低剂量电离辐射诱导小鼠睾丸生精细胞凋亡及P53基因和蛋白表达

目的:研究低剂量电离辐射对小鼠睾丸生精细胞凋亡及 P53 基因和蛋白表达的影响。方法:应用密度梯度离心法分离不同种类生精细胞, 流式细胞术检测其细胞凋亡, 免疫组化法观察生精细胞P53蛋白表达, 原位杂交法观察其 P53 mRNA水平。结果:0.025-0.2 Gy X射线全身照射后, 生精细胞凋亡具有明显的细胞种类规律性。在较低剂量照射(0.025和0.05 Gy)时, 以精原细胞凋亡为主, 随照射剂量增加(0.075-0.2 Gy)逐渐累及精母细胞, 并且前者凋亡率明显高于后者, 很少累及精子细胞和精子。P53蛋白表达主要见于精原细胞和精母细胞, 并且前者阳性率高于后者, 随照射剂量增加, 其阳性率逐渐升高, 而精子细胞和精子阳性率较低; P53 mRNA表达在较低剂量照射(0.025 Gy)时, 主要以精母细胞和精子细胞为主, 随剂量增加(0.05-0.2 Gy)逐渐累及精原细胞。精原细胞和精母细胞 P53 mRNA表达呈明显的剂量依赖性关系, 但精子细胞表现不明显。结论:低剂量电离辐射可选择性诱导小鼠睾丸生精细胞凋亡, 具有明显的剂量和时程效应关系。提示, 这种选择性诱导凋亡调控机制可能与 P53 基因和蛋白表达相关联。     

Immunoexpression of testis-specific histone 2B in human spermatozoa and testis tissue

During mammalian spermatogenesis, the chromatin of the spermatogenic cells is profoundly reorganized. Somatic histones are partly replaced by testis-specific histones. These histones are then replaced by transition proteins and finally by protamines. This series of nucleoprotein rearrangements results in a highly condensed sperm cell nucleus. In contrast to spermatozoa from other species, human spermatozoa still contain a significant amount of histones, including testis-specific histone 2B (TH2B). In the present study it is shown that an antibody targeting tyrosine hydroxylase, which has been found previously to cross-react with rat TH2B, also specifically immunoreacts with human TH2B on Western blots, in immunohistochemistry of human testis tissue, and in immunocytochemistry of decondensed human spermatozoa. In human testis tissue, TH2B immunostaining first apparent in spermatogonia, shows marked variation, especially at the pachytene spermatocyte stage, and then reaches an intense signal in round spermatids. Shortly before spermatid elongation, a portion of the spermatid nucleus, corresponding to the acrosomal region, loses its immunoreactivity. During condensation of the spermatid nucleus, the immunodetectability of TH2B disappears gradually, from the anterior region of the nucleus onwards. At the final stages of spermiogenesis, the immunostaining is completely absent. Immunocytochemical staining of spermatozoa revealed no TH2B immunosignal, but immunostaining was observed when spermatozoa obtained from semen were decondensed to make nuclear proteins accessible to the antibody. There was, however, a striking intercellular variability in the intensity of staining of spermatozoa within an ejaculate. In a population of 35 men attending our Andrology Clinic, we observed interindividual differences in total sperm TH2B content, which showed a significant, although not very pronounced, negative correlation with normal morphology (P = 0.05).      

Spermatogenesis in the dog

The cycle of the seminiferous epithelium of the dog was divided into eight stages, using as criteria the shape of the spermatid nucleus, the location of spermatids and spermatozoa in regard to the basement membrane, the presence of meiotic figures and the release of spermatozoa from the lumen of the tubule. Based upon these criteria, a modification of the eight-stage system of classification of the cycle of the seminiferous epithelium was developed. Cell populations making up each stage are described. The relative frequencies of stages 1 through 8 were 21.9, 12.7, 2.8, 11.5, 8.3, 15.4, 13.3 and 14.0%, respectively. The duration of one cycle of the seminiferous epithelium was 13.6 days (SE ± 0.7), as determined from cells labeled by tritiated thymidine. The absolute durations of stages 1 through 8 were 3.0, 1.7, 0.4, 1.6, 1.1, 2.1, 1.8 and 1.9 days, respectively. The life span of primary spermatocytes was 20.9 days, of secondary spermatocytes 0.5 days, spermatids with round nuclei 10.5 days, spermatids with elongated nuclei up to the time they are released into the lumen, 10.6 days. Counts of the different types of spermatogenic cells in tubular cross sections revealed little or no germ cell degeneration during the two maturation divisions.     

大鼠精子发生和形成过程中溶酶体超微结构和CMPase的细胞化学变化

通过超微结构及胞嘧啶单核苷酸酶（ｃｙｔｉｄｉｎｅ ｍｏｎｏｐｈｏｓｐｈａｔａｓｅ，ＣＭＰａｓｅ）细胞化学方法研究大鼠精子发生和形成过程中溶酶体的动态变化。结果表明：精原细胞中只有极少的溶酶体，精母细胞中溶酶体明显增多；高尔基期精子细胞中出现前顶体泡，高密度多泡体和其他形态的溶酶体，它们呈现ＣＭＰａｓｅ阳性。头帽期时精子细胞顶体系统不断扩大，高密度多泡体等溶酶体已转移至形成中的精子尾附近，并一直停留     

Lectin binding sites on human sperm and spermatogenic cells

Testes of sexually mature men were studied histochemically with 20 fluorescein isothiocyanate-labeled lectins. Based on their pattern of reactivity with intratesticular spermatogenic cells, lectins were divided into five groups: 1) lectins reacting with all spermatogenic cells (Suc. ConA, WGA, LCA, PHA-E, PHA-L, STA, MPA, and RCA-II); 2) lectin reacting with spermatocytes, spermatids, and spermatozoa, but not with spermatogonia (RCA-I); 3) lectins reacting with spermatids and spermatozoa only (BPA, PNA, SBA, and VVA); 4) lectins reacting only with spermatozoa (HPA, GSA-I, UEA-II, and GSA-II); and 5) lectins with no distinct staining of spermatogenic cells (DBA, LBA, and UEA-I). All lectins from groups 1-4 were reactive with ejaculated spermatozoa. On the basis of the staining patterns of the head region of ejaculated spermatozoa, four lectin reactivity groups were defined: 1) lectins reacting with the plasma membrane of the whole head (BPA, WGA, LCA, STA, RCA-II, PHA-E, PHA-L, RCA-I, UEA-II, and GSA-II); 2) lectin reacting with the acrosomal cap and postacrosomal region of the plasma membrane (Suc. ConA); 3) lectin reacting with the acrosomal cap region of the plasma membrane (PNA); and 4) lectins reacting with the midregion of the sperm head in a bandlike manner (HPA, VVA, SBA, GSA-I, and MPA). These data provide a map of lectin binding sites on human testicular spermatogenic cells and ejaculated spermatozoa and show that the distribution of glycoconjugate domains of spermatogenic cell changes during differentiation and maturation.     

DOG1 immunohistochemical staining of testicular biopsies is a reliable tool for objective assessment of infertility

Testicular biopsy may be a component of the work-up of male infertility. However, no reliable diagnostic tools are available for objective quantitative assessment of spermatogenic cells. It is well known that MAGE-A4 is selectively expressed in spermatogonia and our group has previously demonstrated that DOG1 differentially stains germ cells. Therefore, we performed DOG1 and a double stain cocktail (DOG1 and 57b murine monoclonal anti-MAGE-A4) immunohistochemical stains on 40 testicular infertility biopsies (10 each with active spermatogenesis, Sertoli cell-only, hypospermatogenesis, and maturation arrest), 25 benign seminiferous tubules from radical orchiectomies, and 5 spermatocytic tumors (ST). In biopsies/resections with active spermatogenesis, DOG1 stained spermatocytes and spermatids and was absent in spermatogonia, while MAGE-A4 stained spermatogonia and primary spermatocytes (weak). In hypospermatogenesis, DOG1 highlighted decreased spermatocytes/spermatids and MAGE-A4 highlighted decreased spermatogonia. DOG1 staining confirmed decreased to absent spermatocytes in maturation arrest and MAGE-A4 staining established the presence of preserved spermatogonia in all cases. All STs were negative for DOG1 and positive for MAGE-A4, while all Sertoli cell-only cases were negative for DOG1 and the double stain cocktail. In conclusion, we confirmed that DOG1 is expressed in spermatocytes and spermatids and MAGE-A4 highlights primarily spermatogonia. Usage of these stains facilitates confirmation of maturation arrest, assessment of the percentage of testis involvement in hypospermatogenesis and identification of mixed patterns. Finally, this study supports that the differentiation of STs is more closely related to spermatogonia than the more mature spermatocytes.     

The proliferation of spermatogonia in normal and pathological human seminiferous epithelium: an immunohistochemical study using monoclonal antibodies against Ki-67 protein and proliferating cell nuclear antigen

The quantitative distribution pattern of Ki-67 protein and proliferating cell nuclear antigen (PCNA) immunoreactivity was studied in human testis biopsies. In normal seminiferous epithelium Ki-67 is expressed in nuclei of spermatogonia, while PCNA additionally occurs in nuclei of primary spermatocytes. The staining pattern of spermatogonia is as follows (Ki-67-positive/PCNA-positive): 26.6 +/- 12.4%/46.3 +/- 9.5%. No stage-dependent differences were found. Biopsies with mixed atrophy (score < or =7) showed a significant (P < 0.05) decrease of immunopositive spermatogonia to 19.9 +/- 3.0%/31.4 +/- 5.7% (score 1) with minimal variation between different samples (score 7 to 1). Associated with defined histological defects such as hypospermatogenesis (hyp), spermatogenic arrest at the level of spermatids (sda), spermatocytes (sca) or spermatogonia (sga), however, there was a significant (P < 0.05) decrease of Ki-67 staining in tubules showing hyp (28.6 +/- 8.8%), sda (25.6 +/- 9.3%), sca (23.7 +/- 9.3%) and sga (16.2 +/- 6.0%) and of PCNA staining in sca (32.2 +/- 11.8%) and sga (20.0 +/- 9.5%), respectively. The decrease of immunoreactive spermatogonia did not correspond to elevation of follicle stimulating hormone (FSH). These data demonstrate that the low spermatogenic efficiency in infertile men is not only due to postmeiotic events, but also to a decrease in the meiotic activity of spermatogonia, and is not related to serum FSH.      

Genetics: Disturbances of nuclear condensation in human spermatozoa: search for mutations in the genes for protamine 1, protamine 2 and transition protein 1

During spermiogenesis, the successive replacement of the somatichistones by basic proteins, the transition proteins and protamines,allows normal sperm nuclear condensation. It was suggested thatdisturbances in nuclear condensation may result in male infertility.Here we report the first molecular analysis of the structureof three genes which code for germ cell-specific nuclear proteins,namely protamine 1 (PRM1), protamine 2 (PRM2) and transitionprotein 1 (TNP1) in infertile men with disturbed sperm chromatincondensation. In 36 infertile men whose spermatozoa showed apositive reaction with aniline blue, which is an indicationfor the presence of histones in the nuclei, the complete nucleotidesequences of the coding regions and 5’ and 3‘ untranslatedregions of the three genes were evaluated. In addition, 10 infertilepatients with oligoasthenoteratozoospermia were studied in thesame way, as well as nine infertile patients whose spermatozoashowed a reduction of the protamine 2 content. We did not detectany mutation in the three genes in any of the patients. We assumethat the disturbances in the sperm chromatin condensation ofour patients, and those described in the literature, are notprimarily due to mutations in the genes for PRM1, PRM2 and TNP1.     

Demonstration of thiamine pyrophosphatase in human germ cells and Sertoli cells, a histochemical study

The distribution of thiamine pyrophosphatase (TPPase) was studied in the human testicular biopsy tissue with the help of a modified histochemical gel method. The spermatogonia showed a large round strongly positive reaction zone in the supranuclear region. The pachytene spermatocytes exhibited a large semicircular TPPase reaction zone around the nucleus directing towards the lumen. The early round spermatids were characterized by a small, round, and weak supranuclear reaction zone. The elongated late spermatids were devoid of TPPase activity. The Sertoli cells possessed a "streamer" like formation of strong TPPase activity spreading from basal to apical portion.