hormone (FSH) in the testis and attain functional maturation post-birth to significantly augment germ cell (Gc) division and differentiation at puberty. Despite having an operational microRNA (miRNA) machinery, limited information is available on miRNA-mediated regulation of Sc maturation and male fertility. We have shown before that miR-92a-3p levels decline in pubertal rat Sc. In response to FSH treatment, the expressions of FSH Receptor, Claudin11 and Klf4 were found to be elevated in pubertal rat Sc coinciding with our finding of FSH-induced decline in miR-92a-3p levels. To investigate the association of miR-92a-3p and spermatogenesis, we generated transgenic mice where such pubertal decline of miR-92a-3p was prevented by its overexpression in pubertal Sc under proximal Rhox5 promoter, which is known to be activated specifically at puberty, in Sc. Our in vivo observations provided substantial evidence that FSH-induced decline in miR-92a-3p expression during Sc maturation acts as an essential prerequisite for the pubertal onset of spermatogenesis. Elevated expression of miR-92a-3p in post-pubertal testes results into functionally compromised Sc, leading to impairment of the blood-testis barrier formation and apoptosis of pre-meiotic Gc, ultimately culminating into infertility. Collectively, our data suggest that regulation of miR-92a-3p expression is crucial for Sc-mediated induction of active spermatogenesis at puberty and regulation of male fertility.

Anhydride chemistry-based hexanoylation of polyethylenimine increases transfection efficiency and expression of tagged DNA for therapeutic proteins in cultured cells.

Transfection of nucleic acid molecules into mammalian cells can be facilitated using viral vectors, electroporation, or biocompatible cationic materials. However, safety issues and the requirement of specialized equipment limits the use of viral vectors and physical methods of transfection like electroporation and microinjection, respectively. Biocompatible cationic lipids and polymers like branched-polyethyleneimine (bPEI) have a wide transfection range and are user-friendly in most applications. However, bPEI exhibits low transfection efficiency in most cell types. In the present work, we have crosslinked the hexanoyl group to bPEI using anhydride chemistry to enhance its efficiency as a transfection reagent. The efficient association of hexanoyl group to bPEI was assessed using Fourier-transform infrared spectroscopy and other physicochemical methods. Hexanoyl-modified bPEI (FA6-bPEI) was found to exhibit significantly enhanced transfection efficiency in both cell lines and cultured primary cells, as compared to native bPEI and the commercially available transfection reagent, Lipofectamine 3000. Furthermore, our in vitro studies indicated that FA6-bPEI can be used for robust transfection for increased production of therapeutic proteins in a cell culture-based system. These results suggested that hexanoyl-modified bPEI can serve as an efficient transfection reagent for studies on hard-to-transfect cells and for enhanced production of therapeutic proteins in vitro.

FSH mediated cAMP signalling upregulates the expression of Gα subunits in pubertal rat Sertoli cells.

Follicle Stimulating Hormone (FSH) acts via FSH-Receptor (FSH-R) by employing cAMP as the dominant secondary messenger in testicular Sertoli cells (Sc) to support spermatogenesis. Binding of FSH to FSH-R, results the recruitment of the intracellular GTP binding proteins, either stimulatory Gαs or inhibitory Gαi that in turn regulate cAMP production in Sc. The cytosolic concentration of cAMP being generated by FSH-R thereafter critically determines the downstream fate of the FSH signalling. The pleiotropic action of FSH due to differential cAMP output during functional maturation of Sc has been well studied. However, the developmental and cellular regulation of the Gα proteins associated with FSH-R is poorly understood in Sc. In the present study, we report the differential transcriptional modulation of the Gα subunit genes by FSH mediated cAMP signalling in neonatal and pubertal rat Sc. Our data suggested that unlike in neonatal Sc, both the basal and FSH/forskolin induced expression of Gαs, Gαi-1, Gαi-2 and Gαi-3 transcripts was significantly (p < 0.05) up-regulated in pubertal Sc. Further investigations involving treatment of Sc with selective Gαi inhibitor pertussis toxin, confirmed the elevated expression of Gi subunits in pubertal Sc. Collectively our results indicated that the high level of Gαi subunits serves as a negative regulator to optimize cAMP production in pubertal Sc.

Generation of a tissue-specific transgenic model for K8 phosphomutants: A tool to investigate the role of K8 phosphorylation during skin carcinogenesis in vivo.

Keratin 8/18, the predominant keratin pair of simple epithelia, is known to be aberrantly expressed in several squamous cell carcinomas (SCCs), where its expression is often correlated with increased invasion, neoplastic progression, and poor prognosis. The majority of keratin 8/18 structural and regulatory functions are governed by posttranslational modifications, particularly phosphorylation. Apart from filament reorganization, cellular processes including cell cycle, cell growth, cellular stress, and apoptosis are known to be orchestrated by K8 phosphorylation at specific residues in the head and tail domains. Even though deregulation of K8 phosphorylation at two significant sites (Serine73 /Serine431 ) has been implicated in neoplastic progression of SCCs by various in vitro studies, including ours, it is reported to be highly context-dependent. Therefore, to delineate the precise role of Kereatin 8 phosphorylation in cancer initiation and progression, we have developed the tissue-specific transgenic mouse model expressing Keratin 8 wild type and phosphodead mutants under Keratin 14 promoter. Subjecting these mice to 7,12-dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate-mediated skin carcinogenesis revealed that Keratin 8 phosphorylation may lead to an early onset of tumors compared to Keratin 8 wild-type expressing mice. Conclusively, the transgenic mouse model developed in the present study ascertained a positive impact of Keratin 8 phosphorylation on the neoplastic transformation of skin-squamous cells.

Pubertal down-regulation of Tetraspanin 8 in testicular Sertoli cells is crucial for male fertility.

The alarming decline in sperm count has become a global concern in the recent decades. The division and differentiation of male germ cells (Gc) into sperm are governed by Sertoli cells (Sc) upon their functional maturation during puberty. However, the roles of genes regulating pubertal maturation of Sc have not been fully determined. We have observed that Tetraspanin 8 (Tspan8) is down-regulated in Sc during puberty in rats. However, there has been no in vivo evidence for a causal link between the down-regulation of Tspan8 expression and the onset of spermatogenesis as yet. To investigate this, we generated a novel transgenic (Tg) rat, in which the natural down-regulation of Tspan8 was prevented specifically in Sc from puberty up to adulthood. Adult Tg male rats showed around 98% reduction in sperm count despite having a similar level of serum testosterone (T) as the controls. Functional maturation of Sc was impaired as indicated by elevated levels of Amh and low levels of Kitlg and Claudin11 transcripts. The integrity of the blood testis barrier was compromised due to poor expression of Gja1 and Gc apoptosis was discernible. This effect was due to a significant rise in both Mmp7 and phospho P38 MAPK in Tg rat testis. Taken together, we demonstrated that the natural down-regulation of Tspan8 in Sc during puberty is a prerequisite for establishing male fertility. This study divulges one of the aetiologies of certain forms of idiopathic male infertility where somatic cell defect, but not hormonal deficiency, is responsible for impaired spermatogenesis.

Pubertal orchestration of hormones and testis in primates.

The term "Puberty", socially known as "Adolescence" is the transitional period from juvenile life to adulthood with functional maturation of gonads and genital organs. In this process, some remarkable developmental changes occur in morphology, physiology, and behavior leading to reproductive competence. Despite sufficient levels of gonadotropins (luteinizing hormone [LH] and follicle-stimulating hormone [FSH]), robust spermatogenesis is not initiated during infancy in primates due to the immaturity of testicular Sertoli cells. Recent studies suggest that developmental competence augmenting functional activities of receptors for androgen and FSH is acquired by Sertoli cells somewhere during the prolonged hypo-gonadotropic juvenile period. This juvenile phase is terminated with the re-awakening of hypothalamic Kisspeptin/Neurokinin B/Dynorphin neurons which induce the release of the gonadotropin-releasing hormone leading to reactivation of the hypothalamo-pituitary-testicular axis at puberty. During this period of pubertal development, FSH and LH facilitate further maturation of testicular cells (Sertoli cells and Leydig cells) triggering robust differentiation of the spermatogonial cells, ensuing the spermatogenic onset. This review aims to precisely address the evolving concepts of the pubertal regulation of hormone production with the corresponding cooperation of testicular cells for the initiation of robust spermatogenesis, which can be truly called "testicular puberty."

Sertoli cell specific decline in NOR-1 leads to germ cell apoptosis and reduced fertility.

The somatic component of seminiferous epithelium, the Sertoli cells (Sc) respond to Follicle stimulating hormone (FSH), and Testosterone (T) to produce factors which are necessary for germ cell (Gc) survival and differentiation. Infant Sc do not support spermatogenesis in spite of sufficient hormonal milieu, a situation similar to that found in male idiopathic infertility. Sc maturation during pubertal period involves expression of some genes which may be important for initiation of spermatogenesis. Analysis of differentially expressed genes, one by one, in infant and pubertal Sc might provide useful information about the regulation of spermatogenesis. DNA microarray based analysis of mRNA from 5-days (infant) and 12-days (pubertal) old rat Sc revealed increased expression of Nor-1 by pubertal Sc. NOR-1 is an orphan nuclear receptor involved in maintaining cellular homeostasis and disease. We generated transgenic mice using shRNA cloned under Pem (Rhox5) promoter which is activated at puberty in Sc. Such transgenic mice had reduced Nor-1 expression and increased Tgfß1, Tgfß3, and Smad3 expression. Moreover, an increase in ß-catenin expression was observed in NOR-1 knockdown testes. High ß-catenin in such transgenic mice was found to be associated with disruption of Sc maturation characterized by elevated expression of Anti Mullerian hormone, Cytokeratin 18, and Sox9. This disruption of Sc maturation resulted in Gc apoptosis. Such NOR-1 knockdown mice showed reduced sperm count and litter size. We report for the first time that NOR-1 plays a crucial role in regulating sperm count and male fertility.

Attenuation of macrophage accumulation and polarisation in obese diabetic mice by a small molecule significantly improved insulin sensitivity.

Accumulation and polarization of anti-inflammatory M2 to proinflammatory M1 macrophage in the adipose tissue of obese diabetic mice is an important pathogenic signature. It worsens lipid induced inflammation and insulin resistance. Here we demonstrate that a small molecule, a peroxyvanadate compound i.e. DmpzH [VO(O2)2 (dmpz)] or dmp, could robustly decrease macrophage infiltration, accumulation and their polarization in high fat diet (HFD) induced obese diabetic mice. In searching the underlying mechanism it was revealed that SIRT1 level was strikingly low in the inflamed adipose tissue of HFD mice as compared to mice fed with standard diet (SD). Administration of dmp markedly increased SIRT1 level by inducing its gene expression with a consequent decrease in macrophage population. Elevation of SIRT1 coincided with the decrease of MCP1, Fetuin-A (FetA) and IFNγ. Since MCP1 and FetA drive macrophage to inflamed adipose tissue and IFNγ promotes M2 to M1 transformation, both recruitment and M1 induced inflammation were found to be significantly repressed by dmp. In addressing the question about how dmp induced excess SIRT1 could reduce MCP1, FetA and IFNγ levels, we found that it was due to the inactivation of NFκB because of its deacetylation by SIRT1. Since NFκB is the transcriptional regulator of these molecules, their expressions were significantly suppressed and that caused sharp decline in macrophage recruitment and their polarity to M1. This effected a marked fall in proinflammatory cytokine level which significantly improved insulin sensitivity. dmp is likely to be the first molecule that rescues inflammatory burden contributed by macrophage in obese diabetic mice adipose tissue which causes significant increase in insulin sensitivity therefore it may be a meaningful choice to treat type 2 diabetes.

Evaluation of high-fat high-fructose diet treatment in factor VIII (coagulation factor)-deficient mouse model.

Non-alcoholic fatty liver disease (NAFLD)-like conditions enhance the production and action of clotting factors in humans. However, studies examining the effect of NAFLD due to high-fat high-fructose (HFHF) diet in factor VIII-deficient (haemophilia A) animals or patients have not been reported previously. In this study, we investigated the individual role of factor VIII in the progression of diet-induced NAFLD in the factor 8-/- (F8-/- ) mouse model system and its consequences on the haemophilic status of the mice. The F8-/- mice were fed with HFHF diet for 14 weeks. Physiological, biochemical, haematological, molecular, pathological, and immune histochemical analyses were performed to evaluate the effect of this diet. The F8-/- mice developed hepatic steatosis after 14 weeks HFHF diet and displayed lower energy metabolism, higher myeloid cell infiltration in the liver, decreased platelet count, upregulated de novo fatty acid synthesis, lipid accumulation, and collagen deposition. This study helps to understand the role of factor VIII in NAFLD pathogenesis and to analyse the severity and consequences of steatosis in haemophilic patients as compared to normal population. This study suggests that haemophilic animals (F8-/- mice) are highly prone to hepatic steatosis and thrombocytopenia.

Defective Wnt3 expression by testicular Sertoli cells compromise male fertility.

Testicular Sertoli cells make a niche for the division and differentiation of germ cells. Sertoli cells respond to increased follicle-stimulating hormone (FSH) and testosterone (T) levels at the onset of puberty by producing paracrine factors which affect germ cells and trigger robust onset of spermatogenesis. Such paracrine support to germ cells is absent during infancy, despite Sertoli cells being exposed to high FSH and T within the infant testis. This situation is similar to certain cases of male idiopathic infertility where post-pubertal Sertoli cells fail to support germ cell division and differentiation in spite of endogenous or exogenous hormonal support. Defective Sertoli cells in such individuals may fail to express the full complement of their paracrine repertoire. Identification and supplementation with such factors may overcome Sertoli cells deficiencies and help trigger quantitatively and qualitatively normal differentiation of germ cells. To this end, we compared the transcriptome of FSH- and T-treated infant and pubertal monkey Sertoli cells by DNA microarray. Expression of Wnt3, a morphogen of the Wnt/ß-catenin pathway, was higher in pubertal Sertoli cells relative to infant Sertoli cells. Transgenic mice were generated by us in which Wnt3 expression was curtailed specifically in post-pubertal Sertoli cells by shRNA. Subfertility and oligozoospermia were noticed in such animals with low Wnt3 expression in post-pubertal Sertoli cells along with diminished expression of Connexin43, a gap-junctional molecule essential for germ cell development. We report that the FSH- and T-targetedf Wnt3 governs Sertoli cell-mediated regulation of spermatogenesis and hence is crucial for fertility.

A combinatorial approach for robust transgene delivery and targeted expression in mammary gland for generating biotherapeutics in milk, bypassing germline gene integration.

Protein expression in the milk of transgenic farmed animals offers a cost-effective system for producing therapeutics. However, transgenesis in farmed animals is not only cumbersome but also involves risk of potential hazard by germline gene integration, due to interruptions caused by the transgene in the native genome. Avoiding germline gene integration, we have delivered buffalo ß-casein promoter-driven transgene construct entrapped in virosomes directly in the milk gland through intraductal perfusion delivery. Virosomes were generated from purified Sendai viral membrane, containing hemagglutinin-neuraminidase (HN) and fusion factor (F) proteins on surface (HNF-Virosomes) which initiate membrane fusion, devoid of any viral nucleic acids. Intraductal delivery of HNF-Virosomes predominantly transfected luminal epithelial cells lining the milk duct and buffalo ß-casein promoter of the construct ensured mammary luminal epithelial cell specific expression of the transgene. Mammary epithelial cells expressed EGFP at lactation when egfp was used as a transgene. Similarly, human interferon-γ (hIFN-γ) was expressed in the mammary gland as well as in the milk when hIFN-γ was used as a transgene. This combinatorial approach of using Sendai viral membrane-derived virosomes for entrapment and delivery of the transgene and using buffalo ß-casein promoter for mammary gland specific gene expression provided a better option for generating therapeutic proteins in milk, bypassing germline gene integration avoiding risks associated with animal bioreactor generated through germline gene integration.

Advantages of pulsatile hormone treatment for assessing hormone-induced gene expression by cultured rat Sertoli cells.

In response to various hormonal (follicle-stimulating hormone [FSH] and testosterone [T]) and biochemical inputs, testicular Sertoli cells (Sc) produce factors that regulate spermatogenesis. A number of FSH- and T-responsive Sc-specific genes, necessary for spermatogenesis, have been identified to date. However, the hormone-induced in vitro expression pattern of most of these genes is reported to be inconsistent at various time points in primary rat Sc cultures. As a matter of convenience, cultured Sc are constantly exposed to hormones for a few hours to days in the reported literature, although Sc are exposed to pulsatile FSH and T in vivo. The major aim of the present study is to evaluate the advantage, if any, of the in vitro administration of pulsatile hormone (FSH and T in combination) treatment on gene expression of cultured Sc as compared with that of constant hormone treatment. Pulsatile treatment (a 30-min hormonal exposure every 3 h) mimicking the in vivo condition reveals a more prominent effect of hormones in augmenting gene expression as compared with constant treatment. Our results indicate that the expressions of Stem cell factor (Scf, only responsive to FSH), Claudin11 (only responsive to T) and Transferrin (both FSH- and T-responsive) mRNAs are significantly higher at 12 h upon pulsatile treatment than upon constant hormonal treatment. Maximal expression of relevant genes because of pulsatile treatment with hormones suggests that this protocol provides a more suitable premise for assessing hormone-induced gene expression in isolated Sc than one involving constant exposure to hormones.

Role of antigen presenting cell invariant chain in the development of hepatic steatosis in mouse model.

The role of Invariant chain (CD74 or Ii) in antigen presentation via Antigen Presenting Cells (APC), macrophage recruitment as well as survival, T cell activation and B cell differentiation has been well recognized. However, the aspect of CD74 which is involved in the development of hepatic steatosis and the pathways through which it acts remain to be studied. In this study, we investigated the role of CD74 in the inflammatory pathway and its contribution to development of hepatic steatosis. For this, wild type C57BL/6J and CD74 deficient mice (Ii(-/-) mice) were fed with high fat high fructose (HFHF) diet for 12 weeks. Chronic consumption of this feed did not develop hepatic steatosis, glucose intolerance or change in the level of immune cells in Ii(-/-) mice. Moreover, there was relatively delayed expression of genes involved in development of non alcoholic fatty liver disease (NAFLD) in HFHF fed Ii(-/-) mice as compared to that of C57BL/6J phenotype. Taken together, the data suggest that HFHF diet fed Ii(-/-) mice fail to develop hepatic steatosis, suggesting that Ii mediated pathways play a vital role in the initiation and propagation of liver inflammation.

Constitutive expression of SMAR1 confers susceptibility to Mycobacterium tuberculosis infection in a transgenic mouse model.

BACKGROUND & OBJECTIVES: Studies involving animal models of experimental tuberculosis have elucidated the predominant role of cytokines secreted by T cells and macrophages to be an essential component of the immune response against Mycobacterium tuberculosis infection. The immune activities of CD4+ T cells are mediated in part by Th1 cytokine interferon gamma (IFN-γ) which is produced primarily by T cells and natural killer (NK) cells and critical for initiating the immune response against intracellular pathogen such as M. tuberculosis. Nuclear matrix protein SMAR1 plays an important role in V(D)J recombination, T helper cell differentiation and inflammatory diseases. In this study a transgenic mouse model was used to study the role of SMAR1 in M. tuberculosis infection. METHODS: Wild type BALB/c, C57BL/6, BALB/c-EGFP-SMAR1 and C57BL/6-SMAR1 transgenic mice were infected with M. tuberculosis (H37Rv). A dose of 100 bacilli was used for infection via respiratory route. Bacterial load in lung and spleen of infected mice was determined at 2, 4, 6 and 8 wk post-infection. Gene expression analysis for Th1 cytokines and inducible nitric oxide synthase (iNOS) was performed in infected lung tissues by quantitative reverse transcription (RT)-PCR. RESULTS: SMAR1 transgenic mice from both BALB/c and C57BL/6 genetic background displayed higher bacillary load and susceptibility to M. tuberculosis infection compared to wild type mice. This susceptibility was attributed due to compromised of Th1 response exhibited by transgenic mice. INTERPRETATION & CONCLUSIONS: SMAR1 transgenic mice exhibited susceptibility to M. tuberculosis infection in vivo irrespective of genetic background. This susceptibility was attributed to downregulation of Th1 response and its hallmark cytokine IFN-γ. Hence, SMAR1 plays an important role in modulating host immune response after M. tuberculosis infection.

Adipocyte fetuin-A contributes to macrophage migration into adipose tissue and polarization of macrophages.

Macrophage infiltration into adipose tissue during obesity and their phenotypic conversion from anti-inflammatory M2 to proinflammatory M1 subtype significantly contributes to develop a link between inflammation and insulin resistance; signaling molecule(s) for these events, however, remains poorly understood. We demonstrate here that excess lipid in the adipose tissue environment may trigger one such signal. Adipose tissue from obese diabetic db/db mice, high fat diet-fed mice, and obese diabetic patients showed significantly elevated fetuin-A (FetA) levels in respect to their controls; partially hepatectomized high fat diet mice did not show noticeable alteration, indicating adipose tissue to be the source of this alteration. In adipocytes, fatty acid induces FetA gene and protein expressions, resulting in its copious release. We found that FetA could act as a chemoattractant for macrophages. To simulate lipid-induced inflammatory conditions when proinflammatory adipose tissue and macrophages create a niche of an altered microenvironment, we set up a transculture system of macrophages and adipocytes; the addition of fatty acid to adipocytes released FetA into the medium, which polarized M2 macrophages to M1. This was further confirmed by direct FetA addition to macrophages. Taken together, lipid-induced FetA from adipocytes is an efficient chemokine for macrophage migration and polarization. These findings open a new dimension for understanding obesity-induced inflammation.

Isolation and characterisation of promoters from mouse genome to drive post-meiotic germ cell-specific robust gene expression for functional genomics studies.

The generation of spermatozoa from developing germ cells through mitotic and meiotic divisions is a highly regulated and complex process. Any defect in this process, may lead to subfertility/infertility. The role of different transcripts (mRNA/miRNA/lncRNA) in regulation of the pre-meiotic, meiotic, and post-meiotic stages of spermatogenesis are being proposed based on various multiomics based approaches. Such differential gene-expression is regulated by promoter elements that are activated in a stage specific manner. To determine the role of these differentially expressed transcripts in the process of meiosis, a robust post-meiotic germ cell specific promoter is required. In the present study, we have isolated and characterized the expression of the mouse Proacrosin, SP10, and ELP promoters for driving post-meiotic germ cell specific gene-expression. Promoter regions of all these 3 genes were isolated and cloned to generate mammalian expression vector. The transgene expression in post-meiotic germ cells was assessed in mice using the testicular electroporation method in vitro as well as in vivo, using above promoters. It was also validated in goat seminiferous tubules, in vitro. We have also carried out a comparative analysis of the strength of these promoters to confirm their robustness that indicated Proacrosin to be the most robust promoter that can be useful for diving post-meiotic germ cells specific gene-expression. These promoters can be used to alter gene-expression specifically in post-meiotic germ cells for deciphering the role(s) of germ cell genes in spermatogenic progression or for expressing various genome editing tools for engineering the germ cell genome to understand basis of subfertility/infertility.

Etiology of Male Infertility: an Update.

Spermatogenesis is a complex process of germ cell division and differentiation that involves extensive cross-talk between the developing germ cells and the somatic testicular cells. Defective endocrine signaling and/or intrinsic defects within the testes can adversely affect spermatogenic progression, leading to subfertility/infertility. In recent years, male infertility has been recognized as a global public health concern, and research over the last few decades has elucidated the complex etiology of male infertility. Congenital reproductive abnormalities, genetic mutations, and endocrine/metabolic dysfunction have been demonstrated to be involved in infertility/subfertility in males. Furthermore, acquired factors like exposure to environmental toxicants and lifestyle-related disorders such as illicit use of psychoactive drugs have been shown to adversely affect spermatogenesis. Despite the large body of available scientific literature on the etiology of male infertility, a substantial proportion of infertility cases are idiopathic in nature, with no known cause. The inability to treat such idiopathic cases stems from poor knowledge about the complex regulation of spermatogenesis. Emerging scientific evidence indicates that defective functioning of testicular Sertoli cells (Sc) may be an underlying cause of infertility/subfertility in males. Sc plays an indispensable role in regulating spermatogenesis, and impaired functional maturation of Sc has been shown to affect fertility in animal models as well as humans, suggesting abnormal Sc as a potential underlying cause of reproductive insufficiency/failure in such cases of unexplained infertility. This review summarizes the major causes of infertility/subfertility in males, with an emphasis on infertility due to dysregulated Sc function.

Antigen peptide transporter 1 is involved in the development of fructose-induced hepatic steatosis in mice.

BACKGROUND AND AIM: The purpose of this study is to assess whether the decrease in CD8 cells has any role in the development of non-alcoholic fatty liver disease (NAFLD). In this study, we therefore used antigen peptide transporter 1 (TAP1(-/-)) mice that cannot transport major histocompatibility complex class I antigens onto the cell surface resulting in failure of the generation of CD8 cells. METHODS: Wild-type C57Bl/6J and TAP1(-/-) mice were fed with 30% fructose solution for 8 weeks. The percentage of CD4, CD8 cells in peripheral blood mononuclear cells, and liver were sorted by fluorescence-activated cell sorting in both control and fructose-treated mice. Bodyweight, histopathological changes, oil red O staining, glucose tolerance test, intraperitoneal insulin tolerance test, serum levels of triglycerides, cholesterol, aspartate aminotransferase, and alanine aminotransferase were also evaluated. Quantitative real-time polymerase chain reaction was performed to determine the expression of specific genes involved in development of fatty changes in the liver. RESULTS: Chronic consumption of fructose in TAP1(-/-) mice did not develop NAFLD, insulin resistance, or change in level of CD8 cells. Moreover, there was delay in relative expression levels of genes involved in development of NAFLD in fructose-treated TAP1(-/-) mice. CONCLUSION: Taken together, the data suggest that TAP1(-/-) -deficient mice displayed reduced levels of CD8 cells that have a vital role in the initiation and propagation of liver inflammation and is a casual role in the beginning of fructose-induced liver damage as well as insulin resistance in mice.

Role of apoptosis-inducing factor (Aif) in the T cell lineage.

Multiple checkpoints regulating finely balanced death-versus-survival decisions characterize both thymic development and peripheral homeostasis of T lymphocytes. While exploring the mechanisms of T cell death involved at various stages during the life of a T cell, we have observed and reported a variety of non-redundant roles for apoptosis inducing factor (Aif), a mitochondrial flavoprotein. Aif is ubiquitously expressed in all cell lineages and functions as an NADH oxidase in its mitochondrial location. It is released following the mitochondrial death signals, whereupon it translocates to the nucleus, binds to DNA and causes large-scale DNA fragmentation. During T cell development, Aif is important for developing thymocytes to navigate the double negative (DN)3 to DN4 transition (beta-selection), via its oxidoreductase property which protects the rapidly proliferating cells from death due to reactive oxygen species (ROS). In peripheral mature T cells, Aif deficiency leads to an increased susceptibility of T cell blasts to activation induced cell death (AICD), possibly mediated by its antioxidant function, and decreased sensitivity to neglect-induced death (NID). Thus, Aif seems to have pro-apoptotic and anti-apoptotic roles in the same lineage in different contexts and at different stages. Surprisingly, in the closely related B lymphocyte lineage, Aif deficiency does not result in any abnormality. These findings generate the possibility of specific T cell dysfunction in human disease caused by Aif deficiency, as well as in mitochondriopathies due to other causes. Also, these data raise questions regarding the basis of lineage-specific consequences of the dysfunction/deficiency of apparently ubiquitous molecules.