Sperm treatment affects capacitation parameters and penetration ability of ejaculated and epididymal boar spermatozoa

This work was designed to study how this ability is affected by different sperm treatments routinely used for in vitro fertilization (IVF) assay. In this study, boar sperm samples from epididymal or ejaculated origin were processed by three different methods: left unwashed (NW group), washed in Dulbecco's phosphate-buffered saline supplemented with 0.1% BSA (BSA group), and washed on a Percoll^® gradient (PERCOLL group). After preparation of semen samples, changes in motility patterns were studied by CASA, calcium uptake by spectrofluorimetry, and ROS generation, spontaneous acrosome reaction, and lipid disorder by means of flow cytometry. Finally IVF assays were also performed with the different semen samples and penetrability results evaluated at 2 and 4 h post insemination (hpi). Independently of the sperm treatment, epididymal spermatozoa showed higher values of progressive motility, percentage of live cells with low lipid disorder, and penetration ability at 4 hpi than the corresponding ejaculated spermatozoa. Ejaculated spermatozoa showed higher levels of calcium uptake, ROS generation and percentage of spontaneous acrosome reaction than epididymal sperm. Regarding sperm treatments, PERCOLL group showed the highest values for some motility parameters (linearity of the curvilinear trajectory, straightness, and average path velocity/curvilinear velocity), ROS generation and penetration ability at 2 and 4 hpi; however this same group showed the lowest values for sperm curvilinear velocity and lateral head displacement. From all experimental groups, ejaculated-PERCOLL-treated spermatozoa showed the highest fertilization ability after 2 hpi. Results suggest that capacitation pathways can be regulated by suitable treatments making the ejaculated sperm able to reach capacitation and fertilize oocytes in similar levels than epididymal spermatozoa, although most of the studied capacitation-associated changes do not correlate with this ability.     

Sperm ubiquitination in epididymal feline semen

Ubiquitin is a 8.5-kDa peptide that tags other proteins for proteasomal degradation. It has been proposed that ubiquitination might be responsible for the elimination of defective spermatozoa during transit through the epididymis in humans and cattle, but its exact biological function in seminal plasma has not yet been clarified. In the domestic cat (Felis catus), the percentage of immature, unviable, and abnormal spermatozoa decreases during the epididymal transit, indicating the existence of a mechanism that removes defective spermatozoa. Magnetic cell separation techniques, based on the use of magnetic beads coated with anti-ubiquitin antibodies, may allow the selective capture of ubiquitinated spermatozoa from semen, thus contributing to the identification of a potential correlation between semen quality and ubiquitination process. Moreover, the selective identification of all the ubiquitinated proteins in different epididymal regions could give a better understanding of the ubiquitin role in feline sperm maturation. The aims of this study were as follows: (1) to verify the possibility of separating ubiquitinated spermatozoa with magnetic ubiquitin beads and identify the morphological and acrosomal differences between whole sample and unbound gametes, (2) to characterize all the ubiquitinated proteins in spermatozoa retrieved in the three epididymal regions by a proteomic approach. The data indicated the presence of ubiquitinated proteins in cat epididymal semen. However, a correlation between abnormal and ubiquitinated spermatozoa has not been found, and ubiquitin cannot be considered as a biomarker of quality of epididymal feline spermatozoa. To the author's knowledge, this is the first identification of all the ubiquitinated proteins of cat spermatozoa collected from different epididymal regions. The proteomic pattern allows a further characterization of cat epididymal semen and represents a contribute to a better understanding of the ubiquitin role in feline sperm maturation.     

Response of the epididymal duct in the corpus epididymidis to efferent or epididymal duct ligation in the mouse

Different parts of the epididymal duct were ligated when mice were 90 days old. The mice were killed 1--4 weeks later. PAS-positive materials appeared in the epithelial cells of Segment IV (corpus epididymidis) after ligation of the efferent ducts or at Segment II (middle part of caput) but not when the ligature was distal to Segment II. The inclusions were seen as early as 1 week after ligation and became increased in size and number with time.     

Androgenic control of antagglutinin secretion in the boar epididymal epithelium

Summary Antagglutinin, a specific protein synthesized by the boar epididymis, was secreted by the principal cells of the initial segment, the caput and the corpus, but was not detectable in the caudal cells. Castration completely abolished the synthesis and secretion of antagglutinin in all epididymal cells. Androgen replacement suggests that the epithelial cells from different segments have differential regulatory mechanisms. The proximal zone appeared refractory to exogenous testosterone; the median zone was a typical androgen-dependent region; and the caudal cells, where an unusual secretion of antagglutinin was detected, revealed still a different reaction pattern. It is postulated that these latter cells depend not solely on androgen but also or exclusively on other factors. Our results, which demonstrate a primary role of the Golgi complex in the secretory process in the epididymal cells, also suggest that the apical smooth endoplasmic reticulum may be implicated in the intracellular transport of glycoproteins to the cell surface.     

Biochemical and binding characteristics of boar epididymal fluid proteins

During the passage through the epididymis, testicular spermatozoa are directly exposed to epididymal fluid and undergo maturation. Proteins and glycoproteins of epididymal fluid may be adsorbed on the sperm surface and participate in the sperm maturation process, potentially in sperm capacitation, gamete recognition, binding and fusion. In present study, we separated proteins from boar epididymal fluid and tested their binding abilities. Boar epididymal fluid proteins were separated by size exclusion chromatography and by high-performance liquid chromatography with reverse phase (RP HPLC). The protein fractions were characterized by SDS-electrophoresis and the electrophoretic separated proteins after transfer to nitrocellulose membranes were tested for the interaction with biotin-labeled ligands: glycoproteins of zona pellucida (ZP), hyaluronic acid and heparin. Simultaneously, changes in the interaction of epididymal spermatozoa with biotin-labeled ligands after pre-incubation with epididymal fluid fractions were studied on microtiter plates by the ELBA (enzyme-linked binding assay) test. The affinity of some low-molecular-mass epididymal proteins (12-17 kDa and 23 kDa) to heparin and hyaluronic acid suggests their binding ability to oviductal proteoglycans of the porcine oviduct and a possible role during sperm capacitation. Epididymal proteins of 12-18 kDa interacted with ZP glycoproteins. One of them was identified as Crisp3-like protein. The method using microtiter plates showed the ability of epididymal fluid fractions to change the interaction of the epididymal sperm surface with biotin-labeled ligands (ZP glycoproteins, hyaluronic acid and heparin). These findings indicate that some epididymal fluid proteins are bound to the sperm surface during epididymal maturation and might play a role in the sperm capacitation or the sperm-zona pellucida binding.     

Identification of anti-agglutinin for spermatozoa in epididymal boar plasma

The present report identifies epididymal boar anti-agglutinin and examines its effect on sperm motility. Boar spermatozoa from the cauda epididymidis were washed and incubated in modified Krebs–Ringer bicarbonate at 37°C (5% CO₂ in air). In the samples washed three or five times and then incubated for 3–5 h, higher rates (72–79%) of spermatozoa were associated with one another at the acrosomal region, mainly in groups of 2–5 cells (head-to-head agglutination), and many cells exhibited intensively flagellant and/or circular types of movement but rarely progressive motility. The addition of epididymal plasma or 25 kDa protein purified from it markedly inhibited the occurrence of head-to-head agglutination in washed spermatozoa, whereas heat treatment and subsequent removal of insoluble materials reduced the anti-agglutination activity of epididymal plasma. The percentages of progressively motile cells in the samples incubated with epididymal plasma or 25 kDa epididymal protein rose coincident with the reduction of sperm agglutination. These findings demonstrate that the 25 kDa epididymal protein is an anti-agglutinin for the cauda spermatozoa and that it effectively functions to maintain progressive motility of the cells in vitro. © 1994 Wiley-Liss, Inc.     

Mechanisms regulating spontaneous contractions in the bovine epididymal duct

Muscular autorhythmicity provides propulsion of spermatozoa through the epididymal duct, thereby ensuring sperm maturation. In the present study, the mechanisms underlying the bovine epididymal spontaneous phasic contractions (SCs) were analyzed by using muscle-tension recording and patch-clamp techniques. SCs were recorded from the caput, the corpus, and the proximal cauda region and found to be predominantly myogenic in origin. Removal of the luminal fluid induced a burstlike contraction pattern, and removal of the epithelium, a complete loss of SCs. Application of nifedipine, but not heparin and cyclopiazonic acid, suppressed SCs, indicating that influx of Ca2+ through L-type Ca2+ channels, but not Ca2+ release from intracellular stores, was crucial for maintaining SCs. The prostaglandin-endoperoxide synthase 2 (PTGS2) inhibitor NS-398 caused a region-dependent decrease in SCs and tone. These effects were mimicked by the mitogen-activated protein kinase (MAPK) kinase inhibitor PD-98059. Similarly, the prostaglandin F(2alpha) (PGF(2alpha))-receptor antagonist AL-8810 reduced SC generation, whereas PGF(2alpha) induced SC-like activity in epithelium-denuded segments. Cell-isolation experiments revealed the existence of three morphologically different types of contractile cells, which also showed distinct biophysical properties: typical smooth muscle cells in the cauda, myofibroblast-like cells all along the duct, and atypical muscle cells (ATMs) with filament-like spurs in all regions with SCs. These data suggest that the bovine epididymal autorhythmicity is based on an epithelial PTGS2-dependent release of (an) excitatory prostaglandin(s) and a MAPK-dependent activation of L-type Ca2+ channels in the contractile cells. ATM cells may provide electrical coupling between myofibroblasts, which is essential for the generation of regular myogenic activity.     

Changes in the lipid content of boar sperm plasma membranes during epididymal maturation

Plasma membranes of boar sperm from caput, corpus and cauda of the epididymis were purified by differential- and sucrose-density equilibrium centrifugation and were found to yield a single band at a density of 1.13 g/cm3. This fraction was enriched in acid and alkaline phosphatase, 5'-nucleotidase and (Na+ + K+)-ATPase activities, whereas it contained minimal amounts of hyaluronidase and N-acetylglucosaminidase and no succinic acid dehydrogenase activities. The plasma membrane of caput, corpus and cauda sperm had the same phospholipid/protein and cholesterol/phospholipid ratios but yielded different amounts of protein and individual lipid classes. Several changes in the plasma membrane were observed during transit of sperm through the epididymis. Within the phospholipid class a decrease in the percentage of phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol was detected accompanied by an increase in amount of phosphatidylcholine, sphingomyelin and polyphosphoinositides. In the other lipid classes there was a decrease in the amount of free fatty acid and the major glycolipid. The amount of cholesterol decreased, while the amount of desmosterol and cholesterol sulfate increased. There was an increase in the amount of diacylglycerol. In addition, the changes in the fatty acid composition of the total membrane lipid and each phospholipid were determined. The above changes in the lipid composition of the plasma membrane during epididymal maturation may help to explain the decreased resistance to cold shock and changes in membrane fluidity of sperm during transit in the epididymis.     

Immunocytochemical localization of nuclear protamine in boar spermatozoa during epididymal transit

Protamine was specifically demonstrated in boar spermatozoa collected from the rete testis, caput, corpus and cauda epididymidis and the ejaculate by immunoelectron microscopy, using anti-boar or anti-ram protamine antisera and an indirect post-embedding immunogold technique. Spermatozoa from all collection sites stained after incubation although with different degrees of labelling. Controls were negative. Labelling increased from the rete testis towards the epididymal corpus, where it was most intense, decreasing sharply thereafter. The weakest binding of the assayed antibodies was obtained in the ejaculated spermatozoa but it could be reversed by in-vitro induction of chromatin decondensation with sodium dodecyl sulphate and the metal-chelating EDTA. The finding of a significant decrease in the immunolabelling detected from the corpus epididymidis onwards indicates a critical point for the interaction between DNA and the protamines in boar spermatozoa during the epididymal maturation.     

Effect of culture conditions on the obtention of boar epididymal epithelial cell monolayers

The goal of this study was to investigate the effect of the collagenase digestion time, the initial density of fragments and the culture temperature on the obtention of a boar epididymal epithelial cell culture, which is a useful methodology for the study of epididymal functions. A confluent monolayer of caput, corpus and cauda epididymal epithelial cells was only obtained when an adequate enzymatic digestion of the connective tissue surrounding the epididymal tubule was performed. For the correct digestion of caput and corpus fragments two collagenase digestions of 2 and 1h, respectively, were enough. Cauda fragments, however, needed two collagenase digestions of 3h each. A confluent monolayer of caput, corpus and cauda epididymal epithelial cells was obtained regardless of the initial density tested (15, 30, 60 and 90fragments/well). However, cultures originated from 15 and 30fragments/well showed higher cell concentration during the first 2 weeks of culture than cultures originated from 60 and 90fragments/well. A confluent monolayer of caput, corpus and cauda epididymal epithelial cells was obtained at both 32 and 37 degrees Celsius, but at 32 degrees Celsius cells grew very slowly and confluence was not reached until a week later than it was with cells growing at 37 degrees Celsius. In conclusion, we have observed that the time of digestion with collagenase is an important factor for the successful establishment of boar epididymal cell monolayers, and that the initial density of fragments and the culture temperature should be taken into account.     

Sperm DNA fragmentation in a random sample of the Spanish boar livestock

A collection of 180 chilled boar semen samples, randomly chosen from stocks currently used for routine characterization of standard seminal quality, were studied for DNA fragmentation status using the sperm chromatin dispersion test and the DNA fragmentation index (DFI: percent of abnormal cell versus normal cells for DNA fragmentation) was determined. Values for sperm motility, acrosome status, coiled tails and abnormal head morphology, including presence and position of cytoplasmic droplets were also obtained. The DFI in the whole sample presented a wide range of variation with values oscillating between practically 0% and 47.95% and do not fit to a normal distribution. The most frequent classes (83.3%) presented a DFI lower than a 5%. Significant correlations between sperm DNA fragmentation and sperm motility, acrosome status, frequency of distal droplets, coiled tails and abnormal head morphology, were not observed. However, the presence of proximal cytoplasmic droplets showed a significant correlation with the level of DNA fragmentation observed in the ejaculated spermatozoa.     

Density and distribution of anionic sites on boar ejaculated and epididymal spermatozoa

Current knowledge implies that spermatozoa successively acquire negative surface charges as they migrate through the epididymis. Until recently, however, techniques used were not amenable to statistical analysis. In the present study, a novel approach allowing numerical assessment of negative charge labelling was used in order to determine the density and distribution of anionic sites on ejaculated and maturing spermatozoa collected from six regions of the boar epididymis. Labelling was assessed quantitatively for the three morphologically distinct membrane domains on the sperm head. Statistical analysis revealed that labelling density was highest on efferent duct spermatozoa, declined up to the proximal corpus and then increased again. Densities of anionic sites on distal corpus, proximal cauda and ejaculated sperm cells were similar but significantly below the values obtained for efferent duct spermatozoa. All three sperm membrane domains underwent parallel changes. However, the overall density of negative charges on the postacrosomal segment was significantly higher as compared to the acrosomal plasma membrane. These alterations reflect sperm surface modifications through removal and addition of anionic groups. Since charge interactions are considered to play a pivotal role in sperm-egg interactions, these processes should be viewed as an integral part of sperm maturation.     

Biochemical studies on proacrosin and acrosin from epididymal boar spermatozoa: in vitro translation of boar testicular proacrosin mRNA

An inactive form of acrosin was extracted from epididymal boar spermatozoa utilizing acid pH conditions. When subjected to activation in alkaline environment, this form turns into an enzymatically active species, which exhibits close-related electrophoretic characteristics. Both the precursor and the activated species, when incubated in the presence of thermolysin, give rise to two fastly moving acrosin molecular forms. In order to establish the nature of the true acrosin zymogen, we isolated poly(A+)-RNA from boar testicles, performed its translation in vitro in the presence of [35S]-methionine and reticulocyte lysate, immunoprecipitated the translation products with anti-boar acrosin antibody, and analyzed them by SDS-polyacrylamide gel electrophoresis and autoradiography. A single translation product of molecular weight 55,000 was detected. It is concluded that the polypeptide chain of the boar zymogen is of 55,000; increases in molecular weight are due to post-translational modifications, like glycosylation.     

Erg K+ channels modulate contractile activity in the bovine epididymal duct

The expression and functional role of ether-à-go-go-related gene (erg) K+ channels were examined in the bovine epididymal duct. Sperm transit through the epididymal duct relies on spontaneous phasic contractions (SC) of the peritubular smooth muscle wall. Isometric tension studies revealed SC-enhancing effects of the erg channel blockers E-4031, dofetilide, cisapride, and haloperidol and SC-suppressing effects of the activator NS-1643. In the corpus epididymidis, EC50 values of 32 nM and 8.3 microM were determined for E-4031 and NS-1643, respectively. E-4031 was also able to elicit contraction in epithelium-denuded corpus segments, which lacked SC. In the cauda region, E-4031 and NS-1643 exerted effects on agonist-induced contraction similar to those observed in the proximal duct. Experiments with nifedipine and thapsigargin suggested that the excitatory effects of E-4031 depended mainly on external calcium influx and not on intracellular calcium release. Western blot and RT-PCR assays revealed the expression of both, erg1a and erg1b, in all duct regions. Because erg1b appears to predominate in the epididymal duct, patch-clamp experiments were performed on heterologously expressed erg1b channels to investigate the sensitivity of this splice variant to NS-1643. In contrast to its effects on erg1a, NS-1643 induced a concentration-dependent current increase mainly due to a marked leftward shift in erg1b channel activation by approximately 30 mV at 10 microM, explaining the inhibitory effect of the drug on epididymal SC. In summary, these data provide strong evidence for a physiological role of erg1 channels in regulating epididymal motility patterns.