Heparin-binding proteins from seminal plasma bind to bovine spermatozoa and modulate capacitation by heparin

Bovine spermatozoa that have been exposed to seminal plasma possess more binding sites for heparin than sperm from the cauda epididymis that have not been exposed to accessory sex gland secretions. Seminal plasma exposure enables sperm, following incubation with heparin, to undergo zonae pellucidae-induced exocytosis of the acrosome. In this study, the regulatory role of seminal plasma heparin-binding proteins in capacitation of bovine spermatozoa by heparin was investigated. Plasma membranes from sperm exposed to seminal plasma in vivo or in vitro contained a series of acidic 15-17 kDa proteins not found in cauda epididymal sperm. Western blots of membrane proteins indicated that these 15-17 kDa proteins bound [125I]-heparin. Heparin-binding proteins were isolated by heparin affinity chromatography from seminal plasma from vasectomized bulls. Gel electrophoresis indicated that the heparin-binding peaks contained 14-18 kDa proteins with isoelectric variation, a basic 24 kDa protein, and a 31 kDa protein. Western blots probed with [125I]-heparin confirmed the ability of each of these proteins to bind heparin. Each of these proteins, as well as control proteins, bound to epididymal sperm. The seminal plasma proteins were peripherally associated with sperm since they were removed by hypertonic medium and did not segregate into the detergent phase of Triton X-114. Seminal plasma heparin-binding proteins potentiated zonae pellucidae-induced acrosome reactions in epididymal sperm. However, seminal plasma proteins that did not bind to the heparin affinity column were unable to stimulate zonae-sensitivity. Control proteins, including lysozyme--which binds to both heparin and sperm, were ineffective at enhancing zonae-induced acrosome reactions. These data provide evidence for a positive regulatory role of seminal plasma heparin-binding proteins in capacitation of bovine spermatozoa.     

Lectin-binding sites on the plasma membranes of rabbit spermatozoa: Changes in surface receptors during epididymal maturation and after ejaculation

Modifications in rabbit sperm plasma membranes during epididymal passage and after ejaculation were investigated by used of three lectins: concanavalin A (Con A); Ricinus communis I (RCA(I)); and wheat germ agglutinin (WGA). During sperm passage from caput to cauda epididymis, agglutination by WGA drastically decreased, and agglutination by RCA(I) slightly decreased, although agglutination by Con A remained approximately unchanged. After ejaculation, spermatozoa were agglutinated to a similar degree or slightly less by Con A, WGA, and RCA(I), compared to cauda epididymal spermatozoa. Ultrastructural examination of sperm lectin-binding sites with ferritin- lectin conjugates revealed differences in the densities of lectin receptors in various sperm regions, and changes in the same regions during epididymal passage and after ejaculation. Ferritin-RCA(I) showed abrupt changes in lectin site densities between acrosomal and postacrosomal regions of sperm heads. The relative amounts of ferritin-RCA(I) bound to heads of caput epididymal or ejaculated spermatozoa. Tail regions were labeled by ferritin RCA(I) almost equally on caput and cauda epididymal spermatozoa, but the middle-piece region of ejaculated spermatozoa was slightly more densely labeled than the principal-piece region, and these two regions on ejaculated spermatozoa were labeled less than on caput and cuada epididymal spermatozoa. Ferritin-WGA densely labeled the acrosomal region of caput epididymal spermatozoa, although labeling of cauda epidiymal spermatozoa was relatively sparse except in the apical area of the acrosomal region. Ejaculated spermatozoa bound only a few molecules of ferritin-WGA, even at the highest conjugate concentrations used. Caput epididymal, but not cauda epididymal or ejaculated spermatozoa, bound ferritin-WGA in the tail regions. Dramatic differences in labeling densities during epididymal passage and after ejaculation were not found with ferritin-Con A.     

Capacity of rete testicular and cauda epididymal boar spermatozoa to undergo the acrosome reaction and subsequent fusion with egg plasma membrane

The capacity to undergo the acrosome reaction and subsequent fusion with egg plasma membrane was examined in rete testicular and cauda epididymal spermatozoa from boars. Sperm penetration assay using zona-free hamster eggs demonstrated that the penetration rates for rete testicular spermatozoa preincubated for induction of the acrosome reaction for 2 and 3 h were 55% and 97%, respectively. However, most of the eggs (93%) were penetrated with polyspermy by cauda epididymal cells preincubated for 2 h. Results obtained by the triple-stain technique revealed the percentages of acrosome-reacted spermatozoa in the rete testicular and cauda epididymal samples preincubated for 3 h to be 61% and 74%, respectively. These results indicate that many rete testicular spermatozoa possess the capacity to undergo the acrosome reaction and subsequent fusion with egg plasma membrane in vitro, which appears to be completely established only after sperm transit through at least the proximal part of the epididymis. © 1993 Wiley-Liss, Inc.     

Interaction of isolated components from mammalian sperm and egg

In order to identify those proteins from the plasma membrane of hamster spermatozoa that exhibit an affinity for components of the zona pellucida we have used the Western blot technique. Zonae pellucidae from postovulatory hamster oocytes were solubilized by exposure to an acidic pH and then radiolabeled using the Bolton-Hunter reagent. These ¹²⁵I-zona pellucida proteins retain their immunoreactivity and migrate in three heterogeneous bands when submitted to SDS-PAGE electrophoresis. Membrane proteins from epididymal spermatozoa of mature hamsters were extracted by treatment with Nonidet P-40 and then submitted to electrophoresis (SDS-PAGE). The proteins in the gel were electrophoretically transferred to a nitrocellulose membrane and then probed with the radiolabelled zone pellucida proteins. ¹²⁵I-zona pellucida proteins bind preferentially to a sperm protein with a molecular weight of 26,400 ± 1,400 daltons (n = 9). Using a similar procedure it was shown that this protein also binds ¹²⁵I-Concanavalin A. The interaction between the sperm protein and the ¹²⁵I-zona pellucida proteins shows species specificity as demonstrated by the fact that the hamster ¹²⁵I-zona pellucida proteins do not bind to proteins extracted from ram, bull, and stallion spermatozoa. Whether this sperm protein could be implicated be implicated in the process of sperm-egg interaction is under investigation.     

Changes in binding of a 27-kilodalton chimpanzee cauda epididymal protein glycoprotein component to chimpanzee sperm

Motility patterns of caput epididymal chimpanzee sperm, caput epididymal chimpanzee sperm incubated in vitro with chimpanzee cauda epididymal fluid, and cauda epididymal chimpanzee sperm were assessed quantitatively. Sperm recovered from the caput epididymis showed no motility, whereas sperm recovered from cauda epididymis showed progressive forward motility. After incubation in cauda fluid, approximately 25% of caput epididymal sperm showed some motile activity. Electrophoretic analysis of ¹²⁵I-labeled sperm plasma membrane preparations revealed that the surface of caput epididymal sperm, incubated in cauda fluid, was modified by the appearance of a major protein-glycoprotein surface component with an apparent molecular weight of 27 kilodaltons (kD). THis 27-kD component was not detected on caput epididymal sperm incubated in buffer or in caput fluid. However, it was present in cauda fluid and on cauda epididymal sperm. Binding to caput epididymal sperm was cell specific in that chimpanzee erythrocytes incubated in cauda fluid did not bind this 27-kD cauda fluid component. Motility patterns of ejaculated chimpanzee sperm and of ejaculated chimpanzee sperm incubated in the uterus of adult female chimpanzees also were assessed quantitatively. Ejaculated sperm showed progressive forward motility, whereas in utero incubated ejaculated sperm showed hyperactivated motility typical of capacitated sperm. Electrophoretic analysis of ¹²⁵I-labeled sperm plasma membrane preparations revealed the loss of a 27-kD component from the surface of ejaculated sperm after in utero incubation. No significant change in the ¹²⁵I-distribution pattern was detectable when ejaculated sperm were incubated in buffer. These results suggest that the lumenal fluid component, which becomes adsorbed to the surface of chimpanzee sperm during maturation in the epididymis and which is removed from the surface of mature chimpanzee sperm in the female reproductive tract, affects sperm motility.     

Lactosaminoglycans synthesized by mouse male germ cells are fucosylated by an epididymal fucosyltransferase

We have studied the synthesis of protein-bound carbohydrates in differentiating male germ cells in the mouse. Spermatocytes and spermatids synthesize asparagine-linked and high-molecular-weight glycopeptides as the major classes of protein bound carbohydrates. Asparagine-linked glycopeptides were found to be mainly composed of the complex bi-antennary type as shown by affinity chromatography on concanavalin-A Sepharose; high-molecular-weight glycopeptides were represented by nonfucosylated lactosaminoglycans since they were metabolically labeled with [¹⁴C]glucosamine but not with [³H]fucose, did not bind to DEAE-cellulose, and were susceptible to endo-β-galactosidase. Labeling with galactose oxidase/Na B³H₄ technique demonstrated that lactosaminoglycans were present on the surface of differentiating germ cells and of testicular and epididymal spermatozoa. Since lactosaminoglycans from germ cells and testicular spermatozoa were not retained on a column of fucose-binding lectin, it was concluded that these molecules do not contain fucose. On the other hand, epididymal spermatozoa lactosaminoglycans bound to the lectin and therefore contained fucose. A soluble fucosyltransferase, capable of transferring fucose to germ cell lactosaminoglycans, was found to be present in the epididymis but not in the testis. These data show that developing germ cells synthesize nonfucosylated lactosaminoglycans which are probably preserved throughout spermiogenesis. We suggest that these molecules are fucosylated in vivo by a fucosyltransferase secreted by the epididymal epithelium.     

Amino acids in ram testicular fluid and semen and their metabolism by spermatozoa

1. The testis of the ram secretes considerable amounts of amino acids (200μmoles/day) into the fluid collected from the efferent ducts. The principal amino acid in this testicular fluid is glutamate, which is present in concentrations about eight times those in testicular lymph or in blood from the internal spermatic vein. 2. The concentration of glutamate in seminal plasma from the tail of the epididymis is about ten times that in testicular fluid, and, though glutamate is the major amino acid in ejaculated seminal plasma, its concentration is less than in epididymal plasma. 3. After the intravenous infusion of [U-¹⁴C]glucose, labelled glutamate was found in the testicular fluid. Radioactivity was also detected in alanine, glycine, serine plus glutamine and aspartate. Alanine had the highest specific activity, about 50% of the specific activity of blood glucose. 4. When [U-¹⁴C]glutamate was infused, the specific activity of glutamate in testicular fluid was only about 2% that in the blood plasma. 5. Testicular and ejaculated ram spermatozoa oxidized both [U-¹⁴C]glutamate and [U-¹⁴C]leucine to a small extent, but neither substrate altered the respiration from endogenous levels. 6. No radioactivity was detected in testicular spermatozoal protein after incubation with [U-¹⁴C]glutamate or [U-¹⁴C]leucine. Small amounts of radioactivity were detected in protein from ejaculated ram spermatozoa after incubation with [U-¹⁴C]glutamate. 7. The carbon of [U-¹⁴C]glucose was incorporated into amino acids by testicular spermatozoa; most of the radioactivity occurred in glutamate.     

Changes in the protein composition of rat spermatozoa during maturation in the epididymis

Spermatozoa from the testis and cauda epididymidis were solubilized by detergent treatment and electrophoresis on SDS polyacrylamide gels revealed that the relative amounts of 13 detergent-extractable proteins decreased during passage of spermatozoa through the epididymis, 6 increased, whilst the remainder showed little or no change. Lactoperoxidase-catalysed iodination of plasma membrane proteins showed that the components carrying most of the label in testicular spermatozoa had Mr values of 110 000, 94 000, 84 000, 55 000 and 42 000 whereas on cauda epididymal spermatozoa the Mr values were 47 000, 24 000, 17 000, 14 500 and 13 500. Substantial differences were also noted in the protein composition of rete testis fluid and cauda epididymal plasma. The results support the concept that there is a considerable reorganization of the molecular architecture of the plasma membrane of spermatozoa during maturation in the epididymis.     

Quantitative changes ofRicinus communis agglutinin I andHelix pomatia lectin binding sites in the acrosome of rat spermatozoa during epididymal transit

Summary During passage through the epididymis, spermatozoa undergo a number of changes which result in their acquisition of fertility and motility. Some of the changes that occur include loss of the cytoplasmic droplet and changes in sperm morphology, metabolism and properties of the nucleus and plasma membrane. Changes have also been reported in the acrosomic system of mammalian spermatozoa during their transit through the epididymis. In the present study, the quantitative changes of the glycoconjugate content in the acrosome of rat spermatozoa were examined during their passage through the epididymis using lectin-colloidal gold cytochemistry. Various regions of the epididymis (initial segment, caput, corpus and cauda epididymidis) were fixed by perfusion with 1% or 2% glutaraldehyde buffered in sodium cacodylate (0.1M), dehydrated in ethanol and embedded without osmication in Lowicryl K4M. Lectin-colloidal gold labeling was performed on thin sections usingRicinus communis agglutinin I (RCA I) orHelix pomatia lectin (HPL) to detectd-galactose-andN-acetyl-d-galactosamine-containing glycoconjugates, respectively. The labeling density over the acrosome of the acrosomic system was evaluated as the number of gold particles per m² of profile area using a Zeiss MOP-3 image analyzer. The overall mean labeling densities over the acrosome of spermatozoa for each lectin was estimated from 4 rats and over the four distinct epididymal regions. The mean labeling density of the acrosome with RCA I and HPL showed a similar pattern along the epididymis, although RCA I revealed approximately twice as many gold particles per epididymal region. In either case, there was a significant decrease in the labeling density of the acrosome of spermatozoa between the initial segment or caput epididymidis and cauda epididymidis (p<0.01). A similar decrease was also noted between the initial segment and corpus epididymidis (p<0.01). No change was found between the initial segment and caput epididymidis. Controls showed a virtual absence of labeling. These results suggest that in addition to a multitude of changes occurring to spermatozoa during epididymal transit, there are also significant quantitative changes in the glycoconjugate content within the acrosome.     

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.     

White tea intake prevents prediabetes-induced metabolic dysfunctions in testis and epididymis preserving sperm quality

Prediabetes has been associated with alterations in male reproductive tract, especially in testis and epididymis. Moreover, in vitro studies described a promising action of tea (Camellia sinensis L.) against metabolic dysfunctions. Herein, we hypothesized that white tea (WTEA) ingestion by prediabetic animals could ameliorate the metabolic alterations induced by the disease in testicular and epididymal tissues, preserving sperm quality. WTEA infusion was prepared and its phytochemical profile was evaluated by ¹H-NMR. A streptozotocin-induced prediabetic rat model was developed and three experimental groups were defined: control, prediabetic (PreDM) and prediabetic drinking WTEA (PreDM+WTEA). Metabolic profiles of testis and epididymis were evaluated by determining the metabolites content (¹H-NMR), protein levels (western blot) and enzymatic activities of key metabolic intervenient. The quality of spermatozoa from cauda epididymis was also assessed. Prediabetes increased glucose transporter 3 protein levels and decreased lactate dehydrogenase activity in testis, resulting in a lower lactate content. WTEA ingestion led to a metabolic adaptation to restore testicular lactate content. Concerning epididymis, prediabetes decreased the protein levels of several metabolic intervenient, resulting in decreased lactate and alanine content. WTEA consumption restored most of the evidenced alterations, however, not lactate content. WTEA also improved epididymal sperm motility and restored sperm viability. Prediabetes strongly affected testicular and epididymal metabolic status and most of these alterations were restored by WTEA consumption, resulting in the improvement of sperm quality. Our results suggest that WTEA consumption can be a cost-effective strategy to improve prediabetes-induced reproductive dysfunction.     

Plasma Membrane Glycoproteins of Ciona intestinalis Spermatozoa that Interact with the Egg1

In the ascidian Ciona intestinalis the species-specific interaction between the spermatozoon and the egg occurs between the vitelline coat (VC) of the egg and the plasma membrane of the apical part of the head of the spermatozoa. Concanavalin A (Con A)-binding sites are present on this area of the sperm surface. We used Con A to identify and isolate the spermatozoon plasma membrane components that may be involved in the interaction with the VC. These glycoproteins have been identified on SDS-PAGE of a sperm membrane fraction (SMF) enriched with the extermal proteins, after incubation of the gel with ³H-Con A. Affinity chromatography on Con A-agarose has been used for the purification of sperm plasma membrane proteins with and affinity for the lectin. The biological activity of the Con A-retained fraction was determined with binding and fertilization assays.     

Characterization of cAMP-dependent protein kinase and its endogenous substrate proteins in ram testicular,cauda epididymal,and ejaculated spermatozoa

Mammalian spermatozoa have been shown to possess cAMP-dependent protein kinase (A-PK) and endogenous substrate proteins for this enzyme. A study of the kinase system was undertaken to determine changes that may be associated with sperm maturation by comparing immature testicular with mature cauda epididymal and ejaculated spermatozoa. Absolute activity levels of A-PK, stimulated over a concentration range of 10^?9 to 10^?5 M, was significantly greater in testicular than ejaculated spermatozoa. At an optimal cAMP concentration (10^?6M), testicular spermatozoa had significantly greater amounts of cAMP-dependent protein kinase activity than did cauda or ejaculated spermatozoa. Electrophoretic analysis and autoradiography of NP-40-soluble protein extracts revealed the presence of two substrate proteins (M_r = 62,000 and 44,000) in all three types of spermatozoa. In addition, a phosphoprotein (M_r = 20,000) was detected in mature cauda and ejaculated but not immature testicular spermatozoa. The phosphorylation of these substrate proteins was both dose and time dependent. Examination of cyclic AMP phosphodiesterase activity revealed significantly higher levels in testicular than ejaculated spermatozoa. These results indicate marked alterations in cAMP-modulated protein phosphorylation and dephosphorylation systems in ram spermatozoa during epididymal maturation.     

Changes in plasma membrane glycoproteins of rat spermatozoa during maturation in the epididymis

Glycoproteins on the plasma membrane of testicular and cauda epididymidal spermatozoa have been labeled with galactose oxidase/NaB [3H]4 and sodium metaperiodate/NaB[3H]4, followed by analysis on SDS polyacrylamide gels. The major glycoprotein labeling on testicular spermatozoa has a molecular weight 110,000 whereas on cauda epididymidal spermatozoa greater than 90% of the radio-label is incorporated into proteins of molecular weight 32,000. These 32,000-mol wt X proteins are homologous with proteins of similar molecular weight purified from the epididymal secretion and which have been shown previously to be synthesized in the caput epididymidis under hormonal control. Immunofluorescence revealed that the 32,000-mol wt proteins are present on the flagellum of mature but not immature spermatozoa and that they have a patchy distribution suggesting that they are mobile within the plane of the membrane. The membrane-bound 32,000-mol wt proteins possess hydrophobic domains as revealed by charge-shift electrophoresis and they also label with a lipophilic photoaffinity probe suggesting that they are in contact with the lipid bilayer. The evidence indicates that there is a considerable reorganization of the molecular structure of the plasma membrane of spermatozoa during maturation in the epididymis and that some of the changes are brought about by a direct interaction with epididymal secretory proteins.     

Development changes occurring in the lipids of ram epididymal spermatozoa plasma membrane

Ram spermatozoa were obtained from different regions (caput, corpus, and cauda) of the epididymis and their plasma membrane was removed using a nitrogen cavitation treatment (750 psi, 10 min equilibration at 4 degrees C). Membrane was recovered after sucrose gradient centrifugation and identified using 125I-succinylated concanavalin A (125I-succConA) as a surface marker. Based on fluorescein isothiocyanate-succConA (FITC-succConA) labeling and electron microscopy, cavitation removed plasma membrane from the anterior sperm head in the area overlying the acrosome. Cholesterol was the major sterol in plasma membrane, with desmosterol present in sperm entering the epididymis (caput sperm) but negligible in sperm after epididymal transit (cauda sperm). Ethanolamine and choline phosphoglycerides represented 70-80% of membrane phospholipids, with the ethanolamine fraction decreasing relative to choline phosphoglycerides during epididymal transit. The molar ratio of cholesterol to phospholipid increased in the plasma membrane during maturation. The bulk phospholipid-bound fatty acids consisted primarily of palmitoyl acyl groups (16:0) in caput sperm and docosahexaenoyl acyl groups (22:6) in cauda sperm. The choline phosphoglyceride fraction was purified and analyzed. It consisted of a mixture of ether acyl glycero-3-phosphocholine and diacyl phosphoglyceride, with the dominant acyl residue, at all stages of epididymal maturation, being 22:6 throughout epididymal transit. The significance of these findings relative to acquisition of fertilization capacity by sperm during epididymal maturation is discussed.     

Localization, on the head of ram spermatozoa, of affinity sites for the 64kD androgen dependent epididymal prealbumin

The epididymis of the ram synthesizes under androgenic control of specific 64 kD protein. The purified protein appeared as a single band in SDS polyacrylamide gel electrophoresis. It was labelled and an antiserum was prepared in mice. Results showed localization of receptors sites for 64 kD on the periacrosomal plasma membrane of testicular spermatozoa. The protein itself was found on the periacrosomal area of both epididymal and ejaculated spermatozoa.     

小鼠附睾头精子获得与卵子质膜融合能力的物质基础研究

随着精子在附睾中的转运,它们与卵子质膜的融合能力逐渐增加。怩证明２附睾体和附睾尾的精子均具有相当高的膜融合能力,而附睾头中的精了奶少能与卵子质膜融合,这是还说明附睾头中的精子不具备与云透明带卵子融合的物质条件呢？利用附睾结扎留并延长体外获能时间,可使附睾头远端精子的融合能力明显地提高;在精子培养液中加入ＡＴＰ,并延长精卵共培养时间,也可使一少部分附睾头近端的精子获得与卵子质膜融合的能力。这表明附睾     

Acquisition of zona binding by ram spermatozoa during epididymal passage,as revealed by interaction with rat oocytes

To assess the ability of ram spermatozoa to bind to oocytes, spermatozoa (2.5–200 × 10⁶/500μ1) taken from the rete testis, or from various regions of the epididymis (head, body, and tail) were mixed with cumulus-free heterologous oocytes obtained from immature superovulated rats. After incubation for 30–45 min in Parker 199 Hepes medium at 35°C, testicular spermatozoa were unable to bind to the zona at any of the concentrations used. However, spermatozoa from the middle body of the epididymus were able to bind to the zona and this binding reached a maximum in the distal body and in the tail of the epididymis. The spermatozoa were bound by their heads. Electron microscopy showed that the plasma membrane and the acrosome of the bound sperm remained intact, without any sign of an acrosome reaction.     

Mapping of the human testicular proteome and its relationship with that of the epididymis and spermatozoa

The testis produces male gametes in the germinal epithelium through the development of spermatogonia and spermatocytes into spermatids and immature spermatozoa with the support of Sertoli cells. The flow of spermatozoa into the epididymis is aided by testicular secretions. In the epididymal lumen, spermatozoa and testicular secretions combine with epididymal secretions that promote sperm maturation and storage. We refer to the combined secretions in the epididymis as the sperm-milieu. With two-dimensional-PAGE matrix-assisted laser desorption ionization time-of-flight MS analysis of healthy testes from fertile accident victims, 725 unique proteins were identified from 1920 two-dimensional-gel spots, and a corresponding antibody library was established. This revealed the presence of 240 proteins in the sperm-milieu by Western blotting and the localization of 167 proteins in mature spermatozoa by ICC. These proteins, and those from the epididymal proteome (Li et al. 2010), form the proteomes of the sperm-milieu and the spermatozoa, comprising 525 and 319 proteins, respectively. Individual mapping of the 319 sperm-located proteins to various testicular cell types by immunohistochemistry suggested that 47% were intrinsic sperm proteins (from their presence in spermatids) and 23% were extrinsic sperm proteins, originating from the epididymis and acquired during maturation (from their absence from the germinal epithelium and presence in the epididymal tissue and sperm-milieu). Whereas 408 of 525 proteins in the sperm-milieu proteome were previously identified as abundant epididymal proteins, the remaining 22%, detected by the use of new testicular antibodies, were more likely to be minor proteins common to the testicular proteome, rather than proteins of testicular origin added to spermatozoa during maturation in the epididymis. The characterization of the sperm-milieu proteome and testicular mapping of the sperm-located proteins presented here provide the molecular basis for further studies on the production and maturation of spermatozoa. This could be the basis of development of diagnostic markers and therapeutic targets for infertility or targets for male contraception.     

Localization of the antigotensin-converting enzyme activity in testis and epididymis

Angiotensin-converting enzyme (ACE) has been studied in different reproductive organs of the male rat, in somatic cell lines clonally derived from both rat and mouse testes, and in isolated spermatogenic cells of the mouse. Among the various reproductive organs only testis and epididymis show high levels of enzyme activity. The testicular activity is found mainly in the isolated germinal cells and residual bodies, whereas somatic cell lines contain negligible levels of activity even after addition of hormones and growth factors. Testicular homogenates, spermatogenic cells, epididymal spermatozoa, and spermatozoan cytoplasmic droplets, when fractionated by anion exchange chromatography, contain one major and one minor activity peak, whereas epididymal homogenates and epididymal secretions reveal an additional major activity peak together with the minor peak. All forms of ACE have a similar response to different modifiers, and are equally sensitive to the specific inhibitor N-[(S)-1-(ethoxycarbonyl)-3-phenylpropyl]-L-alanyl-L-proline (Enalapril). The testicular enzyme could provide a useful marker for spermatogenic maturation and/or cytoplasmic processes both in testis and epididymis. The separate epididymal peak is a secretory enzyme that may be responsible for the processing of spermatozoan plasma membrane constituents during epididymal transit, or may have a role in attacking some biologically active compounds.