Fibronectin From Oviductal Cells Fluctuates During the Estrous Cycle and Contributes to Sperm-Oviduct Interaction in Cattle.

During the passage of sperm through the oviduct, spermatozoa bind to the oviductal epithelium and form the oviductal reservoir. This interaction keeps the fertilizing capacity of sperm until ovulation-associated signals induce sperm release from the oviductal epithelium, allowing the transit of spermatozoa to the fertilization site. Fibronectin is a glycoprotein from the extracellular matrix that binds to α5ß1 receptors. Fibronectin has been found to be expressed in the oviduct, whereas α5ß1 has been found to be expressed in the sperm of different species. Fibronectin is involved through α5ß1 in sperm functions. The aim of this work was to study the participation of oviductal fibronectin in the regulation of the sperm-oviduct interaction in cattle. We found that oviductal epithelial cells differentially expressed all mRNA splice variants of fibronectin during the estrous cycle. Fibronectin was localized in the apical region of oviductal epithelial cells and fibronectin levels in the oviductal fluid fluctuated during the estrous cycle. Also, bovine spermatozoa expressed α5ß1. Using in vitro sperm-oviduct co-cultures, we found that spermatozoa were attached to the oviductal epithelium through α5ß1. The incubation of co-cultures with fibronectin induced sperm release from the oviductal cells through α5ß1. The sperm population released from oviductal cells by fibronectin was enriched in motile and capacitated spermatozoa. Based on our in vitro culture system results, we propose that fibronectin and α5ß1 are involved in the sperm-oviduct interaction. Also, an increase in fibronectin levels in the oviductal fluid during the pre-ovulatory period may promote sperm release from the oviductal epithelium in cattle. J. Cell. Biochem. 118: 4095-4108, 2017. © 2017 Wiley Periodicals, Inc.

Potential role of sodium-proton exchangers in the low concentration arsenic trioxide-increased intracellular pH and cell proliferation.

Arsenic main inorganic compound is arsenic trioxide (ATO) presented in solution mainly as arsenite. ATO increases intracellular pH (pHi), cell proliferation and tumor growth. Sodium-proton exchangers (NHEs) modulate the pHi, with NHE1 playing significant roles. Whether ATO-increased cell proliferation results from altered NHEs expression and activity is unknown. We hypothesize that ATO increases cell proliferation by altering pHi due to increased NHEs-like transport activity. Madin-Darby canine kidney (MDCK) cells grown in 5 mmol/L D-glucose-containing DMEM were exposed to ATO (0.05, 0.5 or 5 µmol/L, 0-48 hours) in the absence or presence of 5-N,N-hexamethylene amiloride (HMA, 5-100 µmol/L, NHEs inhibitor), PD-98059 (30 µmol/L, MAPK1/2 inhibitor), Gö6976 (10 µmol/L, PKCα, ßI and µ inhibitor), or Schering 28080 (10 µmol/L, H(+)/K(+)ATPase inhibitor) plus concanamycin (0.1 µmol/L, V type ATPases inhibitor). Incorporation of [(3)H]thymidine was used to estimate cell proliferation, and counting cells with a hemocytometer to determine the cell number. The pHi was measured by fluorometry in 2,7-bicarboxyethyl-5,6-carboxyfluorescein loaded cells. The Na(+)-dependent HMA-sensitive NHEs-like mediated proton transport kinetics, NHE1 protein abundance in the total, cytoplasm and plasma membrane protein fractions, and phosphorylated and total p42/44 mitogen-activated protein kinases (p42/44(mapk)) were also determined. Lowest ATO (0.05 µmol/L, ~0.01 ppm) used in this study increased cell proliferation, pHi, NHEs-like transport and plasma membrane NHE1 protein abundance, effects blocked by HMA, PD-98059 or Gö6976. Cell-buffering capacity did not change by ATO. The results show that a low ATO concentration increases MDCK cells proliferation by NHEs (probably NHE1)-like transport dependent-increased pHi requiring p42/44(mapk) and PKCα, ßI and/or µ activity. This finding could be crucial in diseases where uncontrolled cell growth occurs, such as tumor growth, and in circumstances where ATO, likely arsenite, is available at the drinking-water at these levels.

Participation of the human sperm proteasome in the capacitation process and its regulation by protein kinase A and tyrosine kinase.

The proteasome is a multicatalytic cellular complex present in human sperm that plays a significant role during several steps of mammalian fertilization. Here, we present evidence that the proteasome is involved in human sperm capacitation. Aliquots of highly motile sperm were incubated with proteasome inhibitors MG132 or epoxomicin. The percentage of capacitated sperm, the chymotrypsin-like activity of the proteasome, cAMP content, and the pattern of protein phosphorylation were assayed by using the chlortetracycline hydrochloride assay, a fluorogenic substrate, the cAMP enzyme immunoassay kit, and Western blot analysis, respectively. Our results indicate that treatment of sperm with proteasome inhibitors blocks the capacitation process, does not alter cAMP concentration, and changes the pattern of protein phosphorylation. To elucidate how proteasome activity is regulated during capacitation, sperm were incubated with: 1) tyrosine kinase (TK) inhibitors (genistein or herbimycin A); 2) protein kinase (PK) A inhibitors or activators (H89 and Rp-cAMPS, and 8-Br-cAMP, respectively); or 3) PKC inhibitors (tamoxifen or staurosporin) at different capacitation times. The chymotrypsin-like activity and degree of phosphorylation of the proteasome were then assayed. The results indicate that sperm treatment with TK and PKA inhibitors significantly decreases the chymotrypsin-like activity of the proteasome during capacitation. Immunoprecipitation and Western blot results suggest that the proteasome is phosphorylated during capacitation in a TK- and PKA-dependent pathway. In conclusion, we suggest that the sperm proteasome participates in the capacitation process, and that its activity is modulated by PKs.

Type IV collagen induces down-regulation of steroidogenic response to gonadotropins in adult rat Leydig cells involving mitogen-activated protein kinase.

We have previously shown that type IV collagen (alpha1 (IV) and alpha2 (IV) collagen chains) (Col-IV) inhibits testosterone (T) production by Leydig cells (LC). The aim of this study was to analyze mechanism/s by which Col-IV exerts this effect. No significant differences in the specific binding of hCG to LH/hCG receptors in LC cultured on uncoated or Col-IV coated plates were observed. An inhibition of cAMP production in hCG-stimulated LC cultured on Col-IV was detected. The inhibition exerted by Col-IV on T production in response to hCG was also observed when cells were stimulated with 8Bromo-cAMP. In addition, conversion of steroid precursors to T in LC cultured on uncoated and Col-IV coated plates was similar. On the other hand, we detected an increase of ERK1/2 phosphorylation in hCG-stimulated LC cultured on Col-IV. Genistein added to LC cultures reduced the ability of Col-IV to increase ERK1/2 phosphorylation and reverted the inhibitory effect of Col-IV on T production. An inhibitor of MEK, PD98059 added to LC cultures also reverted the inhibitory effect of Col-IV on T production. A decrease of steroidogenic acute regulatory protein (StAR) expression in hCG-stimulated LC cultured on Col-IV coated plates that could be reverted by addition of PD98059 to the cultures was also demonstrated. All together these results suggest that Col-IV inhibits T production in LC by binding to integrins, activating ERK1/2, decreasing cAMP production and decreasing StAR expression.

Galectin-1, a cell adhesion modulator, induces apoptosis of rat Leydig cells in vitro.

Galectin-1 (Gal-1), a beta galactoside-binding lectin, is involved in multiple biological functions, such as cell adhesion, apoptosis, and metastasis. On the basis of its ability to interact with extracellular matrix (ECM) glycoproteins, we investigated the Gal-1 effect on Leydig cells, which express and are influenced by ECM proteins. In this study, Gal-1 was identified in Leydig cell cultures by immunofluorescence. To gain insight into its biological role, Gal-1 was added to purified rat Leydig cells, under both basal and human chorionic gonadotrophin-stimulated conditions. Substantial morphological changes were observed, and cell viability showed an 80% decrease after 24 h culture. As a functional consequence of Gal-1 addition, testosterone production was reduced in a dose-dependent fashion, reaching a minimum of 26% after 24 h compared with basal values. cAMP showed a similar variation after 3 h. Assessment of DNA hypodiploidy and caspase activity determinations indicated that the reduction in viability and in steroidogenesis was caused by apoptosis induced by Gal-1. Besides, addition of Gal-1 caused Leydig cell detachment. Presence of laminin-1 or lactose prevented the effect of Gal-1, suggesting that the carbohydrate recognition domain is involved in inducing apoptosis. These findings demonstrate a novel mechanism, based on Gal-1 and laminin-1 interaction, which could help us better understand the molecular basis of Leydig cell function and survival control.

Role of type IV collagen in prolactin release from anterior pituitaries of male rats.

We previously demonstrated that laminin, a component of basement membranes, modulates pituitary hormone secretion. In the present study, we evaluated the effect of type IV collagen, another component of this membrane, on the release of prolactin (PRL) by anterior pituitary gland from adult male rats. Hemipituitaries were incubated for 3 h with type IV collagen or antibodies against it and PRL release was studied. Rabbit IgG to type IV collagen at concentrations of 10(-7) - 10(-5) M had a significant stimulatory effect on PRL release, in comparison to normal rabbit serum IgG or medium alone used as controls. Type IV collagen induced a significant inhibitory effect on basal release of PRL at a concentration of 30 microg/mL. A slight decrease in PRL release was detected in thyrotropin-releasing hormone-stimulated hemipituitaries incubated with type IV collagen at all concentrations used. These results suggest that type IV collagen, similar to laminin-1, modulates PRL released from hemipituitaries, in vitro.