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)     

Effect of nitrofurazone on the reproductive organs in adult male mice

Aim: To study the effect of 5-nitro-2-furaldehyde semicarbazone (nitrofurazone), a derivative of nitrofuran, on male reproductive organs of Parkes (P) strain mice. Methods: Mice were given nitrofurazone orally at a dose of 64mg/kg body weight per day, for 10 and 20 days, and were killed 24 h and/or 56 days after the last treatment. Histological appearance of testis, motility and number of spermatozoa in cauda epididymidis, and biochemical indices in epididymis and seminal vesicle were evaluated. Results: Histologically, testis showed marked regressive changes in the seminiferous tubules in mice treated with nitrofurazone. Ten days after treatment, there was much depletion of germ cells in the seminiferous tubules, and the germinal epithelium was lined mainly with Sertoli cells, spermatogonia, spermatocytes, and a few round spermatids; intraepithelial vacuoles and multinucleated giant cells were also observed in tubules. By 20 days, regressive changes in the seminiferous tubules were further pronounced, and pachytene spennatocytes were the most advanced germ cells noticed in the tubules. In severe cases, the tubules were lined with a thin layer of Sertoli cells and spermatogonia. The treatment also caused marked reductions in the motility and number of spermatozoa in the cauda epididymidis, in weight and the level of fructose in the seminal vesicle, and in sialic acid level in the epididymis. Fifty six days after drug withdrawal, the alterations induced in the reproductive organs returned to control levels. Conclusion: Our results suggest that nitrofurazone treatment in P mice induces marked alterations in the male reproductive organs, and that the alterations are reversible following cessation of treatment.     

Stereological study of the effects of vitamin E on testis structure in rats treated with para-nonylphenol

This study was organized to see whether vitamin E, as a strong antioxidant, could affect the abnormalities of testis structure caused by para-nonylphenol （p-NP） during its development. A total of 32 female Wistar rats after mating were divided into four groups （n = 8）： control, vitamin E （100 mg kg^-1 per day）, p-NP （250 mg kg^-1 per day） and p-NP ＋ vitamin E. The rats were treated from the seventh day of pregnancy till the twenty-first day. After weaning, the male pups were divided into the same groups and were treated orally for 90 days. Finally, the right testis was fixed, processed, stained and studied using stereological methods. The weight and volume of testis, volume of seminiferous tubules and its diameter, thickness of the basement membrane, height of the germinal epithelium, total number of types A and B spermatogonia, spermatocyte, spermatid and Sertoli cells were significantly reduced in p-NP group when compared with other groups. Co-administration of vitamin E and p-NP compensated for the adverse effects of p-NP on the above parameters. In addition, treatment with only vitamin E caused a significant increase in diameter, basement membrane thickness and height of germinal epithelium, number of spermatogonia and spermatocytes. Co-administration of vitamin E with p-NP could prevent the adverse effects ofp-NP on the testis structure during its development.     

Expression of germ cell nuclear factor in mouse germ cells and sperm during postnatal period

Aim: To assess the spatial and temporal expression of germ cell nuclear factor (GCNF) in male mouse germ cells during postnatal development and in sperm before and after capacitation. Methods: The indirect immunofluorescence method with anti-GCNF antiserum was used to investigate the GCNF expression in mice at day 8, 10,14, 17, 20, 28, 35, 70, and 420 after birth and in sperm before and after capacitation. Results: With the proceeding of spermatogenesis, GCNF was first detected in the nuclei of spermatogonia and a few early stage primary spermatocytes at day 8, which was increased gradually at day 10 to 14 inclusive. From day 17 to day 20, the GCNF was concentrated in round spermatids, while both spermatogonia and early stage primary spermatocytes became GCNF negative. From day 28 until day 420, strong GCNF expression was shown in round spermatids and pachytene spermatocytes, while spermatogonia, early primary spermatocytes and elongating spermatids were all GCNF negative.In addition, it was also found that GCNF was localized on the acrosomal cap region of spermatozoa and there was a big change in GCNF expression during capacitation, from 98 % GCNF positive before capacitation to about 20 % positive following capacitation. The localization of GCNF in caput and cauda spermatozoa was similar. Conclusion:GCNF may play important roles in spermatogenesis, capacitation and fertilization. (Asian J Androl 2004 Sep; 6: 217-222)     

Sperm motility inhibitory effect of the benzene chromatographic fraction of the chloroform extract of the seeds of Carica papaya in langur monkey, Presbytis entellus entellus

Aim： To assess the contraceptive efficacy of the benzene chromatographic fraction of the chloroform extract of the seeds of Carica papaya in langur monkeys. Methods： The test substance was given p.o. to five monkeys at 50 mg/kg body weight/day for 360 days. Control animals （n = 3） received olive oil as vehicle. Sperm parameters as per World Health Organization standards, sperm functional tests, morphology of testis and epididymis, haematology, clinical biochemistry, serum testosterone and libido were evaluated. Following completion of 360 days treatment the animals were withdrawn from the treatment and the recovery pattern was assessed by semen analysis and sperm functional tests. Results： Total inhibition of sperm motility was observed following 60 days of treatment that continued until 360 days study period. Sperm count, percent viability and percent normal spermatozoa showed a drastic decline following 30 days of treatment. Sperm morphology showed predominant mid piece abnormalities. Sperm functional tests scored in sterile range. Histology and ultrastructure of testis revealed vacuolization in the Sertoli cells and germ cells. Loss of cytoplasmic organelles was evident in spermatocytes and round spermatids. Histology and ultrastructure of epididymis of treated animals were comparable to those of control animals. Hematological and serum clinical parameters and testosterone levels fluctuated within the control range throughout the study period. Recovery was evident following 60--120 days of treatment withdrawal. Conclusion- The results suggest that the benzene chro- matographic fraction of the chloroform extract of the seeds of Carica papaya shows contraceptive efficacy without adverse toxicity, mediated through inhibition of sperm motility. （Asian JAndro12008 Mar; 10： 298-306）     

Stage-specific localization of transforming growth factor β1 and β3 and their receptors during spermatogenesis in men

Aim: To investigate the stage-specific localization of transforming growth factor (TGF) β1 and β3 during spermatogenesis in adult human testis, Methods: The localization of TGFβ1 and β3 was investigated by immunohistochemical staining method employing specific polyclonal antibodies. Results: Both TGFβ1 and β3 and their receptors were preponderant in the Leydig cells. TGFβ1 could not be detected in the seminiferous tubules. TGFβ3 and TGFβ-Receptor (R) Ⅰ were mainly seen in the elongated spermatids, while TGFβ-RⅡ in the pachytene spermatocytes and weak in the spermatogonia, spermatids and Sertoli cells. Only TGFβ-RⅡ was detected in the Sertoli cells.TGFβ3, TGFβ-RⅠ and TGFβ-RⅡ showed a staining pattern dependent upon the stages of the seminiferous epithelium cycle. Conclusion: TGFβ isoforms and their receptors are present in the somatic and germ cells of the adult humantestis, suggesting their involvement in the regulation of spermatogenesis.     

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)     

Immunolocalization assessment of metastasis-associated protein 1 in human and mouse mature testes and its association with spermatogenesis

Aim： To investigate the stage-specific localization of metastasis-associated protein 1 （MTA1） during spermatogenesis in adult human and mouse testis. Methods： The immunolocalization of MTA1 was studied by immunohistochemistry and Western blot analysis. The distribution pattern of MTA1 in mouse testis was confirmed by using quantitative analysis of purified spermatogenic cells. Results： The specificity of polyclonal antibody was confirmed by Western blot analysis. MTA1 was found expressed in the nucleus of germ cells, except elongate spermatids, and in the cytoplasm of Sertoli cells; Leydig cells did not show any specific reactivity. MTA1 possessed different distribution patterns in the two species： in humans, the most intensive staining was found in the nucleus of round spermatids and of primary spermatocytes while in mice, the most intense MTA 1 staining was in the nucleus of leptotene, zygotene and pachytene spermatocytes. In both species the staining exhibited a cyclic pattern. Conclusion： The present communication initially provides new evidence for the potential role of MTA1 in mature testis. In addition, its distinctive expression in germ cells suggests a regulatory role of the peptide during spermatogenesis.     

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.     

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。     

Postmeiotic modifications of spermatogenic cells are accompanied by inhibition of telomerase activity

We investigated whether testicular telomerase activity is due to telomerase expression in all cells or expression in a limited number of cells. Telomerase activity was assayed in highly purified fractions of spermatogonia cells plus primary spermatocytes, secondary spermatocytes plus round spermatids, secondary spermatocytes plus spermatids plus spermatozoa, round spermatids, or spermatozoa prepared from healthy or cryptorchid animals. Telomerase activity was additionally assayed in testicular tissue of prepubertal animals and animals with Sertoli cell only pathophysiology. Telomerase activity was detected in fractions containing primary spermatocytes and/or secondary spermatocytes and/or spermatids. Fractions enriched in round spermatids were positive for telomerase activity. In contrast, spermatozoa or Sertoli cell fractions were negative for telomerase activity. Using the relative telomerase activity assay and the sensitive quantitative telomerase assay to quantify telomerase activity, we showed that induction of cryptorchidism does not result in quantitative alterations in testicular tissue telomerase activity. In addition, elimination of round spermatids does not lead to significant alterations in testicular tissue telomerase activity. The present results suggest that the male gamete telomerase activity is inhibited during spermiogenesis. Furthermore, it appears that spermatogonia/primary spermatocytes are the main sources of telomerase activity in the testis. Received: 29 October 1997 / Accepted: 20 October 1998     

Immunohistochemical localization of calmodulin

The localization of calmodulin in the normal human testis was studied by the indirect immunoperoxidase method. Three different types of fixative were used: phosphate-buffered formalin, Bouin's solution or Carnoy's solution. Immunoreactivity specific for calmodulin was not detectable in the testis fixed in Carnoy's solution. The specimens fixed in phosphate-buffered formalin and in Bouin's solution were stained. The immunostaining for calmodulin was observed in pachytene spermatocytes, secondary spermatocytes and round spermatids but not in spermatogonia, or in pre-leptotene, leptotene and zygotene spermatocytes, elongated spermatids, spermatozoa or Leydig cells. Sertoli cells were not stained or were stained slightly. Among pachytene spermatocytes, the cells at the early stage were barely stained but those at the middle and late stages were slightly and intensely stained, respectively. The present report provides the first confirmation of the localization of calmodulin in the human testis.     

Balance of apoptosis and proliferation of germ cells related to spermatogenesis in aged men

To clarify whether germ cell apoptosis is related to a decrease of germ cells in the aged testis with impaired spermatogenesis, we investigated the apoptotic rate of each germ cell type. Testicular specimens were obtained by orchiectomy from 36 men with advanced prostate cancer and by testicular biopsy from 21 men with obstructive azoospermia, which served as controls. The terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) technique was used to identify apoptosis. As a marker of cell proliferation activity, the expression of Ki-67 was immunohistochemically evaluated. Expression of Bcl-xl, which regulates apoptosis of germ cells, was also immunohistochemically examined. Histologically, except for spermatogonia, the ratios of primary spermatocytes, round spermatids, and elongated spermatids to Sertoli cells were significantly decreased in aged testes. The apoptotic rate in spermatogonia was significantly lower in aged men than it was in controls (0.11% +/- 0.06% vs 0.34% +/- 0.21%). Expression of Ki-67 in spermatogonia was decreased in aged men (18.6% +/- 6.0%) compared with that of controls (24.9% +/- 3.3%), suggesting that germ cell proliferation diminished with aging. Consequently, the balance of spermatogonial proliferation and apoptosis showed no difference between the two groups. This was believed to be one of reasons why spermatogonial numbers in aged testes was similar to those of controls. The apoptotic rate of primary spermatocytes in aged men was significantly elevated compared with that of controls (0.60% +/- 0.54% vs 0.22% +/-0.12%), resulting in a decrease of the number of primary spermatocytes per Sertoli cell. The expression of Bcl-xl was inversely correlated with the apoptotic rate in primary spermatocytes, suggesting that Bcl-xl may be related to the regulation of primary spermatocyte apoptosis. Based on these findings, we conclude that accelerated apoptosis of primary spermatocytes might account for a part of the mechanism of germ cell loss in aging men.     

The effects of ethylene glycol monomethyl ether on testicular histology in F344 rats

Ethylene glycol monomethyl ether (EGME) has been found to produce testicular atrophy in experimental rodents. The studies that follow were designed to determine the testicular cell type(s) most susceptible to EGME administration. For histologic studies, F344 rats were gavaged with 150 mg/kg/day of EGME 5 days per week, and serially sacrificed. In sections from perfusion-fixed tissue, necrotic changes were observed in some meiotic and premeiotic spermatocytes 24 hours after a single dose. Also, nuclear condensation was seen in occasional early pachytene spermatocytes. These effects were magnified after two doses; there were more necrotic pachytene and meiotic spermatocytes than necrotic stage I pachytene spermatocytes. By day 4, testes from all treated animals were affected; there was a pronounced maturation-depletion effect, seen as the absence of round spermatids from tubules in stages I to III. These effects continued to develop at days 7 and 10, leaving only Sertoli cells, spermatogonia, and late stage spermatids populating the epithelium. Other animals were treated similarly, but subject to efferent duct ligation 16 hours prior to sacrifice. Fluid production, as judged by weight gain in the testes after efferent duct ligation, was unaffected by EGME treatment. Analysis of the fluid collected at the rete testis indicated that there was no treatment-related change in the relative amounts of androgen binding protein. The data indicate that the spermatocyte is the primary target cell for the histologic effects of EGME in the testis of F344 rats.     

Chalone or inhibine: control of progenitor spermatogonia in the adult rat by gonadal protein extracts

Adult male rats received one injection (J0) of busulfan (10 mg/kg BW), which is specifically lethal for type A spermatogonia (Jackson and al., 1962). They were subsequently injected daily (J1 - J10) either with a proteinaceous extract of adult ram testis (10 mg of proteins/day) via the subcutaneous route, or with ram testicular fluid (1, 2 mg of protein/day), or with bovine serum albumin (1 mg/day) in order to analyse the resultant modification of the repopulation of seminiferous epithelium by the reserve stem cells (Clermont and Mauger, 1974). At the end of the treatments (J11), the number of type A spermatogonia per tubular cross section was significantly decreased in animals receiving testicular extract of fluid. The number of primary spermatocytes, from preleptotene up to zygotene stages were only diminished by testicular fluid supplementation. Ten days later (J20) the populations of primary spermatocytes, from preleptotene to pachytene stages derived from cells which were type A spermatogonia at the time of injections, were depleted. Type A spermatogonia were only decreased by testicular fluid treatment. Testicular fluid or extracts did not modify body weight nor the weights of other organs. A protein-like factor originating from the testis and present in the testicular fluid can thus modify the population of type A spermatogonia. Its action, however, is not specific for germ cells as Sertoli cells are affected by the administration of testicular extract in prepuberal rats (Hochereau-De-Reviers and Courot, 1978).     

Protection of spermatogenesis against cytotoxic effects of two chemotherapeutic drugs by temporary testicular blood flow interruption in the ram

Summary. Temporary interruption of the testicular blood flow for 1 h after injection of cytostatic drugs has a protective effect on spermatogenesis. This was shown in experiments in which spermatogenesis was evaluated at four weeks after a single intravenous injection of Adriblastina^R (ADR; doxorubicine hydrochloride) or Mitomycin-C-kyowa^R (MIT). Interruption of the blood flow was performed by inflation of an occluder implanted around the testicular artery.
The animals were killed and histological sections prepared 4 weeks after treatment. In all drug-treated animals spermatids were near absence and spermatocytes were decreased in number. Therefore, even after occlusion of the blood flow, the drug doses were high enough to kill not only large numbers of differentiating spermatogonia but also stem cells.
The response of the stem cells to the treatments was evaluated by counting the numbers of A spermatogonia per 100 Sertoli cells in the different groups. Normal numbers of these cells were found after both MIT and ADR, indicating that the stem cell population had responded to the initial cell loss by extra proliferation. However, significantly higher numbers of A spermatogonia were found in the drug-treated animals in which the testicular blood flow was interrupted for 1 h. This indicates that occlusion of the blood flow to the testis for 1 h results in a faster recovery of spermatogenesis than after drug treatment alone.