The development of lateral flow devices for urinary biomarkers to assess kidney health.

Serum creatinine levels are insensitive to real-time changes in kidney function or injury. There is a growing interest in assessing kidney injury by measuring biomarkers in body fluid. From our previous studies, we identified and reported three urinary biomarkers namely Uromodulin (UMOD), Osteopontin (OPN), and Interleukin-9 (IL-9) to be associated with kidney health. The availability of a rapid point-of-care test for these urinary biomarkers will potentially accelerate its applicability and accessibility. In this study, we aimed to develop novel lateral flow device (LFD) for UMOD, OPN and IL-9. We tested paired antibodies using Enzyme Linked Immunosorbent Assay wherein we observed functionality only for UMOD and OPN and not for IL-9. A conjugation buffer pH of 7.8 and 8.5 was found suitable at a detection antibody concentration of 15 µg/mL for LFD development. The developed LFDs were found to quantitatively measure UMOD standard (LLOD of 80,000 pg/mL) and OPN standard (LLOD of 8600 pg/mL) respectively. The LFD was also able to measure human urinary UMOD and OPN with a percent CV of 12.12 and 5.23 respectively.

HIV-1 exploits Hes-1 expression during pre-existing HPV-16 infection for cancer progression.

High Risk Human Papilloma Viruses (HR-HPV) persistently infect women with Human Immunodeficiency Virus-1 (HIV-1). HPV-16 escapes immune surveillance in HIV-1 positive women receiving combined antiretroviral therapy (cART). HIV-1 Tat and HPV E6/E7 proteins exploit Notch signaling. Notch-1, a developmentally conserved protein, influences cell fate from birth to death. Notch-1 and its downstream targets, Hes-1 and Hey-1 contribute to invasive and aggressive cancers. Cervical cancer cells utilize Notch-1 and hyper-express CXCR4, a co-receptor of HIV-1. Accumulating evidence shows that HIV-1 affects cell cycle progression in pre-existing HPV infection. Additionally, Tat binds Notch-1 receptor for activation and influences cell proliferation. Oncogenic viruses may interfere or converge together to favor tumor growth. The molecular dialogue during HIV-1/HPV-16+ co-infections in the context of Notch-1 signaling has not been explored thus far. This in vitro study was designed with cell lines (HPV-ve C33A and HPV-16+ CaSki) which were transfected with plasmids (pLEGFPN1 encoding HIV-1 Tat and pNL4-3 encoding HIV-1 [full HIV-1 genome]). HIV-1 Tat and HIV-1 inhibited Notch-1expression, with differential effects on EGFR. Notch-1 inhibition nullified Cyclin D expression with p21 induction and increased G2-M cell population in CaSki cells. On the contrary, HIV-1 infection shuts down p21 expression through interaction of Notch-1 downstream genes Hes-1-EGFR and Cyclin D for G2-M arrest, DDR response and cancer progression. This work lays foundations for future research and interventions, and therefore is necessary. Our results describe for the first time how HIV-1 Tat cancers have an aggressive nature due to the interplay between Notch-1 and EGFR signaling. Notch-1 inhibitor, DAPT used in organ cancer treatment may help rescue HIV-1 induced cancers. Graphical abstract: The illustration shows how HIV interacts with HPV-16 to induce Notch 1 suppression for cancer progression (Created with BioRender.com). Supplementary Information: The online version contains supplementary material available at 10.1007/s13337-023-00809-y.

High-fat simple carbohydrate feeding impairs central and peripheral monoamine metabolic pathway triggering the onset of metabolic syndrome in C57Bl/6J mice.

BACKGROUND: Previous studies have shown disturbances in an individual monoamine pathway but have not studied metabolic pathways at the onset and progression of metabolic syndrome (MetS). Aims, Settings, and Design: The aim of this study was to investigate the effect of high-fat simple carbohydrate (HFSC) diet on central (hypothalamic) and peripheral (plasma and urine) monoamine metabolic pathways during the development of metabolic syndrome in C57BL/6J mice. MATERIALS AND METHODS: Monoamines were analyzed in the 1st, 2nd, 3rd, 4th, and 5th month after feeding mice the HFSC diet or the control diet using the high performance liquid chromatography (HPLC) system (Shimadzu, Japan). Data was statistically analyzed (by Student's t-test) using Graph Pad Instat Version 3.1. Post statistical analysis, Bonferroni correction was applied to the results of 2nd, 3rd, 4th, and 5th month in order to calculate the correct error in the study. RESULTS: Significantly lower hypothalamic, plasma, and urine dopamine, and higher hypothalamic and plasma levels of norepinephrine and normetanephrine levels were observed in the HFSC diet fed C57BL/6J mice as compared to the control diet fed C57BL/6J mice after 5 months of feeding. No consistent changes were observed in other brain regions. The turnover ratio indicated that the lower dopamine levels in the HFSC diet fed C57BL/6J mice was due to the increased formation of norepinephrine and homovanillic acid. CONCLUSION: HFSC diet impairs the central and peripheral dopaminergic and noradrenergic pathways in mice as evidenced by the disturbances in their hypothalamic, plasma, and urine levels and this might be one of the early factors contributing towards the development of the MetS.

The MADD-3 LAMMER Kinase Interacts with a p38 MAP Kinase Pathway to Regulate the Display of the EVA-1 Guidance Receptor in Caenorhabditis elegans.

The proper display of transmembrane receptors on the leading edge of migrating cells and cell extensions is essential for their response to guidance cues. We previously discovered that MADD-4, which is an ADAMTSL secreted by motor neurons in Caenorhabditis elegans, interacts with an UNC-40/EVA-1 co-receptor complex on muscles to attract plasma membrane extensions called muscle arms. In nematodes, the muscle arm termini harbor the post-synaptic elements of the neuromuscular junction. Through a forward genetic screen for mutants with disrupted muscle arm extension, we discovered that a LAMMER kinase, which we call MADD-3, is required for the proper display of the EVA-1 receptor on the muscle's plasma membrane. Without MADD-3, EVA-1 levels decrease concomitantly with a reduction of the late-endosomal marker RAB-7. Through a genetic suppressor screen, we found that the levels of EVA-1 and RAB-7 can be restored in madd-3 mutants by eliminating the function of a p38 MAP kinase pathway. We also found that EVA-1 and RAB-7 will accumulate in madd-3 mutants upon disrupting CUP-5, which is a mucolipin ortholog required for proper lysosome function. Together, our data suggests that the MADD-3 LAMMER kinase antagonizes the p38-mediated endosomal trafficking of EVA-1 to the lysosome. In this way, MADD-3 ensures that sufficient levels of EVA-1 are present to guide muscle arm extension towards the source of the MADD-4 guidance cue.

Insulin resistance precedes glucose intolerance and hyperleptinaemia in high-fat simple carbohydrate-fed C57BL/6J mice.

INTRODUCTION: Very few systematic studies are done during the onset and progression of metabolic syndrome in suitable animal models. In this paper we present the effect of High-Fat Simple Carbohydrate (HFSC) feed on the metabolic hormones in C57BL/6J mice to understand the sequence of events leading to impairment of glucose homeostasis. MATERIAL AND METHODS: One-month-old male C57BL/6J mice were fed with control (C group) and HFSC (T group) feed (n = 30 each) respectively for five months. The glucose tolerance was studied by Oral Glucose Tolerance Test (OGTT) whereas serum insulin and leptin were quantified using ELISA kits, and serum cortisol was quantified using CLIA kits. RESULTS: Insulin resistance index and HOMA-IR levels were higher in the mice of group T as compared to age-matched mice of group C within one month and significantly higher after and five months of feeding. The total area under the glucose tolerance test curve (AUC) and the insulin curve (AUC ins) was found to significantly increase in the mice of T group as compared to the mice of C group as early as two months of feeding and was elevated after 5 months post feeding. Comparison of the Matsuda index revealed that pancreatic beta cell function was significantly lower in mice of T group as compared to mice of C group by five months of feeding. Leptin levels fluctuated during the 1st-4th month and by the 5th month significant hyperleptinaemia was detected. There was no significant change in cortisol levels in mice of group T as compared to mice of group C after five months of feeding. CONCLUSIONS: HFSC feed induces insulin resistance by the first month and progressively impairs glucose tolerance, resulting in hyperleptinaemia by the fifth month in male C57BL/6J mice. (Endokrynol Pol 2016; 67 (6): 592-598).

Putative molecular mechanism underlying sperm chromatin remodelling is regulated by reproductive hormones.

BACKGROUND: The putative regulatory role of the male reproductive hormones in the molecular mechanism underlying chromatin condensation remains poorly understood. In the past decade, we developed two adult male rat models wherein functional deficits of testosterone or FSH, produced after treatments with 20 mg/Kg/d of cyproterone acetate (CPA) per os, for a period of 15 days or 3 mg/Kg/d of fluphenazine decanoate (FD) subcutaneously, for a period of 60 days, respectively, affected the rate of sperm chromatin decondensation in vitro. These rat models have been used in the current study in order to delineate the putative roles of testosterone and FSH in the molecular mechanism underlying remodelling of sperm chromatin. RESULTS: We report that deficits of both testosterone and FSH affected the turnover of polyubiquitylated histones and led to their accumulation in the testis. Functional deficits of testosterone reduced expression of MIWI, the 5-methyl cap binding RNA-binding protein (PIWIlike murine homologue of the Drosophila protein PIWI/P-element induced wimpy testis) containing a PAZ/Piwi-Argonaut-Zwille domain and levels of histone deacetylase1 (HDAC1), ubiquitin ligating enzyme (URE-B1/E3), 20S proteasome α1 concomitant with reduced expression of ubiquitin activating enzyme (ube1), conjugating enzyme (ube2d2), chromodomain Y like protein (cdyl), bromodomain testis specific protein (brdt), hdac6 (histone deacetylase6), androgen-dependent homeobox placentae embryonic protein (pem/RhoX5), histones h2b and th3 (testis-specific h3). Functional deficits of FSH reduced the expression of cdyl and brdt genes in the testis, affected turnover of ubiquitylated histones, stalled the physiological DNA repair mechanism and culminated in spermiation of DNA damaged sperm. CONCLUSIONS: We aver that deficits of both testosterone and FSH differentially affected the process of sperm chromatin remodelling through subtle changes in the 'chromatin condensation transcriptome and proteome', thereby stalling the replacement of 'dynamic' histones with 'inert' protamines, and altering the epigenetic state of condensed sperm chromatin. The inappropriately condensed chromatin affected the sperm chromatin cytoarchitecture, evident from subtle ultrastructural changes in the nuclei of immature caput epididymal sperm of CPA- or FD-treated rats, incubated in vitro with dithiothreitol.

Sperm phosphoproteome profiling by ultra performance liquid chromatography followed by data independent analysis (LC-MS(E)) reveals altered proteomic signatures in asthenozoospermia.

Sperm motility is an important prerequisite for successful fertilization and is regulated by cyclic AMP activated protein kinase A which phosphorylates flagella proteins like axonemal dynein and initiates motility. Increase in calcium influx reverses this process by dephosphorylation that is mediated by calcineurin. Analyzing the phosphoenriched fractions of spermatozoa lysates from eight normozoospermic-, and asthenozoospermic-samples, respectively, by Nano UPLC-MS(E), the present study investigates the phosphoproteins involved in sperm motility in an attempt to identify the key pathways regulating sperm motility and likely to be altered in spermatozoa of asthenozoospermic individuals. 66 phosphoproteins were differentially regulated in asthenozoospermia. The deregulated proteins comprised predominantly the HSPs, cytoskeletal proteins, proteins associated with the fibrous sheath, and those associated with energy metabolism. EM analysis of these spermatozoa demonstrated significant defects in mitochondria, and fibrous sheath and these defects could be correlated with the altered proteome. Pathway analysis revealed that carbohydrate and energy metabolism, cAMP mediated PKA signaling, PI3K/AKT signaling and pathway regulating actin based motility by Rho were significantly altered indicating that motility in spermatozoa is regulated through the concerted effort of these pathways. The data identified signature molecules that have the potential as biomarkers for diagnosing etiology of asthenozoospermia.

Efficient cryopreservation of testicular tissue: effect of age, sample state, and concentration of cryoprotectant.

OBJECTIVE: To study the effect of age and sample state on cryopreservation of testicular tissue, evaluate toxicity of commonly used cryoprotectants (CPs), and determine their optimal concentration for use. DESIGN: Prospective experimental study. SETTING: Academic research unit. PATIENT(S): Patients with prostate carcinoma undergoing orchidectomy. We also studied immature and adult male Holtzman rats. INTERVENTION(S): Toxicity of CPs before freezing, morphology, and relative viability after freezing were evaluated for rat testicular cell suspensions (CS) and tubular fragments (TUB). Relative viability of adult human testicular CS and TUB after thaw was evaluated. Human TUB were cultured after thaw for 48 hours in medium containing epidermal growth factor (EGF), and effects on viability, morphology, and gene expression were determined. MAIN OUTCOME MEASURE(S): Viability and ploidy were measured with flow cytometry, postthaw cryodamage of immature rat tissue was studied by transmission electron microscopy, cell proliferation and differentiation were evaluated by immunohistochemistry and by real-time polymerase chain reaction. RESULT(S): Immature testicular tissue was more susceptible to toxic assault by CP than adult tissue and displayed cell-specific sensitivity to CP, with glycerol, dimethyl sulfoxide and ethylene glycol being effective in protecting spermatid (1N), spermatogonia (2N) and spermatocyte (4N) populations respectively. Preservation as TUB may be preferred over CS and DMSO is an effective CP for immature and ethylene glycol for adult testicular tissue. CONCLUSION(S): Differential sensitivity of immature testicular tissue to CPs warrants judicious selection of CP on the basis of end application for prepubertal tissue.

MADD-4 is a secreted cue required for midline-oriented guidance in Caenorhabditis elegans.

The netrins and slits are two families of widely conserved cues that guide axons and cells along the dorsal-ventral (D-V) axis of animals. These cues typically emanate from the dorsal or ventral midlines and provide spatial information to migrating cells by forming gradients along the D-V axis. Some cell types, however, extend processes to both the dorsal and ventral midlines, suggesting the existence of additional guidance cues that are secreted from both midlines. Here, we report that a previously uncharacterized protein called MADD-4 is secreted by the dorsal and ventral nerve cords of the nematode C. elegans to attract sensory axons and muscle membrane extensions called muscle arms. MADD-4's activity is dependent on UNC-40/DCC, a netrin receptor, which functions cell-autonomously to direct membrane extension. The biological role of MADD-4 orthologs, including ADAMTSL1 and 3 in mammals, is unknown. MADD-4 may therefore represent the founding member of a family of guidance proteins.

Sperm chromatin protamination: an endocrine perspective.

During spermiogenesis, the elongating rat spermatid chromatin undergoes a gradual process of condensation which is initiated in the round spermatids at "step 7" of cytodifferentation in stage VII and extending to elongated spermatids at "step 19" of cytodifferentiation in stage VIII. The mechanism of chromatin condensation in the elongating spermatids is an elaborate process that encompasses several biochemical and biological aspects culminating in the deposition of protamine in DNA grooves. The protamination of sperm chromatin involves expression and storage of proteins involved in condensation, removal and degradation of nuclear histones and their replacement by transition proteins and protamine 1, transcriptional silencing and DNA repair, reduction of nuclear volume, repackaging of protaminated chromatin in torroids and development of a characteristic head shape and perforatorium. A study was undertaken in my laboratory to delineate the role of follicle stimulating hormone (FSH) and testosterone in the condensation of nuclear chromatin in the elongating spermatids of sexually competent species of rat. Towards this end, sexually competent male Holtzmann rats were treated with 20 mg/Kg/d per os (oral supplementum) of cyproterone acetate and 3 mg/Kg/d i.p (intra peritoneal) of fluphenazine decanoate to induce a functional deficiency in either testosterone or FSH. In both rat models, membrane-impermeable CMA3 fluorescent dye uptake assay for GC-rich prospective sites of DNA protamination, was indicative of insufficiency of protamine 1 in spermatozoa taken from caput epididymides of treated rats whereas a fluorescent TUNEL assay indicated the presence of nicked chromatin strands only in protamine-deficient spermatozoa derived from caput epididymides of fluphenazine-treated rats with functional deficiency of FSH. Western blotting of acid-soluble sperm basic proteins had confirmed the near absence of protamine 1 in treated rat spermatozoa in both models. Electron Microscopic evaluation too revealed fine ultrastructural changes in the nuclear membrane of cyproterone acetate as well as fluphenazine decanoate treated spermatozoa derived from caput epididymides. Electrophoretic analysis of caput sperm nuclear basic proteins substantiated the observations at cellular level and revealed a pattern of abnormal persistence of acid-soluble nuclear basic proteins in both rat models, the levels being more prominent in fluphenazine treated rats. Our studies suggest that adequate levels of both FSH and testosterone could be essential during the stages of spermatidal condensation and led us to hypothesize the existence of an endocrine-regulated molecular mechanism for histone to protamine transition and maintenance of chromatin integrity during chromatin condensation in the testis during spermiogenesis.

Novel biphenyl compound, VMNS2e, ameliorates streptozotocin-induced diabetic nephropathy in rats.

AIM: To study the effect of a new biphenyl synthetic compound showing interactions with the active site of protein tyrosine phosphatase 1B by docking and molecular dynamics, VMNS2e in streptozotocin-induced diabetic nephropathy in rats with various renal function parameters and renal ultrastructure. METHODS: Streptozotocin (55 mg/kg)-induced diabetic rats were orally treated once daily with VMNS2e (30, 60, and 120 mg/kg) for 8 weeks. The body weight and blood glucose levels of the rats were recorded during the study period. After 8 weeks of treatment creatinine clearance, urinary protein, blood urea nitrogen, urinary albumin excretion rate, and insulin levels were measured. An ultrastructure study of the kidney tissue was performed and the glomerular basement membrane thickness was measured. RESULTS: Eight weeks of VMNS2e treatment significantly reduced the fasting blood glucose level, attenuated elevating blood urea nitrogen levels, and reduced glomerular basement membrane thickness. CONCLUSION: It is concluded that VMNS2e treatment at 30 and 60 mg/kg, when given for 8 weeks, partly ameliorated early diabetic nephropathy in diabetic rats.

MADD-2, a homolog of the Opitz syndrome protein MID1, regulates guidance to the midline through UNC-40 in Caenorhabditis elegans.

The body muscles of Caenorhabditis elegans extend plasma membrane extensions called muscle arms to the midline motor axons to form the postsynaptic membrane of the neuromuscular junction. Through a screen for muscle arm development defective (Madd) mutants, we previously discovered that the UNC-40/DCC guidance receptor directs muscle arm extension through the Rho-GEF UNC-73. Here, we describe a gene identified through our mutant screen called madd-2, and show that it functions in an UNC-40 pathway. MADD-2 is a C1-TRIM protein and a homolog of human MID1, mutations in which cause Opitz Syndrome. We demonstrate that MADD-2 functions cell autonomously to direct muscle and axon extensions to the ventral midline of worms. Our results suggest that MADD-2 may enhance UNC-40 pathway activity by facilitating an interaction between UNC-40 and UNC-73. The analogous phenotypes that result from MADD-2 and MID1 mutations suggest that C1-TRIM proteins may have a conserved biological role in midline-oriented developmental events.

Disruption of tubulobulbar complex by high intratesticular estrogens leading to failed spermiation.

Spermiation is the final phase of spermatogenesis leading to release of mature spermatids into the lumen of the seminiferous tubules. Morphologically, it involves a series of events, namely removal of excess spermatid cytoplasm, removal of ectoplasmic specialization, formation of tubulobulbar complex, and final disengagement of the spermatid from the Sertoli cell. Previous studies in our laboratory have shown that administration of 17beta-estradiol at a dose of 100 microg/kg body weight for 10 d resulted in failure of spermiation. This was accompanied by a suppression of FSH and intratesticular testosterone with a concomitant rise in intratesticular 17beta-estradiol. The present study was undertaken to determine the cause of failure and subsequently the molecular events in spermiation. Electron microscopic and confocal studies revealed an absence of tubulobulbar complex in step 19 spermatids after estradiol treatment, highlighting the significance of these structures in spermiation. It was further observed that treatment affected the Sertoli cell cytoskeleton and Arp2/3 complex that is critical for de novo polymerization of actin during tubulobulbar complex formation. In conclusion, the present study reports the role of 17beta-estradiol in inhibiting the formation of tubulobulbar complex, which could be one of the mechanism by which environmental estrogens influence male fertility.

Granulosa cell apoptosis induced by a novel FSH binding inhibitory peptide from human ovarian follicular fluid.

Pituitary gonadotropins, follicle-stimulating hormone and luteinizing hormone, are the key regulators of ovarian folliculogenesis; these are known to be directly or indirectly modulated by many intraovarian factors. Our group has identified and studied one such novel peptide from human ovarian follicular fluid. Its partial N-terminal eight amino acid sequence has been deduced, referred to as octapeptide (OP). OP induces follicular atresia in mice and interferes with normal ovarian function in non-human primates, this action being similar to the native peptide. Thus, in this study, an attempt has been made to elucidate the mechanism of action of the synthetic OP by studying the pathway of follicular atresia in mouse ovary. Changes in granulosa cells were studied using various apoptotic markers by flow cytometry and immunohistochemistry. An increase in apoptotic cell population in atretic- and peptide-treated groups was observed compared with normal controls. Interestingly, both these groups exhibited differences in the apoptotic pathway. Results showed that the mitochondrial pathway was predominant in the atretic group, whereas the Fas-FasL pathway was predominant in the peptide-treated groups. The ultrastructural study also showed apoptotic changes in the OP-treated and atretic groups; the pattern of apoptosis differed at the subcellular level.

Endometrial modifications during early pregnancy in bonnet monkeys (Macaca radiata).

The present study was undertaken to investigate endometrial modifications that occur before embryo invasion in bonnet monkeys (Macaca radiata). These changes were analysed in luminal epithelium, glandular epithelium and stroma of endometrial functionalis on Day 6 post ovulation from pregnant and non-pregnant animals (n = 4 each) by transmission electron microscopy. Distinct features (i.e. loss of columnar shape by epithelial cells, changes in mitochondrial size and diffused apicolateral gap junctions) were observed in the luminal and glandular epithelium in pregnant animals. Stromal compaction was also observed in pregnant animals. Further, immunogold localisation studies demonstrated significantly higher expression (P < 0.05) of oestrogen receptor alpha, an oestrogen-regulated gene, in the glandular epithelium and stroma of the endometrium in pregnant animals compared with non-pregnant animals. Expression of two other genes known to be regulated by oestradiol, namely beta-actin and cyclo-oxygenase-1, were also significantly higher (P < 0.05) in the endometria of pregnant animals. These studies demonstrate marked changes in the endometrium before embryo invasion in bonnet monkeys. These studies also indicate altered oestrogenic activity in the uterine milieu before embryo invasion.

Identification of cellular isoform of oviduct-specific glycoprotein: role in oviduct tissue remodeling?

The oviduct is known to secrete mucins (MUC1 and MUC9) under the influence of ovarian steroids. The secreted form of MUC1 binds gametes in the oviduct, whereas the cellular form seen in breast cancers has been implicated in cell adhesion and morphogenesis. The secreted MUC9 or oviduct-specific glycoprotein (OGP), in addition to being a mucin, belongs to family 18 glycosylhydrolases and is known to bind gametes and embryos in the oviduct. Studies in our laboratory have identified non-muscle myosin IIA (involved in cell shape, polarity, and morphogenesis) as the protein partner to OGP in gametes. In view of the crucial role of the cortical cytoskeleton in the selective internalization of tight junctions (TJs) /adherent junctions (AJs) or apical junctional complex (AJC) in simple epithelial cells during tissue remodeling, the present study has been undertaken to evaluate the existence of a cellular form of OGP in oviductal tissue, which itself undergoes cyclic tissue remodeling. In silico analysis of the deduced amino-acid sequence of OGP has revealed the presence of several conserved motifs; these imply that OGP is a component of multi-protein complexes such as TJs. Corroborative immunoelectron-microscopic analysis in peri-ovulatory oviduct epithelia in the bonnet monkey has revealed the presence of OGP at the TJ. Co-localization studies of OGP and cadherin demonstrate that, whereas OGP is localized at the tonofilaments of the TJs, cadherin is localized at the intercellular space of the AJ. The possible role of OGP in oviductal tissue remodeling is discussed in light of the present findings and those reported in the literature.

Spatial distribution of actin and tubulin in human sperm nuclear matrix-intermediate filament whole mounts-a new paradigm.

Sperm is a highly differentiated cell streamlined for fertilization. The function is thus heavily dependent on the cytoskeletal organization. Conventional methods limit the appreciation and correlation of this intricate cytoskeletal filament network in the context of an entire sperm. Our recent successful localization of nonmuscle myosin IIA on sperm nuclear matrix-intermediate filament (NM-IF) preparations from fertile men by embedment-free electron microscopy (EF-EM), prompted us to investigate the antigenic distribution of two major cytoskeletal proteins-actin and tubulin. The NM-IF preparations were subjected to a cocktail of buffered paraformaldehyde (2%) with a low concentration of glutaraldehyde (0.05%). These proteins were localized by indirect immunogold technique using EF-EM on sperm NM-IF whole mounts. Ultrastructure analysis revealed well preserved centrioles, outer dense fibers, axonemal filaments, and submitochondrial reticulum in the sperm NM-IF. Immunoreactive actin was localized along the length of the sperm whereas beta-tubulin was present in the axoneme alone. The spatial distribution of actin and tubulin in normal human sperm NM-IF reported here together with that of myosin on whole mount offers a powerful technique to understand sperm cytoskeletal supramolecular structure.

N-terminal region of progesterone receptor B isoform in human spermatozoa.

Progesterone is known to act on human spermatozoa by an unidentified membrane receptor. Previous studies have demonstrated the existence of transcripts of conventional progesterone receptor (PR) in sperm RNA; antibodies directed against the C-terminal region of the conventional PR recognize a protein in sperm extracts. The present study aimed at characterizing the sperm PR using probes unique to the N-terminal region of the PR-B isoform. PR-B transcripts that were homologous to the conventional PR were detected in sperm RNA and localized in the midpiece region. Using specific antibody against the N-terminal region of PR-B, strong immunoreactivity was observed on the acrosomal region of digitonin-treated spermatozoa; Western blot analysis revealed a single band of approximately 55 kDa. Immunogold labelling studies using the same antibody localized the protein at the inner acrosomal membrane of testicular spermatids. This antibody blocked the binding of fluorescent-tagged progesterone to digitonin-treated spermatozoa and inhibited the progesterone-mediated kinase activation. The results of the present study gives an insight to speculate that the sperm membrane PR may have homology at the N-terminal region of the conventional PR-B isoform, or the membrane PR protein may share structural motifs that allows progesterone binding and interactions with the antibodies against the conventional PR.

Effect of paternal administration of an antiestrogen, tamoxifen on embryo development in rats.

We have earlier reported that oral administration of tamoxifen causes a dose-dependent reduction in the fertility of adult male rats. The decrease in fertility was mainly due to an increase in pre-implantation loss without an effect on fertilizing ability. During the study, an increased incidence of post-implantation loss of conceptuses sired by tamoxifen-treated male rats was observed. A detailed study was undertaken to investigate dose-related changes in pre- and post-implantation loss and the stage(s) of development at which these losses occurred. The present study demonstrates that tamoxifen treatment produced few normal litters as well as significantly increased pre-implantation loss without affecting the rate of fertilization. Also a significant increase in the number of degenerating embryos at the 2-4-cell stage (days 1-2 of gestation), retrieved from the oviduct/uterus of females mated with tamoxifen-treated males was observed. Histology of the resorbed fetuses, in both control and treated groups, showed presence of trophoblast outgrowth indicative of early placenta formation, which normally occurs on days 8-9 of gestation. The present results suggest that pre-implantation loss occurred at the 2-4-cell stage and the post-implantation loss occurred around days 8-9 of gestation, i.e. around midgestation. The possible effects of paternal tamoxifen treatment on embryogenesis may be due to the reduction of androgens or by the blockage of the estrogen receptor by tamoxifen, thereby affecting germ cell maturation during spermatogenesis.