Absence of RBM expression as a marker of intratubular (in situ) germ cell neoplasia of the testis

RBM (RNA-binding motif) protein is a marker of male germ cells. This protein is encoded by the Azoospermia factor region-b (AZF-b) of the human Y chromosome and is expressed exclusively in the male germ cell line, that is, spermatogonia, spermatocytes, and round spermatids. The authors analyzed the expression of the RBM gene in germ cell tumors and in the seminiferous tubules in the vicinity of these tumors to identify the presence of IGCN. Sections from testicular germ cell tumors of 21 patients were stained with anti-RBM antibody by using an immunohistochemical method. Distal tubules showing spermatogenesis were immunopositive for RBM protein. All of the germ cell tumors studied were completely immunonegative for RBM. Defined areas of IGCN also showed an absence of RBM expression. Tubules with spermatocyte-like cells, which were expected to express RBM, did not express this protein. This result enabled the identification of tubules as being IGCN. RBM is a novel marker consistently expressed in normal male germ cells but not in malignant germ cell tumors or IGCN. Thus, the absence of RBM expression in germ cells provides a new diagnostic tool of preinvasive malignancy of the testis.     

Localisation cellulaire de la sécrétion de l’inhibine B testiculaire

Inhibin B is a testicular peptide hormone that regulates FSH secretion in a negative feedback loop. Inhibin B is a dimer of an α and βB subunit. In adult testis, the cellular site of production of these subunits is still controversial: Leydig cells, Sertoli cells and/or germ cells. The immunohistological localization (monoclonal antibodies anti α and anti βB) of both sub-units and the expression patterns of their mRNA (in situ hybridization with RNA probes) were examined in adult testicular biopsies with normal spermatogenesis or spermatogenetic arrest. In all testes, Sertoli cells and Leydig cells showed positive immunostaining for inhibin α subunit and expressed inhibin α subunit mRNA. Conversely, germ cells expressed the βB peptide (located from pachytene spermatocytes to round spermatids) and the βB subunit mRNA (located from spermatogonia to round spermatids). These results agree with the recent opinion that inhibin B is possibly a joint product of Sertoli cells and germ cells in adult men and it may be used as a serum marker of spermatogenesis.     

The contribution of RNA-binding motif (RBM) antibody to the histopathologic evaluation of testicular biopsies from infertile men

Testicular biopsies of infertile men are often characterized by mixed histologic patterns, with different types of spermatogenic impairments being found in adjacent seminiferous tubules. RNA-binding motif (RBM) is a nuclear protein expressed exclusively in the male germ cell line. We reasoned that RBM might be a useful marker to identify germ cells in testicular sections, particularly in biopsies with mixed histologic phenotype and small focal concentrations of spermatogenesis. Testicular biopsies from azoospermic men were immunohistochemically evaluated for RBM expression. RBM expression was detectable in spermatogonia, spermatocytes, and round spermatids in biopsies of men with obstructive azoospermia and normal spermatogenesis. No specific cell staining was shown in cases of Sertoli-cell-only (SCO) syndrome. In biopsies of patients with spermatogenic disorders, all the germ cells were stained up to and including the stage level of the arrest in spermatogenesis. This approach enabled identification of small focal concentrations of spermatogenesis in a biopsy previously classified as being SCO by hematoxylin and eosin staining. Thus, RBM can be a useful immunohistochemical marker for the specific identification of germ cells and provide greater accuracy in the histopathologic evaluation of testicular biopsies.     

Ki-A10, a germ cell nuclear antigen retained in a subset of germ cell-derived tumors

Monoclonal antibody Ki-A10 recognizes a nuclear antigen of 25 and 22 kd apparent molecular mass, which is abundantly expressed by immature gonocytes, spermatogonia, and spermatocytes, whereas it is absent in spermatids, spermatozoa, oocytes, and normal somatic tissues. In a broad spectrum of human cancers the antibody showed no reactivity except for a small subset of malignant lymphomas. Because of this restricted expression pattern, we examined 173 germ cell tumors and 18 sex cord stromal tumors immunohistochemically to assess the distribution of the Ki-A10 antigen. A strongly positive reaction was found in classic seminomas, dysgerminomas, spermatocytic seminomas, and the germ cell component of gonadoblastomas. Yolk sac tumors presented a heterogeneous reactivity pattern ranging from overall positivity to complete lack of antigen expression, and in three of eight choriocarcinomas, a few clusters of cytotrophoblast cells were strongly labeled. All other tumors, including Leydig and Sertoli cell tumors as well as placental tissue, were negative. Our findings suggest that specific germ cell antigens can be retained in germ cell tumors along particular differentiation pathways. Ki-A10 is the first marker that consistently labels spermatocytic seminoma, further confirming its germ cell origin and suggesting a close relationship to classic seminoma. The antibody may serve for diagnostic purposes and promises new insights into the process of germ cell differentiation and the development of germ cell-derived neoplasia.     

Expression of Bcl-2 family proteins and spontaneous apoptosis in normal human testis

We investigated the frequency of spontaneous apoptosis and expression of the Bcl-2 family of proteins during normal spermatogenesis in man. Testicular tissue with both normal morphology and DNA content was obtained from necro-donors and fixed in Bouin's solution. A TdT-mediated dUTP end-labelling method (TUNEL) was used for the detection of apoptotic cells. Expression of apoptosis regulatory Bcl-2 family proteins and of p53 and p21(Waf1) was assessed by immunohistochemistry. Germ cell apoptosis was detected in all testes and was mainly seen in primary spermatocytes and spermatids and in a few spermatogonia. Bcl-2 and Bak were preferentially expressed in the compartments of spermatocytes and differentiating spermatids, while Bcl-x was preferentially expressed in spermatogonia. Bax showed a preferential expression in nuclei of round spermatids, whereas Bad was only seen in the acrosome region of various stages of spermatids. Mcl-1 staining was weak without a particular pattern, whereas expression of Bcl-w, p53 and p21(Waf1) proteins was not detected by immunohistochemistry. The results show that spontaneous apoptosis occurs in all male germ cell compartments in humans. Bcl-2 family proteins are distributed preferentially within distinct germ cell compartments suggesting a specific role for these proteins in the processes of differentiation and maturation during human spermatogenesis.     

Normal and pathological human testes express hormone-sensitive lipase and the lipid receptors CLA-1/SR-BI and CD36

Numerous studies have demonstrated the important role of cholesterol and cholesteryl esters in tumor cell proliferation and progression of cancer. However, few studies have focused on the role of lipid transporters and lipases in cancer development and progression. The present study examined the expression of hormone-sensitive lipase (HSL) and the scavenger receptors CLA-1/SR-BI and CD36 in normal human testis and in nontumor and tumor testicular disorders by immunohistochemistry and Western blotting analysis. In normal young testes, immunoreaction to CLA-1/SR-BI was found in the spermatid acrosomic vesicle and on the surface of Sertoli and Leydig cells. HSL was detected in spermatogonia, the Golgi region of spermatocytes, the nucleus of spermatids, and the cytoplasm of both Sertoli and Leydig cells. Elderly testes and testes with hypospermatogenesis showed a similar staining pattern to that of normal young testes except for CD36, which was expressed in Sertoli cells. Cryptorchid testes demonstrated intense labeling to HSL and weak labeling to SR-BI in Sertoli cells (nucleus and cytoplasm) and Leydig cells (cytoplasm). Seminiferous tubules with intratubular germ cell neoplasia exhibited intense immunolabeling to the 3 lipid receptors in the surface of neoplastic cells and to HSL in the nucleus. In seminoma and spermatocytic seminoma, neoplastic cells labeled to HSL but failed to stain with antilipid receptors; in the seminiferous tubules at the periphery of the tumour, Charcot-B?ttcher crystalloids of Sertoli cells were strongly positive to CLA-1. Testes with mature teratoma showed a weak reaction to CD36 and SR-BI in some cells of enteric-type glands, and immature teratoma were exclusively immunolabeled with HSL. Western blotting analysis revealed that multiple bands were immunolabeled, with differences seen between normal and pathological testes. The results of this study indicate that the presence of lipid receptors (CLA-1/SR-BI) and hormone-sensitive lipase in Leydig cells suggests a role of these proteins in steroidogenesis. Also, these proteins seem to be involved in spermiogenesis, as their labeling in spermatids suggests. In nonmalignant and malignant pathologies, cholesterol metabolism is probably altered, and HSL labeling in neoplastic germ cell nuclei suggests a still-unknown function of this enzyme, probably related to cell cycle regulation.     

MAGE-1 and MAGE-3 tumor rejection antigens in human germ cell tumors.

The MAGE-1 and MAGE-3 genes are members of the melanoma antigen-encoding gene family. These genes encode for HLA-restricted tumor-associated rejection antigens recognized by cytotoxic T lymphocytes. MAGE-1 and MAGE-3 gene expression has been identified in a number of human malignancies, and MAGE-1 and MAGE-3 antigenic peptides are potential targets for tumor-specific immunotherapy. The only normal tissues known to express these genes are testicular germ cells and placental tissue. The objective of this study was to examine MAGE-1 and MAGE-3 antigens immunohistochemically in testicular germ cell tumors, including seminoma, a germ cell tumor frequently associated with a lymphoid infiltrate. Forty-three germ cell tumors (24 seminomas, six embryonal carcinomas and 13 mixed germ cell tumors), and 10 Leydig cell tumors were selected for study, and standard immunohistochemical techniques were used on formalin-fixed paraffin-embedded tissues using mouse monoclonal antibodies to MAGE-1 (clone M454) and MAGE-3 (clone 57B) antigens. MAGE-1 antigen was identified in 16.6% of seminomas. No embryonal carcinomas, yolk sac tumors, or teratomas contained MAGE-1 protein. MAGE-3 antigen was identified in 41.8% of seminomas, and this protein was not identified in embryonal carcinomas, yolk sac tumors, or teratoma. Spermatogonia and primary spermatocytes contained MAGE-1 and MAGE-3 antigen, and more mature forms, including spermatids, were weakly positive to negative. Leydig cell tumors were negative for MAGE-1 and MAGE-3. In seminoma, the presence of MAGE-1 and MAGE-3 antigens did not correlate with tumor size, tumor stage, the presence of a lymphoid infiltrate, or patient outcome. The low frequency of MAGE-specific HLA alleles in the population, the loss of the HLA class I antigens in neoplastic germ cells, and the finding that the majority of seminomas and all non-seminomatous germ cell tumors lacked MAGE-1 and MAGE-3 antigenic peptides indicate that immunotherapy directed towards MAGE-1 and MAGE-3 antigen is not a likely treatment option for seminoma and nonseminomatous germ cell tumors. The significance of MAGE-1 and MAGE-3 proteins in normal spermatogonia and primary spermatocytes will require additional study.     

Impact of genetic engineering on the understanding of spermatogenesis

To date, about 100 genes have been found, by genetic engineering, to be implicated in spermatogenesis. Primordial germ cells, spermatogonia, spermatocytes I and elongating spermatids are particularly sensitive. Transgenic and knockout mice permit an approach to be made to the question of genetic factors involved in DNA damage repair, thermal injury, sperm chromatin compaction and sex-specific recombination. Knockout mice reveal unexpected functional redundancies of testis-specific genes. This review considers how functional divergences can exist among homologous genes from different species, and to what extent the phenotypes of knockout mice can be similar to those from spontaneous mutations. Additional anomalies in reproductive function have frequently been found in these mice, as were found factors leading to tumour susceptibility and/or various diseases. Finally, knockout mice remind us that, in nearly all cases, hemizygous individuals retain a fertility and a wild-type sperm phenotype, although half of the spermatozoa share a genetic defect. The findings strongly emphasize the importance of understanding epidemiology in male infertility, to identify hereditary forms of impaired spermatogenesis, and to create DNA and pathological germ cell banks.     

Histochemical mapping of glycoconjugates in the testis of the one humped camel (Camelus dromedarius) during rutting and non-rutting seasons

In the present study, the distribution of various sugar residues in the testicular cells of sexually mature camels during rutting and non-rutting seasons was examined employing 10 fluorescein isothiocyanate- (FITC) conjugated lectins. Lectin labeling was restricted to the germ cell lines and interstitial Leydig cells, while the Sertoli cells remained completely unlabeled. Our results revealed the presence of mannose (labeled by lectins PSA, LCA), galactose (labeled by PNA), GalNAc (labeled by HPA), and GlcNAc (labeled by WGA) residues in the camel spermatogonia. However, spermatocytes were only labeled with mannose (PSA, LCA) and GlcNAc (WGA) binding lectins. Binding sites for PSA, LCA and WGA in spermatogonia and spermatocytes were only evident during the rutting season. Although spermatids were exclusively labeled with PNA in the non-rutting seasons, other lectins (PSA, GSA-I, WGA) additionally bound to camel spermatids during the rutting period. Leydig cells and basal lamina of the seminiferous tubules of camel testis were consistently labeled with the mannose- (PSA, LCA) and GlcNAc- (WGA) binding lectins in both seasons, while DBA-labeling was seen in the Leydig cells during rutting period only. In conclusion, the findings of the present study clearly indicate that the camel testis contains a wide range of glycoconjugates (bearing mannosyl, galactosyl and glucosyl residues), and they lack fucosyl residues, both in the active sexual period and in the non-breeding season. The topographical distribution of the sugar moieties in the camel testis may indicate that specific carbohydrate structures are required for spermatogenesis during periods of sexual activity.     

Establishment of the paternal methylation imprint of the human H19 and MEST/PEG1 genes during spermatogenesis

Parental-specific epigenetic modifications are imprinted on a subset of genes in the mammalian genome during germ cell maturation. However, the precise timing of their establishment remains to be determined. Methylation of CpG dinucleotides has been shown to be a part of the parental imprint. We have examined how the methylation pattern characteristic of the paternal allele in germ cells are established during human spermatogenesis. Two representative imprinted genes, H19 and MEST/PEG1, were studied. The experiments were performed using the bisulphite sequencing method on microdissected individual cells at different stages of male germ cell differentiation. We show that both genes are unmethylated in fetal spermatogonia, suggesting that all pre-existing methylation imprints are already erased by this stage. The MEST/PEG1 gene remains unmethylated at all subsequent post-pubertal stages of spermatogenesis, including mature spermatozoa. The methylation of H19 typical of the paternal allele first appears in a subset of adult spermatogonia and then is maintained in spermatocytes, spermatids and mature spermatozoa. Our results suggest that the methylation imprint inherited from the parents is first erased in the male germ line at an early fetal stage. The paternal-specific imprint is re-established only later, during spermatogonial differentiation in the adult testis.     

Expression of CD1d Protein in Human Testis Showing Normal and Abnormal Spermatogenesis

CD1d is a member of CD1 family of transmembrane glycoproteins, which represent antigen-presenting molecules. Immunofluorescent staining methods were utilized to examine expression pattern of CD1d in human testicular specimens. In testis showing normal spermatogenesis, a strong CD1d cytoplasmic expression was seen the Sertoli cells, spermatogonia, and Leydig cells. A moderate expression was observed in the spermatocytes. In testes showing maturation arrest, CD1d expression was strong in the Sertoli cells and weak in spermatogonia and spermatocytes compared to testis with normal spermatogenesis. In Sertoli cell only syndrome, CD1d expression was strong in the Sertoli and Leydig cells. This preliminary study displayed testicular infertility-related changes in CD1d expression. The ultrastructural changes associated with with normal and abnormal spermatogenesis are open for further investigations.     

Expression of CD1d protein in human testis showing normal and abnormal spermatogenesis

CD1d is a member of CD1 family of transmembrane glycoproteins, which represent antigen-presenting molecules. Immunofluorescent staining methods were utilized to examine expression pattern of CD1d in human testicular specimens. In testis showing normal spermatogenesis, a strong CD1d cytoplasmic expression was seen the Sertoli cells, spermatogonia, and Leydig cells. A moderate expression was observed in the spermatocytes. In testes showing maturation arrest, CD1d expression was strong in the Sertoli cells and weak in spermatogonia and spermatocytes compared to testis with normal spermatogenesis. In Sertoli cell only syndrome, CD1d expression was strong in the Sertoli and Leydig cells. This preliminary study displayed testicular infertility-related changes in CD1d expression. The ultrastructural changes associated with with normal and abnormal spermatogenesis are open for further investigations.     

人绒毛膜促性腺激素对切除颌下腺小鼠静止期精母细胞的一些影响

目的：探讨人绒毛膜促性腺激素(HCG)对切除颌下腺小鼠静止期精母细胞表皮生长因子(EGF)的表达和生精功能的影响。方法：采用组织化学和免疫组织化学技术。结果：EGF、分布在小鼠睾丸间质细胞、精原细胞、静止期精母细胞、细线期精母细胞和精子细胞。去颌下腺组EGF阳性反应静止期精母细胞数量明显减少，同时相应的生精小管的各级生精细胞也显著减少。与去颌下腺组相比，去颌下腺给药组EGF阳性反应静止期精母细胞数量明显增多，同时相应的生精小管的生精细胞也显著增多。结论：睾丸间质细胞和部分生精细胞能分泌EGF。EGF在生精小管上皮的分布与生精周期有密切关系。静止期精母细胞EGF表达减弱是切除颌下腺导致小鼠少精症的原因之一。HOG可促进静止期精母细胞表达EGF，以调节精子发生过程。     

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.     

Testicular development in cynomolgus monkeys

The testes from 136 male cynomolgus monkeys were examined histopathologically in order to investigate the relationship between the development of spermatogenesis and testis weight, age, and body weight. At Grade 1 (immature), Sertoli cells and spermatogonia were the only cell classes in the testis. At Grade 2 (pre-puberty), no elongated spermatids were observed in the testis, although a few round spermatids and small lumen formation were observed. At Grade 3 (onset of puberty), all classes of germ cells were observed in the testis, although seminiferous tubule diameters and numbers of germ cells were small. Slight debris in the epididymis was observed in almost all animals. At Grade 4 (puberty), almost complete spermatogenesis was observed in the seminiferous tubules and it was possible to ascertain the spermatogenesis stage as described by Clermont, although tubule diameters and numbers of germ cells were small. There was less debris in the epididymis than at Grade 3. At Grade 5 (early adult), complete spermatogenesis was observed in the seminiferous tubules. At Grade 6 (adult), complete spermatogenesis in the seminiferous tubules and a moderate or large number of sperm in the epididymis were observed. Moreover, sperm analysis using ejaculated sperm was possible. Logistic regression analysis showed that testis weight is a good indicator of testicular maturity.     

The genetic control and germ cell kinetics of the female and male germ line in mammals including man.

The female germ line (germ cell lineage, Keimbahn) is provided with only one proliferation wave, the oogenic, whereas male gametogenesis involves two successive waves: prespermatogenic, which corresponds to the female proliferation wave, and spermatogenesis, which is responsible for the immense number of male gametes produced in mature testes. Both male proliferation systems are linked by the transitional or T prospermatogonia. Using the reverse percentage of labelled metaphases method, it has been shown that the first differences between female and male germ cells can be identified by the end of the first wave, when oogonia and multiplying or M prospermatogonia are proliferating. This prenatal first wave of proliferation of male germ cells was also demonstrated in man and ceases around the 22nd week of pregnancy. Spermatogenesis involves a stock of stem cells (stem spermatogonia), a flexibly reacting pool of undifferentiated spermatogonia and several generations of differentiating spermatogonia, which proliferate almost exponentially. Furthermore, it consists of spermatocytes and haploid spermatids transforming into spermatozoa. The oocytes pass through the preleptotene stage, synthesizing DNA, and thereafter traverse the meiotic prophase up to the diplotene stage. In mammals they act as 'pre-embryos' in a similar but to a lesser degree than oocytes of amphibia and insects. The maternal chromosomes are largely responsible for the development of the embryo, the paternal genome for the development of the extra-embryonic tissue. The synthesis of transgenic animals is a powerful weapon in the armoury of geneticists, as has recently been demonstrated: a 14 kb genomic DNA fragment (Sry) is sufficient to induce testis differentiation and subsequent male development when introduced into chromosomally female mouse embryos.     

Atypical germ cells of the testis

Summary It is uncertain whether the so called intratubular atypical germ cells (carcinoma in situ cells) demonstrable in the testicular tissue around different germ cell tumors and in testicular biopsies of patients with impaired fertility are identical with regard to their morphology and further development. Thus atypical germ cells of 18 patients with testicular germ cell tumors and of 3 patients with atypical germ cells in testicular biopsies without tumor were studied by electron microscopy and/or by immunohistochemistry. The atypical germ cells show characteristic alterations distinguishing them from normal germ cells, especially spermatogonia. However, there are no differences between atypical germ cells in the above mentioned groups. Immunohistochemical reactions are negative with anti-alpha-fetoprotein and anti-beta-human-chorionic-gonadotropin, but 6 of the 15 cases are positive with antiferritin. However, this positive reaction occurs in cases in different diagnostic groups. Atypical germ cells of the different groups cannot be distinguished by electron microscopy or immunohistochemical methods, but further investigations vestigations, including cell cultures, may provide more information.