Lectin Staining of Rat Testis and Epididymis: Effect of Cyproterone Acetate and Testosterone

Control and castrated (C) adult rats were treated with cyproterone acetate (CA), testosterone (T) or their combination (CA + T) for 15 days and the affinity of the testicular and epididymal tissue for seven rhodamine-conjugated lectins was analysed in fluorescence microscopy. In the testis CA caused the appearance of degenerating cells in the basal part of the seminiferous epithelium. These cells (spermatogonia, early spermatocytes) appear to be the most vulnerable germinal cells to anti-androgen treatment and this effect could be prevented by simultaneous administration of T. Castration caused a marked reduction in lectin staining of principal and light cells of distal caput (Dcp) and distal cauda (Dcd) epididymidis accompanied by the disappearance of the intratubular sperm mass. In castrated animals CA caused a moderate and T as well as CA + T a marked increase in the stainability of these cells and the appearance of homogeneous secretory material in the tubules. This material gave moderate or strong affinity for most of the lectins. Some minor differences were found in the staining pattern of the cells and the secretory material in Dcp and Dcd as well as after different treatments. This is consistent with local and qualitative differences in the epididymal secretory and absorptive activity, which can be further modified by the hormonal milieu.     

Effect of Castration on Lectin Staining in Rat Epididymis

Seven rhodamine-conjugated lectins (PNA, RCA I, Con A, WGA, UEA I, SBA, DBA) were used to follow the staining pattern of the rat epididymis at different time points after castration. The affinity of the intratubular sperm mass for the lectins increased rapidly with concurrent augmentation of the staining in the principal cells but a decline of the reaction in the light cells. The light cells showed some differences in their response to castration, which was compatible with secretory/absorptive activity in caput and absorptive activity in cauda. The active phase of sperm mass destruction and epithelial involution was accompanied by local accumulation of macrophages and round cells, which also acquired an increased affinity for most of the lectins. It is concluded that the androgen-deprived epididymis is rapidly programmed for autolytic and phagocytic processes, which include the destruction of macromolecules including glycoproteins of the spermatozoa.     

Distribution of Lectin Binding in Rat Testis and Epididymis

Summary: Seven lectins (PNA, RCA I, SBA, Con A, WGA, UEA I, DBA) conjugated with rhodamine were employed to analyse the staining pattern of glycoproteins with varying sugar residues in the testis and epididymis of adult Wistar rats. Some lectins (UEA I, SBA, DBA) gave rather specific staining of the mature acrosome, while others (PNA, RCA I) showed affinity for the early stages of acrosome formation or had a wide affinity for germinal and non-germinal cells and structures (Con A, WGA). In the epididymis the sperm mass had a homogenous staining reaction with some lectins (PNA, RCA I, Con A, WGA, DBA) which also showed a rather strong reaction on the epithelial surface. It was concluded that this reaction is at least partially due to the secretory products synthetized by principal, apical, narrow and light cells of the epididymal epithelium. Some differences in the staining pattern of these cells were recorded indicating specialization of the cells for the production of distinct glycoproteins. The staining pattern of the interstitial and intertubular compartment of the testis and epididymis was also recorded. Zusammenfassung: Die Verteilung der Lektin-Bindung im Hoden und Nebenhoden der Ratte Sieben Lektine (PNA, RCA I, SBA, Con A, WGA, UEA I, DBA) wurden verbunden mit Rhodamin verwendet, um das Darstellungsmuster von Glykoproteinen mit verschiedenen Zuckerresten im Hoden und Nebenhoden erwachsener Wistar-Ratten zu analysieren. Einige Lektine (UEA I, SBA, DBA) gaben eine recht spezifische Darstellung des reifen Akrosoms, wohingegen andere (PNA, RCA I) eine Affinität zu den frühen Stadien der Akrosomformation zeigten oder eine breite Affinität zu Germinal- und Nichtgerminalzellen und -strukturen aufwiesen (Con A, WGA). Im Nebenhoden zeigte die Spermatozoenmasse ein homogenes Darstellungsmuster mit einigen Lektinen (PNA, RCA I, Con A, WGA, DBA), welche auf der epithelialen Oberfläche auch eine recht starke Reaktion aufwiesen. Es wurde daraus geschlossen, daβ diese Reaktion zumindest teilweise auf die sekretorischen Produkte, die in den Haupt-, Apikal-, Schmal- und Hellzellen des Nebenhodens synthetisiert werden, zurückzuführen ist. Einige Unterschiede im Darstellungsmuster dieser Zellen wurden aufgezeichnet, die die Spezialisierung der Zellen für die Produktion bestimmter Glykoproteine anzeigen. Das Darstellungsmuster des interstitiellen und intertubulären Anteils des Hodens und Nebenhodens wurde ebenfalls aufgezeichnet.     

Circulating and luminal testicular factors affect LRP-2 and Apo J expression in the epididymis following efferent duct ligation

Apolipoprotein J (clusterin or sulfated glycoprotein-2) has been shown to be secreted by the epididymal principal cells, whereupon it binds to sperm in the lumen. Apolipoprotein J also is endocytosed by principal cells along the epididymis. Recently, it has been demonstrated that low-density lipoprotein receptor-related protein-2 (LRP-2) mediates the endocytosis of Apo J and is present in the epididymis. The purpose of the present study was to determine the factors regulating the synthesis of these 2 proteins in various experimentally treated animals. The epididymides of adult rats were fixed with Bouin's fluid and examined with anti-Apo J and anti-LRP-2 antibodies by a light microscope immunocytochemical method. In normal adult animals, expression of Apo J was evident in principal cells of all epididymal regions except the proximal initial segment. Diffuse cytoplasmic staining indicated Apo J secretion. Reactive apical vesicles, presumably endosomal in nature, suggested endocytosis of Apo J. Lipoprotein receptor-related protein-2 expression was solely apical in nature and was seen as an intense apical band in principal cells of all regions except the proximal and distal initial segment and distal caput regions of the epididymis. Hypophysectomy, up to 28 days after the procedure, did not affect expression of Apo J or LRP-2 in principal cells along the entire epididymis. Orchidectomy, with or without testosterone replacement at all time intervals examined, also did not affect LRP-2 expression along the entire epididymis. This also was noted for Apo J expression in all regions except the proximal initial segment. Thus, expression of these 2 proteins does not appear to be regulated by testicular or pituitary factors. In contrast, bilateral as well as unilateral (intact and ligated sides) efferent duct ligation resulted in dramatic differences in LRP-2 and Apo J expression in principal cells in the various epididymal regions. In the case of LRP-2, a complete absence of reaction was noted in principal cells along the entire epididymis. As for Apo J, expression in the distal initial segment, intermediate zone, and caput region remained unchanged compared with that in normal adult animals, whereas in the corpus and cauda epididymides, results of cytoplasmic staining were negligible. These results suggest that under conditions of efferent duct ligation, a circulating factor emanates from the testis to inhibit expression of LRP-2 and Apo J in these epididymal regions. Furthermore, because Apo J was affected in a region-specific manner, unlike the case for LRP-2, different factors appear to be involved for each protein. These factors may be produced to inhibit proteins from being synthesized by the epididymis in the absence of luminal testicular input and may exist in cases of congenital and pathologic epididymal tubule blockages as well as after vasectomy. In the case of immunostaining for Apo J in the proximal initial segment only, normally unreactive principal cells in control adult animals became intensely reactive after orchidectomy as well as bilateral and unilateral (ligated side only) ligation. As this was not the case for hypophysectomized animals and the intact side of unilateral efferent duct-ligated animals, it is suggested that a testicular factor entering via the lumen of the efferent ducts serves to inhibit Apo J expression in this area. The present data also reveal that after efferent duct ligation, there are circulating factors that inhibit Apo J expression in a region-specific manner (corpus and cauda) and that inhibit LRP-2 expression along the entire epididymis and that these are derived from the testis. Furthermore, the data reveal that a testicular luminal factor appears to inhibit Apo J expression in the proximal initial segment of normal adult animals. Key words: Principal cells, orchidectomy, glycoprotein 330, clusterin, sulfated glycoprotein-2.     

Immunolocalization of the Yb1 subunit of glutathione S-transferase in the adult rat epididymis following orchidectomy and efferent duct ligation

In addition to the maturation of sperm, the epididymis also serves to protect sperm from harmful reactive oxygen species. To this end, various antioxidant enzymes are produced by the epididymis, such as glutathione S-transferases (GSTs), a family of dimeric proteins that catalyze the conjugation of glutathione to various electrophilic compounds, thus providing cellular detoxification. In the present study, the regulation of the Yb(1) subunit of GST was examined in Bouin-fixed epididymides of adult control, orchidectomized (O) rats with or without testosterone (T) supplementation and efferent duct-ligated (EDL) rats using light microscope immunocytochemistry with an anti-Yb(1)-GST antibody. The intensely reactive ciliated cells of the efferent ducts and principal cells of the epididymis showing a checkerboard staining pattern were unaltered in their expression of Yb(1)-GST after all experimental procedures, suggesting their regulation by factors other than of testicular origin. On the other hand, the intense reaction of narrow/apical cells and moderate reaction of basal cells of the proximal initial segment of control animals became negligible in O rats and was not restored with T supplementation. As staining was also absent after EDL, the data suggest that a luminal testicular factor(s), other than androgens, regulates expression of Yb(1)-GST in narrow/apical and basal cells of the proximal initial segment. Although basal cells of the caput and cauda epididymidis were unreactive after all experimental protocols, as also noted in controls, the intensely reactive basal cells of the corpus epididymidis of control animals became unreactive in O animals. However, Yb(1)-GST expression was restored to these cells with T supplementation, and as there was no effect on Yb(1)-GST expression after EDL, the data suggest that circulating testosterone or one of its metabolites regulates expression of Yb(1)-GST in basal cells of the corpus region. Taken together, these data indicate a differential regulation with respect to the expression of Yb(1)-GST in the various cell types and regions of the epididymis.     

Stage and region-specific localization of lipocalin-type prostaglandin D synthase in the adult murine testis and epididymis

Lipocalin-type prostaglandin D synthase in semen has been associated with male fertility, although this relationship is not well defined. To gain insight into potential mechanisms, the objective of the present study was to immunocytochemically localize lipocalin-type prostaglandin D synthase within the testis, efferent ducts, and 4 segments of mouse epididymis. In the testis, immunoperoxidase staining was localized within the Sertoli cells only at stages VI-VIII of the spermatogenic cycle, which is just prior to spermiation. Intense staining was also evident throughout the interstitial tissue, including Leydig cells. The entire epithelium of the efferent ducts, including ciliated and nonciliated cells, was immunoreactive. A distinct pattern of immunostaining for lipocalin-type prostaglandin D synthase was observed in different regions of epididymis, suggesting a possible role in sperm maturation. Staining for lipocalin-type prostaglandin D synthase was strikingly absent in the initial segment. In caput epididymidis, staining was evident throughout the cell cytoplasm of principal cells with some cells more intensely stained than adjacent ones. In the corpus region, overall staining intensity decreased and appeared to be concentrated in the apical region of principal cells, but some cells were completely unreactive. Reaction product in the cauda region was heavily concentrated on microvilli and within the epididymal lumen. In all epididymal regions, expression of lipocalin-type prostaglandin D synthase was specific to epithelial principal cells; no immunoreactivity was apparent in other cell types. The specific localization of lipocalin-type prostaglandin D synthase within the testicular interstitial tissue, Sertoli cells, and principal cells of caput epididymidis strongly suggests that this protein plays an integral role in both the development and maturation of sperm.     

TGF-beta could be involved in paracrine actions in the epididymis of the marmoset monkey (Callithrix jacchus).

The transforming growth factor-beta1 (TGF-beta1) and the transforming growth factor-beta receptor type II (TGF-betaRII) were studied in the epididymis of sexually mature marmoset monkeys (Callithrix jacchus) by immunohistochemical localization of the protein and by polymerase chain reaction (PCR) analysis of the mRNA level. In order to specify reactive cell types, the morphology of all three segments (caput, corpus, and cauda epididymidis) was evaluated by light microscopy. Six different cell types could be distinguished: principal, basal, apical, and clear cells, as well as intraepithelial lymphocytes and macrophages. Using immunohistochemistry, specific staining for TGF-beta1 in the caput was found in 47% of the apical cells, whereas the TGF-betaRII was located in the apical portion of 91% of all principal cells. In the corpus epididymidis, 20% of the apical cells were immunopositive for TGF-beta, and binding of the receptor antibody occurred in 17% of the principal cells (all numbers based on counts of counterstained nuclei). All differences between percentages in the caput and corpus were significant as determined by chi-square test. PCR analysis revealed detectable levels of TGF-beta1 mRNA in the marmoset epididymis. Our results indicate for the first time that TGF-beta1 is synthesized in the marmoset epididymis, possibly in a different subpopulation of epididymal cells than the TGF-beta receptor type II. Thus, TGF-beta might be of functional relevance in the primate epididymis.     

Fine structure of fetal human testis and epididymis.

Observations on the structure of the human fetal testis and epididymis at 16 weeks' gestation have been made with the light and electron microscope. The fetal testis is organized into solid cords surrounded by a well-defined cellular investment. The basement membrane is multilaminated with a highly redundant basal lamina. The germ cells rest on thin processes from adjacent Sertoli cells. Intercellular bridges between centrally located germ cells have been observed. The epididymis is remarkably well developed. The tall pseudostratified epithelium lines a discrete duct with a patent lumen. Stereocilia and cilia are seen on apical surface of the principal cells.     

Non-specific uptake of IgG by rat epididymal tubules in vitro

Tubules isolated from the initial segment, caput and corpus regions of the rat epididymis were incubated for 4 h in medium containing rat IgG or bovine-serum-albumin (10 mg ml-1) and processed for light microscopy immunocytochemistry using gold-labelled anti-rat IgG and silver staining or peroxidase/anti-peroxidase techniques. Distribution of IgG was confined to the peritubular matrix only, with no detectable uptake into the epithelium or lumen. In tubules incubated with rat IgG coupled to gold particles as tracer and fixed for electron microscopy, the IgG-gold complex failed to penetrate to the base of the epithelium. When IgG-peroxidase conjugates were used, sparse enzyme activity was localized in small basal and apical vesicles and multivesicular bodies of the principal cells. However, this uptake could not be inhibited by the presence of a 30-fold excess of non-labelled IgG, and the same distribution of enzyme activity was also observed when tubules were incubated with a similar activity of horseradish peroxidase alone. These findings indicate that there is no detectable receptor-mediated transport of IgG across the epididymal epithelium, although non-selective transfer could occur via fluid-phase endocytosis.     

Cell specificity of aquaporins 0, 3, and 10 expressed in the testis, efferent ducts, and epididymis of adult rats

Aquaporins (AQPs) are transmembrane protein channels that allow the rapid passage of water across an epithelium at a low energy requirement, though some also transport glycerol, urea, and solutes of various sizes. At present, 11 members of the AQP family of proteins have been described in mammals, with several being localized to the testis (AQP-7 and AQP-8), efferent ducts (AQP-1 and AQP-9), and epididymis (AQP-1 and AQP-9) of adult rats. With the discovery of expression of multiple AQPs in different tissues, we undertook a systematic analysis of several other members of the AQP family on Bouin-fixed tissues of the male reproductive tract employing light microscope immunocytochemistry. In the testis, AQP-0 expression in the seminiferous epithelium was restricted to Sertoli cells and to Leydig cells of the interstitial space; no reaction was observed in the efferent ducts or epididymis. In Sertoli cells, a semicircular pattern of staining was noted, with only one fourth or one half of the Sertoli cells of a given tubule showing a reaction product. Furthermore, while Sertoli cells at stages VI-VIII of the cycle showed intense staining, those at stages IX-XIV were least reactive, with Sertoli cells at stages I-V showing intermediate levels of reaction product. The epithelial expression of AQP-10 was restricted to the microvilli of the nonciliated cells and the cilia of the ciliated cells of the efferent ducts; however, the endothelial cells of vascular channels of the efferent ducts and epididymis were also intensely reactive. AQP-3 expression was localized exclusively to the epididymis, where intense staining was noted exclusively over basal cells. Examination of orchidectomized rats revealed that AQP-3 expression was abolished over basal cells and that it was greatly diminished after efferent duct ligation. As the reaction was not fully restored in orchidectomized animals supplemented with high levels of testosterone, we suggest that AQP-3 expression in basal cells is regulated in part by testosterone, in addition to a luminal factor emanating from the testis. Together, the data indicate a cell- and tissue-specific expression for AQP-0, AQP-3, and AQP-10 in the testis, efferent ducts, and epididymis, as well as differential regulating factors for the expression of AQP-3 in basal cells.     

Distribution of glycoconjugates in normal rat ventral prostate and their use as markers of androgen-controlled secretory function in culture

The distribution of glycoconjugates in uncultured and cultured rat ventral prostate was studied by using eight fluorescent lectins. The prostate pieces were cultured in defined medium with or without testosterone for 1-14 days. Each lectin revealed a characteristic binding pattern. Con A, LCA, WGA, and RCA I stained both epithelial and interstitial components. SBA and PNA were specific for the epithelium: SBA stained the region of the Golgi complex; PNA showed the brightest fluorescence in the apical part of the cells representing the region of secretory granules. In culture without testosterone the epithelial cells gradually lost their fluorescence, whereas the stromal fluorescence increased. The basement membrane was disorganized. With testosterone the integrity of the epithelium and stroma was maintained, and the staining pattern of the lectins was in the main similar as in vivo. However, at 14 days a change in the staining pattern of apical cytoplasm with PNA was noted, indicating that in long-term cultures, in addition to testosterone, other hormones and growth factors are necessary to complete especially the last stages of the secretory process in the epithelial cells.     

Cell- and region-specific localization of lysosomal and secretory proteins and endocytic receptors in epithelial cells of the cauda epididymidis and vas deferens of the adult rat.

The epithelial cells lining the cauda epididymidis and vas deferens are active in endocytosis and have an abundance of lysosomes and a well-characterized secretory apparatus. However, little is known about the nature of lysosomal proteins contained within lysosomes, the types of receptors on the cell surface, and the types of proteins secreted by these cells. In the present study, cathepsins A, D, B, and sulfated glycoprotein (SGP)-1, well-characterized lysosomal proteins, as well as SGP-2, a secretory protein and low-density lipoprotein receptor-related protein-2 (LRP-2), an endocytic receptor, were immunolocalized at the light-microscopic level within epithelial cells of the cauda epididymidis and vas deferens. Principal cells showed numerous intensely reactive lysosomes for cathepsins A, D, and SGP-1 in all regions of the cauda and vas deferens and for cathepsin B only in the cauda epididymidis. Basal cells were intensely reactive for cathepsin A, unreactive for cathepsins D and B, and weakly reactive for SGP-1 in the cauda region. In the vas deferens, these cells were intensely reactive for cathepsin A and SGP-1 and unreactive for cathepsin B; in the case of cathepsin D, basal cells were weakly reactive in the proximal vas deferens but intensely reactive in the middle and distal vas deferens. Clear cells, present in the cauda region and proximal vas deferens, were intensely reactive for cathepsin A, weakly reactive for SGP-1, and unreactive for cathepsins D and B, while narrow cells found mainly in the proximal vas deferens were intensely reactive for cathepsins A, D, and SGP-1 and unreactive for cathepsin B. Thus, the expression of different lysosomal enzymes in the cauda epididymidis and vas deferens is not only cell- but also region-specific, suggesting differences in the type of substrates internalized by these cells. SGP-2, a secretory protein, showed a checkerboardlike staining pattern in the cytoplasm of principal cells of the cauda epididymidis, while the cytoplasm of all principal cells were intensely reactive in the vas deferens. This type of reaction, as well as staining of sperm, suggests that SGP-2 is secreted into the lumen, where it functions in relation to sperm. The endocytic receptor LRP-2 was noted only on the apical surface of principal cells of the cauda and vas deferens and in spherical structures indicative of endosomes suggestive of their role in the uptake of various ligands, including SGP-2, for which it has a high binding affinity. Thus SGP-2 in the cauda and vas deferens is not only secreted but endocytosed by principal cells, suggestive of an active turnover in the lumen. In summary, the epithelial cells of the cauda and vas deferens show marked differences in expression of lysosomal proteins, SGP-2, and LRP-2 suggestive of differences in their functional activity while sperm are stored and protected in these regions.     

Immunolocalization and regulation of cystic fibrosis transmembrane conductance regulator in the adult rat epididymis

Cystic fibrosis is the most common serious autosomal recessive condition in whites, and more than 95% of men with cystic fibrosis are infertile. The cystic fibrosis transmembrane conductance regulator (CFTR), a cyclic adenosine monophosphate (cAMP)-regulated chloride channel, has been localized in the efferent ducts; however, to our knowledge, its expression and regulation in the epididymis by testicular factors have not been examined. In the present study, these parameters were examined immunocytochemically by the light microscope with an anti-CFTR antibody in Bouin-fixed, paraffin-embedded control adult rat epididymides and both orchidectomized adult rats with or without testosterone supplementation and efferent duct-ligated rats sacrificed at different time points. In control animals, a thick dense band of immunoperoxidase reaction product was visualized over the apical plasma membrane of the principal cells but not their microvilli. The apical band was prominent only in the corpus and cauda regions. While there was no CFTR expression in basal cells, clear cells of the corpus and cauda regions showed a weak-to-moderate band of apical plasma membrane staining. An examination of orchidectomized, orchidectomized and testosterone, and efferent duct-ligated rats revealed that CFTR was no longer expressed as an intense band on the apical plasma membrane of the principal cells of the corpus and cauda regions. However, under these conditions, an intense apical/supranuclear reaction was noted in the form of small vesicular structures. Clear cells were unaffected by the different experimental treatments. Together, these data indicate that CFTR is expressed in a cell- and region-specific manner and that, while its synthesis in principal cells is not under the control of testicular factors, targeting to the apical plasma membrane is regulated by a testicular luminal factor.     

Expression profile of Toll-like receptor 4 in rat testis and epididymis throughout postnatal development

Toll-like receptors (TLRs) belonging to pattern recognition receptors are involved in maintaining testicular and epididymal immune homeostasis. The purpose of the current study was to investigate TLR4 expression in rat testis and epididymis throughout postnatal development. Weak staining was detected in peritubular myoid cells and immature Sertoli cells while no staining was observed in gonocytes during prepubertal period. However, TLR4 expression began to appear in spermatocytes in pubertal period and gradually increased in spermatids. An intense staining was observed in steps 5–19 spermatids in post pubertal and mature periods. Similarly, TLR4 expression in the testes steadily increased from pubertal period to mature period. Puberty also caused a significant increase in TLR4 expression in epididymis. TLR4 expression in cauda epididymis was lower as compared to those of other epididymal segments. The majority of epididymal epithelial cells exhibited apical TLR4 expression, whereas basal cells showed intense intracytoplasmic immunoreaction. We detected an intense staining in epididymal smooth muscle cells. The expression levels of TLR4 showed dynamic changes in both spermatogenic cells, and entire testicular and epididymal tissues during postnatal development. These results suggest that TLR4 expression contributes not only to inflammation but also to the development of spermatogenic cells.     

NADPH-diaphorase in glandular cells and nerves and its relation to acetylcholinesterase-positive nerves in the male reproductive tract of man and guinea-pig

The presence of NADPH-diaphorase activity and acetylcholinesterase in the testis, epididymis, vas deferens, seminal vesicle, pelvic plexus, prostate and urethra of man and guinea-pig was investigated with the nitro blue NADPH technique and the thiocholine method, respectively. In human material NADPH-diaphorase activity was found in the Leydig cells, Sertoli cells and the epithelial linings of the rete testis, the excretory ducts, seminal vesicle, prostate and urethra. The guinea-pig material showed staining of the Leydig cells and spermatozoa and similar epithelial staining of the tract as man. Nerves beneath the epithelium and in the muscle layers of cauda epididymis, vas deferens, seminal vesicle, prostate and urethra were also stained. NADPH-diaphorase-positive nerve cells were seen in the pelvic plexus. Some cells also displayed acetylcholinesterase activity but others showed activity for only one of the enzymes or no activity for either enzyme. In the cauda epididymis, vas deferens, seminal vesicle, prostate and urethra acetylcholinesterase-positive nerve fibres formed a plexus beneath the secretory cells. It is concluded that NADPH-diaphorase, generally accepted as a nitric oxide synthase, is present in glandular cells of the male genital tract. The enzyme is also present in nerves, where it is partly co-localized with acetylcholinesterase. Received: 15 January 1997 / Accepted: 18 September 1997     

Postnatal development and regulation of beta-hexosaminidase in epithelial cells of the rat epididymis

beta-Hexosaminidase (Hex) catalyzes the hydrolysis of terminal sugar residues from a number of substrates such as GM2 gangliosides, glycoproteins, glycolipids, and glycosaminoglycans. As an enzyme present in lysosomes of epithelial cells of the adult rat epididymis, it serves to degrade substances endocytosed from the epididymal lumen. In this way, it modifies and creates a luminal environment where sperm can undergo their maturational modifications. In this study, the postnatal developmental pattern of expression of Hex was examined in animals from days 7-56. In addition, the role of testicular factors on Hex expression in the different cell types and regions of the epididymis of adult rats was examined in orchidectomized and efferent duct-ligated rats. Both parameters were examined on Bouin-fixed epididymides in conjunction with light microscope immunocytochemistry. At postnatal day 7, the epithelium of the entire epididymis was unreactive for anti-Hex antibody. By day 21, narrow and clear cells of their respective regions became reactive, whereas basal cells became reactive only by day 29. Principal cells displayed only an occasional reactive lysosome at day 21, several by day 29, and numerous reactive lysosomes by day 39, comparable to the region-specific distribution noted for 90-day-old animals, and at an age when high androgen levels are attained. Thus, postnatal onset of Hex expression varies according to the different cell types of the epididymis, suggesting different regulatory factors. This finding was confirmed from studies employing adult orchidectomized and efferent duct-ligated adult rats. Indeed, in all experimental animals, Hex immunostaining in narrow, clear, and basal cells was intense and comparable to control animals. In contrast, there was a notable absence of lysosomal staining in principal cells at all time points after orchidectomy, which was restored, however, following testosterone replacement. No effect on Hex expression was observed in efferent duct-ligated animals. Taken together, the data suggest that Hex expression in lysosomes of principal cells is regulated by testosterone or one of its metabolites. However, the expression of Hex being independent of testicular factors in narrow, clear, and basal cells of adult animals, but occurring at different time points during postnatal development, suggests that different regulatory factors are responsible for onset of Hex expression in these cell types during development.     

急性热应激对性成熟雄性小鼠睾丸、附睾、输精管中热休克蛋白70表达的影响

目的:探讨急性热应激对性成熟雄性小鼠睾丸、附睾、输精管中热休克蛋白70(heat shock protein 70,HSP70)表达的影响。方法:将32只8周龄雄性小白鼠随机均分为4组,饲养7d后,进行热应激处理,温度控制在(39±0.5)℃,时间分别为0.5、1和3h。应激后立即采血,分离血清测定谷草转氨酶(GOT)含量。一侧附睾制备精子悬液,用于计算精子密度和顶体畸形率;另一侧附睾、睾丸、输精管用于免疫组化研究。结果:应激后,小鼠体重、睾丸系数、顶体畸形率变化不显著(P>0.05),附睾系数和精子密度有不同程度的下降,GOT含量急剧升高(P<0.01)。随着应激时间的延长,小鼠精子密度呈递减趋势,顶体畸形率呈上升趋势。应激时间最短的0.5h组小鼠体重、睾丸系数、附睾系数的降幅反而最大。免疫组化法观察发现,HSP70在性成熟小鼠睾丸、附睾、输精管中均有表达。正常状态下,HSP70在睾丸组织间质细胞中少量表达,应激后分布于间质细胞核,此外在精母细胞核与精子细胞核中也有大量分布;附睾中HSP70主要分布于主细胞质,基细胞和亮细胞中没有表达,应激后附睾体的纤毛细胞中也发现大量棕色颗粒;输精管中HSP70主要定位在基细胞质,主细胞中不表达。随着应激时间的延长,HSP70在睾丸、附睾中的表达量明显升高,而在输精管中的增幅不明显。结论:急性热应激对性成熟雄性小鼠的生殖系统造成了损伤;HSP70在睾丸、附睾、输精管中的表达与定位具有区域特异性和细胞特异性,提示其可能参与精子的发生与成熟;HSP70在应激状态下表达量大幅上升的作用可能在于保护细胞免受高热损伤。     

Alterations in the testis and epididymis associated with loss of function of the cystatin-related epididymal spermatogenic (CRES) protein

Cystatin-related epididymal spermatogenic protein (CRES) or cystatin 8 (Cst8 gene) is a member of the cystatin superfamily of cysteine protease inhibitors. It differs from typical cystatins because it lacks consensus sites for cysteine protease inhibition and exhibits reproductive-specific expression. In the present study, we examined CRES expression within the testes, efferent ducts, and epididymides of normal mice by light microscope immunolocalization. Alterations to these tissues in male mice lacking the Cst8 gene (Cst8(-/-2)) were also characterized by histomorphometry and electron microscopy. In the normal testis, CRES was localized exclusively in mid and late elongating spermatids. In the efferent ducts, CRES was localized to the apical region of the epithelial cells suggestive of localization in the endosomes. In the initial segment of the epididymis, principal cells showed supranuclear and luminal reactions. In the cauda region, CRES was present exclusively as aggregates in the lumen and was detected in clear cells. Compared with wild-type mice (Cst8(+/+)), older (10-12 months) Cst8(-/-) mice had modest but statistically significant reductions in tubular, epithelial, and/or luminal profile areas in the testis and epididymis. By electron microscopy, some Cst8(-/-) tubules in the testis were normal in appearance, but others showed a vacuolated seminiferous epithelium, degenerating germ cells, and alterations to ectoplasmic specializations. In the epididymal lumen, abnormally shaped sperm heads and tails were noted along with immature germ cells. In addition, principal cells contained numerous large irregularly shaped lysosomes suggestive of disrupted lysosomal functions. In both the testis and epididymis, however, these abnormalities were not apparent in younger mice (4 months), only in the older (10-12 months) Cst8(-/-) mice. These findings suggest that the altered testicular and epididymal histology reflects a cumulative effect of the loss of CRES and support a role for CRES in maintaining the normal integrity and function of the testis and epididymis.     

Localization of cellular retinol-binding protein and cellular retinoic acid-binding protein in the rat testis and epididymis

The distribution of cellular retinol-binding protein (CRBP) and cellular retinoic acid-binding protein (CRABP) in rat testis and epididymis was examined by the peroxidase-antiperoxidase immunolocalization technique. In the testis, cellular retinol-binding protein was localized exclusively in the Sertoli cells. Staining varied with the stages of the seminiferous epithelium cycle and was maximal prior to the maturation divisions. Cellular retinoic acid-binding protein was localized exclusively in the germinal cells in the adluminal compartment. The results suggest that retinoic acid may be the retinoid form used by the germinal cells, and that Sertoli cells may use the cellular retinol-binding protein to transfer retinol from the basal to the adluminal compartment. In the epididymis, cellular retinol-binding protein was localized in the cytoplasm and stereocilia of the principal cells in the proximal caput epididymidis, while cellular retinoic acid-binding protein was localized in the spermatozoa and the stereocilia of the principal cells throughout the epididymis and in the epithelial cells of the distal vas deferens. Sperm staining intensity decreased from the initial segment to the cauda. The presence of high levels of cellular retinol-binding protein in the epithelial cells and high levels of cellular retinoic acid-binding protein in the spermatozoa of the caput epididymidis, known to be involved in the synthesis and secretion of factors necessary for sperm maturation, suggests that vitamin A may have a role in this process.