Identification of antigen in rat spermatogenic cells interacting with an anti-human sperm monoclonal antibody

A monoclonal antibody (MAb) raised against human sperm protein, designated YWK-II, was used to determine the distribution of antigens in rat spermatozoa and rat testicular germ cells. By an indirect immunofluorescent method, the antibody localized over the rat spermatozoal head, except for the postacrosomal region. In paraffin sections of adult and immature rat testis, germ cells, at every developmental stage, and Sertoli cells stained, while interstitial cells and peritubular myoid cells remained unstained. When cocultures of Sertoli and germ cells were tested, only the germ cells stained intensely. Sertoli cells and peritubular myoid cells in cultures did not stain. In the epididymal sections, strong staining occurred with spermatozoa in the lumen and epididymal epithelial cells, with moderate staining in the myoid layers of epididymis. To determine the sperm antigen interacting with the YWK-II antibody, rat spermatozoa proteins were prepared and analyzed by an immunoblot technique. The monoclonal antibody interacted with a single protein, with an estimated molecular weight of 115,000, present in the cauda epididymal spermatozoa. Among the proteins of the caput epididymal spermatozoa, however, the antibody interacted with a major and a minor band with molecular weights of 115,000 and 88,000, respectively. On the other hand, with proteins prepared from the membrane fraction of adult and immature rat testis, the antibody reacted with two bands with estimated molecular weights of 88,000 and 115,000. In the lysate prepared from germ cells dissociated from Sertoli-germ cell cocultures, the antibody recognized only the 88,000 protein. The present results show that the YWK-II MAb interacts with two proteins with different molecular weights. The amount of the interacting proteins in spermatozoa varied with their location within the epididymis.     

The antibody from an infertile woman that induces sperm agglutination interacts with rat testicular cells and sperm

The serum obtained from an infertile woman induced a specific head-to-head agglutination of human and rat sperm. The immunoglobulin G (IgG) fraction of the serum was obtained and found to interact with the proteins of rat sperm in testis and epididymis. Using an indirect immunofluorescent method with rat sperm from vas deferens, we determined that the antibody recognized the protein on the convex and concave regions of the acrosome and over the entire tail. However, with testicular spermatozoa, the antibody recognized only the distal end of the tails. In paraffin sections of adult rat testis, sperm tails located at the luminal region of the seminiferous tubules stained intensely. Weak but significant staining also occurred on late spermatids. In the epididymal sections, staining was restricted to spermatozoa in the lumen. On the other hand, sections of testes from 25-day-old rats containing spermatogonia and early spermatocytes had a completely negative reaction. Testicular somatic cells, including Sertoli cells, peritubular myoid cells and interstitial cells, did not stain. To identify the testicular protein interacting with the antibody, adult rat testis proteins were prepared and analyzed by a sodium dodecyl sulfate-polyacrylamide gel electrophoretic (SDS-PAGE) immunoblot technique. The antibody interacted with a protein with an estimated molecular weight of 82,000 in the testicular homogenate and particulate fraction, whereas the reaction was considerably weaker with the testicular cytosol fraction.     

Immunohistochemistry of the cytoskeleton in the excurrent ducts of the testis in birds of the Galloanserae monophyly

The presence, location and degree of immunoexpression of various microfilament (MF) and intermediate filament (IF) systems (actin, cytokeratins, desmin, vimentin) were studied in the excurrent ducts of the testis in sexually mature and active galliform (Japanese quail, domestic fowl, turkey) and anseriform (duck) birds. These proteins were variably expressed between the epithelia and periductal tissue (periductal smooth muscle cell layer and interductal connective tissue) types and between species. Variable heterogeneous co-expression of filament systems was also found in the various duct epithelia and periductal tissue types: co-expression of filament systems was the rule rather than the exception. In the duck, neither vimentin nor cytokeratin was present in any of the tissues, whereas actin and desmin (absent in the rete testis) were co-expressed in the efferent ducts and epididymal duct unit (comprising the ductus conjugens, ductus epididymidis and ductus deferens). Actin, desmin and vimentin were generally co-expressed in the rete testis, efferent ducts and epididymal duct unit of the quail, domestic fowl and turkey, with vimentin being more strongly immunoreactive than actin and desmin in the epididymal duct unit, but more weakly immunoexpressed in the efferent ducts. Cytokeratin was present and co-expressed with actin, desmin and vimentin in the rete testis, efferent ducts and epididymal duct unit of the domestic fowl and turkey, but not in the quail and duck. The periductal smooth muscle cell layer and interductal tissue co-expressed actin, desmin and vimentin variably in all birds. Luminal spermatozoa of both the turkey and duck were immunonegative for all protein systems, whereas those of the quail and domestic fowl co-expressed actin, desmin and vimentin moderately or strongly. The tissues of the reproductive tract of male birds thus contain cytoskeletal protein systems that are variably but mostly co-expressed and whose contractile ability appears necessary and sufficient for transportation through the various excurrent ducts of the voluminous testicular fluid and its high sperm content, characteristic features of male avian reproduction.     

Immunocytochemistry of extracellular matrix in the lamina propria of the rat testis: electron microscopic localization

The distribution of laminin, type IV collagen, heparan sulfate proteoglycan, and fibronectin was investigated in the rat testicular lamina propria by electron microscopic immunocytochemistry. Distinct patterns were observed for each antigen within the extracellular matrix (ECM) layers of the lamina propria. Laminin, type IV collagen, and heparan sulfate proteoglycan all localized to the seminiferous tubule basement membrane. Type IV collagen and heparan sulfate proteoglycan, but not laminin, localized to the seminiferous tubule side of the peritubular myoid cells. All four of the antigens were localized between the peritubular and lymphatic endothelial cells. Failure to localize fibronectin in the ECM layer between the Sertoli and peritubular myoid cells tends to support the concept that adult Sertoli cells do not produce this protein in vivo. Intracellular immunostaining was insufficient to allow unambiguous identification of the cellular source of any of the ECM molecules.     

S-100 protein immunoreactivity in mammalian testis and epididymis

The present study deals with immunohistochemical localization of S-100 protein in mouse, bank vole and pine vole testis and epididymis. S-100 protein immunoreactivity was observed in the endothelia of capillaries and lymphatic sinusoids of pine vole testis. A reaction to S-100 protein of the same intensity as that noted in the endothelia of testicular capillaries was found in myoid cells of pine vole and bank vole seminiferous tubules. Moreover, a positive reaction to S-100 protein was observed in bank vole and mouse Leydig cells. In the epididymis, a weaker reaction to S-100 occurred in smooth muscles of pine vole and mouse epididymal duct. Despite difficult interpretation of physiological role of S-100 protein we suggest that it may be a part of the blood-testis barrier. It may also participate in the processes of transcytosis and contractility; its cellular expression is regulated by local factors. However, location of S-100 is not specific to the representatives of the same order.     

Specificity of sperm-binding Wolffian duct proteins in the rooster and their persistence on spermatozoa in the female host glands

Unlike those of mammals, chicken spermatozoa can develop their fertilizing ability before they leave the testis to pass into the Wolffian duct; moreover, chicken spermatozoa do not require a period of capacitation in the female tract. A question arises, therefore, as to the significance of secretory proteins shown to bind to the surface of chicken spermatozoa as they pass into and through the Wolffian duct. Using anti-Wolffian duct fluid IgG as a probe visualized by immunoperoxidase staining, the present investigation confirms that testicular spermatozoa of quail and turkey as well as chicken do not have any surface determinants in common with those present in Wolffian duct secretions. By contrast, those in the lower portion of the vas deferens display a strong reaction with anti-Wolffian fluid IgG over their entire surface, and immunoprecipitation studies suggest that this reflects the binding of four Wolffian duct proteins. Since a reaction to the antichicken fluid IgG is shown also by mature quail and turkey, but not duck and pigeon spermatozoa, the Wolffian components that coat spermatozoa in birds appear to have a specificity confined to the same order, in this case the Galliformes. Following vaginal or intramagnal insemination, spermatozoa present 48 hours later in the uterovaginal host glands and infundibulum glands, respectively, still reacted strongly. This finding that Wolffian duct components persist on the surface of spermatozoa in the female tract is consistent with the possibility that they have some role in sperm storage or survival in female birds.     

Role of epithelial cells of the male excurrent duct system of the rat in the endocytosis or secretion of sulfated glycoprotein-2 (clusterin)

The localization of sulfated glycoprotein-2 (clusterin; SGP-2) was investigated in the rete testis, efferent ducts, and epididymis of the rat using light (LM) and electron (EM) microscope immunocytochemistry. At the LM level, the epithelial cells of the rete testis and efferent ducts demonstrated an intense immunoperoxidase reaction over their apical and supranuclear regions, and sperm in the lumen of the efferent ducts were unreactive. In the EM, gold particles were found exclusively over the endocytic apparatus of these cells. In the proximal area of the epididymal initial segment, an insignificant immunostaining of epithelial cells and sperm was observed. However, the distal area of the initial segment showed a moderate staining over the epithelial principal cells and sperm, while in the intermediate zone of the epididymis a stronger reaction was observed over these cells. The strongest immunoperoxidase reaction was noted in the caput epididymidis, where it formed a distinct mottled pattern. Thus, while some principal cells were intensely stained, others were moderately or weakly stained; a few were completely unreactive. In the corpus and cauda epididymidis, the staining pattern was similar but not as intense. In the EM, only the secretory apparatus of these cells was found to be immunolabeled with gold particles. Sperm in the lumen of these different regions were also labeled. The epithelial clear cells were unreactive throughout the epididymis. Northern blot analysis substantiated these results and showed the presence of highest levels of SGP-2 mRNA in the caput epididymidis, especially in its proximal area, whereas increasingly lower levels were found in the corpus and cauda epididymidis. In summary, these results suggest that testicular SGP-2 dissociates from the sperm during passage through the rete testis and efferent ducts, where it is endocytosed by the epithelial cells lining these regions. In the epididymis, it is replaced by an epididymal SGP-2 that is secreted by the epithelial principal cells of the epididymis. Furthermore, in the epididymis, the principal cells appear to be in different functional states with respect to the secretion of epididymal SGP-2 within a given region of the duct as well as along the epididymal duct.     

High level of endostatin in epididymal epithelium: protection against primary malignancies in this organ?

Rete testis and epididymis are rare locations for primary tumors or metastasis. Assuming that this may be related to expression level of angiogenic inhibitors, we focused our study on the expression pattern of collagen 18/endostatin. In situ hybridization and immunohistochemistry for collagen 18 and endostatin were carried out on sections of human rete testis and epididymis as well as on epididymal adenoma and human testicular tissue with or without carcinoma in situ (CIS). In situ hybridization revealed strong expression of collagen 18 mRNA in rete testis, efferent ducts and epididymal duct. Immunostaining showed collagen 18 in epithelium and basement membrane as well as in blood vessels of rete testis. Further, in both efferent ducts and epididymal duct, collagen 18 was mainly localized in the basement membrane of these ducts and of the blood vessel wall. Endostatin immunostaining was localized in the epithelium of rete testis, efferent ducts and epididymal duct. This pattern of endostatin staining was absent in epididymal adenoma tissue while tumor associated blood vessels exhibited strong endostatin staining. No endostatin staining was detectable in normal germinal epithelium and CIS cells while Leydig cells exhibited strong endostatin staining. High endostatin expression in epididymis may protect this organ against tumor development. Gene therapeutic strategies providing high expression of endostatin in normal epithelia may be useful to prevent tumor development.     

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

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

Immunohistochemical and quantitative analysis of 5-hydroxytryptamine in the retina of some vertebrates

5-Hydroxytryptamine immunoreactive neurons were found in retinae from chicken, pigeon, frog and goldfish. They were localized among the amacrine cells with a distribution of cell bodies and nerve fibres that varied with the species. In chicken and pigeon, bipolar-like cell bodies were also found in the middle of the inner nuclear layer, sending processes inwards to the inner plexiform layer and outwards to the horizontal cells. The signalling direction of these cells is doubtful. No 5-hydroxytryptamine immunoreactivity was found in retinae from cow, pig, cat, rabbit, guinea-pig, rat or mouse.Quantitative analyses were performed with HPLC on extracts from chicken, pigeon, frog and goldfish retinae. High concentrations were found in goldfish and frog whereas less, about 100 ng/g, was observed in chicken and pigeon.The results suggest that 5-hydroxytryptamine is the transmitter of a set of amacrine cells in cold-blooded vertebrates and perhaps also in birds. The transmitter of the indoleamine accumulating neurons of mammals remains to be further elucidated.     

C-terminal region of teneurin-1 co-localizes with the dystroglycan complex in adult mouse testes and regulates testicular size and testosterone production

Testicular size is directly proportional to fertility potential and is dependent on the integration of developmental proteins, trophic factors, and sex steroids. The teneurins are transmembrane glycoproteins that function as signaling and cell adhesion molecules in the establishment and maintenance of the somatic gonad, gametogenesis, and basement membrane. Moreover, teneurins are thought to function redundantly to the extracellular matrix protein, dystroglycan. Encoded on the last exon of the teneurin genes is a family of bioactive peptides termed the teneurin C-terminal-associated peptides (TCAPs). One of these peptides, TCAP-1, functionally interacts with β-dystroglycan to act as a neuromodulatory peptide with trophic characteristics independent from the teneurins. However, little is known about the localization and relationship between the teneurin-TCAP-1 system and the dystroglycans in the gonad. In the adult mouse testis, immunoreactive TCAP-1 was localized to spermatogonia and spermatocytes and co-localized with β-dystroglycan. However, teneurin-1 was localized to the peritubular myoid cell layer of seminiferous tubules and tubules within the epididymis, and co-localized with α-dystroglycan and α-smooth muscle actin. TCAP-1-binding sites were identified in the germ cell layers and adluminal compartment of the seminiferous tubules, and epithelial cells of the epididymis. In vivo, TCAP-1 administration to adult mice for 9 days increased testicular size, seminiferous and epididymal tubule short-diameter and elevated testosterone levels. TCAP-1-treated mice also showed increased TCAP-1 immunoreactivity in the caput and corpa epididymis. Our data provide novel evidence of TCAP-1 localization in the testes that is distinct from teneurin-1, but is integrated through an association with the dystroglycan complex.     

Immunohistochemical localization of two angiotensin I-converting isoenzymes in the reproductive tract of the male rabbit

The male reproductive tract contains two different isoenzymes of angiotensin I-converting enzyme (ACE), i.e., pulmonary and testicular ACE. The present study shows selectively the cellular distribution of the ACE isoenzymes in the reproductive tract of male rabbit, using indirect immunofluorescence or immunoperoxidase methods. Testicular ACE was found in the seminiferous tubules of the testes in spermatocytes containing mature spermatids, and in spermatids within the epididymal tubular lumen in sexually mature, but not in immature, rabbits. Epididymal tubular cells contained pulmonary ACE. In the young rabbit, epididymal tissue contained more ACE than that in adult rabbit, since ACE was observed in principal cells in addition to basal cells. In mature rabbit, ACE was observed in basal cells only. Strong staining for pulmonary ACE was observed in cells of the vas deferens in both young and adult rabbit. Therefore, synthesis of epididymal ACE, unlike the testicular isoenzyme, was not stimulated by sexual maturation. Enzymatically active ACE in seminal fluid corresponds to the pulmonary isoenzyme. The present study indicates that this seminal fluid ACE may originate from cells of the epididymal tubules, particularly those of the vas deferens. Endothelial cells of blood vessels lying in the interstitium of both testicular and epididymal tissue contained the pulmonary isoenzyme.     

Spatiotemporal patterns of expression of neurotrophins and neurotrophin receptors in mice suggest functional roles in testicular and epididymal morphogenesis.

Several reports have established that the action of neurotrophins is not restricted to the nervous system but can affect a broad range of non-neuronal cells. Nerve growth factor (NGF) is present in adult testis and has been suggested as a potential regulator of meiosis in rat seminiferous epithelium. Here we present an extensive immunohistochemical study on neurotrophins and their receptors (p75 and trk) in the developing mouse testis and epididymis, and in fetal human testis. During the early steps of testicular and epididymal organization in the mouse, strong p75 immunoreactivity is detectable in the gonadal ridge in the mesenchyme that is excluded from the evolving testicular cords, and in the mesenchymal cells of the mesonephros. Later in organogenesis, most of the p75-positive interstitial cells of the testis coexpress neurotrophin-3 (NT-3) and the truncated trk B receptor in a developmentally regulated pattern. Our Western blot data confirm the expression of these molecules. These findings suggest that neurotrophin receptors play a role in early inductive events during critical periods of testicular and epididymal development. During fetal and postnatal histogenesis, an increasing number of NT-3- and p75-positive mesenchymal cells start to express alpha-smooth muscle isoactin, suggesting a role for the so-called neurotrophic system in the differentiation of testicular myoid cells and epididymal smooth muscle cells. In the testis of an 18-wk gestational-age human fetus, immunohistochemical analysis has shown intense immunoreactivity of mesenchymal cells to antibodies for neurotrophin receptors p75, trk A, and trk C, and their ligands NGF and NT-3. In addition, we found that in the human fetal testis, the interstitial cells that are differentiating into peritubular myoid cells are associated with a dense network of nerve fibers. Our data suggest that neurotrophins and their receptors are involved in a multifunctional system that regulates cell differentiation and innervation in the developing testis and epididymis.     

Effects of androgen on androgen receptor expression in rat testicular and epididymal cells: a quantitative immunohistochemical study

Androgen is essential for maintenance of spermatogenesis in the testis and for maturation of spermatozoa in the epididymis. The effects of androgen are mediated through its receptor (AR), the levels of which are, in turn, regulated by androgen. Previous studies have shown that AR concentrations in Leydig and Sertoli cells are differentially regulated during development. The aim of the present study was to determine if cell-type-specific regulation of AR by androgen occurs in testicular and epididymal cells during adulthood. Adult male rats were treated with the LHRH-antagonist Azaline B (100 g/day) by osmotic pump for 1, 2, 3, 4, or 8 wk to suppress endogenous androgen, with identical numbers of intact control animals at each time period. An androgen replacement group was simultaneously treated with the antagonist and a synthetic androgen, 7 alpha-methyl-19-nortestosterone (MENT), during the final 4 wk of the experiment. Levels of nuclear AR protein in specific cell types were quantified by immunohistochemistry in conjunction with computer-assisted image analysis. Levels of AR in testicular cells declined sharply after treatment with the LHRH antagonist. In Sertoli cells, nuclear AR levels decreased to 8% of control (P < 0. 01) after 4 wk treatment; and to 12% and 17% of control (P < 0.01) in Leydig and myoid cells, respectively. Androgen replacement resulted in complete recovery of nuclear AR levels in Sertoli cells (93%, P > 0.05) but in only partial recovery in myoid (69%, P < 0. 01) and Leydig cells (56%, P < 0.01). In the epididymis, tubular epithelial cells and stromal cells differed in their responses to the LHRH antagonist. After 1 wk, nuclear AR levels in caput stromal cells decreased dramatically to 34% of control (P < 0.01) and in cauda stromal cells to 43% (P < 0.01). In contrast, the decline of AR levels in epididymal epithelial cells was not as dramatic as that in stromal cells. After 1 wk, the decline in the caput and cauda was to 87% and 76% of control, respectively. After 8 wk, nuclear AR levels in stromal cells further declined to 1.1% in caput and 1.4% in cauda, whereas in the epithelial cells, a smaller decline in nuclear AR was noted (to 30% in the caput and 45% in the cauda). After androgen replacement with MENT, nuclear AR levels recovered to more than 90% of control in both epididymal cell types. These results indicate that AR levels in the nuclei of adult Sertoli cells depend mainly on the level of androgen, whereas in the adult Leydig and myoid cells, the androgen dependency is more limited. The results also indicate that in the epididymis, stromal cells are more sensitive than epithelial cells to the regulation of AR levels by androgen.     

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.     

De novo biosynthesis and localization of inhibin in marmoset (Callithrix jacchus) and rat epididymis.

Using specific polyclonal antibodies generated against a 13 Kd human testicular inhibin, immunocytochemical localization was carried out in epididymis of intact and castrated marmoset monkey and rat epididymis. Inhibin was found to be present in the cytoplasm of epithelial cells of caput, corpus and cauda epididymis. The intensity of staining and pattern of distribution did not change following castration. Further, the in vitro biosynthesis of inhibin studied by incorporating 3H-leucine and precipitating it with specific antibody indicated maximum biosynthesis in the corpus epididymis in case of marmosets and cauda in case of rats. Following castration in rats, the epididymal tissue still retained the capacity to biosynthesize inhibin. These studies indicate that marmoset and rat epididymis are capable of biosynthesizing/absorbing inhibin and whose synthesis does not depend on androgens.     

Effect of D-valine and cytosine arabinoside on [3H]thymidine incorporation in rat and rabbit epididymal epithelial cell cultures

Epithelial cell enriched primary cultures were established from the rat and the rabbit epididymis. Epithelial cell aggregates, obtained after pronase digestion of minced epididymis, attached to the culture dish and after 72 h in vitro spread out to form discrete patches of cells. These cells have an epithelioid morphology and form a monolayer of closely apposed polygonal cells where DNA synthesis, as judged by [3H]thymidine uptake, is very low. In L-valine medium the nonepithelial cell contamination was no more than 10% in rat and rabbit epididymal primary cultures. The labeling index of rat epididymal cells cultured in D-valine medium was significantly lower than that of cells cultured in L-valine medium. In contrast, the labeling index of rabbit epididymal cells cultured in D-valine medium was significantly higher than that of cells cultured in L-valine medium. Cytosine arabinoside decreased the number of labeled cells in both L-valine and D-valine cultures. From these results, it appears that D-valine is a selective agent for rat epididymal epithelial cells, but not for rabbit epithelial cells, and that cytosine arabinoside is a simple and effective means to control the proliferation of fibroblast-like cells in both rat and rabbit epididymal cell cultures.     

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.