睾丸精曲小管精子发生功能量化评价法分析无精子症患者睾丸生精功能

目的 :分析无精子症患者临床和病理资料 ,研究病理学量化评价睾丸精曲小管精子发生功能的方法的临床意义。　方法 :无精子症患者 112例 ,年龄 2 2～ 4 6 (2 9.0± 4 .4 )岁 ,婚龄 2～ 12 (4 .0± 2 .8)年、病程 2～ 6 (2 .70±1.0 2 )年 ,其中原发性无精子症 96例 ,继发性无精子症 16例 ;梗阻性无精子症 7例。不育症患者精液常规检查 3次确认无精子症 ,检测性激素水平 ,常规消毒下睾丸活检病理检查 ,在高倍镜下计数每个精曲小管中各类生精细胞数 ,测定小管直径、生精上皮高度和固有层厚度 ,按制定的精曲小管精子发生功能 10分 5级分度法加以评分 ,进行统计学分析。　结果 :精曲小管生精上皮 10分分度法评分结果 ,1分 5例 (4 .5 % ) ,2分 38例 (33.9% ) ,3分 2例(1.8% ) ,4分 6例 (5 .4 % ) ,5分 2例 (1.8% ) ,6分 17例 (15 .2 % ) ,7分 6例 (5 .4 % ) ,8分 19例 (17% ) ,9分 10例(8.9% ) ,10分 7例 (6 .3% )。精曲小管精子发生功能 5级分度法结果 ,1级 5例 (4 .5 % ) ,2级 38例 (33.9% ) ,3级 33例 (2 9.5 % ) ,4级 2 9例 (2 5 .9% ) ,5级 7例 (6 .3% )。多元回归分析结果 ,精曲小管精子发生功能分级与生精上皮高度、固有层厚度、精曲小管直径和血清卵泡刺激素 (FSH)具有极显著相关性 (P <0 .0 1)。组合     

Immunohistochemical expression of cyclin A in testicular biopsies of fertile and infertile men: correlation with the morphometry of seminiferous tubules

Cyclin A is a member of the cyclin family of proteins, which are required for both the mitotic and meiotic divisions that characterise spermatogenesis in human and other mammalian species. The data on cyclin A expression in various human spermatogenic disorders and its relationship to the morphology of seminiferous tubules are not well clarified. This study aimed to evaluate the immunohistochemical expression of cyclin A in testicular biopsies of different spermatogenic disorders correlating with the morphology of seminiferous tubules using morphometry tools. Immunohistochemical evaluation of cyclin A was carried out on testicular biopsies obtained from 48 infertile males (nonobstructive azoospermia) and 15 normal subjects together with using semiautomatic morphometric analysis for evaluation of seminiferous tubules. Cyclin A is expressed in 100% of normal and hypospermatogenesis groups and in 80% of maturation arrest group, with complete absence in Sertoli cell only group. In positive cases, cyclin A stained the nuclei of spermatogonia and primary spermatocytes with a higher intensity of expression in normal cases compared with infertile group. Cyclin A expression was significantly associated with the different examined morphometric parameters. Cyclin A is involved in both mitosis and meiosis of human spermatogenesis as it is expressed in spermatogonia and primary spermatocytes. Morphometry of human testis is intimately correlated with the testicular histopathology.     

Immunohistochemical localization of calmodulin in the testes of patients with idiopathic male infertility

The localization of calmodulin in testes of patients with idiopathic male infertility was studied using the indirect immunoperoxidase method. Specimens were obtained by testicular biopsy from 55 patients. They were divided into 26 cases of hypospermatogenesis, 11 cases of maturation arrest (8 of primary spermatocyte arrest and 3 of spermatid arrest) and 18 cases of Sertoli cell-only syndrome. Regardless of the type of testicular pathology, the types of immuno-reactive cell and the intensities of staining were the same as those in the normal testis. That is, staining for calmodulin was first found to be positive in early pachytene primary spermatocytes. It became intense in late pachytene primary spermatocytes and round spermatids. By contrast, elongated spermatids and spermatozoa were not stained. Sertoli cells were stained slightly or not at all. A calmodulin-staining index (CaM-S index) was defined as the proportion of primary spermatocytes that were stained intensely for calmodulin relative to the total number of primary spermatocytes. The indices for the testes of men with complete spermatocyte maturation arrest were significantly lower than those for the testes of normal controls and of men with hypospermatogenesis.
Degenerating late pachytene spermatocytes observed in the testes of men with spermatocyte arrest showed low calmodulin-specific immunoreactivity. Such a decrease in numbers of normal late pachytene spermatocytes might be responsible for the low CaM-S index in cases of complete spermatocyte arrest.     

Spermatogenese

Spermatogenesis takes place within the testicular seminiferous tubules which consist of the peritubular lamina propria and the seminiferous epithelium. The latter is composed of germ cells and somatic Sertoli cells. Sertoli cells trigger germ cell development by mediating follicle-stimulating hormone and androgen hormonal stimuli. Spermatogenesis comprises proliferation of spermatogonia, meiosis of spermatocytes, and differentiation of spermatids into spermatozoa (spermiogenesis). There are six distinct and specific germ cell associations (I–VI). These “stages of spermatogenesis” occur sequentially along the length of a tubule. Different defects in spermatogenesis occur in adjacent seminiferous tubules (mixed atrophy) and are associated with deficits in differentiation of Sertoli cells. Biopsy specimens should be fixed in Bouin’s solution. Diagnosis of preinvasive carcinoma in situ is based on the immunohistochemical demonstration of placental-like alkaline phosphatase (PLAP), which is expressed exclusively in carcinoma in situ cells. Histological evaluation should be performed using a score count system, and the use of histological techniques for protein and mRNA expression. Testicular biopsy should only be performed in accordance with strict indication criteria, and histological evaluation should be carried out in specialist centres, i.e. as recommended by the European Academy of Andrology (EAA).     

Spermatogenesis--physiology and pathophysiology

Spermatogenesis takes place within the testicular seminiferous tubules which consist of the peritubular lamina propria and the seminiferous epithelium. The latter is composed of germ cells and somatic Sertoli cells. Sertoli cells trigger germ cell development by mediating follicle-stimulating hormone and androgen hormonal stimuli.Spermatogenesis comprises proliferation of spermatogonia, meiosis of spermatocytes, and differentiation of spermatids into spermatozoa (spermiogenesis). There are six distinct and specific germ cell associations (I-VI). These "stages of spermatogenesis" occur sequentially along the length of a tubule. Different defects in spermatogenesis occur in adjacent seminiferous tubules (mixed atrophy) and are associated with deficits in differentiation of Sertoli cells. Biopsy specimens should be fixed in Bouin's solution. Diagnosis of preinvasive carcinoma in situ is based on the immunohistochemical demonstration of placental-like alkaline phosphatase (PLAP), which is expressed exclusively in carcinoma in situ cells. Histological evaluation should be performed using a score count system, and the use of histological techniques for protein and mRNA expression. Testicular biopsy should only be performed in accordance with strict indication criteria, and histological evaluation should be carried out in specialist centres, i.e. as recommended by the European Academy of Andrology (EAA).     

Testicular Biopsy and Hormonal Study in a Male with Noonan''s Syndrome

The testicular biopsy study of a 17-year-old male with Noonan's syndrome revealed seminiferous tubules of reduced diameter with hypospermatogenesis. Many spermatocytes underwent degeneration and many spermatids developed abnormal. The Sertoli cells were similar to immature Sertoli cells. Fully differentiated Leydig cells were rare while precursor Leydig cells were numerous. Both gonadotropin and testosterone levels were low, and a lack of response to LH-RH as well as to clomiphene was found. The testicular biopsy performed at 20 years of age revealed a certain maturation of the seminiferous tubules which increased the germ cell number. The abnormalities in the spermatogenesis as well as the immature appearance of Sertoli cells continued. Leydig cells were more numerous and showed a certain development without reaching the normal pattern. Gonadotropin levels were normal while testosterone levels low. The response to LH-RH was increased and the absence of response to clomiphene persisted. These features suggest a delayed puberty.     

抑制素B βB亚单位在不同病理分型的无精子症患者睾丸组织中的表达

目的:探讨抑制素B(INH B)βB亚单位在不同生精功能状态的人睾丸组织中的表达情况。方法:对83例无精子症患者进行睾丸组织病理检查诊断,根据病理形态的不同分为:唯支持细胞综合征型(n=21);生精功能低下型(n=20);生精阻滞型(n=24);生精功能基本正常型(n=18)。选择上述各型结构完整的睾丸组织,分别应用免疫组化法(SP)对血清INH B βB亚单位在不同生精功能状态的睾丸组织,进行定位研究。结果:各型睾丸组织内均存在血清INH B βB的表达,其分布特点为:间质细胞(Leydig cell)和早期生精细胞多为强阳性表达,呈深棕黄色;支持细胞(Sertoli cell)多为阳性表达;而晚期精子细胞和成熟精子未见表达;生精小管管周类肌细胞呈弱阳性表达。结论:INH B可能是睾丸Sertoli细胞和早期生精细胞的一个联合产物。     

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.     

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。     

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.     

Expression of beta-catenin in human testes with spermatogenic defects

beta-catenin is a multifunctional molecule that functions in intercellular adhesion and signal transduction during assembly of AJs between Sertoli cells as well as between Sertoli cells and germ cells. To assess changes in the testicular beta-catenin in male infertility conditions, testicular tissues from obstructive azoospermia with normal spermatogenesis, spermatogenic arrest (SA) and Sertoli cell-only syndrome (SCO) patients were examined for immunohistochemical localization of beta-catenin. In normal spermatogenic tissue, expression of beta-catenin was largely found in the Sertoli cell-germ cell (primarily spermatocytes) contact areas. Interestingly, perinuclear localization of beta-catenin was found in spermatocytes and spermatids. In spermatogenic arrest, beta-catenin in cell contact areas between Sertoli cells and germ cells was greatly decreased, but perinuclear beta-catenin in spermatocytes was not. In SCO, weak or negligible immunoreactivity of beta-catenin was found in cell contacts between Sertoli cells. Nuclear localization of beta-catenin was found in myotubular cells in all samples. Taken together, altered expression of beta-catenin in cell contacts within the seminiferous epithelia in spermatogenic arrest and SCO suggests that interactions between Sertoli cells and germ cell are crucial for expression of beta-catenin, and thus functional development of AJs in seminiferous epithelia in human testis. It should be also emphasized that perinuclear beta-catenin in germ cells may play a specific role in spermatogenesis.     

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     

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.     

Flow cytometric DNA analysis of defined stages of rat seminiferous epithelial cycle during in vitro differentiation

The stages of the rat seminiferous epithelial cycle have been isolated for flow cytometric analysis of DNA and for culture, using transillumination-assisted microdissection. Precise stages have been identified by phase contrast microscopy of live cell squashes from adjacent segments. Each stage of the cycle showed a characteristic flow cytometric pattern with haploid (1C), diploid (2C) and tetraploid (4C) peaks. Stages I to VIII of the cycle showed an additional hypofluorescent (0.25-0.70C) peak due to a reduced dye-binding capacity of maturation phase-spermatids at steps 15 through 19. The appearance of the hypofluorescent haploid peak coincided with the second nucleoprotein transition at step 15 of spermiogenesis and the homogeneous condensation of the chromatin seen in electron microscopy. As a concomitant of the formation of disulphide bonds during epididymal maturation, the fluorescence intensity decreased further to reach a relative value of 0.07C in the cauda epididymidis. The constant 1C peak was raised by round and elongating spermatids (steps 1-14), 2C by spermatogonia, secondary spermatocytes and Sertoli cells, and the 4C peak by primary spermatocytes and spermatogonia at G2 or M phase of the mitotic cycle. The proportion of each peak accurately reflected the relative proportion of cells in most stages of the cycle when compared with morphometric measurements of histologic preparations. DNA flow cytometry is a suitable method for quantitative evaluation of cultured seminiferous tubule segment DNA. Although the relative yield of the meiotic reductive divisions in vitro is comparable with that observed in vivo, steps 9 and 15 of spermiogenesis involving nucleoprotein transitions and spermiation itself did not occur under the present culture conditions.     

Specific localization of the basigin protein in human testes from normal adults, normal juveniles, and patients with azoospermia

Basigin is a transmembrane protein belonging to the immunoglobulin superfamily. Specific localization of the protein in normal human testes, from those of a 2-year-old boy to those of a 50-year-old man, and in testes with Sertoli cell only syndrome and germ cell arrest, is reported. Basigin localization was determined using an immunohistochemical technique with an antibody against human basigin. In the normal adult testes, basigin was detected at the periphery of both spermatocytes older than zygotene and round spermatids. In the juvenile testes, it was expressed in accordance with the appearance of pachytene spermatocytes. In this study, pachytene spermatocytes were detected in an 11-year-old boy. Basigin was not expressed in immature testes with germ cells younger than pachytene spermatocytes, namely in testes from boys aged 2-9 years. In testes from adult patients with Sertoli cell only syndrome, basigin was expressed at the periphery of Sertoli cells, but localization was confined to the adluminal compartment of the seminiferous tubule. In testes with germ cell arrest, the protein was expressed on germ cells from pachytene spermatocytes to step 2 spermatids, where present. The results show that in the normal human testes basigin is expressed with the onset of spermatocyte differentiation. Because human basigin is expressed in adult testes with Sertoli cell only syndrome, the protein seems to be synthesized in Sertoli cells and expression continues after these cells dedifferentiate in the seminiferous epithelium.     

Testicular biopsy in patients with obstructive azoospermia

The present report studies the testicular biopsy lesions (histologic and semiquantitative) in a series of 48 patients with obstructive azoospermia of known etiology (vasectomy, congenital absence of vas deferens, herniorrhaphy, hydrocelectomy, Young's syndrome, and ejaculatory duct obstruction) in order to establish objective testicular data that permit the pathologist to diagnose an obstructive process, which should not be mistaken with a primary testicular lesion. The semiquantitative study included determinations of the average numbers of spermatogonia, primary spermatocytes, young spermatids (Sa + Sb), and differentiated spermatids (Sc + Sd). According to this study, the testes were classified into the following groups: (1) normal testes whose germ cell numbers were within normal limits (27 testes); (2) testes with lesions in the adluminal compartment; these lesions comprise two subgroups: (2a) late sloughing of primary spermatocytes (both spermatid types were greatly reduced in number while the other germ cell types were in normal numbers) (45 testes); and (2b) early sloughing of primary spermatocytes (normal spermatogonial number, reduced number of spermatocytes, and scanty spermatids) (9 testes); and (3) lesions in the basal compartment; these lesions comprise two subgroups: (3a) pure hypospermatogenesis (a proportionate decrease in the numbers of all germ cell types) (8 testes); and (3b) hypospermatogenesis associated with sloughing of primary spermatocytes (decreased numbers of all germ cell types with a very scanty number spermatids) (4 testes). Two testes appeared hyalinized and one testis was removed owing to cryptorchidism. The most frequent testicular lesion observed (alteration in the adluminal compartment of seminiferous tubules) seems to be related to the increase in hydrostatic pressure in the tight compartment formed by seminiferous tubules, rete testis, efferent ducts, the epididymal duct, and the initial portion of the vas deferens. The severity of the lesions is probably related to the cause and span of the obstruction. In addition, two azoospermic men without obstructive azoospermia and whose testicular biopsy study revealed meiotic anomalies (with the subsequent bad prognosis) were also studied for comparison. The semiquantitative study of these patients permitted the differential diagnosis between two lesion types. Testes with meiotic anomalies had a disproportionately elevated number of primary spermatocytes, and an extremely low number of young spermatids.     

Hyperactivation of early steps of spermatogenesis compromises meiotic insufficiency in men with hypergonadotropism. A possible quantitative assay for high FSH/low testosterone availabilities

It has been shown previously that blood plasma elevation of FSH is associated with an impaired function of the seminiferous tubules. In the study presented here quantitative analysis of the seminiferous epithelium was performed in testicular biopsies of men with qualitatively complete spermatogenesis and hypergonadotropism (HG group, n = 6) or normogonadotropism (NG group, n = 9). Hormonal determinations were performed also in eight men with Sertoli cells only (SCO group). Plasma levels of gonadotropins found in HG were comparable with those found in SCO, while mean plasma testosterone levels in these patients were significantly lower than in SCO or NG. A significant decrease in the mean number of spermatids was present in HG, while the mean numbers of B spermatogonia (0.6 +/- 0.2) and preleptotene spermatocytes (0.6 +/- 0.1) were significantly elevated in comparison with NG (0.4 +/- 0.1 and 0.3 +/- 0.2, respectively). In all men with HG a GnRH test (100 micrograms i.v.) was performed and the relative increase of plasma FSH (maximum/basal level) correlated positively with the number of A-pale (r = 0.85, P less than 0.05) and B spermatogonia (r = 0.81, P less than 0.05). In NG group basal levels of FSH correlated with A-pale (r = 0.90, P less than 0.001) and B spermatogonia (r = 0.59). It seems that FSH plays a role to maintain the number and the differentiation rate of spermatogonia in men and is responsible for the hyperactivation of spermatogonia when secreted in excess. Hyperactivation of spermatogonia probably develops to compensate quantitative decrease in gamete production.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spatiotemporal expression patterns of natriuretic peptides in rat testis and epididymis during postnatal development

Natriuretic peptide (NP) family is composed of atrial, brain and C‐type NP (NPPA, NPPB and NPPC). Here, we aimed to investigate NP expression in testis and epididymis during postnatal development. NPPA expression was observed in gonocytes at prepubertal period but in only spermatocytes in pachytene and leptotene/zygotene stage at pubertal period. In prepubertal and pubertal periods, we detected NPPB expression in only Leydig cells. However, NPPC expression was detected in all of the gonocytes and Sertoli cells, spermatocytes and some interstitial cells in prepubertal and pubertal periods. In postpubertal and mature periods, NPPA and NPPB staining were detected in Leydig cells, elongated and round spermatids but not in spermatogonia and spermatocytes. However, we observed NPPC expression in all cells of the seminiferous tubules and Leydig cells in the postpubertal and mature periods. Epididymal epithelium showed intense NPPC expression during postnatal period but weak NPPA and NPPB expression in prepubertal and pubertal periods. The expression of three NPs in the testis significantly increased after puberty. In conclusion, puberty had a significant effect on NP expression in testis. Unlike NPPA and NPPB, expression of NPPC in all cells of the seminiferous tubule suggests that NPPC is effective in each step of spermatogenesis.     

Expression of hyperacetylated histone H4 during normal and impaired human spermatogenesis

Histone-to-protamine exchange in haploid spermatids is preceded by hyperacetylation of core histones resulting in decreased DNA-histone interaction. During normal spermatogenesis, immunohistochemistry with a polyclonal antihyperacetylated histone H4 antibody displayed a strong signal in nuclei of elongating spermatids and, in addition, spermatogonia. Quantitative analysis revealed 98.2 +/- 1.1% of immunopositive spermatids. The percentage of positive spermatids was significantly reduced in infertile men exhibiting at least qualitatively normal spermatogenesis (scores 10-8, 93.1 +/- 6.6%) and impaired spermatogenesis (scores 7-1, 74.9 +/- 23.4%). In seminiferous tubules showing spermatogenic arrest at the level of round spermatids, only 59.5 +/- 16.5% of spermatids were immunopositive for hyperacetylated histone H4. These data demonstrate that the decrease of histone acetylation in spermatids associated with impaired spermatogenesis corresponds with the well known reduction of protamine expression in these cells and confirms the essential role of histone hyperacetylation for correct histone-to-protamine exchange. In seminiferous tubules exhibiting round spermatid maturation arrest, there was an additional signal in nuclei of spermatocytes, suggesting that premature hyperacetylation of histone H4 may result in precocious histone-to-protamine exchange followed by infertility. This is in accordance with data from transgenic mice, where it has been demonstrated that premature expression of protamine-1 results in precocious chromatin condensation followed by sterility.     

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.