Miguel W. Fornés

Hypercholesterolemia impaired sperm functionality in rabbits.

Hypercholesterolemia represents a high risk factor for frequent diseases and it has also been associated with poor semen quality that may lead to male infertility. The aim of this study was to analyze semen and sperm function in diet-induced hypercholesterolemic rabbits. Twelve adult White New Zealand male rabbits were fed ad libitum a control diet or a diet supplemented with 0.05% cholesterol. Rabbits under cholesterol-enriched diet significantly increased total cholesterol level in the serum. Semen examination revealed a significant reduction in semen volume and sperm motility in hypercholesterolemic rabbits (HCR). Sperm cell morphology was seriously affected, displaying primarily a “folded head”-head fold along the major axe-, and the presence of cytoplasmic droplet on sperm flagellum. Cholesterol was particularly increased in acrosomal region when detected by filipin probe. The rise in cholesterol concentration in sperm cells was determined quantitatively by Gas chromatographic-mass spectrometric analyses. We also found a reduction of protein tyrosine phosphorylation in sperm incubated under capacitating conditions from HCR. Interestingly, the addition of Protein Kinase A pathway activators -dibutyryl-cyclic AMP and iso-butylmethylxanthine- to the medium restored sperm capacitation. Finally, it was also reported a significant decrease in the percentage of reacted sperm in the presence of progesterone. In conclusion, our data showed that diet-induced hypercholesterolemia adversely affects semen quality and sperm motility, capacitation and acrosomal reaction in rabbits; probably due to an increase in cellular cholesterol content that alters membrane related events.

Conserved ram seminal plasma proteins bind to the sperm membrane and repair cryopreservation damage

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 < 0.05); however, it did not improve total motility (15 min, P = 0.480; 30 min, P = 0.764; and 45 min, P = 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.

First observations on enzymatic activity and protein content of vesicles separated from rat epididymal fluid

Summary. Fluid of rat cauda epididymidis was obtained by flushing the duct with 0.25 mol l−1 sucrose in 0.01 mol l−1 Tris‐HCl buffer pH 7.4. The fluid was centrifuged at 600 × g for 15 min and the sperm free supernatant was centrifuged at 47 000 × 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 β‐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 l−1 KCl. It was then inferred that β‐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.

Morphological and enzymatic study of membrane-bound vesicles from the lumen of the rat epididymis

Summary The epithelium of caput and cauda epididymidis of the rat was studied with transmission electron microscopy (TEM) and freeze‐fracture techniques. In thin sections of both zones, the tissue consisted mainly of tall columnar cells (principal cells) with long stereocilia. Clusters of small membrane‐bound vesicles were located in the lumen between or immediately over the stereocilia. Freeze‐fracture replicas also displayed groups of smooth‐surface vesicles in the same location. Membrane‐bound vesicles isolated from the lumen of the rat epididymis were studied by TEM. In thin sections, some of them contained an electron dense material and others looked empty. In addition, the hydrolases: β‐galactosidase, N‐acetyl‐glycosaminidase, α‐mannosidase, aryl‐sulfatase and β‐glucuronidase were detectable in pellets of vesicles treated with Triton X‐100. The results presented here indicate the presence of membrane‐bound vesicles observed by two different methodologies in the rat epididymal fluid and demonstrate five glycosidases in their content.

Semen Quality and Sperm Function Loss by Hypercholesterolemic Diet Was Recovered by Addition of Olive Oil to Diet in Rabbit

Fat increment (0.05% cholesterol, chol) in standard diet promoted a significant increase in serum and sperm membrane chol, which ultimately altered membrane-coupled sperm specific functions: osmotic resistance, acrosomal reaction, and sperm capacitation in White New Zealand rabbits. These changes were also associated with a reduction in motility percentage and appearance of abnormal sperm morphology. The present study was carried out to evaluate the effect of dietary olive oil (OO, 7% v/w) administration to several male hypercholesterolemic rabbits (hypercholesterolemic rabbits, HCR) with altered fertility parameters. These HCR males were achieved by feeding normal rabbits with a high-fat diet (0.05% chol). HCR were associated with a modest non-significant increase in body weight (standard diet, 4.08±0.17 Kg, versus high-fat diet, 4.37±0.24 Kg). Hypercholesterolemic rabbits presented a marked decrease in semen volume, sperm cell count, and percentage of sperm motility, associated with a significant increase in sperm cell abnormalities. Moreover, sperm capacitation measured by the characteristic phosphorylated protein pattern in and induced acrosomal reaction were also altered suggesting sperm dysfunction. However, the administration of OO (for 16 weeks) to rabbits that were fed with 50% of the high-fat diet normalized serum chol. Curiously, OO supply succeeded to attenuate the seminal and sperm alterations observed in HCR group. Administration of OO alone did not cause any significant changes in above mentioned parameters. These data suggest that OO administration to HCR male rabbits recovers the loss of semen quality and sperm functionality.

Acrosome content release in streptolysin O permeabilized mouse spermatozoa

Summary. Sperm cell plasma membrane and the outer acrosomal membrane fuse profusely during the acrosome reaction. The process is triggered by extracellular signals that elicit several intracellular events leading ultimately to membrane fusion. We have developed a streptolysin O permeabilizing protocol that selectively affects the spermatozoon plasma membrane without causing a significant loss of the acrosomal content. Most of the acrosomal acid phosphatase remained sperm‐associated even after a 20 min incubation at 37°C. However, the presence of 100 μM Ca2+ in the incubation buffer stimulates the release of the enzyme. The reaction was followed biochemically, measuring the acid phosphatase activity released to the medium and morphologically by the binding of fluorescein isothiocynate‐conjugated peanut agglutinin and by electron microscopy. The results show that the streptolysin O permeabilized spermatozoon is a promising model for studying the complex set of events mediating and regulating the acrosome reaction.

Affinity sites for β‐glucuronidase on the surface of human spermatozoa

Summary. 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 β‐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‐6‐phosphate, phosphomannan fragments from the yeast Hansenula holstii and α‐mannosidase from the Dictyostelium discoideum, suggesting that phospho‐mannosyl 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 β‐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.

Asynchronic steroid activity of Leydig and Sertoli cells related to spermatogenic and testosterone cycle in Phymaturus antofagastensis

The severe environments where Phymaturus lizards inhabit in the Andes highlands and in Patagonia, Argentina, impose restrictions on their reproduction, offering a framework for the development of life history strategies to overcome hard weather conditions. Among them, prolonged female cycles, asynchrony between sexes in receptivity, and sperm storage in males, were described. Asynchrony in the reproductive timing between males and females is a consequence of different energy requirements for gametogenesis, and often imply the existence of cellular mechanisms to enhance fertilization, such as the asynchronic steroid synthesis between testicular compartments, allowing gametogenesis independently of mating. In the present study ultrastructural and hormone assays were combined for the first time in liolaemids. Specifically, morphological features of steroid activity in Leydig and Sertoli cells, and serum testosterone concentrations have been studied in the lizard Phymaturus antofagastensis. Leydig and Sertoli cells presented morphological features characteristic of steroid synthesis during the spermatogenesis, and evident asynchronic steroid production between testicular compartments. Active Sertoli cells and inactive Leydig cells were observed in spring and autumn, while in mid-summer their steroid activity was synchronic in coincidence with maximal abundance of spermatozoa in epididymis. Serum testosterone concentration was at its maximum in mid-summer (126-230 ng ml(-1)), and minimum in late spring (4-24 ng ml(-1)) and early autumn (2-17 ng ml(-1)). In view of these results, P. antofagastensis males show an original approach to adjust their reproductive activity to physiological and environmental constraints at high latitudes and altitudes in the Andean highlands of Argentina.

Morphometrical comparison of human spermatozoa obtained from semen and swim‐up methodology

Summary. Because morphology is regularly established in semen smears, but not in swim‐up spermatozoa, we were interested in comparing some morphological parameters of semen and swim‐up spermatozoa to establish if the cells selected by the swim‐up method were morphologically similar to those considered normal in semen. Normal human semen samples were divided into two aliquots. One of these aliquots was washed by centrifugation with B2 medium and sperm smears were prepared with the resulting pellet as a control. The other aliquot was used to perform swim‐up separation and the spermatozoa from the supernatant were used as experimental smears. Both groups were stained according to the triple stain technique and spontaneous acrosome reaction and viability were determined. Video microscopy and computer‐assisted image processing of live and non‐reacted sperm cells were used to establish morphometrical parameters of the sperm head in both populations. The following set of morphometrical parameters were considered: width, length, width/length ratio, acrosome area, head area, and acrosome area/head area ratio. An increase in head width, a decrease in head length and a subsequent increase of width/length ratio were found in swim‐up cells compared with the control group. A slight increase in acrosome area/head area ratio was also observed in swim‐up spermatozoa. Through the swim‐up methodology we were able to select a subpopulation of oval shaped heads with spermatozoa having a bigger acrosome area in comparison to semen.

Manchette-acrosome disorders during spermiogenesis and low efficiency of seminiferous tubules in hypercholesterolemic rabbit model.

Hypercholesterolemia is a marker for several adult chronic diseases. Recently we demonstrated that sub/infertility is also associated to Hypercholesterolemia in rabbits. Seminal alterations included: abnormal sperm morphology, decreased sperm number and declined percentage of motile sperm, among others. In this work, our objective was to evaluate the effects of hypercholesterolemia on testicular efficiency and spermiogenesis, as the latter are directly related to sperm number and morphology respectively. Tubular efficiency was determined by comparing total number of spermatogenic cells with each cell type within the proliferation/differentiation compartments. We found lower testicular efficiency related to both a decrease in spermatogonial cells and an increase in germ cell apoptosis in hypercholesterolemic rabbits. On the other hand, spermiogenesis–the last step of spermatogenesis involved in sperm shaping–was detaily analyzed, particularly the acrosome-nucleus-manchette complex. The manchette is a microtubular-based temporary structure responsible in sperm cell elongation. We analyzed the contribution of actin filaments and raft microdomains in the arrangement of the manchette. Under fluorescence microscopy, spermatocyte to sperm cell development was followed in cells isolated from V to VIII tubular stages. In cells from hypercholesterolemic rabbits, abnormal development of acrosome, nucleus and inaccurate tail implantation were associated with actin–alpha-tubulin–GM1 sphingolipid altered distribution. Morphological alterations were also observed at electron microscopy. We demonstrated for the first time that GM1-enriched microdomains together with actin filaments and microtubules are involved in allowing the correct anchoring of the manchette complex. In conclusion, cholesterol enriched diets promote male fertility alterations by affecting critical steps in sperm development: spermatogenesis and spermiogenesis. It was also demonstrated that hypercholesterolemic rabbit model is a useful tool to study serum cholesterol increment linked to sub/infertility.