Emilce S. Diaz

Participation of the Human Sperm Proteasome in the Capacitation Process and Its Regulation by Protein Kinase A and Tyrosine Kinase

Abstract 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.

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, βI 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 [3H]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 (p42/44mapk) 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 p42/44mapk and PKCα, βI 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.

Protein phosphatases decrease their activity during capacitation: a new requirement for this event.

There are few reports on the role of protein phosphatases during capacitation. Here, we report on the role of PP2B, PP1, and PP2A during human sperm capacitation. Motile sperm were resuspended in non-capacitating medium (NCM, Tyrodes medium, albumin- and bicarbonate-free) or in reconstituted medium (RCM, NCM plus 2.6% albumin/25 mM bicarbonate). The presence of the phosphatases was evaluated by western blotting and the subcellular localization by indirect immunofluorescence. The function of these phosphatases was analyzed by incubating the sperm with specific inhibitors: okadaic acid, I2, endothall, and deltamethrin. Different aliquots were incubated in the following media: 1) NCM; 2) NCM plus inhibitors; 3) RCM; and 4) RCM plus inhibitors. The percent capacitated sperm and phosphatase activities were evaluated using the chlortetracycline assay and a phosphatase assay kit, respectively. The results confirm the presence of PP2B and PP1 in human sperm. We also report the presence of PP2A, specifically, the catalytic subunit and the regulatory subunits PR65 and B. PP2B and PP2A were present in the tail, neck, and postacrosomal region, and PP1 was present in the postacrosomal region, neck, middle, and principal piece of human sperm. Treatment with phosphatase inhibitors rapidly (≤1 min) increased the percent of sperm depicting the pattern B, reaching a maximum of ∼40% that was maintained throughout incubation; after 3 h, the percent of capacitated sperm was similar to that of the control. The enzymatic activity of the phosphatases decreased during capacitation without changes in their expression. The pattern of phosphorylation on threonine residues showed a sharp increase upon treatment with the inhibitors. In conclusion, human sperm express PP1, PP2B, and PP2A, and the activity of these phosphatases decreases during capacitation. This decline in phosphatase activities and the subsequent increase in threonine phosphorylation may be an important requirement for the success of sperm capacitation.

The Laminin-Induced Acrosome Reaction in Human Sperm Is Mediated by Src Kinases and the Proteasome

The aim of this work was to determine whether laminin (Ln), an extracellular matrix protein, induces the intracellular events that may be involved in producing the acrosome reaction in human sperm. To this end, we evaluated the effect of Ln on tyrosine phosphorylation, intracellular calcium concentration, proteasome activity, and phosphorylation in human sperm. Aliquots of highly motile sperm selected with a Percoll gradient, were incubated with different concentrations of Ln (0–20 μg/ml) for different periods (0–18 h). The percentage of viable acrosome-reacted sperm was evaluated using fluorescein isothiocyanate-labeled Pisum sativum agglutinin and Hoechst 33258 DNA dye. Tyrosine phosphorylation was evaluated by Western blot analysis. The chymotrypsin-like activity of the proteasome was evaluated with a fluorogenic peptide, and intracellular calcium concentration was measured with fura-2. The results indicate that Ln stimulated the acrosome reaction of human sperm in a dose-dependent manner. This increase was drastically inhibited in the presence of herbimycin A, SU6656, and epoxomicin. In addition, Ln increased proteasome activity and phosphorylation; both events were inhibited by herbimycin A and SU6656. Finally, Ln induced an increase in intracellular calcium concentration, which was inhibited by SU6656 and epoxomicin. These results suggest that Ln is able to induce the acrosome reaction. This effect may be mediated by Src kinase and the proteasome, with the consequent induction of a calcium influx.

Fibronectin stimulates human sperm capacitation through the cyclic AMP/protein kinase A pathway

STUDY QUESTION Does fibronectin (Fn) stimulate the sperm capacitation process in humans? SUMMARY ANSWER Fibronectin stimulates human sperm capacitation. WHAT IS KNOWN ALREADY Capacitation is a process that occurs in the oviduct. It has been suggested that some molecules present in the oviductal fluid and cells as well as proteins present in the cumulus oophorus could be involved in the modulation of sperm function and their acquisition of fertilizing capacity. Fibronectin is a glycoprotein that is present in the fluid and the oviduct epithelium, and its receptor (alpha 5 beta 1 integrin) is present in human sperm. When alpha 5 beta 1 (α5β1) integrin binds to fibronectin, intracellular signals similar to the process of sperm capacitation are activated. STUDY DESIGN, SIZE, DURATION Human sperm were selected via a percoll gradient and were then incubated in non-capacitated medium (NCM) or reconstituted capacitated medium (RCM), in the presence or absence of fibronectin for different time periods. A total of 39 donors were used during the study, which lasted 3 years. PARTICIPANTS/MATERIALS, SETTING, METHODS Freshly ejaculated sperm from healthy volunteers were obtained by masturbation. All semen samples were normal according to the World Health Organization parameters. Six approaches were used to determine the effects of fibronectin on sperm capacitation: chlortetracycline (CTC) assay, heterologous co-culture of human sperm with bovine oviductal epithelial cells (BOEC), measurement of cyclic (c) AMP levels, activity of protein kinase A (PKA), phosphorylation of proteins in tyrosine (Tyr) residues, and induction of acrosome reaction with progesterone. MAIN RESULTS AND THE ROLE OF CHANCE When sperm were incubated in RCM in the presence of Fn, we observed differences with respect to sperm incubated in RCM without Fn (control): (i) a 10% increase in the percentage of sperm with the B pattern (capacitated sperm) of CTC fluorescence from the beginning of capacitation (P < 0.001); (ii) an effect on both the concentration of cAMP (P < 0.05) and PKA activity (P < 0.05) during early capacitation; (iii) an increase in the degree of phosphorylation of proteins on tyrosine residues after 60 min of capacitation (P < 0.01); (iv) an increase in the percentage of acrosome-reacted sperm in response to progesterone (P < 0.05); and (v) a decrease in the percentage of sperm attached to BOEC (P < 0.05). Moreover, we noted that the effect of Fn was specific and mediated by alpha 5 beta 1 integrin (P < 0.001). Fn by itself had no effect on sperm capacitation. LIMITATIONS, REASONS FOR CAUTION This study was carried out with sperm from young adult men. Men with abnormal semen samples were excluded. The results cannot be directly extrapolated to other mammalian species. WIDER IMPLICATIONS OF THE FINDINGS Currently, male subfertility has become a huge public health problem, which makes it imperative to develop new treatments. This is a novel discovery that extends our current knowledge concerning normal and pathological sperm physiology as well as events that regulate the process of fertilization. STUDY FUNDING/COMPETING INTERESTS This study was supported by grants from FONDECYT (1130341, E.S.D. and 1120056, P.M.) and FONCYT (PIP 2011-0496, S.P.-M). The authors have no conflicts of interest.

Participation of protein kinases and phosphatases in the progesterone-induced acrosome reaction and calcium influx in human spermatozoa

In human spermatozoa, protein kinases have a role in the acrosome reaction (AR) induced by a variety of stimuli. However, there is disagreement or a lack of information regarding the role of protein kinases and phosphatases in the progesterone (P)‐induced increase in intracellular calcium concentration ([Ca2+]i). In addition, there are no studies regarding the role of Ser/Thr and Tyr phosphatases and there are contradictory results regarding the role of Tyr kinases in the P‐induced acrosome reaction. Here, we performed a simultaneous evaluation of the involvement of protein kinases and phosphatases in the P‐induced acrosome reaction and in the P‐induced calcium influx. Motile spermatozoa were capacitated for 18 h and different aliquots were allocated to treated or control groups and then evaluated for their ability to undergo the acrosome reaction and to increase [Ca2+]i in response to P. The acrosome reaction was evaluated using Pisum sativum agglutinin (PSA)‐FITC, and [Ca2+]i was evaluated using fura 2AM. At all of the concentrations tested, PKA inhibitors significantly reduced the percentage of the P‐induced acrosome reaction (p < 0.001). However, only the highest concentrations of PKA inhibitors reduced the P‐induced calcium influx; lower concentrations of PKA inhibitors did not affect it. Similar results were apparent for PKC inhibitors and for tyrosine kinase inhibitors. None of the Ser/Thr phosphatase inhibitors affected the P‐induced acrosome reaction or the P‐induced calcium influx, except for the PP2B inhibitors that significantly reduced the P‐induced acrosome reaction without affecting calcium influx. Finally, the protein tyrosine phosphatase inhibitors significantly blocked the P‐induced acrosome reaction and reduced the amplitude of the P‐induced calcium transient (p < 0.001) as well as the amplitude of the plateau phase (p < 0.01). The data suggest that protein kinases and possibly PP2B have a role on the acrosome reaction at some point downstream of calcium entry and that Tyr phosphatases have a role on the acrosome reaction upstream of calcium entry.

Fibronectin From Oviductal Cells Fluctuates During the Estrous Cycle and Contributes to Sperm-Oviduct Interaction in Cattle: FIBRONECTIN CONTRIBUTES TO SPERM-OVIDUCT INTERACTION

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 α5β1 receptors. Fibronectin has been found to be expressed in the oviduct, whereas α5β1 has been found to be expressed in the sperm of different species. Fibronectin is involved through α5β1 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 α5β1. Using in vitro sperm–oviduct co‐cultures, we found that spermatozoa were attached to the oviductal epithelium through α5β1. The incubation of co‐cultures with fibronectin induced sperm release from the oviductal cells through α5β1. 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 α5β1 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.