A combined approach facilitates the reliable detection of human spermatogonia in vitro.

STUDY QUESTION: Does a combined approach allow for the unequivocal detection of human germ cells and particularly of spermatogonia in vitro? SUMMARY ANSWER: Based on our findings, we conclude that an approach comprising: (i) the detailed characterization of patients and tissue samples prior to the selection of biopsies, (ii) the use of unambiguous markers for the characterization of cultures and (iii) the use of biopsies lacking the germ cell population as a negative control is the prerequisite for the establishment of human germ cell cultures. WHAT IS KNOWN ALREADY: The use of non-specific marker genes and the failure to assess the presence of testicular somatic cell types in germ cell cultures may have led to a misinterpretation of results and the erroneous description of germ cells in previous studies. STUDY DESIGN, SIZE, DURATION: Testicular biopsies were selected from a pool of 264 consecutively obtained biopsies. Based on the histological diagnosis, biopsies with distinct histological phenotypes were selected (n = 35) to analyze the expression of germ cell and somatic cell markers. For germ cell culture experiments, gonadotrophin levels and clinical data were used as selection criteria resulting in the following two groups: (i) biopsies with qualitatively intact spermatogenesis (n = 4) and (ii) biopsies from Klinefelter syndrome Klinefelter patients lacking the germ cell population (n = 3). PARTICIPANTS/MATERIALS, SETTING, METHODS: Quantitative real-time PCR analyses were performed to evaluate the specificity of 18 selected germ cell and 3 somatic marker genes. Cell specificity of individual markers was subsequently validated using immunohistochemistry. Finally, testicular cell cultures were established and were analyzed after 10 days for the expression of germ cell- (UTF1, FGFR3, MAGE A4, DDX4) and somatic cell-specific markers (SMA, VIM, LHCGR) at the RNA and the protein levels. MAIN RESULTS AND THE ROLE OF CHANCE: Interestingly, only 9 out of 18 marker genes reflected the presence of germ cells and cell specificity could be validated using immunohistochemistry. Furthermore, VIM, SMA and LHCGR were found to reflect the presence of testicular somatic cells at the RNA and the protein levels. Using this validated marker panel and biopsies lacking the germ cell population (n = 3) as a negative control, we demonstrated that germ cell cultures containing spermatogonia can be established from biopsies with normal spermatogenesis (n = 4) and that these cultures can be maintained for the period of 10 days. However, marker profiling has to be performed at regular time points as the composition of testicular cell types may continuously change under longer term culture conditions. LIMITATIONS, REASONS FOR CAUTION: There are significant differences regarding the spermatogonial stem cell (SSC) system and spermatogenesis between rodents and primates. It is therefore possible that marker genes that do not reflect the presence of spermatogonia in the human are specific for spermatogonia in other animal models. WIDER IMPLICATIONS OF THE FINDINGS: While some studies have reported that human SSCs can be maintained in vitro and show characteristics of pluripotency, the germ cell origin and the differentiation potential of these cells were subsequently called into question. This study provides critical insights into possible sources for the misinterpretation of results regarding the presence of germ cells in human testicular cell cultures and our findings can therefore help to avoid conflicting reports in the future. STUDY FUNDING/COMPETING INTEREST(S): This project was supported by the Stem Cell Network North Rhine-Westphalia and the Innovative Medical Research of the University of Münster Medical School (Grant KO111014). In addition, it was funded by the DFG-Research Unit FOR 1041 Germ Cell Potential (GR 1547/11-1 and SCHL 394/11-2), the BMBF (01GN0809/10) and the IZKF (CRA 03/09). The authors declare that there is no conflict of interest. TRIAL REGISTRATION NUMBER: Not applicable.

The RHOX homeobox gene cluster is selectively expressed in human oocytes and male germ cells.

STUDY QUESTION: What human tissues and cell types express the X-linked reproductive homeobox (RHOX) gene cluster? SUMMARY ANSWER: The RHOX homeobox genes and proteins are selectively expressed in germ cells in both the ovary and testis. WHAT IS KNOWN ALREADY: The RHOX homeobox transcription factors are encoded by an X-linked gene cluster whose members are selectively expressed in the male and female reproductive tract of mice and rats. The Rhox genes have undergone strong selection pressure to rapidly evolve, making it uncertain whether they maintain their reproductive tissue-centric expression pattern in humans, an issue we address in this report. STUDY DESIGN, SIZE, DURATION: We examined the expression of all members of the human RHOX gene cluster in 11 fetal and 8 adult tissues. The focus of our analysis was on fetal testes, where we evaluated 16 different samples from 8 to 20 weeks gestation. We also analyzed fixed sections from fetal testes, adult testes and adult ovaries to determine the cell type-specific expression pattern of the proteins encoded by RHOX genes. PARTICIPANTS/MATERIALS, SETTING, METHODS: We used quantitative reverse transcription-polymerase chain reaction analysis to assay human RHOX gene expression. We generated antisera against RHOX proteins and used them for western blotting, immunohistochemical and immunofluorescence analyses of RHOXF1 and RHOXF2/2B protein expression. MAIN RESULTS AND THE ROLE OF CHANCE: We found that the RHOXF1 and RHOXF2/2B genes are highly expressed in the testis and exhibit low or undetectable expression in most other organs. Using RHOXF1- and RHOXF2/2B-specific antiserum, we found that both RHOXF1 and RHOXF2/2B are primarily expressed in germ cells in the adult testis. Early stage germ cells (spermatogonia and early spermatocytes) express RHOXF2/2B, while later stage germ cells (pachytene spermatocytes and round spermatids) express RHOXF1. Both RHOXF1 and RHOXF2/2B are expressed in prespermatogonia in human fetal testes. Consistent with this, RHOXF1 and RHOXF2/2B mRNA expression increases in the second trimester during fetal testes development when gonocytes differentiate into prespermatogonia. In the human adult ovary, we found that RHOXF1 and RHOXF2/2B are primarily expressed in oocytes. LIMITATIONS, REASONS FOR CAUTION: While the average level of expression of RHOX genes was low or undetectable in all 19 human tissues other than testes, it is still possible that RHOX genes are highly expressed in a small subset of cells in some of these non-testicular tissues. As a case in point, we found that RHOX proteins are highly expressed in oocytes within the human ovary, despite low levels of RHOX mRNA in the whole ovary. WIDER IMPLICATIONS OF THE FINDINGS: The cell type-specific and developmentally regulated expression pattern of the RHOX transcription factors suggests that they perform regulatory functions during human fetal germ cell development, spermatogenesis and oogenesis. Our results also raise the possibility that modulation of RHOX gene levels could correct some cases of human infertility and that their encoded proteins are candidate targets for contraceptive drug design.

The pluripotency factor LIN28 in monkey and human testes: a marker for spermatogonial stem cells?

Mammalian spermatogenesis is maintained by spermatogonial stem cells (SSCs). However, since evidentiary assays and unequivocal markers are still missing in non-human primates (NHPs) and man, the identity of primate SSCs is unknown. In contrast, in mice, germ cell transplantation studies have functionally demonstrated the presence of SSCs. LIN28 is an RNA-binding pluripotent stem cell factor, which is also strongly expressed in undifferentiated mouse spermatogonia. By contrast, two recent reports indicated that LIN28 is completely absent from adult human testes. Here, we analyzed LIN28 expression in marmoset monkey (Callithrix jacchus) and human testes during development and adulthood and compared it with that in mice. In the marmoset, LIN28 was strongly expressed in migratory primordial germ cells and gonocytes. Strikingly, we found a rare LIN28-positive subpopulation of spermatogonia also in adult marmoset testis. This was corroborated by western blotting and quantitative RT-PCR. Importantly, in contrast to previous publications, we found LIN28-positive spermatogonia also in normal adult human and additional adult NHP testes. Some seasonal breeders exhibit a degenerated (involuted) germinal epithelium consisting only of Sertoli cells and SSCs during their non-breeding season. The latter re-initiate spermatogenesis prior to the next breeding-season. Fully involuted testes from a seasonal hamster and NHP (Lemur catta) exhibited numerous LIN28-positive spermatogonia, indicating an SSC identity of the labeled cells. We conclude that LIN28 is differentially expressed in mouse and NHP spermatogonia and might be a marker for a rare SSC population in NHPs and man. Further characterization of the LIN28-positive population is required.

Misleading and reliable markers to differentiate between primate testis-derived multipotent stromal cells and spermatogonia in culture.

BACKGROUND: Several studies have reported the generation of spermatogonia-derived pluripotent stem cells from human testes. The initial aim of the present study was the derivation of equivalent stem cells from an established and experimentally accessible non-human primate model, the common marmoset monkey (Callithrix jacchus). However, an essential prerequisite in the absence of transgenic reporters in primates and man is the availability of validated endogenous markers for the identification of specific cell types in vitro. METHODS AND RESULTS: We cultured marmoset testicular cells in a similar way to that described for human testis-derived pluripotent cells and set out to characterize these cultures under different conditions and in differentiation assays applying established marker panels. Importantly, the cells emerged as testicular multipotent stromal cells (TMSCs) instead of (pluripotent) germ cell-derived cells. TMSCs expressed many markers such as GFR-α, GPR125, THY-1 (CD90), ITGA6, SSEA4 and TRA-1-81, which were considered as spermatogonia specific and were previously used for the enrichment or characterization of spermatogonia. Proliferation of TMSCs was highly dependent on basic fibroblast growth factor, a growth factor routinely present in germ cell culture media. As reliable markers for the distinction between spermatogonia and TMSCs, we established VASA, in combination with the spermatogonia-expressed factors, MAGEA4, PLZF and SALL4. CONCLUSIONS: Marmoset monkey TMSCs and spermatogonia exhibit an overlap of markers, which may cause erroneous interpretations of experiments with testis-derived stem cells in vitro. We provide a marker panel for the unequivocal identification of spermatogonia providing a better basis for future studies on primate, including human, testis-derived stem cells.

Developmental expression of the pluripotency factor sal-like protein 4 in the monkey, human and mouse testis: restriction to premeiotic germ cells.

SALL4 (sal-like protein 4) is a pluripotency transcription factor, which is highly expressed in embryonic stem (ES) cells and which is essential for mouse preimplantation development. In adult mouse organs, Sall4 mRNA is highly expressed in the testis and ovary, while there is only little or no expression in other organs. There is also a high expression of SALL4 in human testicular germ cell tumors. However, there is as yet no detailed analysis of SALL4 expression during mammalian testicular development. We analyzed SALL4 expression in ES cells, preimplantation embryos, and the developing and adult testis of a nonhuman primate (NHP) species, the common marmoset monkey (Callithrix jacchus). Immunofluorescence revealed SALL4 in the nuclei of marmoset ES cells and preimplantation embryos. Marmoset SALL4 isoform analysis in ES cells and newborn and adult testis by RT- PCR and Western blotting showed two different isoforms, SALL4-A and SALL4-B. Immunohistochemistry localized this transcription factor to the nuclei of primordial germ cells and most gonocytes in the prenatal and early postnatal marmoset testis. In the pubertal and adult testis SALL4 was present in undifferentiated spermatogonia. In the developing and adult human and mouse testis SALL4 expression mimicked the pattern in the marmoset. Adult testes from additional NHP species, the treeshrew, the cat and the dog also exhibited SALL4 in undifferentiated spermatogonia, indicating a conserved expression in the mammalian testis. Taking into account the importance of SALL4 for mouse development, we conclude that SALL4 may play an important role during mammalian germ cell development and is involved in the regulation of spermatogonial proliferation in the adult testis.

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.

Molecular cloning and functional characterization of endogenous recombinant common marmoset monkey (Callithrix jacchus) follicle-stimulating hormone.

BACKGROUND: Common marmoset monkeys (Callithrix jacchus) are readily used in biomedical research. However, superovulation for embryonic stem cell production and developmental research still remain difficult. Inexplicably, follicle-stimulating hormone (FSH) as key player in superovulation has to be administered in extremely high dosages in this non-human primate compared to human. METHODS: To evaluate whether marmoset FSH (cjFSH) is functionally more competent than its human homologue on the marmoset FSH receptor (cjFSHR), we established in vitro a homologous system characterizing homologous and recombinantly produced cjFSH. RESULTS: Upon stimulation of two cell lines stably expressing either the marmoset or the human FSH receptor (cj/hFSHR), respectively, the second messenger signaling of the activated receptor displayed no significant difference in ED(50) values. Thermostability of cjFSH was significantly prolonged by roughly 20% on both FSHRs. CONCLUSION: High FSH dosage in marmoset superovulation cannot be explained by enhanced biopotency of the natural animal's gonadotropin.

Variation in CAG and GGN repeat lengths and CAG/GGN haplotype in androgen receptor gene polymorphism and prostate carcinoma in Nigerians.

Prostate cancer has become the most common cancer in Nigerian men. The growth of the prostate gland depends on circulating androgens and intracellular steroid signalling pathways. The effects of androgens are mediated through the androgen receptor (AR), a nuclear transcription factor encoded by the AR gene. The common polymorphisms, CAG and GGN repeats, in exon 1 of this gene have been implicated as possible risk factors. Thus far, existing supporting data are scanty and none are from sub-Saharan African populations. Therefore, this study investigates the possible association between AR polymorphism repeat length (CAG and GGN) and prostate cancer in Nigerians. A total of 261 subjects (70 with prostate cancer, 68 with benign prostate hyperplasia [BPH], 123 age-matched apparently normal subjects as controls) were studied. CAG and GGN repeats length were determined by fragment length analysis using GeneScan. The CAG repeat length in prostate cancer and in BPH compared to the controls was significantly different (P < 0.05) with reduce length of CAG repeats showing a significant odds ratio (OR) in both cases. However, this was not observed in GGN repeat length, which showed no significant difference between cases and controls (P > 0.05). CAG and GGN haplotype variation showed no significant difference between cases and controls (P > 0.05), except that the haplotypes CAG > or =21 and GGN < or =21 were more common in the control group. The results of this study, the first from sub-Saharan Africa, supports the hypothesis that reduced CAG repeat length is a risk factor for prostate cancer, and also suggests an association with BPH.

Novel genetic aspects of Klinefelter's syndrome.

Klinefelter's syndrome (KS) is the most common chromosome aneuploidy in males, characterized by at least one supernumerary X chromosome. Although extensively studied, the pathophysiology, i.e. the link between the extra X and the phenotype, largely remains unexplained. The scope of this review is to summarize the progress made in recent years on the role of the supernumerary X chromosome with respect to its putative influence on the phenotype. In principal, the parental origin of the X chromosome, gene-dosage effects in conjunction with (possibly skewed) X chromosome inactivation, and--especially concerning spermatogenesis--meiotic failure may play pivotal roles. One of the X chromosomes is inactivated to achieve dosage-compensation in females and probably likewise in KS. Genes from the pseudoautosomal regions and an additional 15% of other genes, however, escape X inactivation and are candidates for putatively constituting the KS phenotype. Examples are the SHOX genes, identified as likely causing the tall stature regularly seen in KS. Lessons learned from comparisons with normal males and especially females as well as other sex chromosomal aneuploidies are presented. In addition, genetic topics concerning fertility and counseling are discussed.

Germ cell dynamics in the testis of the postnatal common marmoset monkey (Callithrix jacchus).

The seminiferous epithelium in the nonhuman primate Callithrix jacchus is similarly organized to man. This monkey has therefore been used as a preclinical model for spermatogenesis and testicular stem cell physiology. However, little is known about the developmental dynamics of germ cells in the postnatal primate testis. In this study, we analyzed testes of newborn, 8-week-old, and adult marmosets employing immunohistochemistry using pluripotent stem cell and germ cell markers DDX4 (VASA), POU5F1 (OCT3/4), and TFAP2C (AP-2γ). Stereological and morphometric techniques were applied for quantitative analysis of germ cell populations and testicular histological changes. Quantitative RT-PCR (qRT-PCR) of testicular mRNA was applied using 16 marker genes establishing the corresponding profiles during postnatal testicular development. Testis size increased during the first 8 weeks of life with the main driver being longitudinal outgrowth of seminiferous cords. The number of DDX4-positive cells per testis doubled between birth and 8 weeks of age whereas TFAP2C- and POU5F1-positive cells remained unchanged. This increase in DDX4-expressing cells indicates dynamic growth of the differentiated A-spermatogonial population. The presence of cells expressing POU5F1 and TFAP2C after 8 weeks reveals the persistence of less differentiated germ cells. The mRNA and protein profiles determined by qRT-PCR and western blot in newborn, 8-week-old, and adult marmosets corroborated the immunohistochemical findings. In conclusion, we demonstrated the presence of distinct spermatogonial subpopulations in the primate testis exhibiting different dynamics during early testicular development. Our study demonstrates the suitability of the marmoset testis as a model for human testicular development.

Idiopathic male infertility is strongly associated with aberrant methylation of MEST and IGF2/H19 ICR1.

Aberrant imprinting in spermatozoa in a subset of infertile men has been postulated to be a risk factor for congenital diseases in children conceived via assisted reproduction techniques (ART). Studies in clinically well characterized large cohorts, however, have been missing. Using bisulfite sequencing, we determined the degree of methylation of the IGF2/H19 imprinting control region 1 (ICR1) and MEST differentially methylated regions in swim-up purified spermatozoa from 148 idiopathic infertile men and 33 normozoospermic controls. All control individuals had a high degree of IGF2/H19 ICR1 and a low degree of MEST methylation. Low sperm counts were clearly associated with IGF2/H19 ICR1 hypomethylation and, even stronger, with MEST hypermethylation. MEST hypermethylation, but not IGF2/H19 ICR1 hypomethylation was found in idiopathic infertile men with progressive sperm motility below 40% and bad sperm morphology below 5% normal spermatozoa. Ageing could be ruled out as a cause for the observed methylation defects. Sequence analysis of the CTCFL gene in peripheral blood DNA from 20 men with severe methylation defects revealed several polymorphisms, but no bona fide mutation. We conclude that idiopathic male infertility is strongly associated with imprinting defects at IGF2/H19 ICR1 and MEST, with aberrant MEST methylation being a strong indicator for sperm quality. The male germ cell thus represents a potential source for aberrant epigenetic features in children conceived via ART.

A common haplotype of protamine 1 and 2 genes is associated with higher sperm counts.

Sperm chromatin compaction in the sperm head is achieved when histones are replaced by protamines during spermatogenesis. Haploinsufficiency of the protamine 1 (PRM1) or PRM2 gene causes infertility in mice. However, the published data remain inconclusive about a role of PRM1/2 variants in male infertility and their association with semen parameters. By full sequence analysis, we assessed the frequency of sequence variations in PRM1 and PRM2 in three groups of Caucasian patients with idiopathic teratozoospermia and normal (n = 88) or reduced sperm concentration (n = 83) and in men with a high percentage of normal sperm morphology and normal concentrations (n = 77). Two rare (c.54G>A and c.102G>T) and one common SNP (c.230A>C) were identified in PRM1. In PRM2, some rare heterozygous mutations and the two common intronic SNPs 298G>C and 373C>A were detected. None of the PRM1/2 variants was associated with teratozoospermia or individually with other semen parameters. However, significant linkage disequilibrium was detected between the common SNPs of PRM1 and PRM2 which formed haplotypes. Analysis of the pooled group (n = 248) revealed that homozygous carriers of the common haplotype ACC had a twofold higher sperm concentration and count than men lacking this haplotype, with sperm counts of heterozygotes for ACC being midway between the homozygotes. This markedly decreased sperm output might either be caused by spermatozoa lacking the ACC haplotype not being viable, or subject to negative selection. In addition, a significant deviation from Hardy-Weinberg-Equilibrium of these SNPs might indicate natural selection in favour of the ACC allele which leads to higher sperm output and therefore better fertility. In conclusion, for the first time we describe an association of a common haplotype formed by PRM1 and PRM2 with sperm output in a large group of men.

Phenotypic variation within European carriers of the Y-chromosomal gr/gr deletion is independent of Y-chromosomal background.

BACKGROUND: Previous studies have compared sperm phenotypes between men with partial deletions within the AZFc region of the Y chromosome and non-carriers, with variable results. In this study, a separate question was investigated, the basis of the variation in sperm phenotype within gr/gr deletion carriers, which ranges from normozoospermia to azoospermia. Differences in the genes removed by independent gr/gr deletions, the occurrence of subsequent duplications or the presence of linked modifying variants elsewhere on the chromosome have been suggested as possible causal factors. This study set out to test these possibilities in a large sample of gr/gr deletion carriers with known phenotypes spanning the complete range. RESULTS: In total, 169 men diagnosed with gr/gr deletions from six centres in Europe and one in Australia were studied. The DAZ and CDY1 copies retained, the presence or absence of duplications and the Y-chromosomal haplogroup were characterised. Although the study had good power to detect factors that accounted for >or=5.5% of the variation in sperm concentration, no such factor was found. A negative effect of gr/gr deletions followed by b2/b4 duplication was found within the normospermic group, which remains to be further explored in a larger study population. Finally, significant geographical differences in the frequency of different subtypes of gr/gr deletions were found, which may have relevance for the interpretation of case control studies dealing with admixed populations. CONCLUSIONS: The phenotypic variation of gr/gr carriers in men of European origin is largely independent of the Y-chromosomal background.

Androgen receptor gene CAG and GGN polymorphisms in infertile Nigerian men.

The human androgen receptor gene (AR) is an important regulator of male sexual development including spermatogenesis. Exon 1 of this gene encodes the N terminal domain, which controls transcriptional activity of the receptor and the two polymorphic repeats CAG and GGN. Many studies have reported association of the expanded CAG repeat length with male infertility, although this is still controversial. The GGN repeat, in contrast, has been less thoroughly studied. Thus far, only scanty studies have been reported from African populations and none from Nigeria. Therefore, we have investigated the possible association between AR polymorphism repeats length (CAG and GGN) and reduced spermatogenesis in infertile Nigerian men (no.=60) consisting of 20 non-obstructive azoospermic and 40 oligozoospermic subjects compared with controls with normozoospermia and proven evidence of fertility (no.=38). In addition, 48 volunteers with normal spermatogenesis were recruited from a German population. CAG and GGN repeats length were determined by fragment length analysis using GeneScan. The CAG and GGN repeats length of infertile compared to fertile populations were not significantly different (p>0.05). We found a unique AR GGN allele distribution with 20-23 GGN repeats predominant in the Nigerian study population. Our results show that CAG and GGN repeats polymorphisms are not a critical index of male infertility. While we do not find a relationship with CAG and GGN repeats haplotypes and male infertility, we report for the first time a unique and wider distribution of the GGN allele in the Nigerian population which is significantly different from the Caucasian population. The functional relevance of this variance to male fertility warrants in-depth elucidation.

Ageing in men with normal spermatogenesis alters spermatogonial dynamics and nuclear morphology in Sertoli cells.

BACKGROUND: Ageing in men is believed to be associated with fertility decline and elevated risk of congenital disorders for the offspring. The previous studies also reported reduced germ and Sertoli cell numbers in older men. However, it is not clear whether ageing in men with normal spermatogenesis affects the testis and germ cell population dynamics in a way sufficient for transmitting adverse age effects to the offspring. OBJECTIVES: We examined men with normal spermatogenesis at different ages concerning effects on persisting testicular cell types, that is the germ line and Sertoli cells, as these cell populations are prone to be exposed to age effects. MATERIAL AND METHODS: Ageing was assessed in testicular biopsies of 32 patients assigned to three age groups: (i) 28.8 ± 2.7 years; (ii) 48.1 ± 1 years; and (iii) 70.9 ± 6.2 years, n = 8 each, with normal spermatogenesis according to the Bergmann-Kliesch score, and in a group of meiotic arrest patients (29.9 ± 3.8 years, n = 8) to decipher potential links between different germ cell types. Besides morphometry of seminiferous tubules and Sertoli cell nuclei, we investigated spermatogenic output/efficiency, and dynamics of spermatogonial populations via immunohistochemistry for MAGE A4, PCNA, CREM and quantified A-pale/A-dark spermatogonia. RESULTS: We found a constant spermatogenic output (CREM-positive round spermatids) in all age groups studied. In men beyond their mid-40s (group 2), we detected increased nuclear and nucleolar size in Sertoli cells, indirectly indicating an elevated protein turnover. From the 7th decade (group 3) of life onwards, testes showed increased proliferation of undifferentiated spermatogonia, decreased spermatogenic efficiency and elevated numbers of proliferating A-dark spermatogonia. DISCUSSION AND CONCLUSION: Maintaining normal sperm output seems to be an intrinsic determinant of spermatogenesis. Ageing appears to affect this output and might provoke compensatory proliferation increase in A spermatogonia which, in turn, might hamper germ cell integrity.

[Genetics of male infertility]. / Genetik der männlichen Infertilität.

Genetic causes of male infertility increase in frequency with decreasing sperm concentration (oligo-/azoospermia). The decision about genetic tests should be made after a complete andrological work-up. Common causes comprise chromosomal aberrations (including Klinefelter syndrome), microdeletions of the AZF loci of the Y chromosome, mutations in the gene responsible for cystic fibrosis (CFTR) causing CBAVD and in genes involved in hypogonadotropic hypogonadism (including Kallmann syndrome). Every genetic investigation should be accompanied by comprehensive genetic counselling to help with the interpretation of results and support the patient/the couple concerning consequences for their family planning and treatment options.

Is the FSHR 2039A>G variant associated with susceptibility to testicular germ cell cancer?

Testicular germ cell cancer (TGCC) is derived from germ cell neoplasia in situ (GCNIS), which arises due to niche disturbances affecting the Sertoli cells. It is believed that exogenous endocrine factors have a crucial role in governing neoplastic transformation but on a strong hereditary background. Follicle-stimulating hormone (FSH) is the major regulatory hormone of the Sertoli cells. FSH signalling-related single-nucleotide polymorphisms (SNPs) have previously been shown to affect FSH action in men at different levels. We aimed to investigate whether three FSH-related SNPs (FSHR 2039A>G, FSHR -29G>A and FSHB -211G>T) are associated with development of TGCC. A total of 752 Danish and German patients with TGCC from two tertiary andrological referral centres were included. Three control groups comprising 2020 men from the general population, 679 fertile men and 417 infertile men, were also included. Chi-squared test was performed to compare genotype- and allele frequencies. Kruskal-Wallis test was performed to compare age at diagnosis. Patients with TGCC had a higher frequency of the A-allele of FSHR 2039A>G compared to the group of fertile men with an AA-genotype frequency of 30.2% vs. 22.0%, respectively, p = 0.002. This variant is associated with higher FSH receptor activity. The distribution of the FSHR 2039A>G did not differ significantly between the patients with TGCC and the infertile or the general population. The frequency of the two other SNPs did not differ between patient with TGCC and any of the control groups. No differences were detected between genotypes and age distribution or histological subtype of the tumours. In conclusion, we observed that a genetic variant associated with FSHR activity may modulate the susceptibility to TGCC.

Y-chromosomal microdeletions and partial deletions of the Azoospermia Factor c (AZFc) region in normozoospermic, severe oligozoospermic and azoospermic men in Sri Lanka.

AIM: To assess for the first time the occurrence of Y chromosomal microdeletions and partial deletions of the Azoospermia Factor c (AZFc) region in Sri Lankan men and to correlate them with clinical parameters. METHODS: In a retrospective study, we analyzed 96 infertile men (78 with non-obstructive azoospermia) and 87 controls with normal spermatogenesis. AZFa, AZFb, AZFc and partial deletions within the AZFc region were analyzed by multiplex polymerase chain reaction (PCR) according to established protocols. RESULTS: No AZFa, AZFb or AZFc deletions were found in the control group. Seven patients in the group of infertile men were found to have deletions as following: one AZFa, two AZFc, two AZFbc and two AZFabc. The relative distribution of these patterns was significantly different compared with that found in the German population. Extension analysis confirmed that the deletions occurred according to the current pathogenic model. gr/gr deletions were found to be equally present both in the patients (n=4) and in the control group (n=4). One b2/b3 deletion was found in the patient group. CONCLUSION: These results suggest that the frequency and pattern of microdeletions of the Y chromosome in Sri Lankan men are similar to those found in other populations and confirm that gr/gr deletions are not sufficient to cause spermatogenetic failure.

[Social freezing - the male perspective]. / "Social freezing" - die männliche Seite.

BACKGROUND: In Germany there is an emerging trend for postponing parenthood due to non-medical, sociocultural reasons. This clearly impacts on the reproductive success due to an age-dependent decrease in fertility. Thus, strategies and techniques are currently discussed which could preserve the female fertility status, among which social freezing (cryopreservation of oocytes) for later fertilization is the most realistic one; however, while there is an intensive discussion on the procedure and timing of oocyte cryopreservation, virtually no attention has been paid to the male side and the aging effects on the male germ cells. AIM: To evaluate the risk paternal age poses for the integrity of germ cells. METHODS: For this review a literature search using PubMed, data from the Federal Statistical Office of Germany, the German in vitro fertilization (IVF) register as well as own data were used. RESULTS: Sperm cell integrity is clearly affected by age both at the genetic as well as at the epigenetic levels. The estimated mutation rate for spermatozoa doubles every 16.5 years. Monogenic and multifactorial diseases are strongly associated with paternal age. Men aged >40 years have an increased risk of passing age-related mutations to their children. CONCLUSIONS: Cryopreservation of spermatozoa is an option for men who postpone planning a family. Genetic counseling is recommended for couples undertaking social freezing and a male age of >40 years.

An alternative interpretation of cellular 'selfish spermatogonial selection'-clusters in the human testis indicates the need for 3-D-analyses.

The 'selfish spermatogonial selection'- model was proposed to explain the paternal age effect (PAE) of some congenital disorders associated with point mutations in male germ cells. According to this, spermatogonia carrying pathogenic mutations gain a selection advantage over non-mutated spermatogonia which leads to an increased number of mutated spermatogonia and consequently spermatozoa over time. Recently, an immunohistochemical approach using the premeiotic marker melanoma antigen family A4 (MAGE A4) was undertaken by the Wilkie group to confirm the presence of microclones of putatively mutated spermatogonia in testes of elderly men. The objective of our study was the age-dependent assessment of testes from men with normal spermatogenesis using MAGE A4 immunohistochemistry to identify and corroborate cellular clusters indicative for 'selfish spermatogonial selection' in our cohort. We analyzed testicular tissues obtained from men with normal spermatogenesis assigned to three age groups [(1) 28.8 ± 2.7 years; (2) 48.1 ± 1 years; (3) 71.9 ± 6.8 years, n/group = 8]. We could detect very similar distribution patterns of MAGE A4-positive cells and the presence of several types of microclusters as reported previously. However, these cellular clusters, indicative for clonal expansion, were not only present in testes from elderly men but also in those from age group 1 and 2. Using graphical three-dimensional modelling, we identified that cross-section directions e.g. longitudinal sections might provoke misleading interpretation of spermatogonial clusters, in particular when the tissue processing is limited. Thus, appropriate fixation and embedding is needed for reliable analysis of testicular sections. We therefore propose a more careful interpretation of such spermatogonial clusters and recommend a 3-D analysis to unequivocally determine 'selfish spermatogonial selection'-manifestations.