Retinoic acid receptor alpha is required for synchronization of spermatogenic cycles and its absence results in progressive breakdown of the spermatogenic process.

Targeted mutagenesis of the retinoic acid receptor alpha (RAR alpha) gene has revealed its essential role in spermatogenesis. Although cells in all stages of spermatogenesis were detected in RAR alpha(-/-) testes, there was an increase in degenerating pachytene spermatocytes and a temporary developmental arrest in step 8-9 spermatids in the first wave of spermatogenesis, a delay in the onset of the second wave, and a temporary arrest in preleptotene to leptotene spermatocytes in the first, second, and third waves. A striking aspect of the mutant phenotype was the failure of spermatids to align at the tubular lumen at stage VIII. Furthermore, there were missing or decreased numbers of the predicted cell types in tubules, and they exhibited a profound asynchrony of mixed spermatogenic cell types. In vivo bromodeoxyuridine labeling revealed a significant decrease in germ cell proliferation in both juvenile and adult RAR alpha(-/-) testes and confirmed the arrest at step 8-9 spermatids. Retinoid signaling through RAR alpha, thus, appears to be critical for establishment of synchronous progression of spermatogenesis and the subsequent establishment of correct cellular associations.     

Induction of apoptosis involving multiple pathways is a primary response to cyclin A1-deficiency in male meiosis.

The meiotic arrest in male mice null for the cyclin A1 gene (Ccna1) was associated with apoptosis of spermatocytes. To determine whether the apoptosis in spermatocytes was triggered in response to the arrest at G2/M phase, as opposed to being a secondary response to overall disruption of spermatogenesis, we examined testes during the first wave of spermatogenesis by terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labeling (TUNEL) staining. We observed enhanced apoptosis coinciding with the arrest point in postnatal day 22 tubules, with no overt degeneration. Along with activation of caspase-3, an increase in the levels and change of subcellular localization of Bax protein was observed in cyclin A1-deficient spermatocytes, which coincided with the detection of apoptosis. As p53 is implicated in the activation of Bax-mediated cell death, we generated mice lacking both cyclin A1 and p53. Although the absence of p53 did not rescue the meiotic arrest, there was a decrease in the number of apoptotic cells in the double-mutant testes. This finding suggested that p53 may be involved in the process by which the arrested germ cells are removed from the seminiferous tubules but that other pathways function as well to ensure removal of the arrested spermatocytes.     

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.     

Damaging Effect of Taxol on Mouse Spermatogenesis

Taxol produced a specific effect on mouse spermatocytes I and on stem spermatogonia, which leads to overall degeneration of spermatocytes I and long lasting disorders of spermatogenesis. Breaks of the axial and lateral elements of the synaptonemal complex, aneuploidies in spermatocytes in early, middle, and late pachytene, and accumulation of cells with associations of sex chromosomes and autosomes were observed, which attested to blockade of spermatogenesis in late pachytene and diplotene of meiosis prophase I. Mesna, a chemoprotective agent, reduced total toxicity and lethal effects of taxol, but did not prevent destruction of the testes.     

Reduced expression of DNMT3B in the germ cells of patients with bilateral spermatogenic arrest does not lead to changes in the global methylation status

DNA methylation events during spermatogenesis have important implications for gamete integrity and transmission of epigenetic information to the next generation. However, the role of DNA methyltransferases in the disorders of human spermatogenesis has not been elucidated. The aim of the present study was to evaluate the expression of DNMT3B, crucial for full germ cell methylation, in testicular germ cells of patients with spermatogenic arrest and to determine whether or not there is an association with the global methylation status. In order to determine the DNMTs expression status at various stages of spermatogenesis, immunohistochemical localization was performed on 16 fertile controls having normal spermatogenesis and 11 patients with bilateral spermatogenic arrest. DNMT3B was expressed in most of the germ cell types in both controls and patients with bilateral spermatogenic arrest. The number of DNMT3B positive preleptotene/zygotene cells and pachytene spermatocytes was significantly lower in patients with bilateral arrest. However, evaluation of 5-methylcytosine, a global methylation marker, in the few matured germ cells of these patients did not reveal altered methylation. In conclusion, the global methylation status of germ cells is not affected by spermatogenic defects in spite of aberrant DNMT3B expression indicating the necessity of proper methylation for full spermatogenesis.     

Human spermatogenesis: basic research and clinical issues]

Histological evaluation of human spermatogenesis suffers from the hazy border line between normal and pathological germ cell development. This border line needs better definition for histological fertility diagnosis and the early detection of germ cell tumors. Testicular biopsies from more than 2,900 patients with fertility disturbances and more than 1,900 patients with testicular tumors were investigated by means of semithin sectioning, different immunocytochemical methods and transmission electron microscopy. Cellular systems of the human testes possess a degree of autonomy from the body. Their morphological and functional heterogeneity reveals characteristics of cells that are not terminally differentiated. In the testis of an adult, fertile man not only the proliferation of spermatogonia, maturation divisions of spermatocytes and differentiation of spermatids take place, but also abortive germ cells, as well as apoptotic and degenerative cells appear. Disturbances of spermatogenesis are defined by the evaluation of quantity and quality of germ cell alterations. Compensatory and non compensatory defects of spermatogenesis may be distinguished. Deficiency of spermatogonial cell types, multilayered spermatogonia, megalospermatocytes, malformed spermatids and single tumor cells in the face of sufficient development of mature spermatids are considered compensatory defects of spermatogenesis. Dominating malformed germ cells or tumor cells accompanied by an arrest or lack of spermatogenesis, however, represent non-compensatory defects of spermatogenesis. In addition, normal organization and function of the microvasculature, Leydig cells and compartmentalizing cells in the intertubular space are prerequisites for spermatogenesis. The neuroendocrine function of Leydig cells may be responsible for regulating the blood flow rate and the permeability to hormones and nutritive substances. Finally, for patients a successful definition of the border line between normal and pathological events of germ cell development may be essential for early detection of germ cell tumors. Therefore, anatomical sciences not only contribute to basic research, advanced diagnostics and therapeutic concepts related to diseases of the male gonad, but also to the improvement of assisted reproduction.     

Testicular alterations in the nonobese diabetic mouse

The nonobese diabetic (NOD) mouse was used as a model for evaluating alterations in testicutar structure. The NOD mouse exhibits diabetic manifestations on an apparently autoimmune basis and thus resembles human type 1 insulin-dependent diabetes mellitus. Testicular samples were obtained from animals with and without diabetic manifestations at various ages. The principal changes noted on histological examination were in the seminiferous tubules, including germ cell degeneration, disruption of spermatogenesis, tubular fibrosis, and intratubular calcification. These effects were diffuse and extensive in animals with severe hyperglycemia. In those with normal and borderline elevated glucose levels, focal tubular damage was found to occur in a patchy distribution. The changes included disorganization of maturation, malorientation of spermatids, and hypospermatogenesis. Electron microscopic studies also indicated effects on Sertoli cells and the peritubular tissue. There was no evidence of mononuclear cell infiltration or vascular abnormalities in any of the testicular specimens. The findings indicate that the NOD mouse may be a useful model for understanding the development of pathological changes in the testis in insulin-dependent diabetes.     

Testis structure in the sys (symplastic spermatids) mouse

Testes of mice with the recessive insertional mutation termed symplastic spermatids (sys) were assessed for structural and developmental abnormalities. Homozygous (sys/sys) males are infertile due to an abnormality in spermatogenesis leading to azoospermia. The major interruption to spermatogenesis occurs when the intercellular bridges that connect round spermatids open prematurely resulting in the formation of symplasts. Symplasts contain as many as 285 nuclei. Development of spermatids within symplasts is arrested just before, or just after, elongation of the spermatid nuclei begins. Symplasts degenerate and appear to be phagocytized by Sertoli cells and by intratubular macrophages. In addition, degeneration of young round spermatids and also spermatocytes occasionally is observed. Spermatocyte degeneration is substantial in some tubules and leaves them depleted of cells other than basal compartment cells. Sertoli cell abnormalities are prominent and include intracellular vacuolation, absence of apical processes surrounding round spermatids, degeneration, and occasional sloughing. Although reduplication and infolding of the basal lamina is also seen, this does not appear as a common phenomenon. The sys phenotype is first manifest in animals between 19 days and 22 days of age. Considerable variability is seen in testis histology of prepubertal animals; some display degenerating pachytene spermatocytes and virtually no Sertoli cell vacuoles, while others display vacuoles without apparent elevated numbers of degenerating spermatocytes. Although this study has not revealed the primary cell type(s) affected by the insertional inactivation event, it is possible that the abnormalities in the Sertoli cells are responsible for germ cell degeneration as it is generally recognized that deficits in the Sertoli cell can result in major germ cell abnormalities but not vice versa.     

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.     

Retinoids and spermatogenesis: lessons from mutant mice lacking the plasma retinol binding protein.

Using Rbp4-null mice as models, we have established for the first time the kinetics of the spermatogenetic alterations during vitamin A deficiency (VAD). Our data demonstrate that the VAD-induced testicular degeneration arises through the normal maturation of germ cells in a context of spermatogonia differentiation arrest. They indicate that retinoic acid (RA) appears dispensable for the transition of premeiotic to meiotic spermatocytes, meiosis, and spermiogenesis. They confirm that RA plays critical roles in controlling spermatogonia differentiation, spermatid adhesion to Sertoli cells, and spermiation, and suggest that the VAD-induced arrest of spermatogonia differentiation results from simultaneous blocks in RA-dependent events mediated by RA receptor gamma (RARgamma) in spermatogonia and by RARalpha in Sertoli cells. They also provide evidence that expression of major RA-metabolizing enzymes is increased in mouse Sertoli cells upon VAD and that vitamin A-deficient A spermatogonia differ from their RA-sufficient counterparts by the expression of the Stra8 gene.     

Changes in seminiferous tubules after vasectomy

The histologic, immunohistochemical, and ultrastructural changes in the seminiferous tubules (ST) of 17 healthy adult males who had been vasectomized between 1 1/2 and 5 years are reported. There was minimal spermatogenesis in 4 cases. 8 of the cases underwent electron microscopy examination and 4 showed evidence of minimal histological spermatogenesis. The ST showed a thickening of the basement membrane and heavy deposits of lipofuchsin in the Sertoli cells (SC) seen as lipid infiltration. The spermatogenic cells presented variable changes characterized by the disorientation of cells, maturation arrest, and premature sloughing. No immune complex deposits were seen. Of the 9 cases followed, pregnancy occurred in 2 following vas reanastomosis; a regenerative capacity of the ST was seen in 6 cases. The follow-up studies also show that the morphological alterations initially produced by the vasectomy have little affect on the appearance of spermatogenesis after vas reanastomosis. Thus, we see that the vasectomy exerts its major effect on the SC and that the damage to these cells alters the testicular environment. However, these changes are apparently reversible following vasovasostomy.     

Fas expression correlates with human germ cell degeneration in meiotic and post-meiotic arrest of spermatogenesis

Degeneration of human male germ cells was analysed by means of light (LM) and transmission electron (TEM) microscopy. The frequency of degenerating cells was correlated with that of Fas-expressing germ cells in human testes with normal spermatogenesis (n = 10), complete early maturation arrest (EMA) (n = 10) or incomplete late maturation arrest (LMA; n = 10) of spermatogenesis. LM analysis of testis sections with normal spermatogenesis indicated that degenerating germ cells were localized in the adluminal compartment of the seminiferous epithelium. TEM showed that apoptotic cells were mostly primary spermatocytes and, to a lesser extent, round or early elongating spermatids. Apoptotic germ cells appeared to be eliminated either in the seminiferous lumen or by Sertoli cell phagocytosis. An increased number of degenerating cells was observed in testes with LMA as compared with normal testes and testes with EMA of spermatogenesis (P < 0.001, Wilcoxon's rank sum test). Comparison of these results with those obtained from immunohistochemistry experiments demonstrated a tight correlation between the number of apoptotic cells and the number of Fas-expressing germ cells (P = 0.001, Spearman's rank = 0.69). These findings suggest that altered meiotic and post-meiotic germ cell maturation might be associated with an up-regulation of Fas gene expression capable of triggering apoptotic elimination of defective germ cells.     

Histologic classification of undescended testes

On the basis of testicular biopsy study in 203 patients and study of a second biopsy specimen from 27 of these patients, prepubertal undescended testes were classified into four categories according to the mean tubular diameter, the tubular fertility index, and the Sertoli cell index. Type I cases (testes with minimal lesions) were characterized by a normal mean tubular diameter and normal tubular fertility and Sertoli cell indexes or slight tubular hypoplasia. This group represented 26 per cent of the undescended testes. The corresponding lesions can be observed from two years of age onward and are probably acquired. After puberty normal spermatogenesis occurs. Type II cases (24 per cent of the undescended testes) included testes with marked germinal hypoplasia as well as slight or marked tubular hypoplasia and a normal Sertoli cell index. After puberty these testes develop a degree of marked hypospermatogenesis, maturation arrest, or Sertoli cells with only isolated spermatogonia and primary spermatocytes. In type III cases (testes with diffuse tubular hypoplasia) the mean tubular diameter and the tubular fertility and Sertoli cell index values were severely reduced. This group represented 33 per cent of the undescended testes, and after puberty most of them showed seminiferous tubules with exclusively adult Sertoli cells. Type IV testes (diffuse Sertoli cell hyperplasia) were associated with a nearly normal mean tubular diameter and variable tubular fertility index values and represented 17 per cent of all the undescended testes. After puberty Sertoli cells do not mature completely, and therefore in spite of the earlier tubular fertility index, the germinal cell line does not reach adult development. Although early orchiopexy prevents tubular fertility index and mean tubular diameter deterioration due to the noxious effects of temperature in type I testes, we believe that there is no such benefit in the other types. These patients may present only slight modifications in these indexes.     

Early effects of vinblastine and vincristine on the rat spermatogenesis: analyses by a new transillumination-phase contrast microscopic method.

Male sterility belongs to the recently recognized complications of cancer chemotherapy and has an increasing importance. Therefore, more information about the mode of action of anticancer drugs on mammalian spermatogenesis is needed. We have developed a technique based on transillumination of living, freshly isolated unstained rat seminiferous tubules, where the cells specifically killed by the drugs are recognized as dull zones. Early stages of cell degeneration can be rapidly analyzed by phase contrast microscopy of living cells. Because the transillumination technique, in addition, permits an accurate recognition of the segments of the seminiferous epithelial wave, the cells representing various stages of the mitotic and meiotic cell cycles during spermatogenesis can be isolated in living state for morphological analysis. Vinblastine and vincristine cause an arrest of mitotic and meiotic divisions to metaphase followed by cell death, which was more rapid after vincristine administration. Both alkaloids had a slight damaging effect on the pachytene spermatocytes. Large doses of both drugs primarily affected the Sertoli cells by destroying their microtubules and mitochondria. Vincristine specifically damaged the acrosomic system and the cytoplasmic bridges of the young spermatids.     

The distribution pattern of cytokeratin and vimentin immunoreactivity in testicular biopsies of infertile men

Testicular biopsies of infertile patients are often characterized by a mixed atrophy, in which different types of spermatogenic lesions are found in adjacent tubules. In order to evaluate a possible involvement of the state of differentiation of the Sertoli cells, the distribution pattern of cytokeratin and vimentin intermediate filaments within the seminiferous epithelium of 228 biopsy specimens with normal spermatogenesis (n=10), mixed atrophy (n= 206) or Sertoli Cell Only Syndrome (n=12) were investigated by means of immunohistochemical techniques. Sertoli cells were regularly found to show vimentin expression in tubules with normal spermatogenesis as well as in tubules with any kind of spermatogenic impairment including SCO. Cytokeratin expression as a marker showing lack of differentiation was common in Sertoli cells of tubules with arrest of spermatogenesis at the level of spermatogonia, and was occasionally associated with arrest at the level of primary spermatocytes or with SCO. Ultrastructural examination of tubules with spermatogonial arrest revealed Sertoli cells with features of typical fetal or prepubertal Sertoli cells, such as round to ovoid nuclei without indentations, stacks of rough ER and spot desmosomes. These data suggest that spermatogenic arrest at the level of spermatogonia might be due to functional impairment of the associated Sertoli cells, which have maintained or regained an undifferentiated state and are not able to initiate or trigger the process of spermatogonial differentiation.     

An azoospermic man with a double-strand DNA break-processing deficiency in the spermatocyte nuclei: case report

BACKGROUND: The mechanisms of meiotic arrest in human spermatogenesis are poorly known. METHODS AND RESULTS: A testicular biopsy from an azoospermic male showed complete spermatogenesis arrest at the spermatocyte stage, asynapsis, lack of formation of the XY body, partial reversion to a mitotic-like division and cell degeneration both at the prophase and at the abnormal cell divisions. Synaptonemal complex analysis showed minor segments of synapsis and mainly single axes. Fluorescent immunolocalization of meiotic proteins showed normal SYCP3, scarcity of SYCP1, null MLH1 foci, about 10 patches of gamma-H2AX, abnormal presence of BRCA1 among autosomal axes, absence of RAD51 in early and advanced spermatocytes and permanence of gamma-H2AX labelling up to the abnormal spermatocyte divisions that are the most advanced stage reached. There are at least six dominions of evenly packed chromatin resembling that of the normal XY body, but no true XY body. CONCLUSIONS: The protein phenotype and the fine structure of the nuclei are compatible with a deficiency of the processing of double-strand DNA breaks in the zygotene-like spermatocytes, but the features of this defect do not agree with Spo11, Sycp1, Atm and Dmc1 null mutations, which give absence of XY body, synapsis disturbances and spermatocyte apoptosis in mice.     

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 prevalence of a YY synaptonemal complex over XY synapsis in an XYY man with exclusive XYY spermatocytes

An infertile XYY man was studied by synaptonemal complex analysis of microspread spermatocytes and by quantitation of germ cells in semithin sections. All the 74 spermatocytes micrographed have an XYY constitution, and the biopsy shows a homogeneous arrest of spermatogenesis at the spermatocyte/young spermatid stages. The overwhelming majority (86%) of spermatocytes showed a Y—Y bivalent plus a univalent X. The Y—Y bivalent is totally synapsed in 48% of the cells. In the remaining cells, the YY bivalent has an average synaptic segment covering 43% of its length that always includes Yp. Another 9% of the spermatocytes showed an XYY trivalent and 4% of the spermatocytes showed univalence of the three gonosomes. Progression through all the pachytene substages was observed in cells with the two main synaptic configurations, but a high level of germ cell death was observed at or immediately after the meiotic divisions. The prevalence of Y—Y synapsis arises from the longer homologous region and the higher speed of pairing between the two Y chromosomes. Germ cell death is probably related to the univalence of the X chromosome. Synaptic competition between three gonosomes seems to be similar to that found in triploid birds but is somewhat different from that of XYY mice.This revised version was published online in November 2005 with corrections to the Cover Date.     

Germ cell degeneration in normal and microwave-irradiated rats: potential sperm production rates at different developmental steps in spermatogenesis

Germ cell degeneration in 14 normal and 14 microwave-irradiated, adult (400-500 gm), Sprague-Dawley rats was compared by evaluating potential sperm production rates at different developmental steps in spermatogenesis. Following 9 days of irradiation at 1.3 GHz (6 hours/day at 6.3 mW/gm using 1-mu sec pulsewidth at 600 pulses/second) or sham treatment, rats were killed at 6.5, 13.0, 26.0, or 52.0 days following treatment. Testes were perfused with 2% glutaraldehyde, embedded in Epon, and sectioned at 0.5 micron for morphometric analyses. Plasma LH and FSH concentrations were determined by radioimmunoassay from blood collected on the day of death. Considering nuclear size, percentage of nuclei in the parenchyma, and life span of different cells, potential daily sperm production was determined for type B spermatogonia, preleptotene or pachytene primary spermatocytes, or spermatids with round nuclei. No differences (P greater than .05) in parameters tested were found among time periods following irradiation. With the possible exception of sperm production per testis (P less than .05) based on pachytene spermatocytes, microwave irradiation had no effect on the parameters evaluated. No degeneration was detected in spermatogenesis when potential sperm production rates were determined either from type B spermatogonia to spermatids or from type B spermatogonia to a posttesticular approximation of sperm production rate. Thus, it appears that regulation of sperm production rates must take place during spermatogonial mitoses, since once the number of type B spermatogonia is determined, there is essentially no subsequent alteration in sperm production potential in normal or irradiated adult rats.