Purification of rat epididymal proteins `D' and `E', demonstration of shared immunological determinants, and identification of regional synthesis and secretion

Two acidic secretory epididymal glycoproteins, protein D of 27000 daltons and protein E of 28000 daltons, have been purified and antisera prepared against each separately. Both proteins were found to share common immunological determinants when tested by double immunodiffusion and tandem crossed immunoelectrophoresis. By selective immunoprecipitation, protein D was shown to be synthesized and secreted by all regions of the epididymis with the exception of the initial segments. In contrast, the synthesis and secretion of protein E was restricted to the corpus and proximal cauda.     

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.     

Carbohydrate composition of specific rat epididymal protein

Previous results demonstrated that androgen-dependent rat specific epididymal proteins (SEP) were bound to spermatozoa during their maturation in the epididymis. This paper describes the purification of glycoprotein DE, which constitutes 40% of SEP, and the identification and semiquantitative determination of the sugars forming its oligosaccharide chain. Affinity chromatography on Sepharose-Concanavalin A produced a sample of D-E 95% pure in which 10.5 g of sugar were present per 100 g protein. The percentual composition of the oligosaccharide was D-mannose 19%; D-galactose 3%; N-acetyl-D-glucosamine 33%; N-acetyl-neuraminic acid 31% and D-glucose 13%.     

Paracrine regulation of Leydig cells by the seminiferous tubules

Testes of adult control and unilateral cryptorchid rats were fixed by vascular perfusion. The cell profile area of peritubular Leydig cells surrounding tubules in different stages of spermatogenesis, and the cell profile area of perivascular Leydig cells were determined. The size of peritubular Leydig cells was dependent on which type of tubulus the cells were surrounding. Some peritubular Leydig cells, especially those surrounding stages VII–VIII (88.1 ± 7.1 μm², mean ± SD, n = 6 rats), were larger than perivascular Leydig cells (69.3 ± 5.9 μm²). The size of Leydig cells surrounding stages IX–XIV was similar to that of perivascular cells. In the abdominal testes no spermatogenic cycle was present and the sizes of peritubular and perivascular Leydig cells were equal (63.0 ± 5.1, vs 66.7 ± 7.3 μm², mean ± SD, n = 5 rats). It is suggested that the tubules and the spermatogenic cycle locally modulate Leydig cell activity and that Leydig cell malfunction in abdominal testes may be due to a decreased stimulatory influence from the damaged tubules.     

Differential expression and regulation of integral membrane protein 2b in rat male reproductive tissues

Aim： To examine the expression and regulation of integral membrane protein 2b （Itm2b） in rat male reproductive tissues during sexual maturation and under different treatments by in situ hybridization. Methods： Testis, epididymis, and vas deferens were collected on days 1-70 to examine Itm2b expression during sexual maturation. To further examine the regulation of Itm2b, adult rats underwent surgical castration and cryptorchidism. Ethylene dimethane sulfonate and busulfan treatments were carried out to test the regulation of Itm2b after destruction of Leydig cells and germ cells. Results： In testis, Itm2b expression was moderately detected in the adluminal area of seminiferous cords on days 1-10, and detected at a low level in the spermatogonia on days 20 and 30. The Itm2b level was markedly increased in Leydig cells from day 20 to day 70. In epididymis and vas deferens, Itm2b was detected from neonate to adults, and the signal gradually increased in accordance with sexual maturation. Itm2b expression was significantly downregulated in epididymis and vas deferens of castrated rats, and strongly stimulated when castrated rats were treated with testosterone. Cryptorchidism led to a significant decline of Itm2b expression in testis and caput epididymis. Itm2b expression in epididymis and vas deferens was significantly decreased after the Leydig ceils were destroyed by ethylene dimethane sulfonate. Busulfan treatment produced no obvious change in Itm2b expression in epididymis or vas deferens. Conelusion： Our data suggested that Itm2b expression is upregulated by testosterone and might play a role in rat male reproduction.     

Acidification of epididymal fluid in the boar

The present study describes the measurement of pH made in vivo in the rete testis fluid and in different regions of the boar epididymis. Furthermore, samples of whole ejaculates, semen fractions, testicular (ductuli efferents/rete testis), epididymal and deferential fluids collected from the same fertile boars, were analysed for their acid/base status with an automatic blood gas analyser. A pH gradient of activity was found between the fluid entering the ductus epididymis (pH 7.2) and the region of sperm storage at the cauda (pH 6.5). A significantly lower concentration of bicarbonate ion was found in the cauda epididymidis (3-4 mM) compared to rete testis fluid (30 mM), which might be related to the quiescence of the spermatozoa. A significant increase in extracellular pH and bicarbonate concentration occurred at ejaculation, the bicarbonate levels being 9-10-fold higher in the semen fraction rich in seminal vesicle fluid, where sperm showed higher motility, than in the cauda epididymis.     

Differentiation of the rat seminiferous tubules between 13 and 19 days of age

The differentiation of rat seminiferous tubules have been studied in 13 to 19 days old rats. Testicular weight and tubular cross-sectional area were more than doubled during this period. The percentage of tubules with more than 10 primary spermatocytes increased from 4% to 90%, and the lanthanum excluding ability of the inter-Sertoli cell junctions (the blood-testis barrier) showed a similar increase but two days later. ABP production in vitro increased more than twentyfold from day 13 to day 19 of age. It is concluded that the differentiation of Sertoli cell function and the appearance of primary spermatocytes are temporally correlated which supports the assumption that the function of the Sertoli cells is important for initiation and maintainance of spermatogenesis.     

Transformation,migration and outcome of residual bodies in the seminiferous tubules of the rat testis

Experiments were performed to study the transformation, migration and outcome of residual bodies (RBs) in the seminiferous tubules of the rat testes. One part of the testes from adult Sprague–Dawley rats was used to generate paraffin sections to observe RBs and RB precursors through specific staining, and the other part of the testes was used to generate ultrathin sections to observe RBs under a transmission electron microscope. Deep blue particles of different sizes were observed in some seminiferous tubules through specific staining for RBs and RB precursors. These particles first appeared in the seminiferous tubules at stage I of the spermatogenic cycle, and after spermiation, the particles travelled rapidly towards the deeper region of the seminiferous epithelium and soon appeared close to the basement membrane of the seminiferous tubule. All of the particles in the tubules disappeared at stage IX. Using transmission electron microscopy, components of different electron densities were observed in the RBs on the surface of the seminiferous epithelium, all of which gradually formed in the cytoplasm of spermatozoon in later stages of spermiogenesis. After the spermatozoa were released, the RBs in the epithelium travelled quickly to the edge of the tube and were gradually transformed into lipid inclusions. These lipid inclusions ultimately became lipidlike particles. The lipidlike particles were discharged into the interstitial tissue. RBs initiate their own digestive process before their formation during spermiation in the rat testes. After spermiation, the RBs transform into lipid inclusions and finally into lipidlike particles. These lipidlike particles can be eliminated from the seminiferous tubules.     

Production and secretion of an interleukin-l-like factor is stage-dependent and correlates with spermatogonial DNA synthesis in the rat seminiferous epithelium

It has been shown previously that the intact adult rat testis produces large amounts of an interleukin-1 (IL-1)-like growth factor. The present study has investigated whether this testicular IL-1-like factor (tIL-1) is produced and secreted differentially by the fourteen stages of the seminiferous epithelial cycle in the rat testis. Seminiferous tubule segments representing defined stages were identified by transillumination-assisted microscopy and isolated by microdissection. Pooled segments were either homogenized and extracted with aqueous buffer or incubated for 24 h to produce conditioned media (CM). The recovered material was then analysed for IL-1 bioactivity in a sensitive murine thymocyte proliferation assay. When divided into four stage groups, extracts of stages II-VI, IX-XII and XIII-I showed equally high IL-1 activity whereas stage group VII-VIII showed much lower activity. More detailed analysis with 10 different stage groups showed that tIL-1 activity was undetectable in extracts of substages VIIab and VIIcd. The same pattern was seen when CM from cultured tubular segments were analysed. Labelling of seminiferous tubules with tritiated thymidine in vitro and analysis by autoradiography revealed that DNA-synthesizing spermatogonia were absent in substages VIIb and VIIc and sparse in substages VIIa and VIId. The results show that tIL-1 activity is produced in a stage-dependent manner and suggest that tIL-1 might be involved in the regulation of spermatogonial proliferation in vivo.     

Local regulation of Leydig cells by the seminiferous tubules. Effect of short-term cryptorchidism

Unilateral cryptorchism was induced in adult rats for 24 h, and its effect on testicular morphology and intratesticular testosterone concentration after hCG-stimulation were studied. In seminiferous, tubules from abdominal testes an increased number of degenerating germ cells was noted in stages XIV-III of the spermatogenic cycle and Sertoli cells contained an increased amount of lipid droplets in stages XIV-VIII. However, germ cells and Sertoli cells from tubules at other stages of the cycle appeared unaffected. In scrotal testes the size of peritubular Leydig cells varied in phase with the spermatogenic cycle. The largest cells were found adjacent to stage VII-VIII and the smallest adjacent to stage XI-XII. In abdominal testes no stage-dependent variation in the size of peritubular Leydig cells was seen. Perivascular Leydig cells were of equal size in abdominal and scrotal testes. The testicular testosterone concentration following stimulation with a low dose of hCG was significantly lower in abdominal testes. It is suggested that the seminiferous tubules locally modulate Leydig cell function and that the stage specific stimulatory influence from stage VII-VIII is rapidly lost during experimental cryptorchidism.     

Regulation of interstitial cell function by seminiferous tubules in intact and cryptorchid rats

The effects of experimental cryptorchidism on seminiferous tubule secretions and interstitial cell testosterone production were studied in vitro. Spent media obtained from incubations of seminiferous tubules (SMST) from cryptorchid rats caused a significant increase in testosterone production when added to interstitial cells isolated from intact rats. The previously noticed inhibitory activity of the SMST from stages VIII–XI of the sperma-togenic epithelial cycle gradually disappeared after the induction of experimental cryptorchidism. SMST obtained from both sham-operated or cryptorchid rats stimulated basal testosterone production when added to interstitial cells from cryptrochid rats. SMST from rats had been cryptorchid for 7, 14 and 28 days stimulated testosterone production when added to interstitial cells prepared from intact animals. Seminiferous tubules from cryptorchid rats therefore appear to be the source of a heat stable, trypsin-resistant factor with an apparent molecular weight of between 5000 and 10 000 daltons which stimulates testosterone production when added to interstitial cells in vitro. Its activity could not be blocked by an LRH antagonist. This factor enhances both basal and LH-stimulated secretion of testosterone in contrast to the inhibitory activity which involves only a partial blockade of LH-dependent steroidogenesis.     

Exploration for testicular remnants: implications of residual seminiferous tubules and crossed testicular ectopia

PURPOSE: Testicular remnants identified during exploration for cryptorchidism contain vascularized fibrous nodules at the termination of the vas deferens, hemosiderin, calcification, a pampiniform plexus or occasionally residual seminiferous tubules that may contain germ cells. An absent testis lacks the features of testicular remnants. To our knowledge testicular remnants have not been described in a crossed ectopic location. We reviewed orchiectomy specimens obtained at exploration for a nonpalpable testis to characterize the features of testicular remnants, including the frequency of seminiferous tubules, germ cells and crossed ectopia, as well as to clarify the diagnostic criteria for testicular remnants. MATERIALS AND METHODS: From 1990 to mid 2000 medical records and histological slides from 101 boys with nonpalpable testes who had undergone inguinal exploration and orchiectomy were reviewed. RESULTS: Of the 71 testicular remnants identified 7 (9.8%) contained residual tubules, of which 4 (5.6%) contained germ cells. In 4 boys the testis was deemed absent but 3 did not undergo laparoscopic exploration. There were 2 ectopic remnants (2.8%) on the contralateral side-the pelvis or in the scrotum. Both crossed remnants demonstrated dissociation of the testis from the vas/epididymis which remained on the correct side associated with a pampiniform plexus. No müllerian remnants were encountered. CONCLUSIONS: Adequate exploration for nonpalpable testis requires laparoscopy with visualization of the contralateral pelvic region because an ectopic remnant may be dissociated from the vas/epididymis and vessels. Identification of a pampiniform plexus, vas and spermatic vessels may not be a reliable indicator of a testicular remnant. Continued removal of testicular remnants is warranted because at least 9.8% contain residual viable tubules.     

Effect of testicular capsulotomy on lipid droplets in the seminiferous tubules of rats

Aim: In order to reveal the histochemical alteration that might occur during the processes of the spermatogenic disruption induced by testicular capsulotomy, the location and alteration of lipid droplets in the seminiferous tubules were observed in the present study. Methods: Osmium tetroxide was used to demonstrate the lipid droplets in the seminiferous tubules of capsulotomized and sham-operated control testes. Results: In the seminiferous tubules of the sham-operated rat testes, many small lipid droplets were located close to the basement membrane of the seminiferous tubules. But for the capsulotomized testes, the lipid droplets in the seminiferous tubules had increased in size and number, with many lipid droplets migrated towards the lumen of the tubules. Conclusion: The results indicated that a progressive fatty degeneration occurred in the seminiferous tubules after testicular capsulotomy.     

Stage-specific degeneration of germ cells in the seminiferous tubules of non-obese diabetic mice

The cause of fertility problems in insulin-dependent diabetes is largely unknown. To evaluate the role of autoimmunity-associated phenomena in the testis as a possible cause of the derangement in spermatogenesis, the stage-specific apoptosis of germ cells in the insulitis phase of pre-diabetes was quantified in the testes of non-obese diabetic (NOD) mice. The seminiferous epithelium of normal BALB/c and NOD mice contained cells positive for in-situ end-labelling (ISEL) of DNA. ISEL-positive germ cells formed clusters in the seminiferous epithelium of the NOD mice in marked contrast to the seminiferous epithelium of the BALB/c mice, which contained only individual cells positive for ISEL. ISEL-positive cells were present in the basal and luminal compartments of the epithelium. Ultrastructural analysis and demonstration of externalized phosphatidyl serine confirmed that the cells were undergoing apoptosis. The ultrastructurally apoptotic cells included spermatogonia, spermatocytes and spermatids. In cytological squash preparations of segments of seminiferous tubules from NOD mice aged 17–20 weeks, the number of ISEL-positive cells/mm tubule was significantly lower in segments at stages I–II of the seminiferous epithelial wave but higher at stages III–IV in comparison to BALB/c mice. The numbers of ISEL-positive cells/mm tubule in the other stages were similar in the two strains of mice. Analysis of ³²P-3' -end labelled DNA from the testes showed that the BALB/c mice had relatively more DNA fragmentation than did the NOD mice. These data suggest that autoimmune insulitis in the NOD mice is associated with increased amounts and abnormal stage distribution of apoptosis in the seminiferous epithelium, resulting in derangement of spermatogenesis.     

On the presence of prostatic secretion protein in rat seminal fluid

The copulating plug collected from the tip of the penis from rats immediately after decapitation contains a protein very similar and probably identical to PSP (prostatic secretion protein); this protein has earlier been purified from rat prostatic cytosol and characterized. The protein present in the copulating plug interacts with [³H]estramustine and binds to the antibody raised against rat PSP. The concentration of the protein in the copulating plug is 400 ng/mg of total protein, when measured using the radio immunoassay technique developed earlier for measurement of PSP in rat prostate. The [³H]estramustine–protein complex formed in a preparation of the copulating plug has an apparent molecular weight of about 50,000 and a sedimentation coefficient of about 3S when analyzed using sucrose density gradient centrifugation. The complex was retained on Concanavallin-A Sepharose indicating that the protein is a glycoprotein. Binding of the complex was also observed on hydroxylapatite and DEAE-Sephadex columns, from which it ws eluted at 0.18 M KC1. Light microscope autoradiograms of rat sperms incubated with ¹²⁵I-labeled PSP indicated that PSP is bound to all parts of the sperms. A macromolecule interacting with the PSP-antibodies is also present in human seminal fluid but at a concentration considerably lower than in rat seminal fluid. The present study shows that a macromolecule probably identical to prostatic secretion protein is present in the copulating plug from the rat. The biological role of this protein in normal male fertility is discussed.     

Chronic stress affects tyrosine phosphorylated protein expression and secretion of male rat epididymis

Chronic stress (CS) is shown to decrease the semen quality with changed expression of tyrosine phosphorylated (TyrPho) proteins in testicular and seminal tissues. However, the alterations of such proteins and fluid contents in the epididymis, producing sperm maturation factors, have never been reported. Sixteen adult rats were randomly divided into 2 groups (n = 8). The control animals were not subjected to stressors whereas CS rats were immobilised within restraint cage (4 hr/day) before cold forced-water swimming (15 min/day) for 60 days. Corticosterone, testosterone, blood glucose level (BGL), malondialdehyde (MDA) and biochemical components in epididymal fluid were assayed. Expressions of heat shock protein 70 (HSP-70), androgen receptor (AR) and TyrPho protein were investigated in epididymal tissue and fluid. Significantly, CS increased the corticosterone and BGL but decreased testosterone and epididymal substance levels. MDA level in tail epididymal fluid and HSP-70 expression in both regions of epididymal tissues and fluids, except in head epididymal fluid of CS were increased. Epididymal tissues showed the decrease of AR expression. Presence and changes of many TyrPho proteins were observed in CS. In conclusion, CS could affect functional proteins particularly TyrPho in epididymis, resulted in low semen quality.     

【原创论文】小鼠杀菌渗透增强性蛋白起源于睾丸及附睾表达并定位于精子中

杀菌渗透增强性蛋白（BPI）是具有抗革兰氏阴性菌活性的内源性杀菌蛋白。在本研究中,我们通过自行制备的多克隆抗体,检测了BPI蛋白在小鼠出生后睾丸及附睾组织中的表达,以及在附睾精子头部的亚细胞定位。实验结果表明,睾丸和附睾均独立表达BPI基因。在附睾中,自起始段至尾部,BPI蛋白的表达水平递减,并逐步特异性富集于亮细胞的胞质中。在顶体反应前的顶体基质内可见BPI蛋白,应起源于睾丸表达;顶体反应后,可见BPI蛋白分布于整个精子头部质膜表面,尤其是赤道板区域,可能有睾丸或附睾表达的两种起源。我们的研究结果提示,BPI蛋白可能参与顶体反应前后精子质膜结构的调控,并参与后续的精卵融合过程。     

Local differences in Leydig cell morphology in the adult rat testis: evidence for a local control of Leydig cells by adjacent seminiferous tubules

In order to test the hypothesis that Leydig cell function in the adult rat testis is influenced by the surrounding tubules, Leydig cell morphology was compared in different types of interstitial areas. Triangular interstitial areas surrounded by 3 cross-sectioned tubules in nearly the same stage of spermatogenesis were chosen for quantitative light microscopy. It was found that the volume density of Leydig cells in such areas was about 30%, except when the surrounding tubules were in stages IX-X or XI-XII, when it was only about 20%. This variation in total Leydig cell mass seemed to be due to a variation in Leydig cell size and not in Leydig cell number. The largest Leydig cell profile area, 118 pL 6 μm² (mean pL SE n = 6 rats), was observed when the surrounding tubules were in stages VII-VIII, i.e. just prior to sperm release. The smallest Leydig cells were seen when the surrounding tubules were in stages IX-X and XI-XII (68 pL 3 and 66 pL 4 μm²). The present results indicate that there may be a Leydig cell cycle in the adult rat testis, which is regulated by the adjacent tubules.