Purification and Characterization of a Sperm Motility Inhibiting Factor from Caprine Epididymal Plasma

Several studies have been reported on the occurrence of sperm motility inhibiting factors in the male reproductive fluids of different mammalian species, but these proteins have not been adequately purified and characterized. A novel sperm motility inhibiting factor (MIF-II) has been purified from caprine epididymal plasma (EP) by Hydroxylapatite gel adsorption chromatography, DEAE-Cellulose ion-exchange chromatography and chromatofocusing. The MIF-II has been purified to apparent homogeneity and the molecular weight estimated by Sephacryl S-300 gel filtration is 160 kDa. MIF-II is a dimeric protein, made up of two subunits each having a molecular mass of 80 kDa as shown by SDS-PAGE. The isoelectric point of MIF-II is 5.1 as determined by chromatofocusing and isoelectric focusing. It is a heat labile protein and maximal active at the pH 6.9 to 7.5. The sperm motility inhibiting protein factor at 2 µg/ml (12.5 nM) level showed maximal motility-inhibiting activity. The observation that the epididymal plasma factor lowered the intracellular cAMP level of spermatozoa in a concentration-dependent manner suggests that it may block the motility of caprine cauda spermatozoa by interfering the cAMP dependent motility function. The results revealed that the purified protein factor has the potential of sperm motility inhibition and may serve as a vaginal contraceptive. The antibody raised against the MIF-II has the potential for enhancement of forward motility of cauda-spermatozoa. This antibody may thus be useful for solving some of the problems of male infertility due to low sperm motility

Identification of Membrane Antigens of Goat Epididymal Spermatozoa

Purified goat sperm plasma membrane was used as antigen to raise the antibody in rabbit. ?Lsing this antisera four groups of antigenic membrane polypeptides are determined in caput and cauda epididymal sperm. The immuno-responsiveness of the polypeptides in caput and cauda sperm differs signifi- cantly. In case of cauda epididymal sperm, the polypeptides of region A (96ma, 82KDa, 78KDa, 6SKDa) and region D (24KDa, 2OKDa, 1SRDa) are highly immunores- ponsive whereas in case of caput epididymal sperm the same antisera recognized the polypeptides of region R, C and D. By surface labelling with lactoperoxi- dase iodination and subsequent immunoprecipitation in the iodinated cell extract we demonstrate eight of these above polypeptides (96KDa, 82KDa, 6SJCDa, SOKDa, 29KDa, 24KDa, 2OKDa and 1SKDa) as surface antigen. The 96KDa, R2RDa and 6WDa surface polypeptides are highly immunoresponsive than the other lower molecular weight surface antigens in cauda epididymal goat spermatozoa

Homologous Liver Parenchymal Cell-Cell Adhesion Mediated by an Endogenous Lectin and its Receptor

Many studies have implicated cell-surface lectins in heterologous cell-cell adhesion, but little is known about the participation of lectins in cellular adhesion in homologous cells. Here, we show the development of a cell model for investigating the direct role of a cell-surface lectin in homologous cell-cell adhesion. Parenchymal cells were isolated from caprine liver using a perfusion buffer, and dispersed in a chemically defined modified Ringer’s solution. These cells undergo autoagglutination in the presence of Ca2+. The autoagglutinated cells can be dissociated specifically with D-galactose (50 mM), which also inhibits the liver cell autoagglutination event. The blood serum protein fetuin has no effect on liver cell autoagglutination, whereas desialylated fetuin (100 μM), with its terminal D-galactose residue, showed a high affinity for blocking the autoagglutination event. The data demonstrates the occurrence of a Ca2+-dependent D-galactose-specific lectin and a lectin receptor on the parenchymal cells. Furthermore, it shows that the observed autoagglutination event is caused by the interaction of the cell-surface lectin with its receptor on the neighbouring homologous cells. The data supports the view that homologous cell-cell contact in mammalian tissues is triggered by such lectin-receptor interaction and that the previously reported cell-surface adhesive proteins serve as a secondary force to strengthen cell adhesion. This cell model could be extremely useful for investigating the direct role of cell-surface lectin and its receptor in homologous cell adhesion in a variety of tissues under normal and pathological conditions

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, Ca2+\p=n-\calmodulin, phosphatidylserine\p=n-\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 spermatozo

Goat sperm membrane: lectin-binding sites of sperm surface and lectin affinity chromatography of the mature sperm membrane antigens

The cell surface glycoproteins of goat epididymal maturing sperma~r;zoa have been investigated using lectins as surface probes that interact with specific sugars with high a~,aity. Concanavalin A (ConA) and wheat-germ agglutinin (WGA) showed high affinity for mature cauda epidi~ymal sperm ~gg[utination, whereas RCA,, kidney beans lectin and peanut agglutinin caused much lower or little aggluth,~fion of the cells. The mature sperm exhibited markedly higher efficacy than the immature caput epididymal sperm fo)r binding both ConA and WGA, as evidenced by sperm agglutination and the binding of the fluorescence isothiocyanate (FITC)-Iabeiled lecfins. FITC-ConA binds uniformly to the entire mature sperm surface whereas FITC.WGA binds to the acrosemai cap region of the head. The FITC-RCA 2 mainly labelled the posterior head of mature cauda sperm. However. no WGA-speciflc glycoprotein receptors could be detected in sperm plasma membrane (PM) by WGA-Sepharose affinity chromatography. The data implied that the epididymal sperm maturation is associated with a marked increase in the ConA/WGA receptors and that WGA receptors may be glycolipids rather than glyco~r~teins. Analysis of the ConA receptors of cauda sperm PM identified by ConA-S:pbarose affinity chromatography and subsequent resolution in SDS-PAGE demonstrated the presence of five glycopolypeptides of different concentrations (98, 96, 43, 27 and 17 kDa) of goat sperm membrane. The immunobbt of these ConA-specific glycopeptides with anti-sperm membrane antiserum showed that 98- and 96-kDa receptors are immunor~spo~sive

Stimulation of Forward Motility of Goat Cauda Epididymal Spermatozoa by a Serum Glycoprotein Factor

Blood sera of humans, rats, goats, and buffalo have been shown to possess a forward motility-stimulating factor (FMSF) that markedly sti,nula:ed goat cauda epididymal sperm forward motility, as assayed by a microscopic method in the presence of epididymal plasma (1.2mg protein/mI) that had sufficient anti-sticking activity to eliminate the possibility of cell-sticking artifacts in motility assays. The spec (fic activity of FMSF was greatest in buffalo blood serwn compared to the sera of the other species. Buffalo serum at a concentration as low as 8.5 mg protein/mi induced forward motility in nearly 45% of the cells. The buffalo serum FMSF was heat-stable, nondialyzable,and sensitive to the action of trypsin. Purified proteins - casein, serum albumin, oval bwnin, myoglobin, and [3-lactoglobulin - showed little or relatively low FMSF activity. FMSF is a glycoprotein, as it binds with high affinity to concanavalin A-agarose. A major portion of the serum protein (approx. 70%) did not bind to the affinity matrix, and this unretained serum protein fraction showed little FMSF activity. The FMSF activity of buffalo serum was confirmed by estimating sperm forward motility specrophotome:ricaliy: an objective method of assessing sperm motility

Identification and Characterization of a Sperm Motility Promoting Glycoprotein From Buffalo Blood Serum

Early investigators reported the occurrence of unidentified protein factors in biological fluids that may regulate sperm motility essential for fertility potential. This study reports for the first time purification of a forward motility stimulating protein (FMSF-I), to apparent homogeneity, from a biological fluid (buffalo blood serum) and its characterization. FMSF-I is the major motility protein of buffalo serum: a rich source of the factor. FMSF showed high protein specificity and affinity for activating forward motility of goat cauda epididymal spermatozoa. The motility promoter at 0.5 mM level showed maximal activity when nearly 60%–70% of spermatozoa expressed forward motility. It is a 66 kDa monomeric acidic protein rich in aspartate, glutamate, and leucine with isoelectric point of 3.7. FMSF: aMg2þ-dependent protein binds to concanavalin A-agarose and the glycoprotein nature of FMSF has been confirmed by PAS staining. The factor lost activity completely when treated with a-mannosidase showing that the sugar part of the protein is essential for its biological activity. FMSF has no species specificity for its motility-activating potential. Sperm surface has specific receptors of FMSF, which is strongly immunogenic. The factor is present in testis and epididymis although liver is its richest source. Motility promoting efficacy of FMSF is markedly higher than the well-known non-protein motility activators: theophylline and bicarbonate or their combination. FMSF is a physiological activator of sperm motility and as a slaughterhouse byproduct it has potentiality for solving some of the problems of animal breeding, conservation of endangered species, and human infertility: a global social problem

Lipid changes Of goat sperm plasma membrane during epididymal maturation

Highly purified plasma membranes of maturing goat caput-, corpus- and cauda-epididymal spermatozoa were isolated by aqueous two-phase polymer methods and their lipid constituents were analysed, Phospholipid (app~'ox. 75% w/w), neutral lipid (approx. 15% w/w) and glycolipid (approx. 10% w/w) were the major sperm membrane lipids. "l'here was a significant decrease in the total lipids (approx. 25% w/w), phospholipid {approx. 30% w/w) and glycotipid (approx. 80% w/w) contents of sperm membrane during epididymal maturation. On the contrary, the mature cauda-sperrn membrane showed greater (approx. 50% w/w) neutral lipid content than that of the immature caput sperm. Phosphatidylcholine (PC), phosphatidylethanolamine (PE) and sphingomyelin were the phospholipids of the sperm membrane, the former two being the major lipids. Both PC and PE fractions consisted of three species - diacyl, alkylacyl and alkenylacyl forms, the last one being lhe dominant species in both PC and PIE. Of all the phospholipids, diacyl PE decreased most strikingly (approx. 65% w/w) during sperm maturation. The neutral lipid fraction contained sterols, wax esters,l-O alkyl-2,3-diacylglycerol, triacylglycerol and fail?, acids. Sterols represented nearly 75% w/w of the neutral lipids and cholesterol was the major component (approx. 95% w/w) of the sterol fraction. The sperm maturity was associated with marked increase of sterol {approx. 60% w/w) and steryl ester (approx. 200% w/w) and decrease (approx. 50-65% w/w) of the other membrane-bound neutral iipids, The glyeolipid was identified as monogalaetosyldiacylglycerol. The fatty acid profile of the various membrane lipids underwent marked alteration during the epididymal transit of the male gametes. Cholesterol/phospbolipid and saturated/unsaturated fatty acid ratios increased greatly in the maturing sperm membrane. The altered lipid profile of ',he ma~nre sperm membrane leads to changes in its fluidity that play an important role in determining the structure and functions of the biomembrane

Synchronous Modulation of Cell Surface Lectin and Its Receptor in a Homologous Cell Population: A Novel Mechanism of Cellular Regulation

Testicular immotile sperm undergo maturation during epididymal transit when these cells pass through caput, corpus, and cauda-epididymal regions. Maturing goat spermatozoa specifically at the distal corpus epididymal stage show head-to-head autoagglutination when incubated in vitro in a modified Ringer's solution. Here, we show the biochemical mechanism of autoagglutination event and its functional significance. A lectin-like molecule located on sperm surface specifically interacts with its receptor of the neighboring homologous cells to cause autoagglutination. Lectin is a Ca++-dependent galactose-specific protein. Failure of the pre- and post-distal corpus sperm to show autoagglutination is due to lack of lectin-like molecule and its receptors, respectively. Maturing sperm at distal corpus stage acquire lectin-like molecule followed by sharp disappearance of its receptor, and this event is synchronously associated with the initiation of sperm forward motility that is essential for fertilization in vivo. Lectin and its receptor isolated from sperm plasma membrane showed high efficacy for blocking autoagglutination phenomenon. The data are consistent with the view that synchronous modulation of homologous cell surface lectin and their receptors constitutes a novel mechanism for cellular regulation by generating waves of signals by manipulating lectin–sugar-dependent “self-talk” and cell–cell “cross-talk”

Phospholipid Asymmetry of goat Sperm Plasma-Membrane during Epididymal Maturation

The p~s~~oI~p~d~ and their fatty acids of the inner and outer plasma membrane feaBets of the rn~i~in~ goat cap&-, corpusand caud~-cp~~~rna~ ~~er~~ozoa were ana&zed by treating the intact s~e~~to~a with phospbo~pas~ C and ~K~~~oben~~ne sulphonate, The inner and outer membrane showed marked differences in the pho~pholipjd corn~s~t~o~ at all stages of cp~didymal sperm ma~ra~o~. The outer memlxm~ was rich in ~~lospbat~~ic~o~~~ (PC> and sp~~~~~e~o fSPf_f) whereas the inner leaflet was dominated by phosphatidylecb~olamine (FE& ~tbougb the ratio of FE/ PC in the inner rnembr~~ was similar in both the mature cauda sperm and the ~rnrna~~rec ap& sperm, it decreased s~~~~~c~t~~in sperm ~~dcrgoi~ mat~lration in the corpus-epididymis. The dis~~~b~tio~o f the saturated and ~sat~~ated fatty acids in the phospbo~~p~df ractions of both the membrane Leaflets undereat profound allocations during the ~~idid~~l ~~~t~~ation. The data demonstrate as~rne~ of phosphol~~ids and their fatty acids in the sperm inner and outer plasma rnernb~a~~s and this lipid asymmetry is greatly altered during ~~idid~a~ maturity of the male gamete