Development of stage-specific paracrine regulation of Leydig cells by the seminiferous tubules

The size of peritubular Leydig cells surrounding tubules in different stages of the spermatogenic cycle was determined in 43- and 47-day-old male rats. A stage-dependent variation in the size of peritubular Leydig cells was not present in 43-day-old rats, but by 47 days those Leydig cells closely adjacent to tubules at stages VII-VIII were larger than others. At 43 days of age spermatogenesis had developed up to step 18 spermatids in late stage VI tubules. At 47 days of age the first mature sperm had just been released from the seminiferous epithelium, and consequently the first wave of the spermatogenic cycle was completed. Tubules at stages VII-VIII therefore acquire the ability to influence surrounding Leydig cells when they contain step 19 spermatids. It remains to be shown whether this maturation step is due to inherent maturation of the Sertoli cells or if step 19 spermatids specifically modulate Sertoli cell function.     

7-Alpha-hydroxytestosterone in seminiferous tubules of rat testis

Production of 4-androstene-7 alpha,17 beta-diol-3-one (7 alpha-hydroxytestosterone) from testosterone was measured in seminiferous tubule and interstitial fractions of rat testis. In adults, specific activity of the 7 alpha-hydroxylase was about 10 times higher in interstitial cells than in seminiferous tubules, but tubule production was a significant portion of the total. Seminiferous tubule 7 alpha-hydroxylase was not detectable in weanlings. Also, we confirmed that 7 alpha-hydroxytestosterone inhibits the activity of the 5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase active on testosterone and suggest a role in maturational changes.     

Assessment of protein synthesis and secretion by rat seminiferous and epididymal tubules in vivo

In vivo microperifbsion and micropuncture were used to study tubule protein synthesis and proluminal secretion by the male reproductive tract in vivo . Somniferous and caput and cauda epididymal tubules were perihsed for 3 h. with [³⁵S]-methionine. Perifused interstitial fluid (IF), lumen fluid (LF), and tubule extract (TE) were collected. Proteins were separated by SDS-PAGE, and autoradiograms were developed.
Trichloroacetic acid precipitable proteins in each fluid were determined and a protein synthesis index (PSI) was calculated. PSI values demonstrated that the cauda epididymis synthesized less protein in vivo than did either seminiferous or caput tubules.
Seminiferous tubules synthesized and secreted into the tubule lumen a relatively constant panel of proteins. Epididymal tubules synthesized and secreted proteins in a region-specific manner. In the caput epididymis the most prominent secreted bands were consistent with the heavy and light chains of epididymal clusterin. In the cauda epididymis, the most prominent synthesized and secreted protein was a 25 kDa protein consistent with the protein D. The above approach to studying protein synthesis and secretion will allow direct study of the physiological and path physiological effects on this important epithelial hnction in vim     

大鼠曲细精管生殖细胞体外共培养和精子发生过程的观察

目的建立体外长期共培养体系和观察方法,为精子发生过程的研究提供细胞模型。方法采用大鼠曲细精管生殖细胞及支持细胞共培养的方法,对睾丸生精细胞作显微镜观察。结果共培养的支持细胞和生精细胞在体外存活超过6个月。在共培养期间,观察到精母细胞、圆形精子细胞和长形精子细胞。结论在不添加任何细胞因子和生长因子的情况下,大鼠曲细精管生殖细胞长期增生分化,不断产生精子细胞。这一结果暗示组织块和共生的支持细胞可为生殖细胞的增生和分化提供必需的细胞因子。该方法为体外研究精子发生过程提供了实验依据。     

Identification of proteins secreted by human osteoblastic cells in culture.

To better understand the biochemistry of matrix-forming cells, we developed a simple and reproducible procedure for the isolation and identification by N-terminal sequencing of proteins secreted by cells into culture medium and applied this procedure to the analysis of the major Coomassie blue-staining proteins under 100 kD that are secreted from three different human osteoblastic cell cultures. The major proteins secreted by normal human osteoblasts from adult trabecular bone were identified by N-terminal sequencing to be gelatinase, osteonectin, the C-terminal propeptides of the alpha 1 and alpha 2 chains of type I collagen, tissue inhibitor of metalloproteinase 1 (TIMP-1), and beta 2-microglobulin. The amounts of each of these proteins secreted into medium over a 24 h interval did not change over the 7 consecutive days of culture under serum-free conditions, which indicates that this pattern of protein secretion is not significantly affected by the serum-free conditions needed for protein identification by this method. In addition, radioimmunoassay for bone gla protein (BGP), a marker for osteoblast phenotype, revealed that BGP secretion remained high over 7 days of culture under serum-free conditions and was comparable to the rate of BGP secretion in control cultures with 10% serum. The major proteins secreted by MG-63 cells were identified by N-terminal sequencing to be gelatinase, a novel 40 kD human bone protein we termed YKL-40, TIMP-1, the recently discovered TIMP-2, and beta 2-microglobulin. Further studies revealed that YKL-40 is the only protein detectable by Coomassie staining of SDS gels of MG-63 media proteins that is induced by extended time at confluence or by treatment with 1,25-(OH)2D3. The apparent absence of detectable Coomassie-stained bands corresponding to the C-terminal propeptides of collagen in the medium of MG-63 cells suggests that these transformed cells may not be a good model for bone matrix formation. The major proteins secreted by normal fetal osteoblastic cells were identified by N-terminal sequencing to be osteonectin and the C-terminal propeptides of the alpha 1 and alpha 2 chains of type I collagen. Gelatinase and TIMP could not be detected among the conditioned medium proteins by these methods. These observations indicate that fetal osteoblasts primarily express proteins that are matrix constituents and adult human osteoblasts secrete, in addition to these, proteins that could function in matrix turnover.     

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.     

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.     

Effects of lithium carbonate on rat seminiferous tubules: an ultrastructural study

Lithium salts are commonly used for treatment of bipolar disorder but prolonged treatment with therapeutic doses induces substantial toxic effects. In the present study we examined the effects of lithium carbonate on the ultrastructure of rat seminiferous tubules. Rats were exposed to lithium carbonate at doses of 35 mg/kg/day for 21 days. After lithium treatment, the tunica propria widened and folded together with convolutions of the basement membrane, myoid cells and lymphatic endothelium. In the seminiferous epithelium loss of germ cell attachment and appearance of expanded intercellular spaces between spermatogenic cells were observed. Early stages of spermatogenic cells showed nuclear protrusions or swellings because of an extensive enlargement of the outer nuclear membrane. Round spermatids exhibited abnormally shaped acrosomes and dilation of the subacrosomal space. Many abnormal, degenerated late spermatids with random orientation were seen towards the basal and adluminal compartments of the seminiferous epithelium. In addition, spermatids exhibited alteration in F-actin bundle ectoplasmic specialization and contained many mitochondria-associated granular bodies.     

Production of lactate and tissue plasminogen activator in vitro by seminiferous tubules obtained from adult unilaterally cryptorchid rats

Pieces of seminiferous tubules obtained from adult unilaterally cryptorchid rats were incubated in vitro and the effect of FSH on the secretion of lactate and tissue-plasminogen activator (t-PA) was studied. Tubules from abdominal testes secreted less lactate than scrotal tubules. On the other hand, the basal secretion of t-PA was similar but the FSH-stimulated t-PA secretion was larger from abdominal than from scrotal tubules. These results indicate a more specific dysfunction of the Sertoli cells in cryptorchidism than earlier recognized.     

Investigation on the histopathological effects of thyroidectomy on the seminiferous tubules of immature and adult rats

INTRODUCTION: This study aimed to investigate the histopathological effects of thyroidectomy on both immature and adult rat testes. MATERIALS AND METHODS: Male albino Wistar rats, 4 weeks old and weighing between 45 and 55 g, were used for this study. The experimental groups were as follows: 2-week control group (group I); 2-week thyroidectomy group (group II); 4-week control group (group III); 4-week thyroidectomy group (group IV); 6-week control group (group V), and 6-week thyroidectomy group (group VI). The control groups included both sham-operated and untreated rats. In groups II, IV and VI, total thyroidectomy was performed under ether anesthesia in all rats at 4 weeks of age. The rats were killed in the 2nd, 4th and 6th weeks, respectively, following the thyroidectomy. The testes of each animal were evaluated histologically. RESULTS: In group II, spermatogenesis progressed to meiosis but round spermatids were found to be decreased and pachytene spermatocytes were observed to be increased when compared to group I. Giant pachytene spermatocytes were seen. There were also many degenerated cells of intermediate origin in the seminiferous epithelium. In groups IV and VI, spermatogonia and primary spermatocytes were normal in appearance, but there was widespread degeneration of the other spermatogenic cells. In addition, some closed lumina covered by degenerated and dead cells were observed. In group II, the mean outer diameter, luminal diameter and area occupied by seminiferous epithelium decreased by 19.74, 32.18, and 28.12%, respectively. In group IV, these data decreased by 23.9, 16.52, and 48.5%, respectively, and in group VI, by 21.10, 19.76 and 40.29%, respectively, when compared with the control groups. These data were statistically significant (p < 0.001). CONCLUSIONS: Thyroid hormones could have a marked influence on the seminiferous tubules of both immature and adult rats, and their permanent lack results in a depression in seminiferous tubule growth as shown by the reduced outer and luminal diameters and area occupied by the seminiferous epithelium, which could give rise to degenerative changes in the spermatogenic cells of thyroidectomized rats. In addition, all these changes could also result from both the inability of Sertoli cells to support spermatogenic cells and the diminished levels of GH and FSH.     

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.     

Cellular regulation of follicle-stimulating hormone (FSH) binding in rat seminiferous tubules

Stage-specific binding of follicle-stimulating hormone (FSH) was measured in rat seminiferous tubules. The binding in single-point assays was over 3-fold higher (P less than 0.05) in stages XIII to I than in stages VI to VII of the epithelial cycle. No difference was found between the equilibrium association constants (Ka) of FSH binding in stages XIV to IV (10 +/- 1.9 X 10(9) 1/mol) and VII to VIII (9.2 +/- 0.6 X 10(9) 1/mol, mean +/- SEM, n = 5). In another experiment, the testes were dosed locally with 3 Gy of 4 MV x-irradiation to selectively lower the number of spermatogonia. After irradiation, FSH binding in staged seminiferous tubule segments was measured when the desired types of spermatogenic cells were reduced in number. Seven days after irradiation when differentiating spermatogonia and preleptotene spermatocytes were reduced in number, FSH binding was decreased in all stages of the cycle, but the cyclic variation remained. Seventeen days after irradiation when intermediate and type B spermatogonia and spermatocytes up to diplotene of stage XIII showed low numbers, FSH binding was decreased in all stages of the cycle and the stage-dependent variation disappeared. At 38 days when pachytene spermatocytes and early spermatids were reduced in number, similar results were found. But at 52 days postirradiation when all spermatids were low in number, FSH binding was slightly elevated compared with days 17 and 38. There were no significant differences in serum FSH or LH levels between irradiated and non-irradiated animals. These findings suggest that all spermatogenic cell types may stimulate FSH binding in the Sertoli cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Tamoxifen induced multinucleated cells （symplasts） and distortion of seminiferous tubules in rat testis

Aim: To evaluate the effect of tamoxifen citrate on male reproductive system of rat. Methods: Groups of male rats were gavaged with tamoxifen at doses of 200 mg·kg-1·d-1, 400 mg·kg-1·d-1 or 800 mg·kg-1·d-1 in 0.1 mL olive oil for 10 consecutive days. Controls were treated with 0.1 mL olive oil. Rats were anesthetized and killed on d 3, d 15 or d 35 after the last dose. Testes were collected, processed for paraffin embedding, sectioned at 5 μm thickness, stained with H&E and analyzed microscopically. Results: There was a dose-dependent increase in the occurrence of seminiferous tubular distortion with germinal cell sloughing. The highest dose increased the number of multinucleated giant cells on d 3 and d 15. Conclusion: Tamoxifen citrate induces multinucleated giant cells and germinal epithelial sloughing in a dose-dependent manner and these changes are detrimental to male fertility.     

De novo morphogenesis of seminiferous tubules from dissociated immature rat testicular cells in xenografts

Testicular development is initiated with the differentiation of Sertoli cells in the embryonic gonad. The aggregation of Sertoli cells is crucial for the generation of testicular cords and thus for the first sign of male gonadal development. To date, functional testicular tissue has not yet been generated in vitro. The objective of this study was to explore the de novo morphogenesis of testicular tissue from isolated postnatal rat testicular cells using a combination of in vitro culture and ectopic xenografting. Immature rat testicular cells were cultured in either a 2-dimensional (laminin-coated coverglass) or a 3-dimensional (extracellular matrix gel) culture system. Whereas testicular cells cultured on laminin showed a slow morphogenetic cascade resulting in cord formation after about 10 days of culture, cells cultured on extracellular matrix gel assembled to a network of cordlike structures within several hours after plating and formed spherical cell aggregates at day 3. Further progression of the morphogenetic cascade was not obtained in either the 2- or the 3-dimensional culture system. In contrast, structures resembling immature testicular tissue were obtained after xenografting of extracellular matrix gel-enclosed spherical testicular cell aggregates. The grafts were vascularized and contained elongated seminiferous tubules. Histologic analysis revealed the presence of a basement membrane, a histologically normal interstitium containing putative Leydig cells, the establishment of tubule lumen, and the integration of few putative spermatogonia into the seminiferous epithelium. We conclude that immature rat testicular cells carry the full potential to generate all somatic components of a testis in xenografts, thus opening fascinating pathways to study testicular organogenesis.     

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.     

In-vitro contractility of human seminiferous tubules in response to testosterone,dihydrotestosterone and estradiol

Summary The effects of steroids (testosterone, dihydrotestosterone and estradiol) on human seminiferous tubules in vitro were ascertained by recording the intratubular pressure with a servonull micropressure measuring device. We describe here the first response of the human seminiferous tubule to steroids. Testosterone and dihydrotestosterone had a biphasic effect on tubular contractility. Higher doses of both testosterone and dihydrotestosterone induced contractions of the seminiferous tubules whereas lower doses of these compounds induced relaxation. Estradiol (10^-9 M to 10^-6 M) induced relaxation of the seminiferous tubules in a dose-dependent manner. The results from these experiments suggested that steroids may be involved in the control of contraction of the human seminiferous tubule and may regulate the movement of spermatozoa from the tests.