Mónica H. Vazquez-Levin

Spermatozoa capture HIV-1 through heparan sulfate and efficiently transmit the virus to dendritic cells.

Semen is the main vector for HIV-1 dissemination worldwide. It contains three major sources of infectious virus: free virions, infected leukocytes, and spermatozoa-associated virions. We focused on the interaction of HIV-1 with human spermatozoa and dendritic cells (DCs). We report that heparan sulfate is expressed in spermatozoa and plays an important role in the capture of HIV-1. Spermatozoa-attached virus is efficiently transmitted to DCs, macrophages, and T cells. Interaction of spermatozoa with DCs not only leads to the transmission of HIV-1 and the internalization of the spermatozoa but also results in the phenotypic maturation of DCs and the production of IL-10 but not IL-12p70. At low values of extracellular pH (∼6.5 pH units), similar to those found in the vaginal mucosa after sexual intercourse, the binding of HIV-1 to the spermatozoa and the consequent transmission of HIV-1 to DCs were strongly enhanced. Our observations support the notion that far from being a passive carrier, spermatozoa acting in concert with DCs might affect the early course of sexual transmission of HIV-1 infection.

Evidence of the presence of calcium/calmodulin-dependent protein kinase IV in human sperm and its involvement in motility regulation

The mechanisms involved in the regulation of mammalian sperm motility are not well understood. Calcium ions (Ca2+) have been suggested to play a key role in the maintenance of motility; nevertheless, how Ca2+ modulates this process has not yet been completely characterized. Ca2+ can bind to calmodulin and this complex regulates the activity of multiple enzymes, including Ca2+/calmodulin-dependent protein kinases (CaM kinases). Results from this study confirmed that the presence of Ca2+ in the incubation medium is essential for maintaining human sperm motility. The involvement of CaM kinases in Ca2+ regulation of human sperm motility was evaluated using specific inhibitors (KN62 and KN93) or their inactive analogues (KN04 and KN92 respectively). Sperm incubation in the presence of KN62 or KN93 led to a progressive decrease in the percentage of motile cells; in particular, incubation with KN62 also reduced sperm motility parameters. These inhibitors did not alter sperm viability, protein tyrosine phosphorylation or the follicular fluid-induced acrosome reaction; however, KN62 decreased the total amount of ATP in human sperm. Immunological studies showed that Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) is present and localizes to the human sperm flagellum. Moreover, CaMKIV activity increases during capacitation and is inhibited in the presence of KN62. This report is the first to demonstrate the presence of CaMKIV in mammalian sperm and suggests the involvement of this kinase in the regulation of human sperm motility.

Spermatozoa capture HIV-1 through heparan sulfate and efficiently transmit the virus to dendritic cells

Results Flow cytometry showed that heparan sulfate is expressed in spermatozoa. Heparan sulfate plays an important role in the capture of HIV-1, as demonstrated by the inhibitory effect induced by heparine (50 U/ml) (>70% capture inhibition, n = 15) and heparinase II pre-treatment of the spermatozoa (>50% capture inhibition, n = 6). By contrast, treatment with the inhibitor of mannose receptor mannan (5 mg/ml) slightly inhibited virus attachment (> 20% capture inhibition, n = 10). Spermatozoa-attached viruses were efficiently transmitted to DCs through a cellto-cell contact-dependent mechanism. Fluorescence, confocal and electronic microscopy showed that this process was associated to the internalization of a fraction of the spermatozoa. This interaction also resulted in the phenotypic maturation of DCs (up-regulation of CD80, CD86, CD40, CD83 and CCR7), and the production of IL-10 but not IL-12p70. Finally, we found that acidic extracellular pH levels, similar to those found in the vaginal mucosa after sexual intercourse, increased more than four times (n = 12) the binding of HIV-1 to the spermatozoa and the subsequent transmission of HIV-1 to DCs.

Male immunologic infertility: Sperm performance on in vitro fertilization

Abstract Objective : To analyze sperm performance in a group of patients with male immunologic infertility treated with IVF-ET. Design : Retrospective clinical study. Setting : Patients attending a private IVF clinic. Patient(s) : The study group comprised seven men with significant levels of surface-bound antisperm antibodies treated in nine IVF cycles. The control group comprised nine couples with female tubal infertility and no indication of male factor infertility treated on the same cycle. Intervention(s) : None. Main Outcome Measure(s) : Fertilization rate, early embryonic development, implantation, and clinical pregnancy rate (PR). Result(s) : Forty-six (44.2%) of 104 inseminated oocytes were fertilized in the study group compared with 65 (84.4%) of 77 in the control group, which was a significant difference. Surfacebound antisperm antibodies significantly inhibited early embryonic cleavage in the study group (13 [28.3%] of 46 embryos with at least 3 blastomeres) compared with the control group (41 [63.1%] of 65 embryos, with at least 3 blastomeres). The percentage of good-quality embryos (grades 1 and 2) was similar in the study and control groups (71.7% and 78.5%, respectively). The percentage of poor-quality embryos (grade 4 and two pronuclei) was higher in the study group compared with the control group (13.9% versus 9.2%, respectively); however, the difference was not significant. The implantation rate and clinical PR were lower in the study group (3% and 11%, respectively) compared with the control group (9.5% and 44%, respectively), but the difference was not statistically significant. Conclusion(s) : The fertilization rate and early embryonic cleavage of human embryos was found to be reduced significantly in patients with high levels of surface-bound antisperm antibodies. Moreover, embryonic quality and the PR may be compromised by the presence of significant levels of surface-bound antisperm antibodies.

Glucose-regulated protein 78 (Grp78/BiP) is secreted by human oviduct epithelial cells and the recombinant protein modulates sperm-zona pellucida binding.

OBJECTIVE To determine the secretion of Grp78 by human oviduct epithelial cells, its association to spermatozoa, and its involvement in gamete interaction. DESIGN Prospective study. SETTING Basic research laboratory. SUBJECT(S) Semen samples obtained from normozoospermic volunteers. Tubal tissue provided by patients undergoing hysterectomies. Oocytes collected from women undergoing IVF-ET. INTERVENTION(S) Analysis of Grp78 expression and secretion by oviductal tissue. Gamete incubation with recombinant Grp78 (rec-Grp78). MAIN OUTCOME MEASURE(S) Assessment of protein expression and secretion by immunohistochemistry and Western immunoblotting, respectively. Evaluation of rec-Grp78 binding to human spermatozoa by immunocytochemistry, and analysis of its effect upon gamete interaction using the hemizona assay. RESULT(S) Grp78 was found in the surface of oviduct epithelial cells. Soluble Grp78 was detected in oviductal fluids from women in the periovulatory period and in oviductal tissue conditioned medium. Rec-Grp78 was able to bind to the sperm acrosomal cap, and its presence during gamete interaction led to a decrease in the number of spermatozoa bound to the zona pellucida (ZP). When calcium ions from the incubation medium were replaced by strontium, rec-Grp78 enhanced sperm-ZP interaction. CONCLUSION(S) Grp78 is expressed and secreted by oviduct epithelial cells. The protein would bind to the gametes and may modulate their interaction in a calcium-dependent manner.

Molecular bases of endometrial cancer: New roles for new actors in the diagnosis and the therapy of the disease

Endometrial carcinoma (EC) is the most commonly diagnosed gynecologic malignancy in the western world. The majority of these cancers are curable, but a subset about 15-20% of endometrial tumors exhibits an aggressive phenotype. Based on clinic-pathological and molecular characteristics, EC has been classified into two groups: Type I estrogen-dependent adenocarcinomas, which have a good prognosis and an endometrioid histology, and Type II or non-estrogen-dependent EC associated with poor prognosis and non-endometrioid histology. EC develops as a result of a stepwise accumulation of alterations that seem to be specific of each histological type. However, more knowledge is needed to better understand the differences in the biology and the clinical outcome of EC. We would like to highlight the need to explore new potential biomarkers of EC as a tool for the detection and monitoring of aggressive endometrial tumors that, at the same time, will allow us to develop novel and more selective molecular targeted therapies against EC.

Anandamide Capacitates Bull Spermatozoa through CB1 and TRPV1 Activation

Anandamide (AEA), a major endocannabinoid, binds to cannabinoid and vanilloid receptors (CB1, CB2 and TRPV1) and affects many reproductive functions. Nanomolar levels of anandamide are found in reproductive fluids including mid-cycle oviductal fluid. Previously, we found that R(+)-methanandamide, an anandamide analogue, induces sperm releasing from bovine oviductal epithelium and the CB1 antagonist, SR141716A, reversed this effect. Since sperm detachment may be due to surface remodeling brought about by capacitation, the aim of this paper was to investigate whether anandamide at physiological concentrations could act as a capacitating agent in bull spermatozoa. We demonstrated that at nanomolar concentrations R(+)-methanandamide or anandamide induced bull sperm capacitation, whereas SR141716A and capsazepine (a TRPV1 antagonist) inhibited this induction. Previous studies indicate that mammalian spermatozoa possess the enzymatic machinery to produce and degrade their own AEA via the actions of the AEA-synthesizing phospholipase D and the fatty acid amide hydrolase (FAAH) respectively. Our results indicated that, URB597, a potent inhibitor of the FAAH, produced effects on bovine sperm capacitation similar to those elicited by exogenous AEA suggesting that this process is normally regulated by an endogenous tone. We also investigated whether anandamide is involved in bovine heparin-capacitated spermatozoa, since heparin is a known capacitating agent of bovine sperm. When the spermatozoa were incubated in the presence of R(+)-methanandamide and heparin, the percentage of capacitated spermatozoa was similar to that in the presence of R(+)-methanandamide alone. The pre-incubation with CB1 or TRPV1 antagonists inhibited heparin-induced sperm capacitation; moreover the activity of FAAH was 30% lower in heparin-capacitated spermatozoa as compared to control conditions. This suggests that heparin may increase endogenous anandamide levels. Our findings indicate that anandamide induces sperm capacitation through the activation of CB1 and TRPV1 receptors and could be involved in the same molecular pathway as heparin in bovines.

Calcium requirements for human sperm function in vitro

OBJECTIVE To determine extracellular calcium (Ca(2+)) requirements for the maintenance of human sperm function in vitro. DESIGN Prospective study. SETTING Basic research laboratory. PATIENT(S) Normozoospermic volunteers provided fresh semen samples; follicular fluid (human FF) and oocytes were collected from women undergoing IVF-ET. INTERVENTION(S) Spermatozoa were incubated for </=18 hours in media containing different CaCl(2) concentrations (maximum, 2.5 mM [control]). MAIN OUTCOME MEASURES Protein tyrosine phosphorylation patterns, development of hyperactivated motility, induction of the acrosome reaction (AR) in response to human FF, and sperm interaction with homologous zona pellucida (ZP). RESULT(S) Cells maintained for 18 hours in medium containing >/=0.1 mM of Ca(2+) were able to undergo the AR when exposed to human FF in the presence of 2.5 mM of Ca(2+). Calcium concentrations of >/=0.22 mM were sufficient to reach protein tyrosine phosphorylation levels and hyperactivated motility values similar to those of controls. Higher Ca(2+) concentrations (>/=0.58 mM) were required to produce maximum human FF-induced AR in previously capacitated cells and to obtain an adequate sperm-ZP binding. CONCLUSION(S) Different steps of the fertilization process have distinctive Ca(2+) requirements. Whereas 0.22 mM of Ca(2+) is sufficient for the development of some capacitation-related events, human FF-induced AR and sperm-ZP interaction require 0.58 mM of this cation.

ETV5 transcription factor is overexpressed in ovarian cancer and regulates cell adhesion in ovarian cancer cells

Epithelial ovarian cancer is the most lethal gynecological malignancy and the fifth leading cause of cancer deaths in women in the Western world. ETS transcription factors are known to act as positive or negative regulators of the expression of genes that are involved in various biological processes, including those that control cellular proliferation, differentiation, apoptosis, tissue remodeling, angiogenesis and transformation. ETV5 belongs to the PEA3 subfamily. PEA3 subfamily members are able to activate the transcription of proteases, matrix metalloproteinases and tissue inhibitor of metalloproteases, which is central to both tumor invasion and angiogenesis. Here, we examined the role of the ETV5 transcription factor in epithelial ovarian cancer and we found ETV5 was upregulated in ovarian tumor samples compared to ovarian tissue controls. The in vitro inhibition of ETV5 decreased cell proliferation in serum‐deprived conditions, induced EMT and cell migration and decreased cell adhesion to extracellular matrix components. ETV5 inhibition also decreased cell–cell adhesion and induced apoptosis in anchorage‐independent conditions. Accordingly, upregulation of ETV5 induced the expression of cell adhesion molecules and enhanced cell survival in a spheroid model. Our findings suggest that the overexpression of ETV5 detected in ovarian cancer cells may contribute to ovarian tumor progression through the ability of ETV5 to enhance proliferation of ovarian cancer cells. In addition, upregulation of ETV5 would play a role in ovarian cancer cell dissemination and metastasis into the peritoneal cavity by protecting ovarian cancer cells from apoptosis and by increasing the adhesion of ovarian cancer cells to the peritoneal wall through the regulation of cell adhesion molecules.

ETV5 cooperates with LPP as a sensor of extracellular signals and promotes EMT in endometrial carcinomas

Endometrial carcinoma (EC) is the most frequent among infiltrating tumors of the female genital tract, with myometrial invasion representing an increase in the rate of recurrences and a decrease in survival. We have previously described ETV5 transcription factor associated with myometrial infiltration in human ECs. In this work, we further investigated ETV5 orchestrating downstream effects to confer the tumor the invasive capabilities needed to disseminate in the early stages of EC dissemination. Molecular profiling evidenced ETV5 having a direct role on epithelial-to-mesenchymal transition (EMT). In particular, ETV5 modulated Zeb1 expression and E-Cadherin repression leading to a complete reorganization of cell–cell and cell–substrate contacts. ETV5-promoted EMT resulted in the acquisition of migratory and invasive capabilities in endometrial cell lines. Furthermore, we identified the lipoma-preferred partner protein as a regulatory partner of ETV5, acting as a sensor for extracellular signals promoting tumor invasion. All together, we propose ETV5-transcriptional regulation of the EMT process through a crosstalk with the tumor surrounding microenvironment, as a principal event initiating EC invasion.