Male Germ Cell Differentiation Research Articles

Impaired Fertility and Spermiogenetic Disorders with Loss of Cell Adhesion in Male Mice Expressing an Interfering Rap1 Mutant

The guanosine trisphosphatase Rap1 serves as a critical player in signal transduction, somatic cell proliferation and differentiation, and cell-cell adhesion by acting through distinct mechanisms. During mouse spermiogenesis, Rap1 is activated and forms a signaling complex with its effector, the serine-threonine kinase B-Raf. To investigate the functional role of Rap1 in male germ cell differentiation, we generated transgenic mice expressing an inactive Rap1 mutant selectively in differentiating spermatids. This expression resulted in a derailment of spermiogenesis due to an anomalous release of immature round spermatids from the seminiferous epithelium within the tubule lumen and in low sperm counts. These spermiogenetic disorders correlated with impaired fertility, with the transgenic males being severely subfertile. Because mutant testis exhibited perturbations in ectoplasmic specializations (ESs), a Sertoli-germ cell-specific adherens junction, we searched for expression of vascular endothelial cadherin (VE-cadherin), an adhesion molecule regulated by Rap1, in spermatogenic cells of wild-type and mutant mice. We found that germ cells express VE-cadherin with a timing strictly related to apical ES formation and function; immature, VE-cadherin-positive spermatids were, however, prematurely released in the transgenic testis. In conclusion, interfering with Rap1 function during spermiogenesis leads to reduced fertility by impairment of germ-Sertoli cell contacts; our transgenic mouse provides an in vivo model to study the regulation of ES dynamics.

Molecular biological features of male germ cell differentiation.

Somatic cell differentiation is required throughout the life of a multicellular organism to maintain homeostasis. In contrast, germ cells have only one specific function; to preserve the species by conveying the parental genes to the next generation. Recent studies of the development and molecular biology of the male germ cell have identified many genes, or isoforms, that are specifically expressed in the male germ cell. In the present review, we consider the unique features of male germ cell differentiation. (Reprod Med Biol 2007; 6: 1-9).

MAGE-A1, GAGE and NY-ESO-1 cancer/testis antigen expression during human gonadal development

Cancer/testis antigens (CTAs) are expressed in several cancers and during normal adult male germ cell differentiation. Little is known about their role in fetal development of human germ cells. We examined expression of the CTAs MAGE-A1, GAGE and NY-ESO-1 in fetal gonads by single and double immunohistochemical staining. We found that GAGE was expressed in the primordial germ cells of the gonadal primordium, whereas MAGE-A1 and NY-ESO-1 were first detected in germ cells of both testis and ovary after sexual differentiation was initiated. The number of positive germ cells and the staining intensity of all three CTAs peaked during the second trimester and gradually decreased towards birth in both male and female germ cells. In oocytes, MAGE-A1 expression terminated around birth, whereas NY-ESO-1 expression persisted through the neonatal stage and GAGE expression was maintained until adulthood. The population of GAGE-expressing male and female germ cells partially overlapped the population of OCT4-positive cells, whereas MAGE-A1 and NY-ESO-1 were clearly expressed only by OCT4-negative cells. Our results suggest that MAGE-A1 and NY-ESO-1 are associated with highly proliferating germ cells, whereas GAGE proteins have a more general function in germ cells unrelated to any specific developmental stage. The recognition of differential cellular expression of GAGE, MAGE-A1, NY-ESO-1 and OCT4 may help define biologically distinct germ cell subpopulations.

Post-meiotic Shifts in HSPA2/HSP70.2 Chaperone Activity during Mouse Spermatogenesis

HSPA2 (formerly HSP70.2) is a testis-specific member of the HSP70 family known to play a critical role in the completion of meiosis during male germ cell differentiation. Although abundantly present in post-meiotic cells, its function during spermiogenesis remained obscure. Here, using a global proteomic approach to identify genome-organizing proteins in condensing spermatids, we discovered an unexpected role for HSPA2, which acquires new functions and becomes tightly associated with major spermatid DNA-packaging proteins, transition proteins 1 and 2. Hence, HSPA2 is identified here as the first transition protein chaperone, and these data shed a new light on the yet totally unknown process of genome-condensing structure assembly in spermatids.

Tudor-related proteins TDRD1/MTR-1, TDRD6 and TDRD7/TRAP: Domain composition, intracellular localization, and function in male germ cells in mice

The germ-line cells of many animals possess a characteristic cytoplasmic structure termed nuage or germinal granules. In mice, nuage that is prominent in postnatal male germ cells is also called intermitochondrial cement or chromatoid bodies. TDRD1/MTR-1, which contains Tudor domain repeats, is a specific component of the mouse nuage, analogously to Drosophila Tudor, a constituent of polar granules/nuage in oocytes and embryos. We show that TDRD6 and TDRD7/TRAP, which also contain multiple Tudor domains, specifically localize to nuage and form a ribonucleoprotein complex together with TDRD1/MTR-1. The characteristic co-localization of TDRD1, 6 and 7 was disrupted in a mutant of mouse vasa homologue/ DEAD box polypeptide 4 ( Mvh/ Ddx4), which encodes another evolutionarily conserved component of nuage. In vivo over-expression experiments of the TDRD proteins and truncated forms during male germ cell differentiation showed that a single Tudor domain is a structural unit that localizes or accumulates to nuage, but the expression of the truncated, putative dominant negative forms is detrimental to meiotic spermatocytes. These results indicate that the Tudor-related proteins, which contain multiple repeats of the Tudor domain, constitute an evolutionarily conserved class of nuage components in the germ-line, and their localization or accumulation to nuage is likely conferred by a Tudor domain structure and downstream of Mvh, while the characteristic repeated architecture of the domain is functionally essential for the differentiation of germ cells.

Tdrd1/Mtr-1 , a tudor -related gene, is essential for male germ-cell differentiation and nuage/germinal granule formation in mice

Embryonic patterning and germ-cell specification in mice are regulative and depend on zygotic gene activities. However, there are mouse homologues of Drosophila maternal effect genes, including vasa and tudor, that function in posterior and germ-cell determination. We report here that a targeted mutation in Tudor domain containing 1/mouse tudor repeat 1 (Tdrd1/Mtr-1), a tudor-related gene in mice, leads to male sterility because of postnatal spermatogenic defects. TDRD1/MTR-1 predominantly localizes to nuage/germinal granules, an evolutionarily conserved structure in the germ line, and its intracellular localization is downstream of mouse vasa homologue/DEAD box polypeptide 4 (Mvh/Ddx4), similar to Drosophila vasa-tudor. Tdrd1/Mtr-1 mutants lack, and Mvh/Ddx4 mutants show, strong reduction of intermitochondrial cement, a form of nuage in both male and female germ cells, whereas chromatoid bodies, another specialized form of nuage in spermatogenic cells, are observed in Tdrd1/Mtr-1 mutants. Hence, intermitochondrial cement is not a direct prerequisite for oocyte development and fertility in mice, indicating differing requirements for nuage and/or its components between male and female germ cells. The result also proposes that chromatoid bodies likely have an origin independent of or additional to intermitochondrial cement. The analogy between Mvh-Tdrd1 in mouse spermatogenic cells and vasa-tudor in Drosophila oocytes suggests that this molecular pathway retains an essential role(s) that functions in divergent species and in different stages/sexes of the germ line.

Proteome profile changes during mouse testis development

Spermatogenesis is a process of terminal differentiation that results in the formation of mature sperm. In the first wave of this differentiation in the mouse testis, different cell types appear in the seminiferous epithelium at specific times. These cytological changes must be accompanied by changes in protein expression patterns. The aim of the present study was the comparative analysis of proteomic profiles of the soluble proteins expressed at different stages of mouse testis development (8, 18 and 45 postnatal days). Conspicuous variations in their accumulation (representing up or downregulation) were detected over the course of development. Using mass spectrometry (MALDI-TOF), 44 proteins or variant forms were identified. Proteins with redox or antioxidant activity were identified in high proportions; others involved in lipid and carbohydrate metabolic pathways, as well as a number of proteins or isoforms not previously characterized in testis were also detected. These results contribute to identify changes in soluble protein associated to the complex process of male germ cell differentiation.

Capillary morphogenesis gene (CMG)‐1 is among the genes differentially expressed in mouse male germ line stem cells and embryonic stem cells

We recently established a technique to expand male germ line stem (GS) cells in long-term culture without losing their spermatogenic capacity. To gain insight into the genetic program of these cells, we compared the mRNA expression profile of GS cells with that of embryonic stem (ES) cells using DNA microarrays. We found 79 genes that were upregulated in GS cells compared to ES cells, including synaptonemal complex protein-1, deleted in azoospermia-like, ubiquitin-conjugating enzyme E2B, and ubiquitin carboxy-terminal hydrolase L1, all of which are functionally important for spermatogenesis. In addition, we identified a cDNA encoding the mouse ortholog of capillary morphogenesis gene (CMG)-1. CMG-1 transcripts were predominantly produced in spermatogonia and spermatocytes in mouse testis. When CMG-1 expression was attenuated in a mouse spermatocyte-derived cell line, GC-2spd(ts), by a target-specific short interfering RNA, the morphology of the cells was changed and the expression of cyclin D2 was abrogated. A reporter assay using a genomic region upstream of the mouse cyclin D2 gene revealed that this downmodulation occurs at the transcriptional level. We detected FLAG-tagged CMG-1 protein in the nuclei of transfected COS7 cells, suggesting that CMG-1 may play a unique role in the transcriptional regulation of the cyclin D2 gene. The upregulated GS genes identified in this study will provide useful information for the future investigation of spermatogonial stem cells and the early phase of male germ cell differentiation.

Infertility Caused by Polymorphisms or Mutations in Spermatogenesis‐Specific Genes

Spermiogenesis Germ cells differentiate and function to transmit the parental genome to descendant generations. All of the attributes of germ cells that distinguish them from somatic cell lines are related to this prime function. Germ cells differentiate by either oogenesis or spermatogenesis to form eggs and sperm, respectively. Meiosis occurs exclusively in the formation of eggs and sperm and is indispensable for maintaining the species (Tanaka and Baba, 2005). Another function specific to dividing germ cells is maintenance of chromosome telomere length (Tian et al, 2005). Telomere length changes gradually through successive mitotic somatic cell divisions, and this ultimately results in cell death. In male germ cells, many dramatic morphological changes occur during spermatogenesis, especially in haploid spermatids after meiotic division (Russell et al, 1990). In various mammals, male germ cell differentiation proceeds actively and continuously in the testis after puberty, and sperm are produced throughout adulthood (Russell et al, 1990). One month of male cell differentiation in mice (2 months in humans) is required for completion of spermatogonial stem cell proliferation and differentiation, meiosis, generation of haploid germ cells, and morphogenesis of the developing sperm in somniferous tubules (Russell et al, 1990). After meiotic division (during the process of haploid germ cell differentiation, or spermiogenesis), the rounded spermatids undergo marked morphological changes to become sperm: the nucleus assumes a compact shape, the mitochondria are rearranged, the flagellum forms, and the acrosome is generated. During this period of differentiation, which takes about 5 to 6 weeks in humans (Clermont, 1963; Heller and Clermont, 1963) and 2 to 3 weeks in mice (Oakberg, 1956), haploid germ cells do not divide, but morphogenesis occurs, indicating that some regulatory mechanism arrests the cell cycle. Searching for functional changes in genes and gene products involved in male infertility would increase our understanding of the causes of this problem, and perhaps lead to treatment in some cases. Comprehensive isolation and analysis of haploid germ cell–specific genes showed that a large number of specific molecules are involved in spermiogenesis (Tanaka et al, 1994, 2002b; Fujii et al, 2002). The most straightforward strategy for elucidating the mechanism of spermatogenesis is to identify and characterize differentiation-specific molecules and their associated genes in germ cells.

Zygote arrest 1 gene in pig, cattle and human: evidence of different transcript variants in male and female germ cells

BackgroundZygote arrest 1 (ZAR1) is one of the few known oocyte-specific maternal-effect genes essential for the beginning of embryo development discovered in mice. This gene is evolutionary conserved in vertebrates and ZAR1 protein is characterized by the presence of atypical plant homeobox zing finger domain, suggesting its role in transcription regulation. This work was aimed at the study of this gene, which could be one of the key regulators of successful preimplantation development of domestic animals, in pig and cattle, as compared with human.MethodsScreenings of somatic cell hybrid panels and in silico research were performed to characterize ZAR1 chromosome localization and sequences. Rapid amplification of cDNA ends was used to obtain full-length cDNAs. Spatio-temporal mRNA expression patterns were studied using Northern blot, reverse transcription coupled to polymerase chain reaction and in situ hybridization.ResultsWe demonstrated that ZAR1 is a single copy gene, positioned on chromosome 8 in pig and 6 in cattle, and several variants of correspondent cDNA were cloned from oocytes. Sequence analysis of ZAR1 cDNAs evidenced numerous short inverted repeats within the coding sequences and putative Pumilio-binding and embryo-deadenylation elements within the 3'-untranslated regions, indicating the potential regulation ways. We showed that ZAR1 expressed exclusively in oocytes in pig ovary, persisted during first cleavages in embryos developed in vivo and declined sharply in morulae and blastocysts. ZAR1 mRNA was also detected in testis, and, at lower level, in hypothalamus and pituitary in both species. For the first time, ZAR1 was localized in testicular germ cells, notably in round spermatids. In addition, in pig, cattle and human only shorter ZAR1 transcript variants resulting from alternative splicing were found in testis as compared to oocyte.ConclusionOur data suggest that in addition to its role in early embryo development highlighted by expression pattern of full-length transcript in oocytes and early embryos, ZAR1 could also be implicated in the regulation of meiosis and post meiotic differentiation of male and female germ cells through expression of shorter splicing variants. Species conservation of ZAR1 expression and regulation underlines the central role of this gene in early reproductive processes.

The chromatoid body of male germ cells: Similarity with processing bodies and presence of Dicer and microRNA pathway components

The chromatoid body is a perinuclear, cytoplasmic cloud-like structure in male germ cells whose function has remained elusive. Here we show that the chromatoid body is related to the RNA-processing body of somatic cells. Dicer and components of microRNP complexes (including Ago proteins and microRNAs) are highly concentrated in chromatoid bodies. Furthermore, we show that Dicer interacts with a germ cell-specific chromatoid body component, the RNA helicase MVH (mouse VASA homolog). Thus, chromatoid bodies seem to operate as intracellular nerve centers of the microRNA pathway. Our findings underscore the importance of posttranscriptional gene regulation and of the microRNA pathway in the control of postmeiotic male germ cell differentiation.

Expression of DNMT3A transcripts and nucleolar localization of DNMT3A protein in human testicular and fibroblast cells suggest a role for de novo DNA methylation in nucleolar inactivation

Transcriptional silencing during differentiation of human male germ cells and serum starvation of human fibroblasts is controlled by epigenetic mechanisms that involve de novo DNA methylation. It is associated with high expression of different transcripts of the DNA methyltransferase 3A (DNMT3A) gene that encode two isoforms with de novo methyltransferase activity and one without catalytic activity. Western blots revealed that DNMT3A protein (with catalytic domain) is present at low levels in several tissues and at increased levels in testicular cells and growth-arrested fibroblasts. Immunofluorescence experiments localized DNMT3A to discrete nucleolar foci in B spermatogonia and resting fibroblasts. The data here suggest a role for de novo DNA methylation in nucleolar inactivation.

Signaling events during male germ cell differentiation: Update, 2006

The intracellular transduction of exogenous and cell-autonomous stimuli triggers the transformation of a multipotent stem cell, the spermatogonion, into a highly differentiated, motile and fertile cell, the spermatozoon. This differentiation process is mediated by cell-cell contact and via key players including hormones, growth factors, and cytokines. Female hormones, estrogens and progestins, play a role in the production and functionality of spermatozoon. New findings, however, reconsider the direct action for estrogens on male germ cells while progestins work through non-canonical receptors. Similarly, testosterone, the male hormone, besides acting through its receptor expressed in the somatic cells of testis, seems to work by means of non-classical mechanisms. The recent identification of growth factors, transcriptional regulators, and media for in vitro growth of spermatogonial stem cells should now make it feasible to unravel the entire spermatogenic process. A peculiar feature of the meiotic cycle is the maintenance of condensed chromatin so that DNA duplication is prevented and reduction of genome is achieved. Recently, molecular mechanisms that lead to such a condensation have been discovered. Junctional intercellular complexes between Sertoli and germ cells are critical for coordinating spermatogenesis. Molecular players involved in such cell-cell communication have been identified in Sertoli cells. Now, there is also a need for unravelling the germ cell molecules involved. These issues are the major topics which are discussed here with the goal to suggest a possible answer.

Tissue-Specific TAFs Counteract Polycomb to Turn on Terminal Differentiation

Polycomb transcriptional silencing machinery is implicated in the maintenance of precursor fates, but how this repression is reversed to allow cell differentiation is unknown. Here we show that testis-specific TAF (TBP-associated factor) homologs required for terminal differentiation of male germ cells may activate target gene expression in part by counteracting repression by Polycomb. Chromatin immunoprecipitation revealed that testis TAFs bind to target promoters, reduce Polycomb binding, and promote local accumulation of H3K4me3, a mark of Trithorax action. Testis TAFs also promoted relocalization of Polycomb Repression Complex 1 components to the nucleolus in spermatocytes, implicating subnuclear architecture in the regulation of terminal differentiation.

RNF17, a component of the mammalian germ cell nuage, is essential for spermiogenesis

Nuages are found in the germ cells of diverse organisms. However, nuages in postnatal male germ cells of mice are poorly studied. Previously, we cloned a germ cell-specific gene named Rnf17, which encodes a protein containing both a RING finger and tudor domains. Here, we report that RNF17 is a component of a novel nuage in male germ cells--the RNF17 granule, which is an electron-dense non-membrane bound spherical organelle with a diameter of 0.5 mum. RNF17 granules are prominent in late pachytene and diplotene spermatocytes, and in elongating spermatids. RNF17 granules are distinguishable from other known nuages, such as chromatoid bodies. RNF17 is able to form dimers or polymers both in vitro and in vivo, indicating that it may play a role in the assembly of RNF17 granules. Rnf17-deficient male mice were sterile and exhibited a complete arrest in round spermatids, demonstrating that Rnf17 encodes a novel key regulator of spermiogenesis. Rnf17-null round spermatids advanced to step 4 but failed to produce sperm. These results have shown that RNF17 is a component of a novel germ cell nuage and is required for differentiation of male germ cells.

Infertility with Defective Spermiogenesis in Mice Lacking AF5q31, the Target of Chromosomal Translocation in Human Infant Leukemia

AF5q31 (also called MCEF) was identified by its involvement in chromosomal translocation with the gene MLL (mixed lineage leukemia), which is associated with infant acute lymphoblastic leukemia. Several potential roles have been proposed for AF5q31 and other family genes, but the specific requirements of AF5q31 during development remain unclear. Here, we show that AF5q31 is essential for spermatogenesis. Although most AF5q31-deficient mice died in utero and neonatally with impaired embryonic development and shrunken alveoli, respectively, 13% of AF5q31-deficient mice thrived as wild-type mice did. However, the male mice were sterile with azoospermia. Histological examinations revealed the arrest of germ cell development at the stage of spermiogenesis, and virtually no spermatozoa were seen in the epididymis. AF5q31 was found to be preferentially expressed in Sertoli cells. Furthermore, mutant mice displayed severely impaired expression of protamine 1, protamine 2, and transition protein 2, which are indispensable to compact the haploid genome within the sperm head, and an increase of apoptotic cells in seminiferous tubules. Thus, AF5q31 seems to function as a transcriptional regulator in testicular somatic cells and is essential for male germ cell differentiation and survival. These results may have clinical implications in the understanding of human male infertility.

TGFβ superfamily members in spermatogenesis: setting the stage for fertility in mouse and Drosophila

Male germ cell development involves a tightly controlled sequence of differentiation switches, from the time that this lineage is specified in the embryo to the moment of sperm release for transport from the testis. Recent research findings and technological advances have allowed key mediators of developmental switches to be identified, and several members of the TGFbeta superfamily of ligands have been implicated at distinct points of male gamete formation and spermatogenesis. This paper reviews the multiple points of control mediated by TGFbeta superfamily signalling molecules during differentiation of the mammalian male germ cell. Comparisons have been made with the Drosophila testis for which genetic analysis has yielded new information concerning the roles of TGFbeta signalling in early germ cell differentiation.

PHGPx in spermatogenesis: how many functions?

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a unique intracellular enzyme that directly reduces lipid hydroperoxides in membranes and has the ability to use protein thiol groups as donor substrates. Three isoforms of PHGPx have so far been identified, namely, a mitochondrial, a cytosolic and a nuclear variant. This article is focused on recent evidence demonstrating that (1) mitochondrial and nuclear PHGPx isoforms display a different pattern of expression during male germ cell differentiation; (2) different PHGPx isoforms play specific and independent functions during sperm maturation. The data are discussed in light of the idea that PHGPx is a moonlighting protein, changing roles depending on the intracellular localization, expression in a specific cell type and different partners which it interacts with.

The Nuclear Import of TAF10 Is Regulated by One of Its Three Histone Fold Domain-Containing Interaction Partners

TFIID, comprising the TATA box binding protein (TBP) and 13 TBP-associated factors (TAFs), plays a role in nucleation in the assembly of the RNA polymerase II preinitiation complexes on protein-encoding genes. TAFs are shared among other transcription regulatory complexes (e.g., SAGA, TBP-free TAF-containing complex [TFTC], STAGA, and PCAF/GCN5). Human TAF10, a subunit of both TFIID and TFTC, has three histone fold-containing interaction partners: TAF3, TAF8, and SPT7Like (SPT7L). In human cells, exogenously expressed TAF10 remains rather cytoplasmic and leptomycin B does not affect this localization. By using fluorescent fusion proteins, we show that TAF10 does not have an intrinsic nuclear localization signal (NLS) and needs one of its three interaction partners to be transported into the nucleus. When the NLS sequences of either TAF8 or SPT7L are mutated, TAF10 remains cytoplasmic, but a heterologous NLS can drive TAF10 into the nucleus. Experiments using fluorescence recovery after photobleaching show that TAF10 does not associate with any cytoplasmic partner but that once transported into the nucleus it binds to nuclear structures. TAF10 binding to importin beta in vitro is dependent on the coexpression of either TAF8 or TAF3, but not SPT7L. The cytoplasmic-nuclear transport of TAF10 is naturally observed during the differentiation of adult male germ cells. Thus, here we describe a novel role of the three mammalian interacting partners in the nuclear localization of TAF10, and our data suggest that a complex network of regulated cytoplasmic associations may exist among these factors and that this network is important for the composition of different TFIID and TFTC-type complexes in the nucleus.

Regulated nucleocytoplasmic transport in spermatogenesis: a driver of cellular differentiation?

This review explores the hypothesis that regulation of nucleocytoplasmic shuttling is a means of driving differentiation, using spermatogenesis as a model. The transition from undifferentiated spermatogonial stem cell to terminally differentiated spermatozoon is, at its most basic, a change in the repertoire of expressed genes. To effect this, the complement of nuclear proteins, such as transcription factors and chromatin remodelling components must change. Current knowledge of the nuclear proteins and nucleocytoplasmic transport machinery relevant to spermatogenesis is consolidated in this review, and their functional linkages are highlighted not only as a means of regulating nuclear protein composition, but also as a key mechanism regulating gene transcription and hence cell fate. Through this, we hypothesize that male germ cell differentiation is mediated through regulation of nuclear transport machinery components, and thereby of the access of critical factors to the nucleus. The importance of nucleocytoplasmic trafficking to male germ cell differentiation is discussed, using the sex-determining factors Sry and SOX9, cell cycle regulators, CREM and cofactors and the Smads as specific examples, together with the roles in gametogenesis for particular nuclear transport factors in Caenorhabditis elegans and Drosophila.