The structure and formation of rolled and crested bull spermatozoa

The ejaculate of a Friesian bull contained three abnormalities: rolled, crested, and knobbed spermatozoa. The rolled form had marked lateral curvature of the head, occasionally forming a complete tube. All three forms were of testicular origin and it is likely that the first two were developmentally related.     

Ascorbic acid induces spectrin reorganization in bull epididymal spermatozoa

The acrosome, a complex organelle, plays a key regulatory role in the sperm–egg interaction. We have previously shown that ascorbic acid affects both motility and spectrin protein patterns in sperm. In this study, we further characterized the changes in spectrin in sperm challenged with ascorbic acid, using SDS-PAGE, western blots, and immunofluorescence. Ascorbic acid shifts spectrin to a higher-molecular-weight species based on western blot studies. This shift in the spectrin band correlates with a striking series of changes in spectrin immunofluorescence patterns. Upon ascorbic acid challenge, spectrin localization changes, eventually resulting in the formation of vesicles. These vesicles can reach sizes up to five times the original volume of the sperm cell and sometimes show multiple spikes. These findings indicate that a novel process is taking place in the acrosome upon ascorbic acid challenge and suggest that the cytoskeleton may be a useful target for studying and hopefully controlling the sperm–egg interaction. This revised version was published online in July 2006 with corrections to the Cover Date.     

Epididymal compounds and their influence on the metabolism and survival of spermatozoa

Epididymal fluid, which is derived from testicular fluid, contains several unusual compounds. Little information is available on the composition of the testicular fluid of primates, but the fluid of the ram, bull, boar, and rat contains high concentrations of inositol and certain amino acids. Analyses have been made of epididymal fluid collected from the cauda epididymis of the Rhesus monkey and several nonprimate species (e.g., ram, bull, dog, stallion, rabbit, guinea pig, rat, and hamster), but similar information on the human is lacking. Cauda epididymal fluid appears to be similar in composition from one mammalian species to another. However, the epididymal plasma differs considerably from blood, lymph, and other extracellular fluids. The environment of spermatozoa in the epididymis is, therefore, highly specialized, and presumably in some way contributes to the prolonged survival of spermatozoa in this organ, and provides substrates for the metabolism of the spermatozoa. The chief characteristics of the cauda epididymal plasma are the low concentration of inorganic ions and the high levels of several unusual organic constituents namely, glycerylphosphorylcholine, carnitine, sialic acid, amino acids, glycosidases, and phosphatases. At least one antifertility compound, namely, orally administered α-chlorohydrin, appears to be concentrated in the epididymis. Studies on laboratory animals, domestic species, and man, suggest that it inhibits enzymes of the glycyolytic pathway in spermatozoa, and this may be the basis for its antifertility activity.     

Spirulina platensis extract addition to semen extender enhances cryotolerance and fertilizing potentials of buffalo bull spermatozoa

The objective of this study was to investigate the effect of Spirulina platensis extract (SPE) addition to the freezing extender on freezability, lipid peroxidation, ultrastructure alterations and fertilizing potentials of frozen-thawed buffalo bull spermatozoa. Semen samples were collected with artificial vagina from five adult fertile bulls and diluted with Tris-base extender containing SPE (1, 5, 10 and 20 μg/mL) or without SPE (control). Diluted semen was cooled to 4 °C throughout one hour and frozen in 0.25 mL straws: prior to being stored in liquid nitrogen. Cryopresreved spermatozoa were assessed for post-thawing sperm motility, viability, acrosomal integrity, ultrastructure changes, antioxidant activities, lipid peroxidation and fertility rate. The current results clearly indicated that adding 10μg/mL SPE to the freezing extender significantly improved (P< 0.05) post-thawing motility and decrease the percentage of acrosomal damage (51.67±6.02% and 16.33±1.46%, respectively) compared with the control (28.33±4.41% and 26.33±1.77%, respectively). Moreover, addition of 10 μg/mL SPE to the semen extender significantly diminished (P< 0.05) MDA concentration (10.66±2.40 nmol/10⁹) compared with the control (22.66±4.26 nmol/10⁹). Therefore, the present results revealed that addition of 10μgl/mL SPE to the freezing extender might improve semen quality and reduce cryodamage of the buffalo bull spermatozoa.     

The zymogen form of acrosin in testicular,epididymal, and ejaculated spermatozoa from ram

The sperm-specific proteinase acrosin (EC 3.4.21.10) is found in spermatozoa as a zymogen. We have looked for different forms of this zymogen in testicular, epididymal, and ejaculated spermatozoa from ram and have compared total sperm extracts made immediately after cell disruption with extracts made later from isolated sperm heads. We have concluded that the autoactivatable zymogen form, known generally as proacrosin, is the only form of acrosin within intact mature ram spermatozoa; no other zymogen form was detected, although lower levels of proacrosin were found in some samples of testicular spermatozoa. From studies of the activation process, it appears that ram proacrosin is truly autoactivatable; no evidence could be found for the involvement of any auxiliary enzyme. Estimations of the molecular weight of proacrosin using gel chromatography (60,000) and SDS-polyacrylamide gel electrophoresis (51,300) indicated that the zymogen is monomeric. Comparison with the molecular weight of ram acrosin (44,000 or 40,000, using the two respective methods) indicated that a single acrosin molecule is derived from each zymogen molecule. The sperm acrosin inhibitor (molecular weight 11,000 or 8,000) was present in testicular spermatozoa as well as in ejaculated spermatozoa; there was no evidence that it was produced as a result of zymogen activation.     

Identification of the bull sperm p80 protein as a PH-20 ortholog and its modification during the epididymal transit

We have identified an 80 kDa protein in ejaculated bull spermatozoa (p80) which is found in acrosomal and post-acrosomal areas of the head. It has a hyaluronidase activity and shares homologies with PH-20, a sperm surface glycoprotein involved in sperm-egg interaction. The aim of the present study was to characterize bull sperm p80 protein at the nucleic and amino acid levels to determine whether it is the bovine PH-20 ortholog. The complete nucleotide sequence determined by RT-PCR, 3' and 5' RACE show that bull p80, displays identity with the PH-20 nucleotide and amino acid sequences. Messenger RNA and protein expressions determined by Northern blot and immunohistochemistry revealed that the protein is testicular (expressed in spermatocytes and spermatids). The localization of p80 on spermatozoa, determined by indirect immunofluorescence using a monoclonal antibody, shows the protein in acrosomal and post acrosomal areas of the head with an increase in the signal intensity as sperm progress through the epididymis. Post-translational modifications of the protein were investigated during the epididymal maturation by Western blot on protein extracts from sperm collected in the caput, corpus and cauda portions of bull epididymis. Glycolysation status of sperm p80 protein on proteins from ejaculated and epididymidal sperm was investigated. Result show that the glycosylation status is modified as spermatozoa migrate through the epididymis. Hyaluronidase activity evaluated in protein extracts from spermatozoa of the three different epididymal sections revealed that the activity is higher at pH 7 than 4 and is not affected by epididymal maturation. These data strongly suggest that p80 is the bovine PH-20.     

Simultaneous detection of X‐ and Y‐bearing bull spermatozoa by double colour fluorescence in situ hybridization

Double colour fluorescence in situ hybridization with sex chromosome probes was applied on sperm cells of five Swedish Holstein‐Friesian bulls. It was demonstrated that cosmids with strong fluorescence signals and scraped chromosomes can successfully be used as markers in this type of study. X and Y segregated as expected according to a 1:1 ratio, and there were no interindividual variations. There was a tendency for there to be more Y‐ than X‐bearing spermatozoa, but this bias was assumed to be due to the markers used. Disomic spermatozoa occurred with a frequency of more than 0.1 % (0.067% XX, 0.029% YY, and 0.029% XY), which is considerably lower than the frequency in humans. Diploid sperm cells occurred with a frequency of 0.05 %. Mol. Reprod. Dev. 53:407–412, 1999. © 1999 Wiley‐Liss, Inc.     

Control of respiration and of motility in ejaculated bull spermatozoa

The relations between motility and respiration were studied in ejaculated bull spermatozoa respiring with lactate. Motility was quantitatively evaluated by a turbidimetric procedure as percentage of cells moving per minute from the bottom of the cuvette into the light path. For selective inhibition of ATP-consuming reactions including motility or of mitochondrial respiration, vanadate or cyanide, respectively, were used. Both inhibitors were found to produce proportional changes in motility and respiration. The simultaneous changes in motility and respiration were linked to shifts in the cellular ATP/ADP ratio. Partial uncoupling of respiration in vanadate-inhibited cells gave similar relations between respiration and ATP/ADP ratios as stepwise inhibition of ATP-utilizing reactions by vanadate. Presuming saturation kinetics with respect to the ATP/ADP ratio, half maximum constants of 1.7 and 4.7 for the ATP/ADP ratio and maximum values of about 130% and 300% (in comparison to untreated cells) were estimated for motility and respiration, respectively. Respiration showed a much steeper dependence on the ATP/ADP ratio than motility resulting in an apparent cooperativity coefficient of 2.9. From these dependences on the ATP/ADP ratio, the shares in the control of ATP turnover in untreated cells were estimated. At sufficient supply with substrate, more than 80% of control were excreted by motility and other ATP-utilizing reactions, the rest by mitochondrial ATP production, i.e., the reactions of oxidative phosphorylation.     

Activity of exoglycosidases in ejaculated spermatozoa of boar and bull

The activity of exoglycosidases in extracts from freshly ejaculated boar and bull spermatozoa with 0.2% Brij-35/2% acetic acid was measured. The results show that beta-N-acetylhexosaminidase, beta-galactosidase and alpha-mannosidase are the major glycosidases; much higher levels of activity were found in boar spermatozoa than in bull spermatozoa. When compared on a per spermatozoon basis, the ratios of the activities of beta-N-acetylhexosaminidase, beta-galactosidase and alpha-mannosidase in boar spermatozoon relative to those in bull spermatozoon were approximately 13000:1, 1700:1 and 400:1, respectively. Liberation of these glycosidases from bull spermatozoa by treatment with phosphatidylinositol-specific phospholipase C (PI-PLC) was low, in contrast to liberation of alpha-mannosidase from boar spermatozoa previously found by the same means. The possibility that the exoglycosidases present in large amounts in boar spermatozoa play a role in the process of binding to the zona pellucida glycoprotein of the egg is discussed.     

Peroxidase activities in bull spermatozoa

The present study was carried out to determine the localization of peroxidase activity in bull spermatozoa. 3,3′-Diaminobenzidine (DAB) was used as a substrate for revealing peroxidase activity, and light and electron microscopic analysis of the results obtained was performed. Peroxidase activity was detected in the mitochondria of the middle piece and the outer acrosomal membrane. Catalase was excluded as an enzyme, catalyzing the detected peroxidase activity. Concerning the biochemical properties of bull sperm peroxidases, peroxidase activity was found to be manifested in a large pH range, 4–10.5. Bull sperm peroxidase activity appeared to be temperature sensitive and azide sensitive and could be readily inhibited by phenylhydrazine. Electrophoretic analysis of the proteins from bull sperm extracts separated in a Davis-Ornstein system of 7% polyacrylamide gel, followed by the determination of peroxidase activity on the polyacrylamide gels, revealed that all 14 sperm protein fractions available on the gel possessed peroxidase when benzidine was used as a substrate. The possible reasons for the electrophoretic heterogeneity of bull sperm peroxidases are discussed. © 1994 Wiley-Liss, Inc.     

Changes in mitochondrial protein composition during testicular differentiation in mouse and bull

The morphology of testicular mitochondria changes markedly during spermatogenesis from a form normally seen in somatic cells to a “germ cell” form in which the matrix is diffuse and vacuolated and finally to a form with a condensed matrix seen in spermatozoa. Colloidal silica gel gradients and high-resolution, two-dimensional gel electrophoresis were used to define the changes in density and polypeptide composition that occur in testicular mitochondria during spermatogenesis. Similar densities were observed for mitochondria isolated from the same bovine or murine tissue, but mitochondria from different tissues usually had different densities. Mitochondria from testis of calf, bull, or sexually mature mouse had densities of 1.06 gm/cm³ while liver mitochondria were more dense, having a density of 1.09 gm/cm³. “Somatic-type” testicular mitochondria from calf and “germ cell-type” mitochondria from sexually mature mouse or bull had similar densities, 1.06 gm/cm³, while the density of mitochondria from ejaculated spermatozoa differed, ρ = 1.08 gm/cm³. Analysis of polypeptide composition of somatic and germ cell mitochondria from testes of prepuberal and sexually mature animals and from highly enriched populations of pachytene primary spermatocytes and round spermatids revealed a staining pattern of mitochondrial proteins that was markedly constant throughout development with most polypeptides being conserved and a few specific spots changing in abundance. Marked differences were detected, however, when mitochondria from ejaculated spermatozoa were compared with those from testis with many minor and major polypeptides missing and several new polypeptides present at high concentration.     

Analysis of lipid and protein components of ejaculated bull sperm surface and seminal plasma

Surface components of ejaculated bull sperm were radiolabeled by enzymatic iodination with lactoperoxidase and Na¹²⁵I. The sperm were lysed and the labeled components analyzed on SDS-7.5% polyacrylamide gels. Electrophoresis of solubilized radioactivity resolved six components with approximate molecular weights of 77, 61, 44, 36, 24, and 15 kilodaltons. To identify components that might be adsorbed to the bull sperm surface from seminal secretions, seminal plasma was labeled. Electrophoresis of labeled seminal plasma resolved four components with approximate molecular weights of 74, 33, 24, and 15 kilodaltons, each of which comigrated with a labeled sperm surface component. To identify the chemical composition of the radiolabeled components, labeled sperm surface and labeled seminal plasma were submitted to isopycnic density gradient centrifugation in cesium chloride. The ¹²⁵I incorporated into bull sperm surface separated into two discrete areas of radioactivity, one having a density characteristic of protein and the other, of lipid. Iodinated seminal plasma banded in one discrete area that had a density characteristic of protein. Electrophoretic analysis of each area of radioactivity recovered from the gradients demonstrated that five of the six sperm surface and all of the seminal plasma components were in the protein fractions. The 15-kilodalton sperm surface component banded as a lipid, whereas the 15-kilodalton seminal plasma componènt banded as a protein.     

Lipid composition and metabolism in testicular and ejaculated ram spermatozoa

1. Spermatozoa collected directly from the testis of the conscious ram contain 25% more phospholipid than ejaculated spermatozoa. The concentration of lecithin, phosphatidylethanolamine and ethanolamine plasmalogen was greater in testicular spermatozoa; little difference was observed in choline plasmalogen. Both types of spermatozoa had significant amounts of cardiolipin and alkyl ether phospholipid. 2. The fatty acids in the phospholipid extracted from testicular spermatozoa have a very high content of palmitic acid. The phospholipids of ejaculated spermatozoa contained less palmitic acid, but more myristic acid. 3. Ejaculated spermatozoa contained less acyl ester and cholesterol. It is suggested that lipids are a source of substrate for spermatozoa during their passage through the epididymis. 4. Testicular spermatozoa when incubated with [U-¹⁴C]glucose incorporated more radioactivity into the glycerol part of the phospholipid and neutral lipid fractions than did ejaculated cells. The distribution of radioactivity in the individual phospholipids and neutral lipids was similar for both cell types. No radioactivity was detected in choline plasmalogen, which accounted for approx. 40% of the total phospholipid. 5. Testicular spermatozoa incorporated more radioactivity from glucose into formate than into acetate, whereas a higher proportion of radioactivity was found in acetate in ejaculated cells. 6. The implications of these lipid changes in the process of spermatozoal maturation are discussed.     

Ultrastructural studies on the epididymal spermatozoa in the rhesus monkey

Ultrastructural studies on the spermatozoa in different regions of the epididymis of the rhesus monkey have shown that the process of sperm maturation is associated with the caudad migration of the cytoplastmic droplet, a reduction in the volume of the cytoplasmic droplet, and an obvious wrinkling of the plasma membrane surrounding the head of the spermatozoa. These changes are completed by the time the spermatozoa reach the distal-middle segment of the epididymis. The present studies also indicate that spermatozoa are incorporated into the intraepithelial cells in the epidymis. This finding suggests that spermiophagy is a normal occurrence in the epidymis of rhesus monkey.     

Basic nuclear proteins in testicular cells and ejaculated spermatozoa in man

Human testis was shown to contain a specific histone, TH2B, having the same electrophoretic mobility as rat TH2B. Testicular and ejaculated human sperm still possessed histones at 50% and 15% of the total basic nuclear proteins, respectively. Comparison of the electrophoretic patterns of histones from human testis, testicular sperm and ejaculated sperm implied that the histones may be removed in the order H2A and H1 before H3, H4 and H2B before TH2B. TH2B which is the major histone fraction in ejaculated sperm has no longer a strong affinity to DNA. TH2B in sperm nuclei could be separated from other basic nuclear proteins by Bio-Gel P-10 column chromatography and its amino acid composition is similar to that of rat TH2B, although no cysteine residue was found.     

Purification and characterization of two plasma membrane domains from ejaculated bull spermatozoa

Plasma membranes were detached from ejaculated bull spermatozoa by a brief sonication in a moderately hypotonic medium, and the released plasma membranes were partially purified by differential centrifugation. The resulting fraction was enriched 8- and 15-fold in alkaline phosphatase and 5' nucleotidase activities, respectively, compared with the starting sonicated spermatozoa. This total plasma membrane fraction was separated into two distinct fractions by equilibrium density centrifugation on a continuous linear sucrose gradient. Two peaks of light scattering material were formed at densities of 1.117 and 1.148 g/ml. The denser peak contained most of the protein of the plasma membrane fraction, whereas nearly all the concanavalin A binding activity was found in the lighter peak. The two bands had distinctly different polypeptide compositions when analyzed by SDS PAGE. Polyclonal antibodies were raised in rabbits against a major integral membrane glycoprotein of each fraction (Mr of 92,000 in the light peak and 98,000 in the dense peak). The two antigens were detected on the surface of intact spermatozoa by indirect immunofluorescence microscopy. The 92-kD protein (present in the lighter band) was detected only on the plasma membrane of the acrosomal and anterior postacrosomal regions of the head. The 98-kD antigen, present in the heavier band, was localized to the surface of the postacrosomal region of the head, to the principal piece of the tail, and to the connecting piece between the head and tail. The exclusive localization of the 92-kD polypeptide to the surface of the anterior portion of the head was confirmed by immunoelectron microscopy. These data show that the two fractions isolated on the sucrose gradient originate from different regions of the sperm cell plasma membrane.     

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.     

Use of zona-free hamster ova to assess sperm fertilizing ability of bull and stallion

Zona-free hamster ova interacted with bull and stallion spermatozoa after treatment of ejaculated semen to capacitate the sperm cells. Sperm conditioning by prolonged incubation in BWW medium (18–26 hr) prior to insemination was effective for capacitation of bull and stallion sperm. Preincubation of bull sperm for 70 or 105 min in defined medium (DM) with NaCl content elevated to result in 350 m0sM/kg also led to penetration of hamster vitelli. More rapid sperm conditioning was possible and higher proportions of interacting vitelli followed insemination with bull or stallion sperm exposed to high ionic strength DM (380–390 m0sM/kg) for 10 min before incubation in isotonic DM prior to insemination, the treatment adopted in subsequent work. Initial efforts to assess relative fertilizing ability of freshly ejaculated semen from two fertile bulls (A and B) in A1 usage resulted in uniformly high ( > 90%) levels of sperm-vitelli interaction (for both) when the hamster ova employed resulted from superovulation with PMSG and HCG. Following use of ova from untreated hamsters sperm samples of bull A and bull B interacted with 53.8% and 84.9% (P < 0.05) of zona-free hamster ova, respectively. Conception data (60–90 day nonreturn rates) resulting from A1 with semen collected during the same interval but processed and stored in liquid nitrogen prior to use revealed an inverse relationship to proportions of vitelli interacting with fresh sperm; nonreturn rates were 69.3% and 66.3% for bull A and bull B, respectively. A similar treatment effected capacitation of frozen-stored bull semen to enable sperm-vitelli interaction. These findings encourage additional efforts to correlate testing of processed semen with fertility.     

The effects of carnitine,glycerylphosphorylcholine, caffeine,and egg yolk on the motility of rat epididymal spermatozoa

Experiments were performed to further the understanding of epididymal processes involved in the acquisition of sperm motility. Samples of luminal contents were collected by micropuncture from four regions of the rat epididymis. These samples were incubated in various diluents to observe the effects of the diluents on sperm motility. Consonant with previous reports, 40 mM glycerylphosphorylcholine (GPC) and 60 mM DL-carnitine reduced overall motility scores of cauda epididymidal spermatozoa but did not prevent normal initiation of motility. Additionally, control sperm cells and cells treated with carnitine could reinitiate full motility after becoming immotile. Spermatozoa treated with GPC could not reinitiate motility. The sperm cells in our system thus react to GPC and carnitine in fundamentally different ways, the exact nature of which remains to be determined. Spermatozoa from the distal caput epididymidis evidenced high motility scores when diluted in a 5% egg yolk + 10 mM caffeine diluent. It was demonstrated, however, that the subjective appearance of full motility in these immature cells was not supported by actual progressive motility as measured in an assay of linear distance traveled. It was concluded that neither 10 mM caffeine, 5% egg yolk, nor their combination was sufficient to induce progressive motility in immature rat spermatozoa.     

Metabolomic fingerprinting of bull spermatozoa for identification of fertility signature metabolites

The objective of the study was to identify the fertility‐associated metabolites in bovine spermatozoa using liquid chromatography‐mass spectrometry (LC‐MS). Six Holstein Friesian crossbred bulls (three high‐fertile and three low‐fertile bulls) were the experimental animals. Sperm proteins were isolated and protein‐normalized samples were processed for metabolite extraction and subjected to LC‐MS/MS analysis. Mass spectrometry data were processed using iMETQ software and metabolites were identified using Human Metabolome DataBase while, Metaboanalyst 4.0 tool was used for statistical and pathway analysis. A total of 3,704 metabolites belonging to various chemical classes were identified in bull spermatozoa. After sorting out exogenous metabolites, 56 metabolites were observed common to both the groups while 44 and 35 metabolites were found unique to high‐ and low‐fertile spermatozoa, respectively. Among the common metabolites, concentrations of 19 metabolites were higher in high‐fertile compared to low‐fertile spermatozoa (fold change > 1.00). Spermatozoa metabolites with variable importance in projections score of more than 1.5 included hypotaurine, d ‐cysteine, selenocystine. In addition, metabolites such as spermine and l ‐cysteine were identified exclusively in high‐fertile spermatozoa. Collectively, the present study established the metabolic profile of bovine spermatozoa and identified the metabolomic differences between spermatozoa from high‐ and low‐fertile bulls. Among the sperm metabolites, hypotaurine, selenocysteine, l ‐malic acid, d ‐cysteine, and chondroitin 4‐sulfate hold the potential to be recognized as fertility‐associated metabolites.