In vitro initiated sperm forward motility in caput spermatozoa: weak and transient.

Testicular spermatozoa during journey through epididymis acquire forward motility, which is essential for fertility. To understand the biochemistry of sperm motility initiation, various initiation media have been developed that permitted high level of motility induction (55-60%) in the immature caput-spermatozoa in presence of activating principles: theophylline, bicarbonate and epididymal plasma (EP) when analysed microscopically. Here, we show for the first time using caprine model that stability and quality of in vitro-induced motility in the caput spermatozoa is insignificant in contrast to naturally induced motility in mature cauda spermatozoa. In vitro-induced motility of the immature spermatozoa was lost completely upon the removal of these activators by centrifugation. Selective withdrawal of either EP or HCO(3) by dilution retains 50-60% of the in vitro-induced motility. Spectrophotometric analysis revealed that in vitro-induced vertical motility in immature spermatozoa is too little when compared to mature spermatozoa. In in vitro-initiated caput spermatozoa, cyclic adenosine monophosphate level becomes doubled but lesser than cauda spermatozoa. This revelation concludes that scientific knowledge generated over the years on the basis of in vitro initiation method is insignificant and needs improvisation to delineate biochemical regulation of sperm motility which in turn has remarkable potential in wide biological fields, especially in infertility treatment.

Role of the major ecto-phosphoprotein in sperm flagellar motility using a cell electroporation method.

Previous studies from our laboratory have identified MPS, a 100-kDa protein, as the major phosphoprotein substrate of caprine sperm ecto-cyclic AMP independent protein kinase. In this study the isolated (32)P-labelled MPS has been incorporated into mature caprine (Capra indicus) cauda-epididymal spermatozoa with the help of cell electroporation technique to investigate the effect of MPS on sperm flagellar motility. The optimum conditions for electroporation of sperm cells consisted of exposure of 0.2 ml of sperm cells (2 x 10(8)/ml) to external electric field of intensity 1.5 kV/cm and capacitation of 25 microF at 4 degrees C and post-pulse incubation at 37 degrees C for 1 hr. when nearly 50% of the cells lost motility. Scanning electron micrographs (SEM) demonstrate the formation of micro-pores and local osmotic swelling in the electroporated spermatozoa. MPS incorporation was maximal when its concentration was 30 microg/ml (300 pmol) in the medium and when the post-pulse incubation time was 60 min. At maximum (75%) MPS incorporation, total and forward motility increments were also maximum: 34% (P < 0.01) and 32% (P < 0.01), respectively. The subcellular fractionation data show that major portion of the introduced MPS was bound to the plasma-membrane of spermatozoa. The 32P-labelled electrophoresed intact spermatozoa lost radioactivity due to the action of the endogenous ecto-phosphoprotein phosphatase. Therefore MPS is primarily localised on the sperm external surface leaving its phosphate group(s) oriented in the extracellular medium. The data provided further evidence to strengthen the view that MPS is an ecto-phosphoprotein and that it plays an important role in the regulation of sperm flagellar motility.

Shedding off specific lipid constituents from sperm cell membrane during cryopreservation.

Membrane damage is one of the main reasons for reduced motility and fertility of sperm cells during cryopreservation. Using a model system of sperm cryopreservation developed in our laboratory, we have investigated the detailed changes due to cryopreservation in the plasma membrane lipid composition of the goat epididymal sperm cells. Total lipid and its components, i.e., neutral lipids, glycolipids and phospholipids decreased significantly after cryopreservation. Among neutral lipids sterols, steryl esters and 1-O-alkyl-2,3-diacyl glycerols decreased appreciably, while among phospholipids, major loss was observed for phosphatidyl choline and phosphatidyl ethanolamine. Unsaturated fatty acids bound to the phospholipids diminished while the percentage of saturated acids increased. The cholesterol:phospholipid ratio enhanced and the amount of hydrocarbon, which was unusually high, increased further on cryopreservation. The data indicates that profound increase of the hydrophobicity of the cell membrane is one of the major mechanisms by which spermatozoa acquire potential to resist or combat stress factors like cryodamage. The results are compatible with the view that for survival against cryodamage, sperm cells modulate the structure of their outer membrane by shedding off preferentially some hydrophilic lipid constituents of the cell membrane.

Identification of goat sperm ecto-cyclic AMP independent protein kinase substrate localized on sperm outer surface.

We have demonstrated the location of a cyclic AMP independent serine/threonine protein kinase (ecto-CIK) on the outer surface of mature goat spermatozoa. We purified and characterized the major physiological protein substrate (MPS) of ecto-CIK. 32P-labeled membrane proteins phosphorylated by endogenous ecto-CIK of intact cauda-epididymal spermatozoa was solubilized with 1% Triton X-100 and then fractionated by following several chromatographic techniques like Sephacryl S-300 molecular sieve chromatography, DEAE-cellulose ion-exchange chromatography and chromatofocussing. The MPS of ecto-CIK has been purified to apparent homogeneity and it was found to be a monomeric protein of 100 kDa. Three isoforms of MPS have been found with pI of 6.37, 6.05, and 5.14 and all these isoforms served as the specific substrate of ecto-CIK. The ecto-kinase has nearly 30 times greater affinity for MPS as compared to casein the most potent exogenous protein substrate. Addition of MPS (pI 5.14) antibody caused head-to-head sperm agglutination. The Fv/Fab fragment of anti-MPS caused significant inhibition of sperm motility. The data show that MPS is an ecto-protein localized on the sperm head. MPS may thus play an important role for the regulation of sperm-egg interaction.

Isolation and identification of a novel motility-inhibiting factor from goat cauda sperm plasma membrane.

A motility inhibiting factor (MIF) in sperm plasma membrane of mammalian spermatozoa (goat) has been demonstrated. This factor has been purified to apparent homogeneity by Sepharose-6B affinity chromatography and DEAE-cellulose ion-exchange chromatography. The molecular weight of the isolated factor has been estimated as 98 kDa by molecular sieving and analytical HPLC. SDS-polyacrylamide gel electrophoresis of MIF gave a single band of 100 kDa, indicating that the factor is a monomer. MIF is a thermo-stable factor and it inhibited the spermatozoa motility in a dose dependent manner. It is a glycoprotein as it binds with high affinity to Sepharose-6B and the affinity matrix-bound factor can be eluted with D-galactose. Data show that the motility inhibiting activity is lost completely when treated with beta-galactosidase indicating that its sugar side chain is essential for its activity. Addition of MIF antibody caused significant enhancement of forward motility of the caput and cauda-spermatoza. This antibody may thus be useful for solving some of the problems of human infertility due to low sperm motility. The motility inhibiting protein may also be useful as a vaginal contraceptive.

Effect of dextrans on cryopreservation of goat cauda epididymal spermatozoa using a chemically defined medium.

Experiments were carried out to investigate the cryoprotecting potential of dextrans (ranging from 10 to 2000 kDa) using a synthetic model system developed recently in this laboratory. Goat spermatozoa from the cauda epididymidis were extracted in a chemically defined medium (modified Ringer's solution) and assayed for motility using a phase-contrast microscope. The sperm cells were subjected to a freezing protocol in a computer-controlled biofreezer (cooling at 0.25 degrees C min(-1) to 5 degrees C; 5 degrees C min(-1) to -20 degrees C; 20 degrees C min(-1) to -100 degrees C) and plunged into liquid nitrogen. The frozen cells were thawed rapidly at 37 degrees C in a thermostatic waterbath. In the absence of dextran, all the spermatozoa lost their motility. The cryoprotecting efficacy of each dextran was found to be biphasic in nature. Initially, as the concentration of dextran was increased, the recovery of sperm motility also increased and reached an optimum value; however, with further increases in dextran concentration, the recovery of motility decreased sharply. Of all the sugar polymers tested, 10 kDa dextran showed the highest cryoprotecting efficacy, whereas the 2000 kDa sugar polymer was the least active. Dextrans of 10, 40, 73, 173, 252, 500 and 2000 kDa offered maximum cryorecovery of forward motility to the extent of approximately 23%, 21%, 19%, 18%, 16%, 15% and 8%, respectively. Optimum concentrations of these dextrans for cryoprotection of sperm motility were 8.42, 2.50, 1.09, 0.37, 0.31, 0.10 and 0.04 mmol l(-1), respectively. It thus appears that each dextran has a characteristic cryoprotection profile. The data show that the cryoprotecting efficacy and optimum cryoprotecting concentrations of dextrans are inversely related to their molecular mass. Dextran also served as a significant cryoprotectant in the presence of glycerol (0.87 mol l(-1)) and dimethyl sulphoxide (0.76 mol l(-1)), which are well known cryoprotectants; the action of the combined cryoprotectants was almost additive. The presence of glycerol or glycerol plus dimethyl sulphoxide caused a significant reduction (from 8.42 to 6.27 mmol l(-1)) in the optimum concentration of dextran. In the presence of the three cryoprotectants, recovery of sperm motility was as high as 58% (forward motility) and 60% (total motility).

Effect of amino acids on goat cauda epididymal sperm cryopreservation using a chemically defined model system.

Studies were carried out to analyze the cryoprotecting efficacy of several amino acids by use of a chemically defined synthetic medium (modified Ringer's solution) and goat cauda epididymal sperm as the model system. Motile goat cauda sperm dispersed in the synthetic medium were subjected to a freezing protocol in a computer-controlled bio-freezer, cooling 0.25 degrees C x min(-1) to 5 degrees C, 5 degrees C x min(-1) to -20 degrees C, and 20 degrees C x min(-1) to -100 degrees C, prior to being plunged into liquid nitrogen. In the absence of amino acids, sperm cells completely lost their flagellar motility. Of all the amino acids tested, l-alanine showed maximal cryoprotection potential. l-Alanine at 135 mM offered optimum cryoprotection potential: recovery of sperm forward motility and total motility were 14 +/- 2% and 19 +/- 2%, respectively. l-Glutamine, l-proline, and glycine at optimum concentration (100-150 mM) cryopreserved approx. 11-17% total motility of the sperm cells, whereas amino acids such as l-arginine, l-lysine, and l-histidine offered little cryoprotection (0-5%) to the cells. Increasing the amino acid concentration beyond the optimum level sharply decreased the recovery of the sperm motility, which therefore showed a biphasic cryoprotection profile. Addition of amino acids enhanced (approx. 7-10%) the cryoprotection efficacy of the well-known cryoprotectants glycerol and a combination of glycerol and dimethyl sulfoxide. The presence of glycerol caused a marked reduction (from 100-150 mM to 20-70 mM levels) in the optimal cryoprotective concentration of the amino acids. The combined cryoprotecting action of glycerol, dimethyl sulfoxide, and amino acids provided motility recovery as high as 52%. The observation that amino acids and dimethyl sulfoxide had an additive effect in augmenting the cryoprotecting potential of glycerol suggests that the mechanism of their action is different from that of glycerol. This cocktail of cryoprotectants may be useful for cryopreservation of semen of various species.

Alteration of goat sperm ecto-phosphoprotein phosphatase activity and its distribution on the sperm surface during epididymal maturation.

Phosphoprotein phosphatase (ecto-PPase) of goat epididymal sperm outer surface showed a significant increase in its activity at the initial stage of epididymal sperm maturation (up to the proximal corpus region) followed by a sharp fall towards the terminal phase of the maturation event. PPase activity showed nearly the same profile when estimated in intact cells as well as in isolated sperm plasma membrane. The ecto-PPase was purified to apparent homogeneity by using various biochemical fractionation procedures, such as solubilization with Triton X-100, sephadex gel filtration chromatography, concanavalin A-sepharose affinity chromatography and diethylaminoethyl-cellulose ion-exchange chromatography. The isolated PPase has a molecular mass of approximately 36 kDa and an isoelectric point of 5.95. Sperm surface topography of the enzyme was investigated using fluorescein isothiocyanate-conjugated antibody of the purified PPase. The immunofluorescent studies have demonstrated that the isolated PPase is localized on the external surface of viable sperm. Immunocytochemical studies also revealed a marked topographical alteration of ecto-PPase during epididymal transit of the male gametes. Immunoreactivity was observed all over the surface of caput sperm, but was restricted primarily to the anterior tip of the head in the corpus sperm and to the posterior part of the head in cauda sperm cells. The maturation-dependent decrease in PPase activity was also confirmed by immunofluorescent studies. This remarkable maturation-dependent modification of ecto-PPase activity, as well as its distribution on sperm surface, suggest that the ecto enzyme may play an important role in sperm function by regulating the phosphorylation states of the membrane-associated and reproductive fluid phosphoprotein substrates.

Development of a simple sperm cryopreservation model using a chemically defined medium and goat cauda epididymal spermatozoa.

This investigation was carried out to develop a simple sperm cryopreservation model using a chemically defined synthetic medium (modified Ringer's solution) and mature goat cauda epididymal sperm as the model system. Rates of cooling, freezing, and maximum freezing temperature were manipulated with the help of a computer-controlled programmable biofreezer. Highly motile goat cauda sperm dispersed in a modified Ringer's solution was subjected to the freezing protocol: cooling 0.25 degrees C min(-1) to 5 degrees C, 5 degrees C min (-1) to -20 degrees C, 20 degrees C min(-1) to -100 degrees C, prior to plunging into liquid nitrogen. In the absence of any cryoprotective agent, all of the spermatozoa lost their motility. Addition of glycerol (0.22 to 0.87 M) caused a dose-dependent increase of sperm motility recovery. The highest recovery of forward and total motility was (32 and 35%, respectively) at 0.87 M. Further increase of the glycerol concentration caused a marked decrease in motility. Changes in the cooling rate particularly before and during freezing had a notable effect on the sperm motility recovery. There was no or low recovery (0-18%) of sperm motility when the cells were transferred directly to liquid nitrogen from the initial two cooling stages. The data demonstrate the importance of all of the cooling stages in the cryopreservation of the cells. Like glycerol, dimethyl sulfoxide (Me(2)SO) and ethylene glycol also showed a dose-dependent increase in motility recovery as well as a biphasic curve of cryoprotection. At optimal concentrations, dimethyl sulfoxide (1.00 M) and ethylene glycol (1.29 M) were effective in recovering sperm motility to the extent of 20 and 13%, respectively. Thus these reagents have markedly lower cryoprotection potential than glycerol.

Maturation-dependent modification of the protein phosphorylation profile of isolated goat sperm plasma membrane.

Highly purified plasma membranes, isolated by an aqueous two-phase polymer method from goat epididymal spermatozoa, were found to possess a kinase activity that causes phosphorylation of serine and threonine residues of several endogenous plasma membrane proteins. Cyclic AMP, cyclic GMP, Ca(2+)-calmodulin, phosphatidylserine-diolein, polyamines and heparin had no appreciable effect on this kinase. Autoradiographic analysis showed that the profile of the phosphorylation of membrane proteins by this endogenous cAMP-independent protein kinase underwent marked modulation during the transit of spermatozoa through the epididymis. In caput sperm plasma membrane, 18, 21, 43, 52, 74 and 90 kDa proteins were phosphorylated, whereas, in the corpus and cauda epididymal spermatozoa, a differential phosphorylation pattern was observed with respect to the 90, 74, 21 and 18 kDa proteins. The rate of phosphorylation of the 74 kDa protein decreased markedly during the early phase of sperm maturation (caput to distal corpus epididymides) whereas there was little change in kinase activity in sperm plasma membrane. In contrast, the rates of phosphorylation of the 18 and 21 kDa proteins increased during the terminal phase (distal corpus to distal cauda epididymides) of sperm maturity, although the kinase activity of membrane decreased significantly during this phase. The modulation of the phosphorylated states of these specific membrane proteins may play an important role in the maturation of epididymal spermatozoa.

Partial purification and characterization of a phosphoprotein phosphatase from sperm plasma membrane.

A phosphoprotein phosphatase (PPase M-I) that dephosphorylates serine and threonine residues of histones was isolated from the goat cauda-epididymal sperm plasma membrane and partially characterized. The PPase was solubilized from the sperm membrane by treating it with 0.1 N NaOH at pH 11.4 and the solubilized enzyme was partially purified by concanavalin A-sepharose affinity chromatography and high-performance liquid chromatography (HPLC), revealing it to be a 520-kDa protein. The PPase gave a single protein band in native polyacrylamide gel electrophoresis (PAGE), but in the presence of SDS it resolved into multiple proteins (35-170 kDa) showing that the isolated enzyme contained a few contaminating proteins. The enzyme is a glycoprotein because it binds with high affinity to concanavalin A. It was maximally active at pH 8.0 and its activity was not dependent on bivalent metal ions. The enzyme is a specific phosphatase as it displayed higher affinity for dephosphorylation of large molecular weight phosphate esters. The PPase showed broad substrate specificity for the dephosphorylation of a variety of proteins. The membrane-associated PPase was strongly (70-80%) inhibited by detergents (0.5%) such as Nonidet P-40, Lubrol PX, Triton X-100 and Tween-20. Pyrophosphate (5 mm) and orthovanadate (400 microM) had no significant effect on the activity of the isolated PPase whereas polyamines such as spermine (10 mM) and spermidine (10 mM) slightly inhibited (20%) the enzymatic activity. Inorganic phosphate (10 mM) and NaF (10 mM), the well-known inhibitors of the cytosolic PPases, had no appreciable effect on the activity of PPase M-I, indicating that the membrane-bound PPase is distinct from the cytosolic PPases. The enzyme was radiolabelled when the intact spermatozoa were subjected to lactoperoxidase-mediated radioiodination reaction. The results show that the PPase M-I is an ecto-enzyme that may play an important role in sperm physiology by causing the dephosphorylation of the sperm outer surface phosphoproteins.

Biochemical parameters regulating forward motility initiation in vitro in goat immature epididymal spermatozoa.

An investigation was carried out to analyse the biochemical parameters influencing forward motility (FM) initiation in vitro in the goat caput-epididymal immature spermatozoa. Forward motility was induced in approximately 55% of caput-sperm upon incubation in an alkaline (pH 8.0) modified Ringer's solution containing theophylline (30 mM) (an inhibitor of cyclic AMP phosphodiesterase), dialysed epididymal plasma (EP) and bicarbonate. Both EP and bicarbonate induced sperm motility in a dose-dependent manner, and at saturating doses EP (0.6 mg protein mL(-1)) and bicarbonate (25 mM) induced FM in approximately 38% and 44% of the cells, respectively. The motility-promoting efficacy of EP was attributed to a heat-stable protein termed 'forward motility protein' (FMP). Bicarbonate served as an initiator as well as a stabilizer of FM and its action was not dependent on FMP. FMP can induce FM in the caput-sperm, but it is not essential for sperm motility initiation. Alteration of the medium pH from 6.60 to 8.00 caused a marked increase in the EP or bicarbonate-dependent sperm FM initiation, as well as intrasperm pH. At the physiological pH, bicarbonate served as a much more potent motility activator than FMP, although both the motility promoters showed maximal efficacy at alkaline pH (approximately 7.8). EP as well as bicarbonate elevated the intrasperm cyclic AMP level. Unlike EP, bicarbonate is capable of increasing intrasperm pH. The intrasperm pH increased from 6.54 +/- 0.02 to 6.77 +/- 0.03 during sperm transit from caput to cauda. The data are consistent with the view that FMP activates sperm forward motility by enhancing the intrasperm cyclic AMP level and that extracellular bicarbonate and pH play a vital role in the initiation of sperm FM during the epididymal transit.

The role of phosphocreatine kinase in the motility of human spermatozoa supported by different metabolic substrates.

In the spermatozoa of some species creatine kinase (CK: E.C. 2.7.3.2) is involved in shuttling energy, in the form of creatine phosphate, between the mid-piece mitochondria and flagellum. In this study, the effects of the CK inhibitor dinitrofluorobenzene (DNFB) on human sperm CK activity, motility and ATP concentrations were assessed with different energy substrates. There was a dose-dependent inhibition of CK activity by DNFB but inhibition was incomplete and there was no effect on the percentage of flagellating cells, irrespective of substrate. However, when lactate alone supported the cells DNFB decreased velocities and increased amplitude of head displacement (fewer progressively motile forms were observed), whereas ATP concentrations in spermatozoa were unaltered. Demembranated sperm models could be reactivated by ADP plus creatine phosphate, but not to the extent caused by ATP, and were able to be inhibited by myokinase inhibitors. Increased velocities, linearity (LIN) and beat cross frequency (BCF) were demonstrated for spermatozoa incubated with lactate, in contrast to glucose as sole energy source, and higher velocities and BCF were generated in the presence of both substrates. This suggests that the production of ATP by glycolysis and respiration are independent and complementary. CK is not obligatory for sperm motility but supplements energy provision under certain conditions.

In-vitro initiation of forward motility in testicular spermatozoa.

Initiation of forward motility in vitro was investigated in goat and ram spermatozoa obtained from the rete testis. No forward motility was generated in the immotile testicular spermatozoa when they were incubated in a modified Ringer's solution containing theophylline (30 mM) and epididymal plasma (2 mg protein/ml). However, these reagents induced non-progressive flagellar movement in approximately 25% of spermatozoa. Bicarbonate (25 mM) induced forward motility in approximately 16% of the goat/ram testicular spermatozoa. Theophylline was essential for the bicarbonate-mediated activation of sperm motility, but epididymal plasma had no significant effect on this activation process. Theophylline activated progressive motility in testicular spermatozoa in a dose-dependent manner, the maximum effect occurring after incubation for 10 min with 30 mM theophylline. The initiation profile of in-vitro motility of goat/ram spermatozoa from the caput epididymis closely resembled that of testicular spermatozoa except that induction of motility in the caput spermatozoa was dependent both on bicarbonate and epididymal plasma. The data indicate that, unlike caput epididymal spermatozoa, initiation of motility in testicular spermatozoa is not dependent on motility-promoting protein(s) in epididymal plasma.

Cyclic AMP phosphodiesterase: a regulator of forward motility initiation during epididymal sperm maturation.

The concentrations of cAMP, cAMP phosphodiesterase (PDE) activity, and the effect of theophylline in vitro on the forward motility (FM) of maturing goat epididymal sperm have been analyzed. cAMP levels increase slowly during transit of the cells from the caput to the proximal cauda, although they acquired a minimal degree of forward progression. The last phase of sperm transit (proximal to distal cauda) was associated with a concomitant sharp rise in the level of both cAMP as well as flagellar motility. PDE activity progressively decreased (approximately threefold) during epididymal maturation, being minimal in mature cauda sperm. Theophylline (30 mM), a specific inhibitor of PDE, markedly activated (10-fold or greater) motility of the sperm derived from proximal-corpus, mid-corpus, distal-corpus, and proximal-cauda epididymides. FM of the native mature caudal sperm was similar to that of the theophylline-treated proximal-cauda sperm. The terminal stage of sperm maturity (proximal to distal cauda) was associated with a markedly reduced level of theophylline-dependent motility activation (approximately 50%). The data are consistent with the view that PDE plays an important role in the initiation of motility during epididymal sperm maturation.

Purification and characterization of a protein kinase from goat sperm plasma membrane.

A protein kinase that causes phosphorylation of serine and threonine residues of casein has been partially purified from goat cauda-epididymal sperm plasma membrane and characterized. The kinase, solubilized from the membrane with 1.0% Triton X-100, was purified to 480-fold by using DEAE-cellulose and casein-Sepharose affinity chromatographic techniques. The kinase is a strongly basic protein with pI of 9.5. The enzyme has a molecular mass of 310 kilodaltons as estimated by Sephacryl S-300 gel exclusion. The kinase showed affinity for protein substrates in the order membrane proteins > casein > phosvitin > histone > protamine. The apparent Km values of the kinase for casein and membrane proteins were 1 and 0.15 mg/mL, respectively. The synthetic peptides Kemptide and poly(Glu80Tyr20) did not serve as substrates of the enzyme. ATP, rather than GTP or PP(i), is the donor of phosphate for the phosphorylation reaction. Cyclic AMP and GMP, NaCl (0.25 M), KCl (0.25 M), Ca2+, calmodulin, phosphatidylserine, and muscle protein kinase inhibitor had no appreciable effect on the kinase activity. Heparin (0.5 microgram/mL) showed high affinity for inhibiting only 40% of the kinase activity, whereas polyamines at a relatively high concentration (5 mM) inhibited 40-50% of the enzymic activity. The kinase appears to be distinct from other protein kinases including casein kinases. The activity of the kinase derived from the purified sperm plasma membrane was markedly (approximately 90%) lost when the intact spermatozoa were pretreated with diazonium salt of sulfanilic acid, a membrane nonpenetrating surface probe.(ABSTRACT TRUNCATED AT 250 WORDS)

Phospholipid asymmetry of goat sperm plasma membrane during epididymal maturation.

The phospholipids and their fatty acids of the inner and outer plasma membrane leaflets of the maturing goat caput-, corpus-and cauda-epididymal spermatozoa were analyzed by treating the intact spermatozoa with phospholipase C and trinitrobenzene sulphonate. The inner and outer membrane showed marked differences in the phospholipid composition at all stages of epididymal sperm maturation. The outer membrane was rich in phosphatidylcholine (PC) and sphingomyelin (SPH) whereas the inner leaflet was dominated by phosphatidylethanolamine (PE). Although the ratio of PE/PC in the inner membrane was similar in both the mature cauda sperm and the immature caput sperm, it decreased significantly in sperm undergoing maturation in the corpus-epididymis. The distribution of the saturated and unsaturated fatty acids in the phospholipid fractions of both the membrane leaflets underwent profound alterations during the epididymal maturation. The data demonstrate asymmetry of phospholipids and their fatty acids in the sperm inner and outer plasma membranes and this lipid asymmetry is greatly altered during epididymal maturity of the male gametes.

Purification and characterization of rat epididymal neutral beta-galactosidase and its changes during in vivo development.

A neutral beta-D-galactosidase has been partially purified from rat epididymis and characterized. The enzyme having molecular mass of approximately 50 kilodaltons has been purified 400-fold by using calcium phosphate gel adsorption, DEAE-cellulose chromatography, Sephadex G-100 gel filtration, and concanavalin A--agarose affinity chromatography. Although the neutral enzyme binds to the concanavalin A affinity column, the activity could be eluted with alpha-methyl mannoside only if the buffer contained salt (NaCl) at a concentration as high as 0.3 M. The enzyme was of cytosolic origin, since 90% of the total enzymic activity of the tissue homogenate was recovered in the soluble fraction of these cells. The neutral beta-galactosidase was not dependent on metal ions for its activity and it had a pH optimum of 7.0. Zn2+, p-chloromercuribenzoate, Hg2+, and Pb2+ served as potent inhibitors of the enzyme. There was a marked increase (approximately fourfold) in the specific activity of the neutral beta-galactosidase during sexual maturity of epididymis in vivo.

Maturation-dependent goat epididymal sperm autoagglutination and its inhibition by a glycoprotein factor.

The maturing goat epididymal spermatozoa were isolated from different segments of epididymis and these cells dispersed in a modified Ringer's solution, were incubated at 37 degrees C for 60 min to evaluate their autoagglutination efficacy. Distal corpus-epididymal spermatozoa specifically showed high order of head-to-head autoagglutination property whereas all other sperm cells did not show any detectable aggregation. The goat epididymal plasma has been shown to possess an anti-agglutinin that markedly inhibits sperm agglutination phenomenon and also dissociates the cells from the sperm clusters. Epididymal plasma is the most potent source of the anti-agglutinin which is a heat-stable specific glycoprotein. Like the autoagglutination phenomenon, the initiation of sperm forward progression also starts in the distal-corpus epididymis. The temporal correlation of these two events suggests that sperm autoagglutination may be a prerequisite for the induction of flagellar motility during the epididymal maturity of male gametes.

Occurrence of wax esters and 1-O-alkyl-2,3-diacylglycerols in goat epididymal sperm plasma membrane.

Two unusual lipid classes were detected by thin-layer chromatography in the neutral lipids derived from goat cauda-epididymal sperm plasma membrane. The lipids were identified as wax esters and 1-O-alkyl-2,3-diacylglycerols based on chromatographic properties, identity of their hydrolysis products, and infrared/1H nuclear magnetic resonance spectral evidence. The membrane contained ca. 3 and 5 micrograms/mg protein of wax esters and alkyldiacylglycerols, respectively. The relative proportions of wax esters and alkyldiacylglycerols in the total neutral lipids were 1.5% and 2.4%, respectively. The lipids contained fatty acids with chain lengths of C14 to C22. The major fatty acids of the wax esters were 14:0, 16:0, 16:1 omega 7, 18:0 and 18:1 omega 9. The fatty acids in alkyldiacylglycerol were 16:0, 18:0, 22:5 omega 3 and 22:6 omega 3. Alkyldiacylglycerol was particularly rich in docosahexaenoic acid (22:6 omega 3) representing 30% of the total fatty acids. The alcohols of wax ester were all saturated with C20-C29 carbon chains. The deacylated products derived from alkyldiacylglycerols were identified as hexadecyl, octadecyl and octadec-9'-enyl glycerol ethers.