Expression of cysteine sulfinate decarboxylase (CSD) in male reproductive organs of mice

Cysteine sulfinate decarboxylase (CSD) is the rate-limiting biosynthetic enzyme of taurine, but it is still controversial whether the male reproductive organs have the function to synthesize taurine through CSD pathway. The present study was thus undertaken to detect CSD expression in male mouse reproductive organs by RT-PCR, Western blot and immunohistochemistry. The results show that CSD is expressed both at the mRNA and protein levels in the testis, epididymis and ductus deferens. The relative levels of both CSD mRNA and protein increase from the testis to the epididymis and to the ductus deferens. Immunohistochemical results demonstrate that the main cell types containing CSD are Leydig cells of testis, epithelial cells and some stromal cells throughout the efferent ducts, epididymis and ductus deferens. These results suggest that male genital organs have the function to produce taurine through the CSD pathway, although quantifying the relation of CSD expression to taurine synthesis and the exact functions of taurine in male genital organs still need to be elucidated in future studies.     

Expression of constitutively active Notch1 in male genital tracts results in ectopic growth and blockage of efferent ducts, epididymal hyperplasia and sterility

The Notch signaling pathway is involved in a variety of developmental processes. Here, we characterize the phenotypes developing in the reproductive organs of male transgenic (Tg) mice constitutively expressing the activated mouse Notch1 intracellular domain (Notch1(intra)) under the regulatory control of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR). Tg expression was detected in testis, vas deferens and epididymis by Northern blot analysis. In situ hybridization with a Notch1-specific probe lacked sensitivity to detect expression in normal-appearing cells, but demonstrated expression in hyperplastic epithelial cells of the vas deferens, epididymis and efferent ducts. Tg males from three independent founder lines were sterile. Histological analysis of reproductive organs of young Tg males (postnatal ages 8 and 21) showed no difference compared to those of non-Tg males. In contrast, in adult Tg mice from day 38 onwards, the efferent ducts, the vas deferens and most epididymal segments revealed bilateral epithelial cell hyperplasia with absence of fully differentiated epithelial cells. Electron microscopy confirmed the uniformly undifferentiated state of these cells. Immunohistochemistry with anti-PCNA antibody also revealed enhanced proliferation of Tg epididymis. In adult Tg testis, the different generations of germ cells of seminiferous tubules appeared normal, although some tubules were highly dilated and revealed an absence of early and/or late spermatids. The epithelial cells of the Tg tubuli recti and rete testis were not abnormal, but the rete testis was highly dilated and contained numerous spermatozoa, suggesting a downstream blockage. Consistent with a blockage of efferent ducts often seen at the rete testis/efferent duct interface, spermatozoa were absent in epididymis of all adult Tg mice and in all highly hyperplastic efferent duct tubules of these Tg mice. Such a blockage was visualized by injection of Evans blue dye into the rete testis lumen. Finally, the presence of ectopic hyperplastic efferent duct tubules was observed within the testicular parenchyma itself, outside their normal territory, suggesting that Notch1 signaling is involved in the establishment of these borders. This phenotype seems to represent a novel developmental defect in mammals. Together, these results show that constitutive Notch1 signaling significantly affects the development of male reproductive organs.     

Immunohistochemical localization of taurine in the rat ovary, oviduct, and uterus.

The distribution of the amino acid taurine in the female reproductive organs has not been previously analyzed in detail. The aim of this study was to determine taurine localization in the rat ovary, oviduct, and uterus by immunohistochemical methods. Taurine was localized in the ovarian surface epithelium. The granulosa cells and oocytes of primordial follicles were immunonegative. In primary and antral follicles, taurine was found mainly in theca cells and oocytes, whereas the zona pellucida, antrum, and most granulosa cells were unstained. However, taurine immunoreactivity in theca cells and oocytes decreased during follicular atresia. During corpora lutea development, the number of immunopositive theca lutein cells increased as these cells invaded the granulosa-derived region. Therefore, most luteal cells from the mature corpora lutea were stained. In the regressing corpora lutea, however, taurine staining in luteal cells decreased. In the fimbriae, infundibulum, and uterotubal junction, taurine was localized in most epithelial cells. In the ampullar and isthmic segments, taurine was found in the cilia of most ciliated cells and in the apical cytoplasm of some non-ciliated cells. In the uterus, most epithelial cells were immunopositive during diestrus and metestrus, whereas most of them were immunonegative during estrus and proestrus. Moreover, taurine immunoreactivity in the oviduct and uterus decreased with pregnancy. (J Histochem Cytochem 49:1133-1142, 2001)     

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.     

Immuno-electron microscopic localization of estradiol receptor in cells of male and female reproductive and non-reproductive organs

Summary— The localization of estradiol receptor (ER) in various tissues and their distribution in sub-cellular compartments were studied by means of immunogold-electron microscopic methods using a site-directed polyclonal antibody developed against a peptide from the DNA binding site of ER. This method was used to determine the presence and localization of ER in tissues and cells of male and female reproductive and non-reproductive organs. In the female reproductive tract, endometrial cells and the cells of the corpus luteum were found to contain ER. In non-reproductive organs of both sexes the following cell types showed significant labeling: hepatocytes, epithelial duodenal cells, striated muscle fibers, cells of the proximal convoluted tubules of the kidney, lymphocytes, neurons, and adipose cells. Alveolar epithelial cells were studied only in female specimens and were labeled by the anti-ER. Prostatic and epididymal epithelial cells were found to be labeled in the male reproductive organs. In all these cells a higher density of label was found in the nucleus, especially in the space between the clumps of compact chromatin, as was previously found in epithelial endometrial cells. These results suggest that estradiol exerts its effects through a common nuclear mechanism in cells of male and female reproductive and non-reproductive organs.     

Cellular localization of estrogen receptor beta messenger ribonucleic acid in cynomolgus monkey reproductive organs.

There is now evidence that the recently identified estrogen receptor (ER) beta is more widely distributed in the body than is ER-alpha. In order to gain more information about the role of ER-beta in reproduction, we have investigated by in situ hybridization the localization of mRNA expression of this ER subtype in adult monkey reproductive organs. In the pituitary gland of animals of both sexes, in both the anterior and intermediate lobes, a large number of cells were positive. No specific signal was observed in the posterior lobe. In the ovary, granulosa cells in primary and growing follicles highly expressed ER-beta mRNA. The theca interna cells were also strongly labeled. In some corpora lutea, the luteal cells were strongly labeled, while in other ones, the signal was weak. A hybridization signal was also detected in the ovarian surface epithelium. In the uterus, ER-beta mRNA was found in high concentration in glandular epithelial cells and stromal cells of the endometrium, while weaker labeling was consistently observed in smooth muscle cells. In the mammary gland, labeling was detected in the epithelial cells of acini and interlobular ducts as well as stromal cells. In the testis, specific labeling was detected in the seminiferous epithelium whereas the interstitial Leydig cells were unlabeled. Although it was not possible to clearly identify all the positive cell types, it appears that Sertoli cells as well as the vast majority of germinal cells express ER-beta mRNA. In the prostate, the secretory epithelial cells exhibited a specific autoradiographic reaction while the stromal cells did not show mRNA expression. The epithelial cells of the prostatic urethra showed a strong labeling. No hybridization signal was detected in the seminal vesicles. It then appears quite clear that ER-beta is expressed in a cell-specific manner in all the monkey reproductive organs studied. In the female, the wide distribution of these receptors in the ovary and uterus suggests that ER-beta may play an important role in the mediation of the known effects of estrogen in reproduction functions. In the male testis and prostate, ER-beta has been found in cells that contain very little or no ER-alpha. The role of circulating or locally produced estrogens in the male reproductive system remains to be clarified.     

The cytoskeletal proteins in the contractile tissues of the testis and its excurrent ducts of the passerine bird, Masked Weaver (Ploceus velatus)

The cellular composition of the testicular capsule, seminiferous peritubular tissue, the epithelia as well as periductal muscle cell layers of the excurrent ducts was studied, in sexually mature and active Masked Weaver (Ploceus velatus) birds of the passerine family, Ploceidae. Ultrastructure of the contractile cells in the testicular capsule, peritubular and periductal tissues showed that these cells were smooth muscles of typical morphological characteristics. Variability in the immunohistochemical co-expression of microfilaments and intermediate filaments in the different tissues was evident. Actin and desmin proteins were co-expressed immunohistochemically in the testicular capsule and seminiferous peritubular smooth muscle layer. Actin was singly and very weakly expressed in the rete testis epithelium while cytokeratins and desmin were co-expressed in the epithelium of the excurrent ducts. The periductal muscle layer of all ducts of the epididymis, the ductus deferens as well as the seminal glomus, strongly co-expressed actin and desmin. Vimentin was absent in all cells and tissue types studied. There is clear evidence that the tissues of the male gonad and its excurrent ducts in the Masked Weaver, as has been reported for members of the Galloanserae and Ratitae, contain well-formed contractile tissues whose function would include the transportation of luminal through-flow from the testis into, and through, its excurrent ducts. The microtubule helix in the head and of the mid-piece, of elongating spermatids, as well as of the mature spermatozoa in the various excurrent ducts, including some spermatozoa in the seminal glomus, also co-expressed these three proteins.     

Immunocytochemical localization of type 5 17beta-hydroxysteroid dehydrogenase in human reproductive tissues.

17beta-hydroxysteroid dehydrogenase (17beta-HSD) controls the last step in the formation of all androgens and all estrogens. At least six 17beta-HSD isoenzymes have been identified. The recently cloned Type 5 17beta-HSD transforms 4-dione into testosterone. To gain a better understanding of the role of this enzyme in reproductive tissues, we immunocytochemically localized the enzyme in human male and female reproductive organs. In the ovary of adult premenopausal women (25-40 years of age), immunostaining was found in corpus luteum cells. In the uterus, staining was detected only in the epithelial cells of the endometrium. Immunolabeling was also detected in the mammary gland, a positive reaction being detected in epithelial cells of acini and intralobular ducts as well as in the surrounding stromal cells. In the testis, strong staining was seen in the Leydig cells, and a weak but specific reaction was occasionally detected in Sertoli and germ cells. In the prostate, specific labeling was observed in alveoli and stromal fibroblasts. In alveoli, all the basal cells were generally labeled, whereas the luminal cells exhibited variations in immunoreactivity. In all the reproductive organs examined, specific staining was routinely detected in the walls of blood vessels, including the endothelial cells. These results indicate a cell-specific localization of Type 5 17beta-HSD in the different human reproductive organs, thus suggesting new mechanisms of local androgen and estrogen formation that may play an important physiological role.     

Distribution of the vacuolar H+ atpase along the rat and human male reproductive tract

Luminal acidification in parts of the male reproductive tract generates an appropriate pH environment in which spermatozoa mature and are stored. The cellular mechanisms of proton (H+) secretion in the epididymis and the proximal vas deferens involve the activity of an apical vacuolar H+ ATPase in specialized cell types, as well as an apical Na+/H+ exchanger in some tubule segments. In this study we used Western blotting and immunocytochemistry to localize the H+ ATPase in various segments of the male reproductive tract in rat and man as a first step toward a more complete understanding of luminal acidification processes in this complex system of tissues. Immunoblotting of isolated total cell membranes indicated a variable amount of H+ ATPase in various segments of the rat reproductive tract. In addition to its known expression in distinct cell types in the epididymis and vas deferens, the H+ ATPase was also localized at the apical pole and in the cytoplasm of epithelial cells in the efferent duct (nonciliated cells), the ampulla of the vas deferens and the ventral prostate (scattered individual cells), the dorsal and lateral prostate, the ampullary gland, the coagulating gland, and all epithelial cells of the prostatic and penile urethra. Both apical and basolateral localization of the protein were found in epithelial cells of the prostatic ducts in the lateral prostate and in periurethral tissue. Only cytoplasmic, mostly perinuclear localization of the H+ ATPase was found in all epithelial cells of the seminal vesicles and in most cells of the ventral prostate and coagulating gland. No staining was detected in the seminiferous tubules, rete testis, and bulbourethral gland. In human tissue, H+ ATPase-rich cells were detected in the epididymis, prostate, and prostatic urethra. We conclude that the vacuolar H+ ATPase is highly expressed in epithelial cells of most segments of the male reproductive tract in rat and man, where it may be involved in H+ secretion and/or intracellular processing of the material endocytosed from the luminal fluid or destined to be secreted by exocytosis.     

Taurine levels and localisation in the stratified squamous epithelia

The content and distribution of the amino acid taurine in squamous epithelia were studied using high-performance liquid chromatography and immunohistochemical methods. Quantitative analysis demonstrated that taurine was highly concentrated in the epidermis (5.49 mumol/g fresh tissue in the hairless skin of the hind footpad of the rat), although the values in the isolated stratum corneum were extremely low (< 0.073 mumol/g in the horny layer of the same skin area). No other analysed amino acid (such as glutamate, glutamine, glycine or alanine) showed this specific pattern of distribution. The immunohistochemical study revealed that in the dog and rat epidermis, taurine was present in the keratinocytes of the granular and upper spinous layers. The basal layer, lower spinous layer and stratum corneum were immunonegative. A similar immunostaining pattern was found in the epithelia of the different organs studied: the mouth, tongue and oesophagus of the dog and rat, the rat forestomach and the rat corneal epithelium. Other cell types, such as sebaceous and muscle cells, were immunolabelled. The existence of a circulating pool of taurine in the epidermis (via taurine release from keratinocytes before they reach the horny layer and its uptake by nearby cells) and its possible roles in these cells are discussed.     

Expression of PDE11A in normal and malignant human tissues.

Cyclic nucleotide phosphodiesterase 11A (PDE11A) is the newest member in the PDE family. Although the tissue distribution of PDE11A mRNA has been shown, its protein expression pattern has not been well studied. The goal of this report is to investigate the distribution of PDE11A proteins in a wide range of normal and malignant human tissues. We utilized a polyclonal antibody that recognized all four PDE11A isoforms. Its specificity was demonstrated by Western blot analysis on a recombinant human PDE11A protein and native PDE11A proteins in various human tissues. Immunohistochemistry showed that PDE11A is widely expressed. Various degrees of immunoreactivity were observed in the epithelial cells, endothelial cells, and smooth muscle cells of all tissues examined. The highest expression was in the epithelial, endothelial, and smooth muscle cells of the prostate, Leydig, and spermatogenic cells of the testis, the tubule epithelial cells in the kidney, the epithelial and endothelial cells in the adrenal, the epithelial cells and macrophages in the colon, and the epidermis in the skin. Furthermore, PDE11A expression was also detected in several human carcinomas. Our results suggest that PDE11A might be involved in multiple physiological processes in various organs via its ability to modulate intracellular cAMP and cGMP levels.     

Immunolocalization of aquaporins 1, 2 and 7 in rete testis, efferent ducts, epididymis and vas deferens of adult dog

The transepithelial movement of water into the male reproductive tract is an essential process for normal male fertility. Protein water channels, referred to as aquaporins (AQPs), are involved in increasing the osmotic permeability of membranes. This study has examined the expression of AQP1, AQP2, and AQP7 in epithelial cells in adult dog efferent ducts, epididymis, and vas deferens. Samples of dog male reproductive tract comprising fragments of the testis, initial segment, caput, corpus and cauda epididymidis, and vas deferens were investigated by immunohistochemistry and Western blotting procedures to show the localization and distribution of the AQPs. AQP1 was noted in rete testis, in efferent ducts, and in vessels in the intertubular space, suggesting that AQP1 participated in the absorption of the large amount of testicular fluid occurring characteristically in the efferent ducts. AQP2 expression was found in the rete testis, efferent ducts and epididymis, whereas AQP7 was expressed in the epithelium of the proximal regions of the epididymis and in the vas deferens. This is the first time that AQP2 and AQP7 have been observed in these regions of mammalian excurrent ducts, but their functional role in the dog male reproductive tract remains unknown. Investigations of AQP biology could be relevant for clinical studies of the male reproductive tract and to technologies for assisted procreation. R.F.D. gratefully acknowledges a Fellowship from the Department of Anatomy, Institute of Biosciences, UNESP, Botucatu, SP, Brazil. This work was also funded by FAPESP (Sao Paulo State Research Foundation; grant 04/05578–1 to A.M.O. and grant 04/05579–8 to R.F.D.). This paper is part of the PhD Thesis presented by R.F.D. to the State University of Campinas – UNICAMP, Brazil.     

Morphological changes in the efferent ducts during the main phases of the reproductive cycle of birds

The changes that take place in the efferent ducts during the major phases of the reproductive cycle of birds were studied morphologically using standard histological, morphometric, and ultrastructural methods in prepuberal, sexually mature and sexually active, and sexually mature but sexually inactive domestic fowl (Gallus domesticus), drake (Anas platyrhynchos), and guinea fowl (Numida meleagris). Profound structural and dimensional changes occurred in both segments (proximal and distal) of the efferent ducts and, in particular, in the nonciliated (Type I) cell of the proximal duct of sexually mature but inactive birds. The subapical tubulovacuolar system was markedly atrophic in nonciliated (Types I and II) cells and the numerous round dense globules of Type I cells that normally occurred in sexually active birds were replaced by fewer and more pleomorphic bodies containing lipofuscin granules in sexually resting birds. Lipid droplets, few and extremely large in inactive drakes but numerous and smaller in size in guinea fowls and domestic fowls, occurred in the Type I cell at both infra- and supranuclear levels of resting but not in prepuberal or sexually active birds. Ciliated cells in both segments of the ducts exhibited fewer and less profound phase-dependent changes ultrastructurally. Generally, the Type I cells of the proximal efferent duct appeared to be more sensitive to androgen deprivation than the Type II cell of the distal efferent duct or ciliated cells in both ducts. These morphologically phase-dependent features of the efferent ducts of birds may be used, together with or independent of testicular changes, in the determination of the status of the testis and epididymis of a male bird with regard to the reproductive cycle, especially in seasonally breeding species.     

Effect of sex steroids on the male reproductive organs of the monitor lizard Varanus monitor (Linn.).

Exogenous sex steroids were administered to adult males of the monitor lizard Varanus during the retrogressive and inactive phases of their annual reproductive cycle. Androgen treatment renews spermatogenetic activity and causes an increase in the number of Leydig cells of the testis during the retrogressive phase; during the inactive phase the testicular response to androgen is only slight. In either reproductive phase oestradiol treatment has an inhibitory action on the germ tubules. Progesterone has no effect on the testis in the inactive phase. The vasa deferentia are well developed during the retrogressive phase and thus the effect of androgens is not appreciable. However, during the inactive phase testosterone highly stimulates the deferent ducts. In the inactive phase oestrogen and progesterone also seem to stimulate slightly the deferent tubules; progesterone increases the interstitial tissue of the deferent ducts. Renal sexual segments hypertrophy and become secretory by androgen treatment in either phase of the reproductive cycle, whereas oestrogen and progesterone have no effect. The hemipenes are also stimulated by androgen treatment.     

Localization of androgen and estrogen receptors in rat and primate tissues

There is now evidence that estrogens and androgens are exerting their effects in different tissues throughout the body. In order to determine the sites of action of these steroids, studies have been performed to identify at the cellular level the localization of androgen receptor (AR) and the two estrogen receptor (ER) subtypes, ERalpha and ERbeta, specially in the rat, monkey and human. In the prostate, AR was observed in the secretory and stromal cells. In the testis, Sertoli, Leydig and myoid cells were labelled. In the epididymis and seminal vesicles, both epithelial and stromal cells contained AR. In the ovary, AR was detected in granulosa and interstitial cells. In the uterus, epithelial, stromal and muscle cells were all immunopositive for AR. In the central nervous system, AR-containing neurons were found to be widely distributed throughout the brain. In the mammary gland, epithelial cells in acini and ducts and stromal cells were demonstrated to express AR. In the skin, AR was detected in keratinocytes, sebaceous and sweat glands, and hair follicles. In addition, AR was also found in anterior pituitary, thyroid, adrenal cortex, liver, kidney tubules, urinary bladder, cardiac and striated muscle, and bone. The ER subtypes are in general differentially expressed. While ERalpha has been predominantly found in anterior pituitary, uterus, vagina, testis, liver and kidney, ERbeta is predominant in thyroid, ovary, prostate, skin, bladder, lungs, gastro-intestinal tract, cartilage and bone. In tissues which contain both receptor subtypes, such as ovary, testis and various regions of the brain, a cell-specific localization for each ER subtype has been generally observed. Altogether, the recent results on the cellular localization of sex steroid receptors will certainly contribute to a better understanding of the specific role of these steroids in different target organs.     

Taurine plays an important role in the protection of spermatogonia from oxidative stress

It has been demonstrated that taurine has various physiological functions in the body. We demonstrated that taurine is abundant in the serum, liver, muscle and testis of the Japanese eel (Anguilla japonica). In the eel testis, taurine is found mainly in spermatogonia and is weakly expressed also in the Sertoli cells. We have further found in the eel testis that taurine is actively accumulated via the sodium/chloride-dependent taurine transporter (TauT; SLC6A6), which is expressed in germ cells. In our current study, the effects of taurine on the anti-oxidant response were examined. Taurine was found to promote the total superoxide dismutase (SOD) activity in the testis. Moreover, our results indicate that taurine does not affect the mRNA levels of copper–zinc (Cu/Zn) SOD or manganese SOD, but promotes the translation of Cu/Zn SOD. Overall, our present data suggest that taurine may modulate Cu/Zn SOD at the translational level and thereby may play an important role in the protection of germ cells from oxidative stress.     

Immunohistochemical demonstration of androgen-binding protein in the rat prostatic gland.

Androgen-binding protein (ABP) is one of the best-characterized products of synthesis by the Sertoli cells in the rat. Although the exact physiological role of ABP remains to be determined, it has been widely used to study Sertoli cells and testicular function in this species. Since this protein is the principal carrier for testosterone in rat testis and epididymis, we decided to investigate ABP immunoreactivity (ABP-I) in androgen-dependent organs, including testicle, epididymides, prostate, and seminal vesicles. The location of ABP was investigated by immunohistochemistry using specific antisera against rat ABP. As previously described in the testis, rat ABP-I was identified in the seminiferous tubules within the cytoplasm of the Sertoli cells and the tubular luminae. The epididymis showed ABP-I only in epithelial cells of the proximal caput. We demonstrated ABP-I in the apical portions of epithelial cells of the rat prostate. Short-term castration and/or ligation of the efferent ducts did not suppress prostatic ABP-I. ABP-I was not present in seminal vesicles of control rats nor under any of the experimental conditions used throughout this study. The results also indicate the presence of ABP-I in prostatic epithelium, probably because of a mechanism similar to that described in epididymis. Our data support and enhance the concept that ABP may serve as a transmembrane carrier protein for androgens in androgen target organs in the male reproductive tract.     

Endothelial cell proliferation in male reproductive organs of adult rat is high and regulated by testicular factors

Endothelial cells in the intact adult are, apart from those in the female reproductive organs, believed to be quiescent. Systematic examination of endothelial cell proliferation in male reproductive organs has not been performed and was therefore the aim of the present study. Intact adult rats were either pulse labeled or long-term labeled with bromodeoxyuridine to label proliferating cells. The roles of Leydig cells and testosterone were examined after castration or treatment with the Leydig cell toxin ethane dimethane sulfonate (EDS) and testosterone substitution. After perfusion fixation, all blood vessels remained open and were easily identified. In all male reproductive organs studied, particularly in the testis and epididymis, endothelial cell proliferation was considerably higher than in other tissues such as the liver, brain, and muscle. Proliferating endothelial cells were observed in all types of blood vessels in male reproductive organs, but other characteristics of new blood vessel formation were not seen. High endothelial cell proliferation may reflect a continuous high turnover of endothelial cells rather than classical angiogenesis. In the epididymis, the ventral and dorsolateral prostate lobes, and the seminal vesicles, endothelial cell proliferation decreased after testosterone withdrawal and increased following testosterone treatment. In the testis, endothelial cell proliferation was decreased after Leydig cell depletion but remained low after testosterone substitution. High, hormonally regulated endothelial cell proliferation is not unique to the female but is also seen in the male reproductive organs.