In vitro effects of simulated microgravity on Sertoli cell function

With the advent of space flights questions concerning the effects of microgravity (0G) on human reproductive physiology have received great attention. The aim of this study was to evaluate the influence of 0G on Sertoli cells. A Sertoli cell line from mouse testis (42GPA9) was analyzed for cytoskeletal and Sex Hormone Binding Globilin (SHBG) changes by immunohistochemistry, for antioxidant content by RT-PCR and for culture medium lactate concentrations by protein chemistry. Cells were cultured for 6, 24 and 48 h on a three-dimensional Random Positioning Machine (3D-RPM); static controls (1G) were positioned on the supporting frame. At the end of each experiment, cultured cells were either fixed in paraformaldehyde or lysed and RNA-extracted or used for culture medium lactate measurements as needed. At 0G, Sertoli cytoskeleton became disorganized, microtubules fragmented and SHBG undetectable already after 24 h, with alterations worsening by 48 h. It was evident that various antioxidant systems appreciably increased during the first 24 h but significantly decreased at 48 h. No changes occurred in the 1G samples. Initially, 0G seemed to disturb antioxidant pro- tection strategies allowing the testes to support sperm production, thus generating an aging-like state of oxidative stress. Lactate pro- duction at 0G slightly decreased after 24 h. Further experiments are needed in space to investigate upon steroidogenesis and germ cell differentiation within the testis, to rule out male infertility as a possible consequence, which could be a problem, as life expectancy increases.With the advent of space flights questions concerning the effects of microgravity (0G) on human reproductive physiology have 13 received great attention. The aim of this study was to evaluate the influence of 0G on Sertoli cells. A Sertoli cell line from mouse testis 14 (42GPA9) was analyzed for cytoskeletal and Sex Hormone Binding Globilin (SHBG) changes by immunohistochemistry, for antioxidant 15 content by RT-PCR and for culture medium lactate concentrations by protein chemistry. Cells were cultured for 6, 24 and 48 h on a 16 three-dimensional Random Positioning Machine (3D-RPM); static controls (1G) were positioned on the supporting frame. At the 17 end of each experiment, cultured cells were either fixed in paraformaldehyde or lysed and RNA-extracted or used for culture medium 18 lactate measurements as needed. At 0G, Sertoli cytoskeleton became disorganized, microtubules fragmented and SHBG undetectable 19 already after 24 h, with alterations worsening by 48 h. It was evident that various antioxidant systems appreciably increased during the 20 first 24 h but significantly decreased at 48 h. No changes occurred in the 1G samples. Initially, 0G seemed to disturb antioxidant pro- 21 tection strategies allowing the testes to support sperm production, thus generating an aging-like state of oxidative stress. Lactate pro- 22 duction at 0G slightly decreased after 24 h. Further experiments are needed in space to investigate upon steroidogenesis and germ 23 cell differentiation within the testis, to rule out male infertility as a possible consequence, which could be a problem, as life expectancy 24 increase

Nitric oxide and cyclic nucleotides: Their roles in junction dynamics and spermatogenesis

Spermatogenesis is a highly complicated process in which functional spermatozoa (haploid, 1n) are generated from primitive mitotic spermatogonia (diploid, 2n). This process involves the differentiation and transformation of several types of germ cells as spermatocytes and spermatids undergo meiosis and differentiation. Due to its sophistication and complexity, testis possesses intrinsic mechanisms to modulate and regulate different stages of germ cell development under the intimate and indirect cooperation with Sertoli and Leydig cells, respectively. Furthermore, developing germ cells must translocate from the basal to the apical (adluminal) compartment of the seminiferous epithelium. Thus, extensive junction restructuring must occur to assist germ cell movement. Within the seminiferous tubules, three principal types of junctions are found namely anchoring junctions, tight junctions, and gap junctions. Other less studied junctions are desmosome-like junctions and hemidesmosome junctions. With these varieties of junction types, testes are using different regulators to monitor junction turnover. Among the uncountable junction modulators, nitric oxide (NO) is a prominent candidate due to its versatility and extensive downstream network. NO is synthesized by nitric oxide synthase (NOS). Three traditional NOS, specified as endothelial NOS (eNOS), inducible NOS (iNOS), and neuronal NOS (nNOS), and one testis-specific nNOS (TnNOS) are found in the testis. For these, eNOS and iNOS were recently shown to have putative junction regulation properties. More important, these two NOSs likely rely on the downstream soluble guanylyl cyclase/cGMP/protein kinase G signaling pathway to regulate the structural components at the tight junctions and adherens junctions in the testes. Apart from the involvement in junction regulation, NOS/NO also participates in controlling the levels of cytokines and hormones in the testes. On the other hand, NO is playing a unique role in modulating germ cell viability and development, and indirectly acting on some aspects of male infertility and testicular pathological conditions. Thus, NOS/NO bears an irreplaceable role in maintaining the homeostasis of the microenvironment in the seminiferous epithelium via its different downstream signaling pathways

Steroid sulfatase activity in homogenates, microsomes and purified Leydig cells from adult rat testis

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Connexin expression and gap junctional intercellular communication in human first trimester trophoblast

Connexin (Cx) expression and gap junctional intercellular communication (GJIC) are involved in development and differentiation processes, and recently mutation of connexin genes has been implicated in pathologies. In the human placenta, two distinct differentiation pathways of cytotrophoblastic cells coexist and lead to a fusion phenotype (villous trophoblast) and a proliferative/invasive phenotype (extravillous trophoblast). Here we characterized in situ and in vitro the expression of Cx transcripts and proteins in the villous and extravillous trophoblast of first trimester placenta. In addition, the GJIC functionality was investigated using the gap-fluorescence recovery after photobleaching (gap-FRAP) method. We demonstrated in the villous trophoblast the presence of Cx43 mRNA and of Cx43 protein localized between cytotrophoblastic cells and between cytotrophoblastic cells and syncytiotrophoblast. In vitro, a transient functional gap junctional intertrophoblastic communication was demonstrated during the trophoblast fusion leading to the multinucleated syncytiotrophoblast. During the proliferative process of the extravillous trophoblast, Cx40 is expressed in the proximal part of the cell columns. When cytotrophoblastic cells were cultured on Matrigel(R) for 2 days, alpha5beta1 integrin expression was observed concomitant with the presence of Cx40 mRNA and of Cx40 protein between the cells. No evidence for a GJIC was detected in this induced extravillous phenotype. In addition, Cx32 was detected between some aggregated cells after 72 h of culture. Our data show that the presence of Cx43 allows an inter-trophoblastic GJIC and is associated with the fusion process leading to the villous syncytiotrophoblast and that the presence of Cx40 does not allow GJIC and is associated with the extravillous phenotype

lnvolvement of gap junctional communication and connexin expression in trophoblast differentiation of the human placenta

Gap junctional intercellular communication (GJIC) permits coordinated cellular activities during development and differentiation processes, and its dysfunction or mutation of connexin genes have been irnplicated in pathologies. In the human placenta, two distinct differentiation pathways of cytotrophoblastic cell coexist leading to a double model: fusion phenotype (villous trophoblast) and proliferative/invasive phenotype (extravillous trophoblast). This review focuses on current knowledge on the connexin expression and the irnplication of GJIC in trophoblastic differentiation. Experimental evidence obtained in hurnan placenta demonstrates the involvement of connexin 43-gap junctions in the trophoblastic fusion process and of a connexin switch during the spatially and ternporally controlled proliferation/invasion process

Reg I protein in healthy and seminoma human testis

Regenerating gene (Reg), encodes a secretory protein with growth and differentiation stimulating effects mostly in digestive tissues. Overexpression of Reg proteins and specifically of Reg I, one member of the Reg family, is associated with several human diseases and cancers. In the present study we analyzed the expression of Reg I in normal rodent and human testes where germ cells normally proliferate and differentiate into spermatozoa, and in seminoma testis, the most common cancer of young men. Western blot analyses demonstrated the presence of a specific band at 19 kDa in human and rodent testis extracts. Immunofluorescence and deconvolution microscopy demonstrated that Reg I was present within the seminiferous tubules in both Sertoli and germ cells. By using a Sertoli cell line we demonstrated that Reg I was localized at the plasma membrane even in the absence of contact between neighboring cells and appeared before the tight junction associated protein ZO-1 was revealed at this location. Reg I was strongly expressed in human seminoma testis tissue and in a human tumor germ cell line where the immunoreactive signal was mainly detected at the plasma membrane level. These data showing for the first time the weak presence of Reg I in the normal testis and its strong expression in the testis cancer suggest a potential role of Reg I in normal and neoplastic germ cell proliferation