Functional role of hepatocyte growth factor receptor during sperm maturation

Mammalian spermatozoa acquire motility and fertilizing capacity during their transit through the epididymis. Hepatocyte growth factor (HGF) is a pleiotropic cytokine with potent motogenic capacities that has been identified in different organs, including the mammalian male genital tract. In mice, HGF is present in the testis and, in large amounts, in the distal part of the epididymis. In prepuberal rats, we have demonstrated that HGF is synthesized by the peritubular myoid cells and in men, HGF is present in significant quantities in seminal plasma. It has been suggested that in mice, HGF has a role in initiating sperm motility, whereas in men, no significant correlations between HGF concentration and sperm motility have been found. In the present paper we report that in rats, HGF receptor, c-met, is expressed in testicular and epididymal spermatozoa. Through immunocytochemistry, we have found that c-met is exclusively localized on the head in testicular sperm. A different localization of c-met has been found in sperm isolated from caput and cauda epididymidis. Cells isolated from epididymal caput show a c-met localization exclusively restricted to the head in most cells. In a minority of caput epididymis spermatozoa the receptor is localized both in the cell head and along the flagellum. Spermatozoa isolated from the epididymal cauda were quite homogeneous, showing the receptor localized along the entire cell surface. We also report that HGF is synthesized and secreted by the rat epididymis as indicated by the scatter effect of epididymal cell, homogenate and culture medium on MDCK cells. To clarify whether HGF is involved in the acquisition of sperm motility in the epididymis, its maintenance, or both, spermatozoa isolated from caput epididymidis have been cultured in medium alone or supplemented with HGF. The results obtained indicated that HGF has a positive effect on the maintenance of sperm motility which, in the absence of HGF, significantly decreases during the first hour of culture, whereas it is maintained for at least 3 hours when HGF is present in the culture medium. We also report that HGF does not influence spermatozoa viability as indicated by the cytometrical analysis of propidium iodide-labeled sperm; an equal number of dead cells appeared in control and in HGF-treated preparations. In conclusion, our data strongly support the hypothesis that HGF positively influences sperm motility maintenance during sperm transit through the epididymis, indicating that c-met receptor and its ligand, HGF, have a role in male fertility

Pleiotropic activityof hepathocyte growth factor (HGF) during embryonic mouse testis development .

The hepatocyte growth factor (HGF) is a pleiotropic cytokine whose action is mediated by c-met, a glycoproteic receptor with tyrosine kinase activity which transduces its multiple biological activities including cell proliferation, motility and differentiation. During embryonic development HGF acts as a morphogenetic factor as previously demonstrated for metanephric and lung development. Recently, culturing male genital ridges, we demonstrated that HGF is able to support in vitro testicular cord formation. In the present paper we report the expression pattern of the HGF gene during embryonic testis development and the multiple roles exerted by this factor during the morphogenesis of this organ. Northern blot analysis reveals a positive signal in urogenital ridges isolated from 11.5 days post coitum (dpc) embryos and in testes isolated from 13.5 and 15.5 dpc male embryos. On the contrary HGF mRNA is undetectable in ovaries isolated from 13.5 and 15.5 dpc embryos. Moreover, we demonstrate that HGF is synthesized and secreted by the male gonad and is biologically active. These data indicate a male specific biological function of HGF during embryonic gonadal development. This hypothesis is supported by the in vitro demonstration that HGF acts as a migratory factor for male mesonephric cells which is a male specific event. In addition we demonstrate that during testicular development, HGF acts as a morphogenetic factor able to reorganize dissociated testicular cells which, under HGF stimulation, form a tridimensional network of cord-like structures. Finally, we demonstrate that HGF induces testicular cell proliferation in this way being responsible for the size increase of the testis. All together the data presented in this paper demonstrate that HGF is expressed during the embryonic development of the testis and clarify the multiple roles exerted by this factor during the morphogenesis of the male gonad

Expression and functional role of hepatocyte growth factor receptor (C-MET) during postnatal rat testis development

The met protooncogene encodes the hepatocyte growth factor receptor (HGFR, c-met). C-met, a tyrosine kinase receptor protein, is widely expressed in different cell types including the male reproductive tract. As we recently demonstrated, both c-met messenger RNA and protein are expressed in prebuberal rat testis. The aim of this work was to detect the expression of c-met during postnatal testis development and to study its functional role. Our findings show that in total rat testis e-met is expressed during postnatal life until the sexual maturation of the animals. To evaluate the receptor expression in the different cell types in the testis, homogeneous cell populations of Sertoli and peritubular myoid cells were isolated from the seminiferous tubules of 10- and 35-day-old animals. c-met gene is expressed in myoid cells at the ages considered and its expression decreases with increasing age. By contrast, in Sertoli cells c-met expression is first detectable at 25 days of Life and its expression increases with the increasing age being well evident at 35 days of age. C-met protein was detected by immunocytochemistry and its expression correlates with gene expression. The receptor is functionally active because HGF administration induces morphological changes in myoid cells and in c-met-expressing Sertoli cells. As a consequence of HGF addition, Sertoli cells cultured on reconstituted basement membrane reorganize into cord-like structures that resemble testicular seminiferous cords. The data here reported demonstrate for the first: time that in Sertoli cells c-met expression is developmentally regulated being present and functionally active in postpuberal Sertoli cells. Given that c-met expression persists in myoid cells during postnatal testis development and that in Sertoli cells its expression correlates over time with germ cell differentiation and lumen formation, we conclude that the c-met/HGF system is involved in testis development and function

Vitamin A metabolism in cultured somatic cells from rat testis

Sertoli and peritubular myoid cells, the somatic cells of the seminiferous tubule, support growth and differentiation of developing germ cells. This action strictly depends on the availability of in situ synthesized retinoic acid and we have previously documented the ability of Sertoli, but not peritubular cell extracts, to support the oxidation of retinol to retinoic acid. Using primary cultures of somatic cells treated with a physiological concentration of free retinol, we show here that the same is essentially true also for whole cultured cells. Sertoli cells are capable of producing not only retinoic acid, but are also the major site of retinyl ester (mainly, retinyl palmitate) formation. Compared with retinyl palmitate accumulation, retinoic acid synthesis was both faster and positively influenced by prior exposure to retinol. This increase in retinoic acid synthesis was further augmented by treatment with the retinoic acid catabolic inhibitor liarozole, thus indicating that enhanced synthesis, rather than reduced catabolism, is responsible for such an effect. Myoid cells had a higher capacity to incorporate exogenously supplied retinol, yet retinoic acid synthesis, and even more so retinyl palmitate formation, were considerably lower than in Sertoli cells. Retinoic acid synthesis in myoid cells was not only depressed, but also very little influenced by prior retinol exposure and totally insensitive to liarozole. These data further support the view that myoid cells are involved in retinol uptake from the blood and its transfer to other cells, rather than in metabolic interconversion or long-term storage of vitamin A, two processes that mainly take place in Sertoli cells

Hepatocyte growth factor modulates in vitro survival and proliferation of germ cells during postnatal testis development

The hepatocyte growth factor (HGF) is a pleiotropic cytokine that influences mitogenesis, motility and differentiation of many different cell types by its tyrosine kinase receptor c-Met. We previously demonstrated that the c-Met/HGF systern is present and functionally active during postnatal testis development. We found also that spermatozoa express c-Met and that HGF has a positive effect on the maintenance of sperm motility. In the present paper, we extend our study on the germ cells at different stages of differentiation during the postnatal development of the testis. We demonstrate that c-met is present in rat spermatogonia, pachytene spermatocytes and round spermatids and that HGF significantly increases spermatogonial proliferation in 8- to 10-day-old prepubertal rats. At this age HGF does not affect Sertoli cells and peritubular myoid cells proliferation. In addition, we studied the effect of the factor on germ cell apoptosis and we show that HGF prevents the genii cell apoptotic process. We also studied the effect of HGF on 18- to 20-day-old and 28- to 30-day-old rat testes. At these ages also the factor significantly increases genii cell duplication and decreases the number of apoptotic cells. However, the effect on programmed cell death is higher in the 8- to 10-day-old rats and declines in the older animals. In conclusion, we report that rat genii cells (spermatogonia, pachytene spermatocytes and round spermatids) express c-met and that HGF modulates genii cell proliferating activity and apoptosis in vitro. These data indicate that the c-Met/HGF system is involved in male germ cell homeostasis and, consequently, has a role in mate fertility

Defining phenotypic and functional heterogeneity of glioblastoma stem cells by mass cytometry

Most patients with glioblastoma (GBM) die within 2 years. A major therapeutic goal is to target GBM stem cells (GSCs), a subpopulation of cells that contribute to treatment resistance and recurrence. Since their discovery in 2003, GSCs have been isolated using single-surface markers, such as CD15, CD44, CD133, and α6 integrin. It remains unknown how these single-surface marker-defined GSC populations compare with each other in terms of signaling and function and whether expression of different combinations of these markers is associated with different functional capacity. Using mass cytometry and fresh operating room specimens, we found 15 distinct GSC subpopulations in patients, and they differed in their MEK/ERK, WNT, and AKT pathway activation status. Once in culture, some subpopulations were lost and previously undetectable ones materialized. GSCs that highly expressed all 4 surface markers had the greatest self-renewal capacity, WNT inhibitor sensitivity, and in vivo tumorigenicity. This work highlights the potential signaling and phenotypic diversity of GSCs. Larger patient sample sizes and antibody panels are required to confirm these findings

Association of kidney disease measures with risk of renal function worsening in patients with type 1 diabetes

Background: Albuminuria has been classically considered a marker of kidney damage progression in diabetic patients and it is routinely assessed to monitor kidney function. However, the role of a mild GFR reduction on the development of stage 653 CKD has been less explored in type 1 diabetes mellitus (T1DM) patients. Aim of the present study was to evaluate the prognostic role of kidney disease measures, namely albuminuria and reduced GFR, on the development of stage 653 CKD in a large cohort of patients affected by T1DM. Methods: A total of 4284 patients affected by T1DM followed-up at 76 diabetes centers participating to the Italian Association of Clinical Diabetologists (Associazione Medici Diabetologi, AMD) initiative constitutes the study population. Urinary albumin excretion (ACR) and estimated GFR (eGFR) were retrieved and analyzed. The incidence of stage 653 CKD (eGFR < 60 mL/min/1.73 m2) or eGFR reduction > 30% from baseline was evaluated. Results: The mean estimated GFR was 98 \ub1 17 mL/min/1.73m2 and the proportion of patients with albuminuria was 15.3% (n = 654) at baseline. About 8% (n = 337) of patients developed one of the two renal endpoints during the 4-year follow-up period. Age, albuminuria (micro or macro) and baseline eGFR < 90 ml/min/m2 were independent risk factors for stage 653 CKD and renal function worsening. When compared to patients with eGFR > 90 ml/min/1.73m2 and normoalbuminuria, those with albuminuria at baseline had a 1.69 greater risk of reaching stage 3 CKD, while patients with mild eGFR reduction (i.e. eGFR between 90 and 60 mL/min/1.73 m2) show a 3.81 greater risk that rose to 8.24 for those patients with albuminuria and mild eGFR reduction at baseline. Conclusions: Albuminuria and eGFR reduction represent independent risk factors for incident stage 653 CKD in T1DM patients. The simultaneous occurrence of reduced eGFR and albuminuria have a synergistic effect on renal function worsening