Dazl protein expression in adult rat testis is up-regulated at meiosis and not hormonally regulated

The Y-chromosomal DAZ (deleted in azoospermia) gene and the autosomal Dazl (deleted in azoospermia-like) gene are two crucial factors for the achievement and maintenance of spermatogenesis. Whereas Y-chromosomal DAZ is present in certain primates, it is lacking in rodents and other species. We have investigated the expression of Dazl protein during spermatogenesis in the adult rat testis using immunohistochemistry. Dazl immunoreactivity was found predominantly in the cytosol of primary pachytene spermatocytes. A weaker but clearly detectable signal was present in intermediate and B spermatogonia and in early spermatocytes from preleptotene to zygotene. The highest expression patterns were observed between stages IV and VIII during the spermatogenic cycle when spermatocytes prepare for the first meiotic division. Specific staining could also be observed in step 11-19 elongating spermatids in the acrosome region. Treatment for 42 days with a potent GnRH-antagonist abolished gonadotrophin secretion and led to a regressed testis, lacking most of the advanced germ cell types such as spermatids but still bearing spermatogonia and spermatocytes. No difference in staining pattern for Dazl protein was observed in GnRH antagonist-treated rats despite the lack of gonadotrophins and substantial impairment of the spermatogenic process, indicating that Dazl expression is clearly hormone-independent. The localization and level of Dazl expression suggests an important role in the regulation of the first meiotic stages of spermatogenesis. The hormone independent onset of expression points to an autonomous cell-cycle event in which Dazl seems to be essential for the entry into meiosis. The presence of Dazl in the acrosome region of elongating spermatids might reflect an unknown role of Dazl as a morphogenetic factor during spermiogenesis.     

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)     

Quantitative （stereological） study of incomplete spermatogenic suppression induced by testosterone undecanoate injection in rats

Aim: To evaluate the key lesions in spermatogenesis suppressed partially by testosterone undecanoate(TU) treatment. Methods: Adult male SD rats were treated with vehicle or TU (19 mg/kg) injection (i.m.) every 15 days for 130 days. The numbers of all types of cells (nuclei) in the seminiferous tubules and the interstitial tissue were estimated using a contemporary stereological tool, the optical disector. Results: In response to TU treatment, the numbers of non-type B spermatogonia, type B sperrnatogonia and late elongated spermatids per testis were reduced to 51%, 66% and 14% of the controls, respectively. The conversion ratios from type B spermatogonia to early spermatocytes and pachytene spermatocytes were not significantly affected and the ratios to the later germ cell types fell to 51%-65% of the controls. Less than 1.0 % of immature round spermatids were seen sloughing into the tubule lumen, 4.0% of elongated spermatids retained in the seminiferous epithelium, and about half of the elongated spermatid nuclei appreciably malformed. Leydig cells were atrophied but their number and the peritubular myoid cell number per testis were unchanged. Conclusion: Double inhibition of spermatogenesis (i.e. inhibition at spermiation and spermatogonial conversion to type B spermatogonia), a scenario seen in the monkey and human following gonadotrophin withdrawal, was not sufficiently effective for a complete spermatogenic suppression in the rat after TU treatment, probably due to ineffective inhibition of the Leydig cell population and therefore the intra-testicular test-osterone levels. (Asian J Androl 2004 Dec; 6: 291-297)     

Localisation of RA175 (Cadm1), a cell adhesion molecule of the immunoglobulin superfamily, in the mouse testis, and analysis of male infertility in the RA175-deficient mouse

RA175, a member of the immunoglobulin superfamily, plays an important role in cell adhesion, and RA175 gene-deficient mice (RA175(-/-) ) show oligoastheno-teratozoospermia. To understand the function of RA175, location in the testis and the morphological features of its spermatogenic cells in RA175(-/-) mice were investigated. Immunohistochemical studies revealed that RA175 immunoreactivity was observed on the cell surface of the spermatogenic cells at specific stages. A strong reaction was detected from type A spermatogonia to pachytene spermatocytes at stage IV and from step 6 to step 16 spermatids during spermatogenesis. From pachytene spermatocytes at stage VI to step 4 spermatids, the reaction was not detected by the enzyme-labelled antibody method and was faintly detected by the indirect immunofluorescence method. Abnormal vacuoles in the seminiferous epithelium, showing exfoliation of germ cells, and ultrastructural abnormality of the elongate spermatids were revealed in the RA175(-/-) testes. Other members of the immunoglobulin superfamily such as basigin, nectin-2 and nectin-3, which have an important role in spermatogenesis, were immunohistochemically detected in the RA175(-/-) testis. These observations indicate a unique expression pattern of RA175 in the testis and provide clues regarding the mechanism of male infertility in the testis.     

Expression and phosphorylation of mitogen-activated protein kinases during spermatogenesis and epididymal sperm maturation in mice.

The expression and phosphorylation/dephosphorylation of mitogen-activated protein (MAP) kinases during mouse spermatogenesis and epididymal sperm maturation have been investigated by immunoblotting and immunohistochemical staining with commercially available anti-ERK2 and anti-Active MAPK antibodies. Two forms of MAP kinases, p42ERK2 and p44ERK1, were expressed in a similar amount in spermatogenic cells at different stages. ERK1 and ERK2 were phosphorylated (activated) in early spermatogenic cells from primitive spermatogonia to zygotene primary spermatocytes, while only a small quantity of phosphorylated MAP kinases could be detected in pachytene primary spermatocytes and spermatids. MAP kinase activity in primative spermatogonia and preleptotene primary spermatocytes was the highest among spermatogenic cells. ERK1 and ERK2 were also present in epididymal spermatozoa, and their phosphorylation was increased while spermatozoa pass through epididymis and vas deferens for maturation. It would appear that MAP kinase activation may contribute to the mitotic proliferation of primative spermatogonia, an early phase of spermatogenic meiosis, and, later, sperm motility acquirement.     

二甲磺酸乙烷致间质细胞破坏所引起的成年大鼠睾丸和附睾的组织学变化:形态定量研究

目的:定量研究睾酮分泌剧烈减少所致睾丸和附睾的组织学变化。方法:14只成年 SD 大鼠腹腔内注射二甲磺酸乙烷(EDS,75mg/kg),14只注射生理盐水作为对照。7天后处死各组中的一半动物,过5天后处死另一半。取睾丸和附睾组织块,甲基丙烯酸树脂包埋。用体视学的光学体视框技术估计睾丸内的细胞数,并用其它形态定量研究方法获取另外一些参数。结果:EDS 注射使睾丸内的间质细胞几乎完全消失,但对支持细胞总数没有影响。EDS 注射7天后,生精上皮内可见许多长形精子细胞滞留,附睾管内可见许多圆形精子细胞。EDS 注射12天后,精子细胞和精母细胞的排列明显变疏松,生精细胞之间出现明显的裂隙,裂隙近似放射状朝向生精小管腔;睾丸内的非 B 型精原细胞总数和精母细胞总数与对照组相似,但 B 型精原细胞总数增加59%,而早期(圆形)、中期和晚期(长形)精子细胞总数分别减少37%、72%和52%。结论:EDS 所致精子发生损害主要是(1)精子释放障碍,(2)精子细胞、精母细胞分离并伴有精子形成和成熟分裂障碍。     

The length of the spermatogenic cycle is conserved in porcine and ovine testis xenografts

Xenografting of immature mammalian testis tissue into mice can accelerate sperm production. To determine whether this shortened time to sperm production is because of reduced length of the spermatogenic cycle, we applied bromodeoxyuridine (BrdU) incorporation to analyze the spermatogenic cycle in porcine and ovine testis xenografts. Small testis fragments from newborn pigs and sheep were ectopically grafted into mice. Once complete spermatogenesis was present in grafted tissue, mice were injected with BrdU and grafts were recovered at different time points thereafter. In porcine grafts, the most advanced germ cells labeled 1 hour, 9 days, 12.3 days, and 18 days after BrdU injection were stage 1 preleptotene/leptotene primary spermatocytes, stage 1 pachytene primary spermatocytes, stage 5 newly-formed round spermatids, and late stage 2 elongating spermatids, respectively. In ovine grafts, the most advanced labeled germ cells at 1 hour, 11 days, and 22 days post-BrdU injection were stage 2 preleptotene/leptotene primary spermatocytes, late stage 1 pachytene primary spermatocytes, and stage 2 elongating spermatids, respectively. These results indicate that each spermatogenic cycle in porcine and ovine xenografts lasts approximately 9 and 11 days, respectively, which is similar to their durations in situ. Therefore, the length of the spermatogenic cycle is conserved in porcine and ovine testis xenografts. This is consistent with earlier reports showing that the cycle length is inherent to the germ cell genotype. The shortened time to sperm production in xenografts therefore appears attributable to accelerated maturation of the testicular somatic compartments. Our results suggest that testis xenografts provide a useful model to study the timing of testicular maturation and spermatogenesis in different mammalian species.     

Histological changes of the testis and epididymis in adult rats as a result of Leydig cell destruction after ethane dimethane sulfonate treatment： a morphometric study

Aim： To quantitatively study the histological changes of the testis and epididymis as a result of a drastic reduction of testosterone secretion. Methods： Fourteen adult Sprague-Dawley rats were injected intraperitoneally with ethane dimethane sulfonate （EDS, 75 mg/kg） and the same number of animals were injected with normal saline as a control. At days 7 and 12 （after treatment）, respectively, half of the animals from each group were killed. The testes and epididymides were removed and tissue blocks embedded in methacrylate resin. The cell number per testis was estimated using the stereological optical disector and some other parameters were obtained using other morphometric methods. Results： The EDS treatment resulted in an almost complete elimination of Leydig cells but had no effect on the numbers of Sertoli cells per testis. At day 7 after EDS treatment, many elongated spermatids were retained in the seminiferous epithelium and many round spermatids could be seen in the epididymal ducts. At day 12, a looser arrangement of spermatids and spermatocytes became evident, with apparent narrow empty spaces being formed between germ cells in an approximately radial direction towards the tubule lumen; the numbers （per testis） of non-type B spermatogonia and spermatocytes were similar to controls, whereas that of type B spermatogonia increased by 59%, and that of early round, elongating and late elongated spermatids decreased by 37%, 72% and 52%, respectively. Conclusion： The primary spermatogenic lesions following EDS administration were （i） spermiation failure and （ii） detachment of spermatids and spermatocytes associated with impairment in spermiogenesis and meiosis.     

The spermatogenic cycle in the blue fox (Alopex lagopus): relative frequency and absolute duration of the different stages

The spermatogenic cycle of the blue fox was divided into eight distinct stages, based on an analysis of different cell associations of the seminiferous epithelium. The criteria used for classification of the stages were the type of spermatogonia, the occurrence of meiotic figures, and the shape and location of spermatids. The relative frequencies of the stages I to VIII were 25.7, 9.8, 8.7, 5.9, 13.8, 9.9, 10.6 and 15.5%, respectively. The duration of one cycle of the seminiferous epithelium was 12.0 +/- 0.2 days as determined from the progression of 5-bromo-2-deoxyuridine (BrdU)-labelled cells at various time intervals. The absolute duration of stages I to VIII was calculated to be 3.1, 1.2, 1.0, 0.6, 1.7, 1.2, 1.3 and 1.9 days, respectively. The estimated life span of primary spermatocytes was 19.2 days, of secondary spermatocytes less than 0.6 days, of spermatids with round nuclei 9.2 days and of spermatids with elongated nuclei 8.9 days.     

Regulatory influence of germ cells on sertoli cell function in the pre-pubertal rat after acute irradiation of the testis

While germ cell regulation of Sertoli cells has been extensively explored in adult rats in vivo, in contrast, very little is known about germ cell influence on Sertoli cell function at the time when spermatogenesis begins and develops. In the present study various Sertoli cell parameters (number, testicular androgen binding protein (ABP) and testin, serum inhibin-B and, indirectly, follicle-stimulating hormone (FSH)) were investigated after the exposure of 19-day-old rats to a low dose of 3 Grays of gamma-rays. Differentiated spermatogonia were the primary testicular targets of the gamma-rays, which resulted in progressive maturation depletion, sequentially and reversibly affecting all germ cell classes. Testicular weight declined to a nadir when pachytene spermatocytes and spermatids were depleted from the seminiferous epithelium and complete or near complete recovery of spermatogenesis and testicular weight was observed at the end of the experiment. Blood levels of FSH and ABP were normal during the first 11 days after irradiation, when spermatogonia and early spermatocytes were depleted. While the number of Sertoli cells was not significantly affected by the irradiation, from days 11-66 after gamma-irradiation, ABP production declined and FSH levels increased when pachytene spermatocytes and spermatids were depleted and the recovery of these parameters was only observed when spermatogenesis was fully restored. Comparison of the pattern of change in serum levels of inhibin-B and testicular levels of testin and of germ cell numbers strongly suggest a relationship between the disappearance of spermatocytes and spermatids from the seminiferous epithelium and the decrease in levels of inhibin-B and increase in levels of testin from 7 to 36 days post-irradiation. Levels of testin and inhibin-B were restored before spermatogenesis had totally returned to normal. In conclusion, this in vivo study shows that pre-pubertal Sertoli cell function is under the complex control of various germ cell classes. This control presents clear differences when compared with that previously observed in adult animals and depends on the Sertoli cell parameter of interest, as well as on the germ cell type.     

Antifertility effect of ethanolic extract of Amalakyadi churna in male albino mice

Aim: To evaluate the antifertility activity of the ethanolic extract of Amalakyadi churna by oral administra-tion in male albino mice. Methods: The ethanol extract of Amalakyadi churna at the dose of 250 mg/kg and 400 mg/kg body weight was administered orally for 30 days to adult male mice. On day 31, the mice were sacrificed and the testis and accessory reproductive organs were removed and weighed. The organs were processed for biochemical estimation and histological work. Results: Treatment with Amalakyadi chuma resulted in decrease in the weights of testis and accessory reproductive organs. The diameters of testis, seminiferous tubules and Leydig cell nucleus were decreased. The spermatogenic elements, like spermatogonia, spermatocytes and spermatids in the testis were re-duced significantly as well as the sperm count in cauda epididymis. There was a significant reduction in the protein,glycogen, DNA and RNA contents and the activity of acid phosphatase in the testis of extract treated mice compared with the control. The cholesterol content and the alkaline phophatase activity were increased significantly in treated mice. Conclusion: Amalakyadi churna extract arrests spermatogenesis in male mice without noticeable side effects.(Asian J Andro12003 Sep; 5: 247-250)     

精子发生阻滞不育患者睾丸雄激素受体和热休克蛋白90α的表达

目的 :探讨精子发生阻滞与雄激素受体 (AR)和热休克蛋白 90α(HSP90α)表达的关系。　方法 :应用免疫组化二步法 ,检查 5 7例精子发生阻滞引起的不育患者睾丸活检标本AR和HSP90α的表达 ,并以 15例正常健康人睾丸组织作为对照。　结果 :在正常健康人睾丸组织中 ,AR在精原细胞、精母细胞、圆形精子细胞、支持细胞、间质细胞和肌样细胞的胞核均有表达 ,但各类细胞表达AR的强度有差异。在精子发生阻滞的睾丸组织中 ,AR高表达主要在阻滞细胞水平的胞质 ,即在核周形成一环行特异性免疫反应阳性产物带。HSP90α在正常睾丸组织精原细胞、精母细胞、支持细胞、间质细胞和肌样细胞的胞质表达 ;在精子发生阻滞的睾丸组织中 ,HSP90α为高表达 ,正常睾丸组织和精子发生阻滞的睾丸组织 ,其表达强度存在显著性差异 (P <0 .0 5 )。　结论 :AR表达部位异常可能无法介导雄激素进入核内 ,而不能实现对基因转录或翻译的调节 ,致使生精细胞难以进入细胞周期而呈现阻滞。HSP90α的高表达可能加剧AR稳定性降低 ,进而增强了AR的遍在化反应。     

Construction and preliminary characterization of a series of mouse and rat testis cDNA libraries.

We have constructed a series of 23 cDNA libraries from mouse and rat testicular cells. These include libraries made from whole, intact adult testes; seminiferous tubule cells from adult testes; combined populations of primary spermatocytes from 18-day-old mouse testes; and isolated populations of primitive type A spermatogonia, type A spermatogonia, type B spermatogonia, preleptotene spermatocytes, leptotene plus zygotene spermatocytes, juvenile pachytene spermatocytes, adult pachytene spermatocytes, round spermatids, Sertoli cells from 6-, 8-, 17-, and 18-20-day-old mice, and peritubular cells from 18-20 day old mice, all recovered from outbred white Swiss (CD-1) mice. We also constructed libraries from whole adult testes from five other lines of mice: C57 Bl6/J, C3 HEB, BDF-1, Balb/c, and 129 Sv. Finally, there are two libraries made from populations of Sertoli cells and peritubular cells isolated from testes of 20-day-old Sprague-Dawley rats. Enzymatic dissociation, followed by gradient separation or plating/lysing techniques, was used to prepare populations of specific cell types in purities of 85-98%. cDNAs were synthesized from poly A+ mRNA primed with oligo dT and unidirectionally cloned into the lambda Uni-Zap XR expression vector from Stratagene. Primary titers ranged from 2.1 x 10(5) to 2.9 x 10(8) plaque-forming units, and insert sizes averaged 1.0-1.2 kb. These libraries have been amplified once and submitted to the American Type Culture Collection (ATCC) for distribution to interested investigators. ATCC accession numbers are provided.     

Effects of levonorgestrel butanoate alone and in combination with testosterone buciclate on spermatogenesis in the bonnet monkey

The spermatogenic effects of levonorgestrel butanoate were studied in adult male bonnet monkeys when administered alone and in combination with testosterone buciclate. Levonorgestrel butanoate (0.25, 1.0 and 2.5 mg kg(-1)) given as two injections on days 0 and 60 (groups II, III, IV) resulted in thickening and folding of the basement membrane and disruption of cell associations in groups III and IV (on day 120). In group II, no apparent changes in testicular histology were observed. When these doses of levonorgestrel butanoate were combined with 40 mg of testosterone buciclate (groups V, VI, VII), maximum changes were seen in group VI in which all stages of spermatogenesis were absent on day 120 except for a small number of spermatogonia. The changes caused by lower dose (group V) and higher dose (group VII) of levonorgestrel butanoate were less prominent than in group VI. A significant decrease in the number of dark A (Ad) and B spermatogonia was observed in all groups except for Ad spermatogonia on day 120 in group V, B spermatogonia on day 60 in group IV and B spermatogonia on day 120 in group III. A significant decrease in pachytene spermatocytes was seen on day 120 in groups V only. Early spermatids showed a significant decrease only in groups V and VII on day 120 of treatment. Advanced spermatids were suppressed significantly in group IV on day 60 and in groups IV and V on day 120. These data indicate that levonorgestrel butanoate (1.0 mg kg(-1)) in combination with 40 mg of testosterone buciclate was the most effective treatment in suppressing spermatogenesis. The site of action of this combination regimen is at the level of renewing Ad spermatogonia.     

Expression of p57kip2 in germ cells and Leydig cells in human testis

p57kip2, a KIP family cyclin-dependent kinase (Cdk) inhibitor, blocks the cell cycle by acting on multiple cyclin-Cdk complexes. To investigate the role of p57kip2 in human fertility, the expression of p57kip2 was investigated in testes from normal and obstructive azoospermic male patients who were positive for p57kip2 mRNA. In the seminiferous tubule, strong immunoreactivity of p57kip2 was found in nuclei of early spermatocytes, but not in the spermatogonia. The p57kip2 immunoreactivity in spermatocytes was markedly heterogeneous. Preleptotene spermatocytes showed strong p57kip2 immunoreactivity, but no visible signal was found in late pachytene spermatocytes. Nuclei of the elongating spermatids was also positive for p57kip2 immunoreactivity. Taken together, this suggests that p57kip2 may play a role in the regulation of meiotic progression of early spermatocytes and cell cycle arrest and differentiation of spermatids. p57kip2 immunoreactivity was found in the perinuclear region of the peritubular cells, but not in the Sertoli cells. In Leydig cells, moderate immunoreactivity of p57kip2 was largely found in the cytoplasm, suggesting the noble function of p57kip2 in the differentiation of adult Leydig cells.     

A stereological study of the effects of experimental inguinal cryptorchidism and subsequent orchiopexy on spermatogenesis in adult monkeys

Our previous study demonstrated that experimental intra-abdominal cryptorchidism in adult rabbits for 13 weeks resulted in severe spermatogenic arrest: type A spermatogonia was the only germ cell type seen in the seminiferous epithelium and its number per testis was reduced by 84%. Seven weeks following orchiopexy, the type A spermatogonial number returned to the near-normal range in most animals and spermatogenesis partially recovered (Reproduction 2002, 124, 95-105). This study aimed to determine whether inguinal cryptorchidism would produce less-severe damage to spermatogenesis and whether subsequent orchiopexy would better restore spermatogenesis. Five normal adult male rhesus monkeys (Macaca mulatta) underwent bilateral artificial inguinal cryptorchidism. Half a year later, one testis together with the ipsilateral epididymis were removed from each animal and then unilateral orchiopexy was performed on the contralateral side, with the remaining testis and epididymis being removed another half a year later. A contemporary unbiased and efficient stereological tool, the optical disector, was used to estimate numbers of all types of spermatogenic cells in the testis and spermatozoa in the epididymis. Spermatogenic arrest was induced by cryptorchidism at the stage of spermatogonia (n = 1), spermatocytes (n = 2) or early spermatids (n = 1), with the type A spermatogonial numbers per testis being reduced to 14.8-57.2% of the control average; in one of the five cryptorchid animals, however, spermatogenesis remained normal. Subsequent orchiopexy, which was successfully performed on two animals with cryptorchidism-induced spermatogenic arrest, brought on a full or partial recovery of spermatogenesis. In conclusion, inguinal cryptorchidism induces less severe (in comparison with an intra-abdominal one) and variable damage to spermatogenesis, which is restored, at least in part, by subsequent orchiopexy.     

Screening the stage-specific expression gene from different germ cells of rat

<正> Objective:To screen the stage-specific expression genes from rat spermatogonia,pachytene spermatocytes and round spermatids.Methods:Highly purified spermatogonia were isolated from 9-day-old rats,pachytene spermatocytes and round spermatids from adult rats by sedimentation velocityat unit gravity,using 2%-4% BSA gradient in DMEM/F12 medium.A mRNA differen-tial display method was used for screening the stage-specific expression gene.Results:Nineteen differentially/ expressed cDNA fragments were obtained.Afterexcluding the false positive cDNA fragments by dot blot,13 cDNAs were selected toclone and sequence.To obtain longer cDNAs,six ESTs were used to screen the rattestis λ-zap Ⅱ cDNA library.Two longer cDNA fragments,designated as LY21 andLM66,were obtained.The analysis with DNAMAN software indicated that LY21 had along open reading frame coding 372 amino acids while LM66 had no long open readingframe.LY21 were highly homologous with hnRNP H1.To observe the expression pat-terns of LY21 gene in the testicular cells,we performed in situ hybridization on testissections from adult rats.The LY21 gene expression was found in the spermatogonia andprimary spermatocytes.Conclusion:This study indicated that LY21 gene was associated with spermatogen-esis.Further studies will be needed to explore the function of LY21.     

成年大鼠睾丸Smad1和Smad5的免疫组织化学研究

目的 :探讨骨形态形成蛋白的细胞内信号传导分子Smad1和Smad5蛋白在成年大鼠睾丸内的表达。　方法 :应用免疫组织化学ABC法结合葡萄糖氧化酶 DAB 硫酸镍铵增强技术 ,检测成年大鼠睾丸Smad1和Smad5蛋白的表达和定位。　结果 :Smad1蛋白表达于大鼠睾丸精曲小管的各级生精细胞的胞质内 ,呈淡染的紫蓝色颗粒 ,胞核为阴性 ,免疫染色阳性细胞主要包括精原细胞、初级精母细胞、次级精母细胞和精子细胞 ;而Smad5蛋白阳性染色不明显 ,只有睾丸间质细胞呈弱阳性反应 ,免疫反应阳性物质主要定位于细胞质。　结论 :Smad1蛋白主要表达于睾丸精曲小管各级生精细胞 ,Smad5蛋白表达于间质细胞 ,为揭示TGF β超家族在精子发生中的分子机理提供了直接证据。     

Possible role of elongated spermatids in control of stage-dependent changes in the diameter of the lumen of the rat seminiferous tubule

Adult male rats were treated with a single dose of 650 mg/kg methoxy acetic acid to deplete the seminiferous tubules specifically of pachytene and later spermatocytes. The effect of this treatment and the subsequent maturation-depletion of later germ cell types on the diameter of the seminiferous tubule and its lumen and the area of the seminiferous epithelium were studied in relation to the stages of the spermatogenic cycle. At 21 days after methoxy acetic acid treatment, the diameter of the tubule and the area of the epithelium were reduced below control values at all stages, consistent with the reduced number of early (stage VIII) or late (all other stages) spermatids. Unexpectedly, diameter of the lumen was also reduced at all stages other than VIII, and especially at stage VII. In controls, lumen diameter at stages VII and VIII was increased by approximately 50% compared with earlier and later stages. In rats treated 21 days previously with methoxy acetic acid no change occurred at stage VII (lacking elongated spermatids) while a normal increase did occur at stage VIII (lacking round but not elongated spermatids). At earlier times after methoxy acetic acid treatment when stage VII tubules were depleted of pachytene spermatocytes alone (3 days) or together with early spermatids (7 days), the diameter of the lumen was not significantly different from the control value. These data suggest that lumen diameter may be regulated by elongated spermatids, especially at stages VII and VIII.     

The effect of selective destruction and regeneration of rat Leydig cells on the intratesticular distribution of testosterone and morphology of the seminiferous epithelium

This study was designed to explore the relationship between the intratesticular distribution of testosterone and spermatogenesis by completely destroying the Leydig cells of mature male rats with injection of a single i.p. dose of ethane dimethanesulphonate. After such treatment, testosterone levels in serum, testicular interstitial fluid, seminiferous tubules, and whole testis declined significantly 6 to 24 hours after injection and fell below assay detection limits between 3 and 7 days. At 3 and 7 days, serum LH and FSH levels rose significantly and remained elevated up to 4 and 6 weeks, respectively, in comparison with vehicle-treated controls. Leydig cells disappeared from the interstitium by day 3, but between 2 and 4 weeks postinjection a new generation of fetal-like Leydig cells repopulated the testicular interstitium and, during weeks 6 to 10, were transformed into, or replaced by, Leydig cells with an adult type of morphology. Histologic examination of the seminiferous tubules showed progressive disruption of spermatogenesis between 3 and 14 days post-ethane dimethanesulphonate. The first histologic sign of spermatogenic damage was noted at day 3, with the occurrence of stage-specific degenerating pachytene primary spermatocytes at stages VII to VIII of the spermatogenic cycle. On day 7, these cells and degenerating round, or step 19, spermatids often were observed during stages VII to XI, although qualitatively normal spermatogenesis also was seen in these and all other stages of the cycle. Maximum impairment of spermatogenesis occurred 2 weeks post-ethane dimethane sulphonate, at which time the tubules commonly lacked one or more germ cell generations or, alternatively, showed accumulation of lipid inclusions, extracellular spaces, and variable numbers of degenerating germ cells. Following repopulation of the testis by Leydig cells during weeks 3 and 4, spermatogenesis recovered. By 10 weeks after treatment, qualitatively normal spermatogenesis was seen in the great majority of seminiferous tubules, although a few tubules still remained in which the germ cell complement was severely reduced, and contained only Sertoli cells and spermatogonia.(ABSTRACT TRUNCATED AT 400 WORDS)