Mammalian Male Germ Cells Research Articles

Two RNA binding proteins, ADAD2 and RNF17, interact to form a heterogeneous population of novel meiotic germ cell granules with developmentally dependent organelle association.

Mammalian male germ cell differentiation relies on complex RNA biogenesis events, many of which occur in non-membrane bound organelles termed RNA germ cell granules that are rich in RNA binding proteins (RBPs). Though known to be required for male germ cell differentiation, we understand little of the relationships between the numerous granule subtypes. ADAD2, a testis specific RBP, is required for normal male fertility and forms a poorly characterized granule in meiotic germ cells. This work aimed to understand the role of ADAD2 granules in male germ cell differentiation by clearly defining their molecular composition and relationship to other granules. Biochemical analyses identified RNF17, a testis specific RBP that forms meiotic male germ cell granules, as an ADAD2-interacting protein. Phenotypic analysis of Adad2 and Rnf17 mutants identified a rare post-meiotic chromatin defect, suggesting shared biological roles. ADAD2 and RNF17 were found to be dependent on one another for granularization and together form a previously unstudied set of germ cell granules. Based on co-localization studies with well-characterized granule RBPs and organelle-specific markers, a subset of the ADAD2-RNF17 granules are found to be associated with the intermitochondrial cement and piRNA biogenesis. In contrast, a second, morphologically distinct population of ADAD2-RNF17 granules co-localized with the translation regulators NANOS1 and PUM1, along with the molecular chaperone PDI. These large granules form a unique funnel-shaped structure that displays distinct protein subdomains and is tightly associated with the endoplasmic reticulum. Developmental studies suggest the different granule populations represent different phases of a granule maturation process. Lastly, a double Adad2-Rnf17 mutant model suggests the interaction between ADAD2 and RNF17, as opposed to loss of either, is the likely driver of the Adad2 and Rnf17 mutant phenotypes. These findings shed light on the relationship between germ cell granule pools and define new genetic approaches to their study.

Endocannabinoid system and epigenetics in spermatogenesis and testicular cancer.

In mammals, male germ cell development starts during fetal life and is carried out in postnatal life with the formation of sperms. Spermatogenesis is the complex and highly orderly process during which a group of germ stem cells is set at birth, starts to differentiate at puberty. It proceeds through several stages: proliferation, differentiation, and morphogenesis and it is strictly regulated by a complex network of hormonal, autocrine and paracrine factors and it is associated with a unique epigenetic program. Altered epigenetic mechanisms or inability to respond to these factors can impair the correct process of germ development leading to reproductive disorders and/or testicular germ cell cancer. Among factors regulating spermatogenesis an emerging role is played by the endocannabinoid system (ECS). ECS is a complex system comprising endogenous cannabinoids (eCBs), their synthetic and degrading enzymes, and cannabinoid receptors. Mammalian male germ cells have a complete and active ECS which is modulated during spermatogenesis and that crucially regulates processes such as germ cell differentiation and sperm functions. Recently, cannabinoid receptor signaling has been reported to induce epigenetic modifications such as DNA methylation, histone modifications and miRNA expression. Epigenetic modifications may also affect the expression and function of ECS elements, highlighting the establishment of a complex mutual interaction. Here, we describe the developmental origin and differentiation of male germ cells and testicular germ cell tumors (TGCTs) focusing on the interplay between ECS and epigenetic mechanisms involved in these processes.

Modern tools applied to classic structures: Approaches for mammalian male germ cell RNA granule research.

First reported in the 1800s, germ cell granules are small nonmembrane bound RNA-rich regions of the cytoplasm. These sites of critical RNA processing and storage in the male germ cell are essential for proper differentiation and development and are present in a wide range of species from Caenorhabditis elegans through mammals. Initially characterized by light and electron microscopy, more modern techniques such as immunofluorescence and genetic models have played a major role in expanding our understanding of the composition of these structures. While these methods have given light to potential granule functions, much work remains to be done. The current expansion of imaging technologies and omics-scale analyses to germ cell granule research will drive the field forward considerably. Many of these methods, both current and upcoming, have considerable caveats and limitations that necessitate a holistic approach to the study of germ granules. By combining and balancing different techniques, the field is poised to elucidate the nature of these critical structures.

Dual DNA staining enables isolation of multiple sub-types of post-replicative mouse male germ cells.

During spermatogenesis, mammalian male germ cells undergo multiple developmental processes, including meiosis and post‐meiotic differentiation (spermiogenesis). To understand the transitions between different cellular states it is essential to isolate pure populations of cells at different stages of development. Previous approaches enabled the isolation of cells from different stages of meiotic prophase I, but techniques to sub‐fractionate unfixed, post‐meiotic spermatids have been lacking. Here we report the development of a protocol enabling simultaneous isolation of cells at different stages of meiotic prophase and post‐meiotic differentiation from testes of adult mice. This approach builds on existing fluorescence activated cell sorting protocols designed to purify cells in different stages of meiotic prophase I. By utilizing the specific spectral properties that two different DNA dyes (Hoechst 33342 and SYTO 16) exhibit when bound to chromatin of different stage male germ cells, we obtain highly pure populations of cells in relatively large numbers. This FACS protocol will enable immunocytological and molecular characterization studies of fractionated meiotic and haploid germ cells from both wild type and genetically mutant animals.

KATNB1 is a master regulator of multiple katanin enzymes in male meiosis and haploid germ cell development.

Katanin microtubule-severing enzymes are crucial executers of microtubule regulation. Here, we have created an allelic loss-of-function series of the katanin regulatory B-subunit KATNB1 in mice. We reveal that KATNB1 is the master regulator of all katanin enzymatic A-subunits during mammalian spermatogenesis, wherein it is required to maintain katanin A-subunit abundance. Our data shows that complete loss of KATNB1 from germ cells is incompatible with sperm production, and we reveal multiple new spermatogenesis functions for KATNB1, including essential roles in male meiosis, acrosome formation, sperm tail assembly, regulation of both the Sertoli and germ cell cytoskeletons during sperm nuclear remodelling, and maintenance of seminiferous epithelium integrity. Collectively, our findings reveal that katanins are able to differentially regulate almost all key microtubule-based structures during mammalian male germ cell development, through the complexing of one master controller, KATNB1, with a 'toolbox' of neofunctionalised katanin A-subunits.

Cell-fate transition and determination analysis of mouse male germ cells throughout development

Mammalian male germ cell development is a stepwise cell-fate transition process; however, the full-term developmental profile of male germ cells remains undefined. Here, by interrogating the high-precision transcriptome atlas of 11,598 cells covering 28 critical time-points, we demonstrate that cell-fate transition from mitotic to post-mitotic primordial germ cells is accompanied by transcriptome-scale reconfiguration and a transitional cell state. Notch signaling pathway is essential for initiating mitotic arrest and the maintenance of male germ cells’ identities. Ablation of HELQ induces developmental arrest and abnormal transcriptome reprogramming of male germ cells, indicating the importance of cell cycle regulation for proper cell-fate transition. Finally, systematic human-mouse comparison reveals potential regulators whose deficiency contributed to human male infertility via mitotic arrest regulation. Collectively, our study provides an accurate and comprehensive transcriptome atlas of the male germline cycle and allows for an in-depth understanding of the cell-fate transition and determination underlying male germ cell development.

Spatiotemporal Small Non-coding RNAs Expressed in the Germline as an Early Biomarker of Testicular Toxicity and Transgenerational Effects Caused by Prenatal Exposure to Nanosized Particles.

In recent years, an apparent decline in human sperm quality has been observed worldwide. One in every 5.5 couples suffers from infertility, with male reproductive problems contributing to nearly 40% of all infertility cases. Although the reasons for the increasing number of infertility cases are largely unknown, both genetic and environmental factors can be contributing factors. In particular, exposure to chemical substances during mammalian male germ cell development has been linked to an increased risk of infertility in later life owing to defective sperm production, reproductive tract obstruction, inflammation, and sexual disorders. Prenatal exposure to nanomaterials (NMs) is no exception. In animal experiments, maternal exposure to NMs has been reported to affect the reproductive health of male offspring. Male germ cells require multiple epigenetic reprogramming events during their lifespan to acquire reproductive capacity. Given that spermatozoa deliver the paternal genome to oocytes upon fertilization, we hypothesized that maternal exposure to NMs negatively affects male germ cells by altering epigenetic regulation, which may in turn affect embryo development. Small non-coding RNAs (including microRNAs, PIWI-interacting RNAs, tRNA-derived small RNAs, and rRNA-derived small RNAs), which are differentially expressed in mammalian male germ cells in a spatiotemporal manner, could play important regulatory roles in spermatogenesis and embryogenesis. Thus, the evaluation of RNAs responsible for sperm fertility is of great interest in reproductive toxicology and medicine. However, whether the effect of maternal exposure to NMs on spermatogenesis in the offspring (intergenerational effects) really triggers multigenerational effects remains unclear, and infertility biomarkers for evaluating paternal inheritance have not been identified to date. In this review, existing lines of evidence on the effects of prenatal exposure to NMs on male reproduction are summarized. A working hypothesis of the transgenerational effects of sperm-derived epigenomic changes in the F1 generation is presented, in that such maternal exposure could affect early embryonic development followed by deficits in neurodevelopment and male reproduction in the F2 generation.

Cannabinoid Receptors Signaling in the Development, Epigenetics, and Tumours of Male Germ Cells.

Endocannabinoids are natural lipid molecules whose levels are regulated by specific biosynthetic and degradative enzymes. They bind to and activate two main cannabinoid receptors type 1 (CB1) and type 2 (CB2), and together with their metabolizing enzymes form the “endocannabinoid system” (ECS). In the last years, the relevance of endocannabinoids (eCBs) as critical modulators in various aspects of male reproduction has been pointed out. Mammalian male germ cells, from mitotic to haploid stage, have a complete ECS which is modulated during spermatogenesis. Compelling evidence indicate that in the testis an appropriate “eCBs tone”, associated to a balanced CB receptors signaling, is critical for spermatogenesis and for the formation of mature and fertilizing spermatozoa. Any alteration of this system negatively affects male reproduction, from germ cell differentiation to sperm functions, and might have also an impact on testicular tumours. Indeed, most of testicular tumours develop during early germ-cell development in which a maturation arrest is thought to be the first key event leading to malignant transformation. Considering the ever-growing number and complexity of the data on ECS, this review focuses on the role of cannabinoid receptors CB1 and CB2 signaling in male germ cells development from gonocyte up to mature spermatozoa and in the induction of epigenetic alterations in these cells which might be transmitted to the progeny. Furthermore, we present new evidence on their relevance in testicular cancer.

Evolution of an X-Linked miRNA Family Predominantly Expressed in Mammalian Male Germ Cells.

MicroRNAs (miRNAs) are important posttranscriptional regulators of gene expression. However, comprehensive expression profiles of miRNAs during mammalian spermatogenesis are lacking. Herein, we sequenced small RNAs in highly purified mouse spermatogenic cells at different stages. We found that a family of X-linked miRNAs named spermatogenesis-related miRNAs (spermiRs) is predominantly expressed in the early meiotic phases and has a conserved testis-specific high expression pattern in different mammals. We identified one spermiR homolog in opossum; this homolog might originate from THER1, a retrotransposon that is active in marsupials but extinct in current placental mammals. SpermiRs have expanded rapidly with mammalian evolution and are diverged into two clades, spermiR-L and spermiR-R, which are likely to have been generated at least in part by tandem duplication mediated by flanking retrotransposable elements. Notably, despite having undergone highly frequent lineage-specific duplication events, the sequences encoding all spermiR family members are strictly located between two protein-coding genes, Slitrk2 and Fmr1. Moreover, spermiR-Ls and spermiR-Rs have evolved different expression patterns during spermatogenesis in different mammals. Intriguingly, the seed sequences of spermiRs, which are critical for the recognition of target genes, are highly divergent within and among mammals, whereas spermiR target genes largely overlap. When miR-741, the most highly expressed spermiR, is knocked out in cultured mouse spermatogonial stem cells (SSCs), another spermiR, miR-465a-5p, is dramatically upregulated and becomes the most abundant miRNA. Notably, miR-741−/− SSCs grow normally, and the genome-wide expression levels of mRNAs remain unchanged. All these observations indicate functional compensation between spermiR family members and strong coevolution between spermiRs and their targets.

The kinases male germ cell-associated kinase and cell cycle-related kinase regulate kinesin-2 motility in Caenorhabditis elegans neuronal cilia.

Kinesin-2 motors power anterograde intraflagellar transport (IFT), a highly ordered process that assembles and maintains cilia. However, it remains elusive how kinesin-2 motors are regulated in vivo. Here, we performed forward genetic screens to isolate suppressors that rescue the ciliary defects of OSM-3-kinesin (homolog of mammalian homodimeric kinesin-2 KIF17) mutants in Caenorhabditis elegans. We identified the C. elegans dyf-5 and dyf-18, which encode the homologs of mammalian male germ cell-associated kinase and cell cycle-related kinase, respectively. Using time-lapse fluorescence microscopy, we show that DYF-5 and DYF-18 are IFT cargo molecules and are enriched at the distal segments of sensory cilia. Mutations of dyf-5 and dyf-18 generate elongated cilia and ectopic localization of the heterotrimeric kinesin-2 (kinesin-II) at the ciliary distal segments. Genetic analyses reveal that dyf-5 and dyf-18 are important for stabilizing the interaction between IFT particles and OSM-3-kinesin. Our data suggest that DYF-5 and DYF-18 act in the same pathway to promote handover between kinesin-II and OSM-3 in sensory cilia.

Advanced immunostaining approaches to study early male germ cell development

Mammalian male germ cell development takes place in the testis under the influence of a variety of somatic cells and an incompletely defined paracrine and endocrine influences. Since it is not recapitulated well in vitro, researchers studying spermatogenesis often manipulate the germline by creating transgenic or knockout mice or by administering pharmaceutical agonists/antagonists or inhibitors. The effects of these types of manipulations on germline development can often be determined following microscopic imaging, both of stained and immunostained testis sections. Here, we describe approaches for microscopic analysis of the developing male germline, provide detailed protocols for a variety of immunostaining approaches, and discuss transgenic fluorescent reporter lines for studying the early stages of spermatogenesis.

Germ granule-mediated RNA regulation in male germ cells.

Germ cells have exceptionally diverse transcriptomes. Furthermore, the progress of spermatogenesis is accompanied by dramatic changes in gene expression patterns, the most drastic of them being near-to-complete transcriptional silencing during the final steps of differentiation. Therefore, accurate RNA regulatory mechanisms are critical for normal spermatogenesis. Cytoplasmic germ cell-specific ribonucleoprotein (RNP) granules, known as germ granules, participate in posttranscriptional regulation in developing male germ cells. Particularly, germ granules provide platforms for the PIWI-interacting RNA (piRNA) pathway and appear to be involved both in piRNA biogenesis and piRNA-targeted RNA degradation. Recently, other RNA regulatory mechanisms, such as the nonsense-mediated mRNA decay pathway have also been associated to germ granules providing new exciting insights into the function of germ granules. In this review article, we will summarize our current knowledge on the role of germ granules in the control of mammalian male germ cell's transcriptome and in the maintenance of fertility.

A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. Currently, it allows the discrimination of up to 9 murine germ cell populations with high yield and purity. This high-resolution in discrimination and purification is possible due to unique changes in chromatin structure and quantity throughout spermatogenesis. These patterns can be captured by flow cytometry of male germ cells stained with fluorescent DNA-binding dyes such as Hoechst-33342 (Hoechst). Herein is a detailed description of a recently developed protocol to isolate mammalian testicular germ cells. Briefly, single cell suspensions are generated from testicular tissue by mechanical dissociation, double stained with Hoechst and propidium iodide (PI) and processed by flow cytometry. A serial gating strategy, including the selection of live cells (PI negative) with different DNA content (Hoechst intensity), is used during FACS sorting to discriminate up to 5 germ cell types. These include, with corresponding average purities (determined by microscopy evaluation): spermatogonia (66%), primary (71%) and secondary (85%) spermatocytes, and spermatids (90%), further separated into round (93%) and elongating (87%) subpopulations. Execution of the entire workflow is straightforward, allows the isolation of 4 cell types simultaneously with the appropriate FACS machine, and can be performed in less than 2 h. As reduced processing time is crucial to preserve the physiology of ex vivo cells, this method is ideal for downstream high-throughput studies of male germ cell biology. Moreover, a standardized protocol for multispecies purification of mammalian germ cells eliminates methodological sources of variables and allows a single set of reagents to be used for different animal models.

A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry

A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry

Importance of eIF2α phosphorylation as a protective mechanism against heat stress in mouse male germ cells.

Mammalian male germ cells are exceptionally labile to heat stress. A temporal arrest of translation is one immediate response to heat, which involves heat-induced phosphorylation of eukaryotic initiation factor 2α (eIF2α) to block the formation of the translational initiation complex. Here, we investigated the protective mechanisms against heat stress in mouse male germ cells. All known eIF2α kinases were expressed in lineage- and developmental stage-specific manners in the testis; noteworthy was the presence of Gcn2 (General control nonderepressible 2 kinase) in spermatocytes of all seminiferous tubules. Multiple eIF2α kinases are likely activated upon heat stress in male germ cells. ISRIB (Integrated stress response inhibitor) was then used to determine the events downstream of eIF2α phosphorylation. ISRIB significantly reduced the rate of stress granule formation in spermatocytes at early-stage (III-IV) seminiferous tubules, and induced a number of apoptotic germ cells at late-stage (XI-XII) seminiferous tubules near the onset of meiosis. Thus, stress granule formation is a downstream event of eIF2α phosphorylation that may not directly protect cells from apoptosis, at least in spermatocytes of seminiferous tubules in early stages. Mol. Reprod. Dev. 84: 265-274, 2017. © 2017 Wiley Periodicals, Inc.

Ultrastructural localization and distribution of Nardilysin in mammalian male germ cells.

BackgroundNRD convertase, also termed Nardilysin, is a Zn++ metalloendopeptidase that specifically cleaves the N-terminus of arginine and lysine residues into dibasic moieties. Although this enzyme was found located within the testis, its function in male reproduction is largely unknown. In addition, the precise distribution of this enzyme within germ cells remains to be determined.MethodsTo answer these questions, we developed an immuno-gold electron microscopy analysis to detect Nardilysin at ultrastructural level in mice. In addition, we performed a quantitative analysis of these gold particles to statistically estimate the distribution of Nardilysin in the different subcellular compartments of differentiating late spermatids/spermatozoa.ResultsExpression of Nardilysin in wild-type mice was restricted to germ cells and markedly increased during the last steps of spermiogenesis. In elongated spermatids, we found the enzyme mainly localized in the cytoplasm, more precisely associated with two microtubular structures, the manchette and the axoneme. No labelling was detected over the membranous organelles of the spermatids. To test whether this localization is dependent of the functional microtubules organization of the flagella, we analysed the localization into a specific mouse mutant ebo/ebo (ébouriffé) known to be sterile due to an impairment of the final organization of the flagellum. In the ebo/ebo, the enzyme was still localized over the microtubules of the axoneme and over the isolated cytoplasmic microtubules doublets. Quantification of gold particles in wild-type and mutant flagella revealed the specific association of the enzyme within the microtubular area of the axoneme.ConclusionsThe strong and specific accumulation of Nardilysin in the manchette and axoneme suggests that the enzyme probably contributes either to the establishment of these specific microtubular structures and/or to their functional properties.

Induction of DNA Methylation by Artificial piRNA Production in Male Germ Cells

Global DNA demethylation and subsequent de novo DNA methylation take place in mammalian male embryonic germ cells [1-3]. P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs), which are germline-specific small RNAs, have been postulated to be critically important forde novo DNA methylation of retrotransposon genes, and many proteins, including PIWI family proteins, play pivotal roles in this process [4-6]. In the embryonic mouse testis, two mouse PIWI proteins, mouse PIWI-like (MILI) and mouse PIWI2 (MIWI2), are involved in the biogenesis of piRNAs through the so-called ping-pong amplification cycle [7-10], and long single-stranded RNAs transcribed from the gene regions of piRNA clusters have been proposed to be the initial material [11-16]. However, it remains unclear whether transcription from the piRNA clusters is required for the biogenesis of piRNAs. To answer this question, we developed a novel artificial piRNA production system by simple expression of sense and antisense EGFP mRNAs in embryonic male germ cells in the piRNA biogenesis phase. EGFP expression was silenced by piRNA-dependent DNA methylation, indicating that concomitant expression of sense and antisense RNA transcripts is necessary and sufficient for piRNA production and subsequent piRNA-dependent gene silencing. In addition, we demonstrated that this artificial piRNA induction paradigm could be applied to an endogenous gene essential for spermatogenesis, DNMT3L [3, 17, 18]. This study not only provides novel insights into the molecular mechanisms of piRNA production, but also presents an innovative strategy for inducing epigenetic modification in germ cells.

Differences in the ovine HSP90AA1 gene expression rates caused by two linked polymorphisms at its promoter affect rams sperm DNA fragmentation under environmental heat stress conditions.

Heat shock (HS) is one of the best-studied exogenous cellular stresses. Almost all tissues, cell types, metabolic pathways and biochemical reactions are affected in greater or lesser extent by HS. However, there are some especially thermo sensible cellular types such as the mammalian male germ cells. The present study examined the role of three INDELs in conjunction with the -660G/C polymorphism located at the HSP90AA1 promoter region over the gene expression rate under HS. Specially, the -668insC INDEL, which is very close to the -660G/C transversion, is a good candidate to be implied in the transcriptional regulation of the gene by itself or in a cooperative way with this SNP. Animals carrying the genotype II-668 showed higher transcription rates than those with ID-668 (FC = 3.07) and DD-668 (FC = 3.40) genotypes for samples collected under HS. A linkage between gene expression and sperm DNA fragmentation was also found. When HS conditions were present along or in some stages of the spermatogenesis, alternative genotypes of the -668insC and -660G/C mutations are involved in the effect of HS over sperm DNA fragmentation. Thus, unfavorable genotypes in terms of gene expression induction (ID-668GC-660 and DD-668GG-660) do not produce enough mRNA (stored as messenger ribonucleoprotein particles) and Hsp90α protein to cope with future thermal stress which might occur in posterior stages when transcriptional activity is reduced and cell types and molecular processes are more sensible to heat (spermatocytes in pachytene and spermatids protamination). This would result in the impairment of DNA packaging and the consequent commitment of the events occurring shortly after fertilization and during embryonic development. In the short-term, the assessment of the relationship between sperm DNA fragmentation sensitivity and ram’s fertility will be of interest to a better understanding of the mechanisms of response to HS and its consequences on animal production and reproduction performance.

Telomerase activity and telomere length in male germ cells.

Telomeres are located at the outermost ends of all eukaryotic chromosomes and provide for the maintenance of genomic stability and integrity during the life span of organisms. The length of telomeres shortens due to each round of DNA replication, genotoxic insults, and/or reactive oxygen species. To counteract this shortening, certain types of cells, including stem cells, male/female germline cells, granulosa cells, early embryos, and most cancerous cells, express an enzyme known as telomerase, which has the potential of restoring the shortened telomeres. Presence of telomerase activity in the male germ cells ensures maintenance of telomere length at maximum levels during spermatogenesis despite telomere attrition due to DNA replication or other genotoxic factors. In this review, telomerase activity and telomere length in mammalian male germ cells during spermatogenesis are evaluated in detail based on the studies in this field. Also, the relationship between telomerase activity/telomere length and development of male infertility is comprehensively discussed.

Small Non-Coding RNAs in Mammalian Male Germ Cells and Their Implications for Male Infertility

Global rise in male infertility as a result of dysfunction of male germ cells has been pointed out by many investigations. Therefore, it is essential to elucidate the molecular mechanisms involving in the development of sperm, which rely on phase-specific gene expression that is regulated by myriads of small non-coding RNAs at transcriptional, post-transcriptional and epigenetic level. Recent advancement in small non-coding RNAs mainly including siRNAs, miRNAs and piRNAs has determined several pathways, which synergically regulate the process of male germ cell development. However, the aberrant expression of them is associated with dysfunction of male germlines, such as sperm arrest or apotosis, which further leads to male infertility. In this review, we focus mainly on the biogenesis and functions of these transcripts in the regulation of mammalian male germ cell development, elucidating the mechanisms between their dysregulation and related dysfunctions of sperm and providing some basic informations for diagnosis and treatment of male infertility.