Milene Kong

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.

Gonadotropin-Releasing Hormone-Stimulated Sperm Binding to the Human Zona Is Mediated by a Calcium Influx

Abstract The mechanism by which GnRH increases sperm-zona pellucida binding in humans was investigated in this study. We tested whether GnRH increases sperm-zona binding in Ca2+-free medium and in the presence of Ca2+ channel antagonists. We also examined the GnRH effect on the intracellular free Ca2+ concentration ([Ca2+]i). Sperm treatment with GnRH increased sperm-zona binding 300% but only when Ca2+ was present in the medium. In Ca2+-free medium or in the presence of 400 nM nifedipine, 80 μM diltiazem, or 50 μM verapamil, GnRH did not influence sperm-zona binding. GnRH increased the [Ca2+]i in the sperm in a dose-dependent manner. The maximum effect was reached with 75 nM GnRH. The GnRH-induced increase in [Ca2+]i was fast and transient, from a basal [Ca2+]i of 413 ± 22 nM to a peak value of 797 ± 24 nM. The GnRH-induced increase in [Ca2+]i was entirely due to a Ca2+ influx from the extracellular medium because the increase in [Ca2+]i was blocked by the Ca2+ chelator EGTA and by the Ca2+ channel antagonists nifedipine and diltiazem. These antagonists, however, were not able to inhibit the progesterone-activated Ca2+ influx. On the contrary, T-type calcium channel antagonists pimozide and mibefradil did not affect GnRH-activated Ca2+ influx but inhibited the progesterone-activated Ca2+ influx. Finally, the GnRH-induced Ca2+ influx was blocked by two specific GnRH antagonists, Ac-D-Nal1-Cl-D-Phe2-3-Pyr-D-Ala3-Arg5-D-Glu(AA)6-GnRH and Ac-3,4-dehydro-Pro1,-p-fluoro-D-Phe2, D-Trp3,6-GnRH. These results suggest that GnRH increases sperm-zona binding via an elevation of [Ca2+]i through T-type, voltage-operated calcium channels.

Sperm binding to the human zona pellucida and calcium influx in response to GnRH and progesterone

Summary.  In this study the effect of the sequential exposure of spermatozoa to progesterone and gonadotrophin‐releasing hormone (GnRH) upon zona binding and the intracellular free Ca2+ concentration was evaluated. Sperm aliquots were treated as follows: (a) 0.7 µmol l−1 progesterone or 0.1% DMSO (progesterone solvent) followed by 50 nmol l−1 of GnRH; (b) 50 nmol l−1 of GnRH or distilled water (GnRH solvent) followed by 0.7 µmol l−1 of progesterone. Additional aliquots were incubated with DMSO or distilled water (controls) and with 0.7 µmol l−1 of progesterone or 50 nmol l−1 of GnRH. All treatments were for 5 min. Motile spermatozoa were incubated in modified Tyrodes medium, at 37 °C, 5% CO2, 10 × 106 spermatozoa ml−1, for 4.5 h. Intracellular Ca2+ concentration and sperm‐zona binding was evaluated using fura 2 and the hemizona assay, respectively. GnRH and progesterone increased sperm‐zona binding and the Ca2+ concentration. Regarding zona binding, the effect of GnRH was significantly greater when the spermatozoa had been previously treated with progesterone (progesterone→GnRH=185 ± 116 zona‐bound spermatozoa versus DMSO→GnRH=99 ± 15, P < 0.001). On the other hand, previous treatment with GnRH did not modify their subsequent response to progesterone (GnRH→progesterone= 114 ± 19 zona‐bound spermatozoa versus distilled water→progesterone=108 ± 22, NS). The results regarding intracellular Ca2+ showed a similar pattern. These findings suggest a priming effect of progesterone upon a GnRH‐induced increase in sperm‐zona binding and intracellular Ca2+.

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.