Departamento De Morfo Fisiología

Glycosidases secreted by the epididymis become bound to the surface of spermatozoa during their transit through the epididymal duct. They are believed to play a role in mammalian fertilization. In the present report, we demonstrate that P-glucuronidase binds to the surface of ejaculated human spermatozoa with high affinity and in a saturable manner. The binding is Ca2+independent, inhibited by either mannose-6phosphate, phosphomannan fragments from the yeast Hansenula holstii and a-mannosidase from the DicQostelium discoideum, suggesting that phosphomannosyl receptors are involved in the recognition of the enzyme. The catalytic site of the enzyme is not involved in the binding. The localization of the P-glucuronidase binding-sites is restricted to the surface of the sperm head. These results suggest that the spermatozoa could be the target for glycosidases present in the seminal plasma.

Fluid of rat cauda epididymidis was obtained by flushing the duct with 0.25 mol 1-' sucrose in 0.01 mol 1-' Tris-HC1 buffer'pH 7.4. The fluid was centrifuged at 600 x g for 15 min and the sperm free supernatant was centrifuged at 47000 x g for 1 h. The sediments observed with the electron microscope consisted of a heterogeneous population of membrane-bound vesicles similar to those seen in the intact organ. In the sediment containing the vesicles the activity of P-galactosidase was mostly unavailable for the substrate showing a high degree of latency: the activity became soluble after a treatment with 0.5% saponin. The activity of N-acetyl-galactosaminadase instead, was mainly available for the substrate and soluble in buffer containing 0.6 mol 1-' KCl. It was then inferred that P-galactosidase is located inside vesicles with no or little affinity for the membrane, while N-acetylglucosaminadase is bound to the external surface of vesicles. Supernatants and precipitates from suspensions of vesicles in buffered 0.5% saponin were analysed for proteins by gel electrophoresis. The electrophoretic patterns of the sediments were very different from those of supernatants and showed a number of bands greater than that of the latter. The vesicles are believed to arise from the epididymal epithelium, but their physiological role is unknown.

The molecular basis of mammalian sperm capacitation, defined as those biochemical and functional changes that render the sperm competent to fertilize the egg, is poorly understood. This extratesticular maturational process is accompanied by the activation of a unique signal transduction pathway involving the cAMP-dependent up-regulation of protein tyrosine phosphorylation presumably through the activation of protein kinase A (PK-A). We demonstrate in this report that capacitation of cauda epididymal mouse sperm in vitro was accompanied by a time-dependent increase in PK-A activity. This increase in PK-A activity did not occur in a medium that does not support capacitation. While PK-A catalytic and RI/RII regulatory subunits, as well as PK-A enzyme activity, were found in both the Triton X-100-soluble and -insoluble fractions of the sperm, the increase in PK-A activity accompanying capacitation was associated with enzyme activity found in the soluble fraction. Moreover, the regulatory and catalytic subunits of PK-A were observed by indirect immunofluorescence to be present throughout the head, midpiece, and principal piece of the sperm. Thus, PK-A appears to be functional in multiple compartments of this highly differentiated cell. A fraction of the Triton X-100-insoluble PK-A is presumably tethered by AKAP82, the major protein of the fibrous sheath of the sperm flagellum which anchors and compartmentalizes PK-A to the cytoskeleton via the RII subunit of PK-A. Using various recombinant truncated AKAP82 constructs in a gel overlay assay, the RII subunit-binding domain of this protein was mapped to a 57-amino-acid residue region at its N-terminus. Computer analysis revealed a 14-amino-acid region that resembled the RII-binding domains of other A Kinase Anchor Proteins. A synthetic peptide corresponding to this domain inhibited AKAP82-RII binding in a gel overlay assay, providing further support that AKAP82 is an anchoring protein for the subcellular localization of PK-A in the mouse sperm fibrous sheath. This work, along with previous findings that cAMP is a key intermediary second messenger in regulating protein tyrosine phosphorylation and capacitation, further supports the importance of PK-A in these processes and necessitates a further understanding of the contribution of both the soluble and insoluble forms of PK-A, as well as AKAP82, to sperm function. ᭧ 1997 Academic Press

The acrosome reaction is a specialized exocytotic process. In the mouse there is compelling evidence that receptor-mediated activation of GTP-binding proteins by factors in the zona pellucida of oocytes is a central event in the acrosome reaction. Several reagents are able to affect GTP-binding proteins directly, bypassing the receptor-ligand step for activation. We have assessed the effect of several of these compounds on human spermatozoa, monitoring cell vitality and the acrosome reaction simultaneously using the triple-stain technique. GTP@ and aluminium fluoride complexes promote sperm activation very efkiently; amphiphilic peptides capable of activating Go and Gi, also elicit the acrosome reaction. The results indicate that activation of heterotrimeric GTP-binding proteins is sufficient to trigger acrosome exocytosis in human spermatozoa.

Las caveolinas son dominios de membrana plasmática que participan en el secuestro de lípidos y proteínas. Las caveolas tienen un papel pivotal en el metabolismo de lípidos, regulación del crecimiento, transducción de señales y apoptosis. Las caveolinas interactúan y regulan los heterodímeros de la proteína G. Las caveolinas 1 (cav-1) y 2 se expresan abundantemente en fibroblastos y células diferenciadas de músculo liso y esquelético, y en endoteliocitos y adipocitos.

Whole seminal plasma (SP) enhances the function and fertility of frozen/thawed ram sperm. The objective of the current study was to investigate whether SP proteins capable of binding to molecules from the sperm plasma membrane were conserved among ram breeds, and whether these proteins were sufficient to overcome cryopreservation-induced reductions in sperm quality. Whole ram SP, obtained from rams of various breeds, improved progressive motility of frozen/thawed sperm at all times evaluated (P Ͻ 0.05); however, it did not improve total motility (15 min, P ϭ 0.480; 30 min, P ϭ 0.764; and 45 min, P ϭ 0.795). To identify SP proteins responsible for this effect, a new method was developed to retain SP proteins that bound specifically to the sperm membrane by immobilization of sperm membrane proteins. These proteins specifically bound to the sperm surface, especially the acrosomal region. Lactotransferrin, epididymal secretory protein E1, Synaptosomal-associated protein 29, and RSVP-20 were identified (mass spectrometry) in this fraction. The retained SP proteins fraction repaired ultrastructural damage of frozen/thawed sperm and, with the addition of fructose, significantly improved motility of frozen/thawed sperm. We concluded that SP proteins that bound to the sperm membrane were conserved among ram breeds, and that when added to frozen/thawed semen (along with an energy source), they repaired ram sperm damage and enhanced sperm motility.

Phymaturus zapalensis inhabits harsh thermal environments in the steppe of Patagonia, Argentina, characterized by climate conditions that impose constraints on reproduction, providing an appealing model to study the role of steroid hormones in the regulation of seasonal reproductive events. Males of P. zapalensis exhibited a postnuptial spermatogenic cycle with spermiation in mid-spring in synchrony with female ovulation time when mating occurs, followed by testicular recrudescence, but do not show sperm reservoir during hibernation period in winter. Females of P. zapalensis can reproduce annually or biennially. Here, we studied the steroidogenic functions of testicular compartments of P. zapalensis by analysing serum testosterone and ultrastructure related to steroidogenic activity in Sertoli and Leydig cells, as a possible mechanism for the synchronization of male and female reproductive cycles. The testosterone cycle resembles the gonadal cycle in P. zapalensis previously described by morphology and histology of testes. Testosterone concentration is highest in mid-spring and lowest in early summer, with an initial recovery at the beginning of a new spermatogenic cycle in late summer and early autumn. Ultrastructural morphological features indicative of steroidogenic activity in Leydig and Sertoli cells were observed during the spermatogenic cycle. Evidence of temporal asynchrony in steroidogenic activity between compartments were found in males captured in summer and autumn, while synchronous activity was found during mating in spring. Temporal separation of steroidogenic activity serves to synchronize male and female cycles in P. zapalensis and assures the adjustment of reproductive activity to physiological and environmental constraints.