Impact of epididymal maturation on the tyrosine phosphorylation patterns exhibited by rat spermatozoa

As mammalian spermatozoa migrate through the epididymis, they acquire functionality characterized by the potential to express coordinated movement and the competence to undergo capacitation. The mechanisms by which spermatozoa gain the ability to capacitate during epididymal transit are poorly understood. The purpose of this study was to investigate the impact of epididymal maturation on the signal transduction pathways regulating tyrosine phosphorylation, because this process is thought to be central to the attainment of a capacitated state and expression of hyperactivated motility. Western blot and immunocytochemical analyses demonstrated that epididymal maturation in vivo is associated with a progressive loss of phosphotyrosine residues from the sperm head. As cells pass from the caput to the cauda epididymis, tyrosine phosphorylation becomes confined to a narrow band at the posterior margin of the acrosomal vesicle. Epididymal maturation of rat spermatozoa was also associated with an acquired competence to respond to high levels of intracellular cAMP by phosphorylating tyrosine residues on the sperm tail. Immature caput spermatozoa were incapable of exhibiting this response, despite the apparent availability of cAMP and protein kinase A. These findings help to clarify the biochemical changes associated with the functional maturation of spermatozoa during epididymal transit.     

Protein thiols in spermatozoa and epididymal fluid of rats.

Thiol (SH) oxidation to disulphides (SS) is thought to be involved in sperm chromatin condensation and tail structure stabilization, which occur during maturation of spermatozoa. Previously developed procedures, using the fluorescent labelling agent monobromobimane (mBBr), enabled us to study the thiol-disulphide status of spermatozoa. Electrophoretic separation of labelled sperm proteins from the caput and cauda regions showed that during maturation thiol oxidation occurs in many protein fractions from the tail and that the magnitude of oxidation differs between proteins. Among the protein bands, one major band (MPB), probably a dense fibre constituent, is quantitatively prominent. N-Ethylmaleimide (NEM) or mBBr alkylation (of intact spermatozoa) changes the mobility of the caput MPB, but not that of the cauda MPB. The results indicated that the altered mobility of MPB is mainly due to a change in its shape, possibly resulting from the alkylation of a few critical SH groups. Epididymal fluid proteins contain both SH and SS. The thiol and disulphide content of the various epididymal proteins appears similar, although some diminution in fluorescence is seen in epididymal fluid proteins from the cauda region as compared with those from the caput region. The prominent changes in thiol status occur in the spermatozoa.     

Impact of epididymal maturation, ejaculation and in vitro capacitation on tyrosine phosphorylation patterns exhibited of boar (Sus domesticus) spermatozoa

Mammalian spermatozoa acquire functionality during epididymal maturation and ability to penetrate and fertilize the oocyte during capacitation. The aim of this study was to investigate the impact of epididymal maturation, ejaculation and capacitation on phosphotyrosine content of sperm proteins. Western blot, immunocytochemical and flow cytometry analyses demonstrated that epididymal maturation in vivo is associated with a progressive loss of phosphotyrosine residues of the sperm head followed by a subtle increase after in vitro capacitation. As cells pass from caput to cauda epididymis, tyrosine phosphorylation becomes confined to a triangular band over the posterior part of midacrosome region, whereas in vitro capacitation causes a spread labeling over the whole head. Different bands with phosphotyrosine residues were detected during epididymal maturation and after in vitro capacitation: 1) 93, 66 and 45 kDa bands with specific phosphotyrosine expression in immature spermatozoa; 2) 76, 23 and 12 kDa bands with specific phosphotyrosine expression in mature spermatozoa, being significantly increased in their expression after in vitro capacitation; 3) 49, 40, 37, 30, 26 and 25 kDa constitutive bands that increased their phosphotyrosine expression after maturation and/or in vitro capacitation; and 4) 28 and 20 kDa bands with a specific phosphotyrosine expression in in vitro capacitated spermatozoa. These results provided integral novel data of expression and location of phosphotyrosine residues during epididymal maturation, ejaculation and in vitro capacitation of boar spermatozoa. Two new constitutive proteins bands of 26 and 25 kDa with phosphotyrosine residues were also identified.     

Tyrosine phosphorylation as evidence of epididymal cauda participation in the sperm maturation process of Corynorhinus mexicanus bat

The bat Corynorhinus mexicanus provides an interesting experimental model for the study of epididymal sperm maturation because after spermatogenesis and the regression of the testes, this bat stores sperm in the epididymal cauda for several months. Earlier research conducted by our group suggested that sperm maturation in this species must be completed in the caudal region of the epididymis. One of the major signal transduction events during sperm maturation is the tyrosine phosphorylation of sperm proteins. The aim of the present study was to comparatively evaluate tyrosine phosphorylation in spermatozoa obtained from the caput, corpus and cauda of the epididymis during the sperm storage period. The maturation status of the sperm was determined by the percentage of capacitation and tyrosine phosphorylation in sperm obtained from the epididymis. The highest proportion of tyrosine phosphorylation was registered after the sperm had reached the cauda epididymis during the middle of the storage period. In conclusion, in Corynorhinus mexicanus and most likely in other chiropteran species with an asynchronous male reproductive pattern, epididymal sperm maturation ends in the caudal region of the epididymis and is related to the time that the sperm remains in the epididymis before mating activity.     

Effects of caput ligation on rat sperm and epididymis: protein thiols and fertilizing ability.

Mammalian spermatozoa mature while passing through the epididymis. Maturation is accompanied by thiol oxidation to disulfides. In rats, sperm become motile and fertile in the cauda. We have previously demonstrated that rat caput sperm contain mostly thiols and that upon passage from the corpus to the cauda epididymidis, sperm protein thiols are oxidized. The present work was undertaken to study the role of the regions of the epididymis in sperm maturation as reflected in the thiol status, fertility, and motility of the spermatozoa. The distal caput epididymidis of mature albino rats was ligated on one side. After 5 days, sperm were isolated from the ligated caput and from caput and cauda of the control side. Thiol groups in sperm, epididymal luminal fluid (EF), and epididymal tissue were labeled using the fluorescent thiol-labeling agent monobromobimane. After ligation, changes were observed in a) sperm proteins, sperm nuclear proteins, and epididymal fluid by electrophoresis; b) epididymal tissues by histochemistry; c) progressive motility by phase microscopy; and d) fertilizing ability after insemination into uteri of immature females. We found that after ligation, caput sperm thiols, especially protamine thiols, are oxidized, rendering them similar to mature sperm isolated from the cauda epididymidis. Spermatozoa from ligated caput epididymidis gain progressive motility and partial fertilizing ability. Morphology of epithelial cells of ligated caput is similar to that of cauda cells. However, other changes in caput EF and epithelium induced by ligation render the ligated caput epididymidis different from either control caput or cauda. Hence, sperm thiol oxidation, along with the development of fertilizing ability, can occur in sperm without necessity for sperm transit through the corpus and cauda epididymidis.     

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.     

Development of the signalling pathways associated with sperm capacitation during epididymal maturation

As spermatozoa mature within the epididymis they acquire the potential for capacitation and ultimately fertilization. In biochemical terms, the former is reflected in the progressive activation of a signal transduction pathway characterized by cAMP-mediated induction of phosphotyrosine expression on the sperm tail. In this study, we have examined the cellular mechanisms controlling this maturational event. Caput epididymal spermatozoa exhibited tyrosine phosphorylation on the sperm head that was largely unresponsive to cAMP and not significantly impaired by removal of extracellular HCO(3) (-). In contrast, caudal epididymal spermatozoa exhibited low levels of phosphorylation on the sperm head, yet responded dramatically to cAMP by phosphorylating a new set of proteins on the sperm tail via mechanisms that were highly dependent on extracellular HCO(3) (-). The impact of extracellular HCO(3) (-) depletion on caudal cells was not associated with a significant change in the redox regulation of cAMP but could be fully reversed by buffering the intracellular pH with N-Tris[Hydroxymethyl]methyl-3-amino-propanesulfonic acid (TAPS). The pattern of tyrosine phosphorylation was also profoundly influenced by the presence or absence of added extracellular calcium. In the presence of this cation, only caudal spermatozoa could respond to increased extracellular cAMP with tyrosine phosphorylation of the sperm tail. However, in calcium-depleted medium, this difference completely disappeared. Under these conditions, caput and caudal spermatozoa were equally competent to exhibit phosphotyrosine expression on the sperm tail in response to cAMP. These results emphasize the pivotal role played by calcium and HCO(3) (-) in modulating the changes in tyrosine phosphorylation observed during epididymal maturation.     

The effects of shRNA-mediated gene silencing of transcription factor SNAI1 on the biological phenotypes of breast cancer cell line MCF-7

Developing spermatozoa require a series of posttesticular modifications within the luminal environment of the epididymis to achieve maturation; this involves several surface modifications including changes in plasma membrane lipids, proteins, carbohydrates, and alterations in the outer acrosomal membrane. Epididymal maturation can therefore allow sperm to gain forward motility and fertilization capabilities. The objective of this study was to identify maturation-dependent protein(s) and to investigate their role with the production of functionally competent spermatozoa. Lectin blot analyses of caput and cauda sperm plasma membrane fractions identified a 17.5 kDa wheat germ agglutinin (WGA)-binding polypeptide present in the cauda sperm plasma membrane not in the caput sperm plasma membrane. Among the several WGA-stained bands, the presence of a 17.5 kDa WGA-binding polypeptide band was detected only in cauda epididymal fluid not in caput epididymal fluid suggesting that the 17.5 kDa WGA-binding polypeptide is secreted from the cauda epididymis and binds to the cauda sperm plasma membrane during epididymal transit. Proteomic identification of the 17.5 kDa polypeptide yielded 13 peptides that matched the sequence of peroxiredoxin-5 (PRDX5) protein (Bos Taurus). We propose that bovine cauda sperm PRDX5 acts as an antioxidant enzyme in the epididymal environment, which is crucial in protecting the viable sperm population against the damage caused by endogeneous or exogeneous peroxide.     

Dynamics of the thiol status of rat spermatozoa during maturation: analysis with the fluorescent labeling agent monobromobimane

Mammalian spermatozoa undergo maturation as they pass through the epididymis. Maturation is accompanied by the oxidation of thiols to disulfides. Disulfides are probably involved in sperm chromatin condensation and tail structure stabilization. In this work, we used the fluorescent thiol-labeling agent monobromobimane to determine the changes occurring in thiols and disulfides in rat sperm heads and tails during maturation. Spermatozoa were obtained from testis, epididymis (caput, corpus, cauda, and vas deferens), and ejaculate. Intact spermatozoa were labeled with monobromobimane, with or without pretreatment with dithiothreitol. Labeling was evaluated microscopically, and quantitative analysis was carried out spectrofluorimetrically with labeled globin used as a standard. Samples were also analyzed by gel electrophoresis. The total amount of thiols and disulfides remained the same during the entire period of sperm maturation (26 +/- 0.5 nmoles thiols + disulfides/10(6) spermatozoa). However, the reactive thiols decreased markedly between the corpus and the cauda (from greater than 90% of total in testis and 75% in corpus to about 25% in cauda), with little or no further change in vas deferens and ejaculated sperm. Trypsin treatment followed by sucrose gradient was used to separate the heads from the tails. Thiols comprised 84% of the total SH + SS in the heads and 74% in the tails of caput spermatozoa, decreasing to 14% and 45%, respectively, in cauda sperm. Thus, the decrease in reactive thiols involved both heads and tails-oxidation to disulfides being very marked in the head. Electrophoresis revealed that oxidation of thiols to disulfides occurred in many protein fractions during maturation in the epididymis.     

Transformation of sperm histone during formation and maturation of rat spermatozoa.

Changes of chromosomal basic proteins of rats have been followed during transformation of spermatids into spermatozoa in the testis and during maturation of spermatozoa in the epididymis. Rat testis chromatin has been fractionated on the basis of differing sensitivity to shearing, yielding a soluble fraction and a condensed fraction. The sperm histone is found in the condense fraction. Somatic-type histones are found in both fractions. The somatic-type histones in the condensed fraction contains much more lysine-rich histone I, than does the somatic-type histones in the soluble fraction. This may suggest that the lysine-rich histone I is the last histone to be displaced during the replacement of somatic-type histones by sperm histone. After extensive shearing followed by sucrose centrifugation, the condensed portion of testis chromatin can be further fractionated into two morphologically distinctive fractions. One is a heavy fraction possessing an elongated shape typical of the head of late spermatids. The other is a light fraction which is presumably derived from spermatids at earlier stages of chromatin condensation and which is seen as a beaded structure in the light microscope. Sperm histone of testis chromatin can be extractable completely by guanidinium chloride without a thiol, wheras 2-mercaptoethanol is required for extraction of sperm histone from caput and cauda epididymal spermatozoa. The light fraction of the condensed testis chromatin contains unmodified and monophospho-sperm histone. The sperm histones of the heavy fraction is mainly of monophospho and diphospho species, whereas unmodified and monophosphosperm histones are found in caput and cauda epididymal spermatozoa. Labeling of cysteine sulfhydryl groups of sperm histone releases by 2-mercaptoethanol treatment shows that essentially all of the cysteine residues of sperm histone in testis chromatin are present as sulfhydryl groups, while those of sperm histone isolated from mature (cauda epididymal) spermatozoa are present as disulfide forms and approximately 50% of the cysteine residues of sperm histone obtained from caput epididymal spermatozoa are in disulfide forms. These results suggest that phosphorylation of sperm histone is involved in the process of chromatin condensation during transformation of spermatozoa in the epididymis.     

cSrc is necessary for epididymal development and is incorporated into sperm during epididymal transit

Changes that occur to mammalian sperm upon epididymal transit and maturation render these cells capable of moving progressively and capacitating. Signaling events leading to mammalian sperm capacitation depend on the modulation of proteins by phosphorylation and dephosphorylation cascades. Recent experiments have demonstrated that the Src family of kinases plays an important role in the regulation of these events. However, sperm from cSrc null mice display normal tyrosine phosphorylation associated with capacitation. We report here that, despite normal phosphorylation, sperm from cSrc null mice display a severe reduction in forward motility, and are unable to fertilize in vitro. Histological analysis of seminiferous tubules in the testes, caput and corpus epididymis do not reveal obvious defects. However, the cauda epididymis is significantly smaller, and expression of key transport proteins in the epithelial cells lining this region is reduced in cSrc null mice compared to wild type littermates. Although previously, we and others have shown the presence of cSrc in mature sperm from cauda epididymis, a closer evaluation indicates that this tyrosine kinase is not present in sperm from the caput epididymis, suggesting that this protein is acquired by sperm later during epididymal maturation. Consistent with this observation, cSrc is enriched in vesicles released by the epididymal epithelium known as epididymosomes. Altogether, these observations indicate that cSrc is essential for cauda epididymal development and suggest an essential role of this kinase in epididymal sperm maturation involving cSrc extracellular trafficking.     

Posttranslational processing of PH-20 during epididymal sperm maturation in the horse.

It is generally accepted that spermatozoa become functionally mature during epididymal transit. The objective of this study was to determine whether the cellular location of equine PH-20 is modified during epididymal transit and, if so, the mechanism for such modification. Sperm were isolated from caput and cauda epididymal regions from stallions undergoing castration (n = 7) and used as whole sperm cell or subjected to nitrogen cavitation for isolation of plasma membrane proteins. Both caput and cauda sperm and sperm protein extracts were subjected to N-deglycosylation, O-deglycosylation, or trypsinization. The SDS-PAGE and Western blot analysis using a polyclonal anti-equine PH-20 IgG were performed in sperm extracts, and indirect immunofluorescence on whole sperm was also performed to determine the cellular distribution of plasma membrane PH-20 following similar treatments (deglycosylation or trypsinization). Hyaluronan substrate gel electrophoresis was performed to detect hyaluronidase activity in SDS-PAGE proteins. Western blots revealed significant differences in electrophoretic migration of PH-20 proteins from caput and cauda epididymal sperm. No effect was seen from deglycosylation treatments on the Western blot pattern; caput protein extracts exposed to trypsin showed the same band pattern as extracts from the cauda epididymis. N-deglycosylation resulted in the loss of hyaluronidase activity of sperm from both epididymal regions, whereas O-deglycosylation or trypsinization did not affect hyaluronidase activity. In caput epididymal sperm, the PH-20 protein is distributed over the entire sperm head; in cauda epididymal sperm, it is restricted to the postacrosomal region. No effect from deglycosylation on the cellular distribution of PH-20 was observed; however, treatment with trypsin changed the cellular distribution of PH-20 in caput sperm similar to that of the distribution of cauda sperm. These results suggest that PH-20 distribution during epididymal maturation is dependent on proteolytic trypsin-like mechanisms and, possibly, on complementary membrane-associated factors.     

Intra-acrosomal 155,000 dalton protein increases the antigenicity during mouse sperm maturation in the epididymis: A study using a monoclonal antibody MC101

We found an intra-acrosomal antigen of about 155,000 daltons (155 kDa) in a survey using the monoclonal antibody MC101 raised against mouse cauda epididymal spermatozoa. Morphological studies by means of indirect immunofluorescence and immunogold electron microscopy localized the antigen to the cortex region of the anterior acrosome. Avidin biotin complex immunocytochemistry initially demonstrated a faint signal at the anterior acrosome in the testis spermatozoa that increased in intensity as the sperm moved toward the distal epididymis. This incremental immunoreactivity was also confirmed by immunoblotting following one-dimensional SDS-PAGE. The 155 kDa protein band was immunostained, and it was much more intense in the cauda epididymal than in the caput and corpus epididymal spermatozoa. Only a trace or no immunostain was evident in the caput or testis spermatozoa. The antigen localization did not change during passage through the epididymis, being confined at the cortex region of the anterior acrosome. The epididymal epithelial cells were not immunostained. These findings suggested that the 155 kDa protein is biochemically modified, further implying that the biochemical alteration of intra-acrosomal material is involved in sperm maturation in the epididymis. © 1995 wiley-Liss, Inc.     

Differences between epididymal and ejaculated sperm characteristics in donkey

Spermatozoa acquire their motility and fertilizing ability during their passage through the epididymal canal. In the epididymal caput and corpus spermatozoa undergo several biochemical and metabolic changes while the cauda of the epididymis should be considered as the primarily site for storage of the spermatozoa. In the horse spermatozoa from cauda epididymis were collected and frozen, and the fertility of semen assessed. However, no studies have detailed semen characteristics of spermatozoa collected from the cauda epididymis in the jackass. In this study sperm characteristics of spermatozoa in the cauda epididymis of the donkey was reported and a comparison with ejaculated spermatozoal characteristics was performed. Samples from 10 Martina Franca jackasses were collected and analyzed for viability (Propidium iodide/Sybr-14? fluorescent stain), mitochondrial activity (Mitotraker? fluorescent stain), objective motility characteristics (by Computer Assisted Sperm Analyzer - CASA) and morphology. A higher viability and mitochondrial activity in the cauda epididymis samples were reported in this paper. Samples reported in this paper were identified and the percentage of total and progressive spermatozoa was comparable, but trajectories were more rapid (higher VCL) with less progressiveness (higher ALH and lower STR and LIN) in the cauda epididymis. Sperm morphology showed a pronounced variability between jackasses, with comparable values for all morphological subclasses. In this study the loss of the distal cytoplasmic droplets happen close to or after ejaculation because the percentage fell to nearly 0% after ejaculation. As suggested for bulls, the presence of a similar percentage in sperm with proximal cytoplasmic droplet in epididymal and ejaculated semen is likely to indicate a failure in the maturation process.     

Creatine phosphokinase in domestic cat epididymal spermatozoa

Mammalian spermatozoa that have not completed final testicular sperm maturation have residual cytoplasm and increased creatine phosphokinase (CK) content. This study determined: (1) if CK could be detected by immunostaining cat spermatozoa from the caput, corpus, and cauda epididymis, (2) fluctuations in the proportions of spermatozoa with mature or immature CK-staining patterns during epididymal sperm transit, and (3) how well sperm maturity (as determined by a CK marker) correlated with testicular or epididymal dysfunctions associated with morphological sperm abnormalities. One epididymis was collected from each of 37 cats after orchiectomy and processed immediately to allow sperm morphology evaluations on a 'regional' basis. Sperm released from the contralateral epididymis were evaluated for motility, sperm membrane integrity, and immunostaining with CK-B antibodies. Proportions of spermatozoa with malformed or detached heads, proximal droplets and acrosomal or midpiece abnormalities decreased (P < 0.05) from the caput to the cauda epididymis. In contrast, proportions of spermatozoa that were motile, membrane-intact or with flagellar abnormalities or distal droplets increased (P < 0.05) from the caput to cauda region. Percentages of spermatozoa with an immature CK-staining pattern also decreased (P < 0.05) with epididymal transit (which differs from that reported for the human and stallion). There was no correlation (P > 0.05) between sperm morphology and the CK-staining patterns. In summary, the results reveal that some specific sperm malformations in the domestic cat are of testicular origin, whereas others develop during epididymal transit.     

The localization of protein carboxyl-methylase in sperm tails

Protein carboxyl-methylase (PCM), an enzyme known to be involved in exocytotic secretion and chemotaxis, has been studied in rat and rabbit spermatozoa. PCM activity and its substrate methyl acceptor protein(s) (MAP) were demonstrated in the supernate after solubilization of the sperm cell membrane by detergent (Triton X-100). A protein methylesterase that hydrolyzes methyl ester bonds created by PCM was demonstrated in rabbit but not in rat spermatozoa. This enzyme was not solubilized by nonionic detergent. The specific activities of PCM in rat spermatozoa from caput and cauda epididymis were similar and lower than that found in testis. By contrast, MAP substrates were low in testis and increased in parallel with sperm maturation in the epididymis. Multiple MAP were demonstrated in spermatozoa by polyacrylamide gel electrophoresis. The pattern of these proteins was similar in spermatozoa from different portions of the reproductive tract. Fractionation of heads and tails of rat spermatozoa on sucrose gradients indicated that PCM was found exclusively in the tail fraction, whereas MAP was detected both in head and tail fractions. The presence of all the components of the protein carboxyl-methylation system in spermatozoa and the localization of PCM and some of its substrates in the sperm tail are consistent with their involvement in sperm cell motility.     

Changes in rat sperm membrane glycosidase activities and carbohydrate and protein contents associated with epididymal transit

Rat spermatozoa were recovered from the caput, corpus, and cauda epididymides and assayed for glycosidase activity, total nonamino (neutral) carbohydrate, and protein content. The activities of beta-glucosidase, beta-galactosidase, beta-N-acetylglucosaminidase, and beta-N-acetylgalactosaminidase were fluorometrically assayed in spermatozoa and membrane-enriched fractions. Except for beta-glucosidase, the activities of the glycosidases based on protein content were greatest in whole sperm and membrane-enriched fractions obtained from the cauda epididymides. Based on sperm concentration, however, glycosidase activities increased proceeding from the caput to the corpus epididymides, then declined from the corpus to the cauda epididymides. Analyses of nonamino carbohydrate and protein content based on sperm number indicated regional trends similar to those of glycosidase activity. Total nonamino carbohydrate and protein content were highest in corpus sperm, and lowest in cauda sperm. These data indicate major quantitative changes in cell surface carbohydrate as spermatozoa traverse the epididymis. A positive correlation for the membrane-enriched fraction between increasing glycosidase activity and decreasing carbohydrate and protein content suggests that glycosidases may play a significant role in modifying the spermatozoon surface during epididymal transit and maturation.     

Identification and validation of mouse sperm proteins correlated with epididymal maturation

Sperm need to mature in the epididymis to become capable of fertilization. To understand the molecular mechanisms of mouse sperm maturation, we conducted a proteomic analysis using saturation dye labeling to identify proteins of caput and cauda epididymal sperm that exhibited differences in amounts or positions on two-dimensional gels. Of eight caput epididymal sperm-differential proteins, three were molecular chaperones and three were structural proteins. Of nine cauda epididymal sperm-differential proteins, six were enzymes of energy metabolism. To validate these proteins as markers of epididymal maturation, immunoblotting and immunofluorescence analyses were performed. During epididymal transit, heat shock protein 2 was eliminated with the cytoplasmic droplet and smooth muscle γ-actin exhibited reduced fluorescence from the anterior acrosome while the signal intensity of aldolase A increased, especially in the principal piece. Besides these changes, we observed protein spots, such as glutathione S-transferase mu 5 and the E2 component of pyruvate dehydrogenase complex, shifting to more basic isoelectric points, suggesting post-translational changes such dephosphorylation occur during epididymal maturation. We conclude that most caput epididymal sperm-differential proteins contribute to the functional modification of sperm structures and that many cauda epididymal sperm-differential proteins are involved in ATP production that promotes sperm functions such as motility.     

Plasma membrane-associated protein tyrosine phosphatase activity in hamster spermatozoa

Plasma membranes of caput and cauda epididymal spermatozoa of hamster exhibited protein phosphatase activity. This membrane-associated protein phosphatase was identified as a protein tyrosine phosphatase based on its ability to hydrolyse a substrate specific for PTPase, by inhibition of its activity with a specific inhibitor of PTPase (sodium orthovanadate) and by the inability to inhibit its activity with calyculin, okadaic acid, trifluoperazine, calcium, EGTA, and EDTA, which are specific inhibitors of other protein phosphatases, namely PP-1, PP-2A, PP-2B, and PP-2C respectively. The specific activity of the protein tyrosine phosphatase both in the caput and cauda epididymal sperm plasma membranes was similar, implying that the enzyme may not be solely responsible for the differential phosphorylation of membrane proteins observed during maturation (Uma Devi et al. 1997. Mol Reprod Dev 47:341-350). Thus the significance of the PTPase activity in epididymal maturation still remains to be determined. The membrane-associated PTPase may not be essential for acquisition of motility. However, it appears that the activity is essential for the sustenance of motility since sodium orthovanadate, which specifically inhibits PTPase activity, also inhibits motility of spermatozoa and decreases the overall velocity of the spermatozoa by decreasing the average path velocity, straight line velocity, curvilinear velocity, and amplitude of lateral head displacement of the treated spermatozoa.     

Ontogeny of tyrosine phosphorylation-signaling pathways during spermatogenesis and epididymal maturation in the mouse

The objectives of this study were to map the ontogeny of tyrosine phosphorylation signal transduction pathways during germ cell development and to determine their association with the differentiation of a functional gamete. Until testicular germ cells differentiate into spermatozoa, cAMP-induced tyrosine phosphorylation is not detectable. Entry of these cells into the epididymis is accompanied by sudden activation of the tyrosine phosphorylation pathway, initially in the principal piece of the cell and subsequently in the midpiece. In the caput and corpus epididymides, the potential to express this pathway is inhibited by the presence of calcium in the extracellular medium. However, calcium has no effect on the expression of this pathway in caudal epididymal sperm. The competence of these cells to phosphorylate the entire sperm tail, from the neck to the tail-end piece, is accompanied by a capacity to exhibit hyperactivated motility on stimulation with cAMP. A distinctly different pattern of tyrosine phosphorylation, involving the acrosomal domain of the sperm head, is invoked as spermatozoa enter the caput epididymis, and phosphorylation remains high until these cells enter the distal corpus and cauda. The proportion of cells exhibiting this form of tyrosine phosphorylation is not affected by extracellular calcium or cAMP but is negatively correlated (R2 = 0.99) with their ability to acrosome-react. However, this relationship is not causative. Our findings indicate that the development of functional spermatozoa is accompanied by carefully orchestrated changes in tyrosine phosphorylation, controlled by independent regulatory mechanisms in distinct subcellular compartments of these highly specialized cells.