Haploid Male Germ Cells Research Articles

Testis atrophy and reduced sperm motility in transgenic mice overexpressing c-FLIP L

To study the effect of c-FLIP overexpression in testicular germ cells. A novel transgenic mouse model overexpressing the apoptotic modulator c-FLIP in the testis was generated. Animal facility and university research laboratory. Transgenic mice overexpressing the long isoform of c-FLIP (c-FLIP(L)) under the transcriptional control of a 400 bp long regulatory region of the Stra8 promoter. Spermatozoa motility and testis histological, immunohistochemical, and Western blot analyses were carried out in transgenic and control derived specimens. Testis morphology, sperm motility, and germ cell apoptosis were assayed. Stra8 promoter was found to activate the ectopic overexpression of c-FLIP(L) in round and elongated spermatids. As a consequence of such overexpression, a dramatic loss of germ cells was observed, resulting in testicular atrophy associated with reduced sperm motility. The data show that c-FLIP(L) forced expression in haploid male germ cells has detrimental effects on spermatogenesis and sperm quality and reveal a possible mechanism underlying the onset of testicular atrophy.

Functional Analysis of Male Mouse Haploid Germ Cells of Various Differentiation Stages: Early and Late Round Spermatids Are Functionally Equivalent in Producing Progeny1

Spermiogenesis is a complex process consisting of three main phases: the round, elongating, and elongated spermatid phases. Although the germ cells acquire a haploid set of paternal chromosomes after meiosis, how functional these male haploid germ cells are as male gametes at various differentiation stages has remained unclear. We selectively injected specific steps of haploid male germ cells into oocytes and assessed the function of the zygotes. Applying the transillumination technique using acrosin-green fluorescent protein transgenic mice, we succeeded in selecting four types of haploid male germ cells for microinsemination: early round spermatids (steps 2-3), late round spermatids (steps 7-8), elongating spermatids (steps 9-10), and elongated spermatids (step 16). The microinsemination technique revealed that the early and late round spermatids had similar developmental abilities in producing progeny, indicating that the nuclear status of newly generated round spermatids was similar to that of late round spermatids. An increased birthrate of progeny was first observed in steps 9-10 of elongating spermatids, but the frequency was slightly lower than that of the elongated spermatids. These results indicated that the transition from steps 7-8 of round spermatids to steps 9-10 of elongating spermatids is a key step in changing the nuclear status of male gametes in producing progeny.

Haploid male germ cell‐ and oocyte‐specific Mbd3l1 and Mbd3l2 genes are dispensable for early development, fertility, and zygotic DNA demethylation in the mouse

Genome-wide erasure of CpG methylation occurs along the paternal pronucleus in fertilized oocytes. This process involves an active, replication-independent enzymatic step, which has remained enigmatic. MBD3L1 and MBD3L2 are two mammalian homologues of the methyl-CpG-binding protein genes MBD2 and MBD3 that arose from recent gene duplication events. Expression of Mbd3l1 occurs specifically in haploid male germ cells. Mbd3l2 expression is restricted to metaphase II oocytes and zygotes making both proteins candidates for the zygotic demethylation process. Neither of these genes was able to promote reactivation of a methylation-silenced reporter gene. We created Mbd3l1 and Mbd3l2 knockout mice, which were viable and fertile. We show that demethylation of the paternal pronucleus in Mbd3l1-/- and Mbd3l2-/- mice is identical to that in wild-type controls. These data suggest that Mbd3l1 and Mbd3l2 are not involved in genome-wide demethylation of paternal genomes in mouse zygotes and are dispensable for normal development.

Nuclear regulator Pygo2 controls spermiogenesis and histone H3 acetylation

Mammalian spermiogenesis, a process where haploid male germ cells differentiate to become mature spermatozoa, entails dramatic morphological and biochemical changes including remodeling of the germ cell chromatin. Proteins that contain one or more plant homeodomain (PHD) fingers have been implicated in the regulation of chromatin structure and function. Pygopus 2 (Pygo2) belongs to a family of evolutionarily conserved PHD finger proteins thought to act as co-activators of Wnt signaling effector complexes composed of β-catenin and LEF/TCF transcription factor. Here we analyze mice containing hypomorphic alleles of pygopus 2 ( Pygo2 or mpygo2) and uncover a β-catenin-independent involvement of the Pygo2 protein in spermiogenesis. Pygo2 is expressed in elongating spermatids at stages when chromatin remodeling occurs, and block of Pygo2 function leads to spermiogenesis arrest and consequent infertility. Analysis of spermiogenesis in Pygo2 mutants reveals reduced expression of select post-meiotic genes including protamines, transition protein 2, and H1fnt, all of which are required for germ cell chromatin condensation, and drastically altered pattern of histone H3 hyperacetylation. These findings suggest that Pygo2 is involved in the chromatin remodeling events that lead to nuclear compaction of male germ cells.

Cloning and expression profiling of testis-expressed piRNA-like RNAs

Using a novel small RNA cloning method, we identified 630 piRNA-like RNAs (pilRNAs) from the mouse testis, and 498 of them are novel. These pilRNA genes were mapped to all chromosomes as 71 clusters, and the majority of them ( approximately 84%) are derived from intergenic, intronic, and exonic sequences. One of the structural characteristics for pilRNAs is that a single locus can encode numerous homologous pilRNAs with overlapping sequences. Hundreds or even thousands of pilRNAs from a single pilRNA gene cluster are all produced from a single long transcript. Expression profiling for 64 pilRNAs revealed that approximately 14% of all the pilRNAs analyzed displayed a ubiquitous expression pattern, although the majority of ( approximately 86%) pilRNAs were preferentially or exclusively expressed in meiotic and haploid male germ cells of the testis. Our semiquantitative analyses also suggest that the testis is the organ with the highest expression of pilRNAs both in number and in abundance. The large number, high abundance, unique genomic locations, and biogenesis all suggest that pilRNAs have important regulatory roles not only in spermatogenesis but also in other biological processes.

Epigenetics in Male Germ Cells

Epigenetics in Male Germ Cells

A Developmentally Regulated Chaperone Complex for the Endoplasmic Reticulum of Male Haploid Germ Cells

Glycoprotein folding is mediated by lectin-like chaperones and protein disulfide isomerases (PDIs) in the endoplasmic reticulum. Calnexin and the PDI homologue ERp57 work together to help fold nascent polypeptides with glycans located toward the N-terminus of a protein, whereas PDI and BiP may engage proteins that lack glycans or have sugars toward the C-terminus. In this study, we show that the PDI homologue PDILT is expressed exclusively in postmeiotic male germ cells, in contrast to the ubiquitous expression of many other PDI family members in the testis. PDILT is induced during puberty and represents the first example of a PDI family member under developmental control. We find that PDILT is not active as an oxido-reductase, but interacts with the model peptide Delta-somatostatin and nonnative bovine pancreatic trypsin inhibitor in vitro, indicative of chaperone activity. In vivo, PDILT forms a tissue-specific chaperone complex with the calnexin homologue calmegin. The identification of a redox-inactive chaperone partnership defines a new system of testis-specific protein folding with implications for male fertility.

Evidence for the Involvement of Testicular Protein CRISP2 in Mouse Sperm-Egg Fusion1

CRISP2, originally known as Tpx-1, is a cysteine-rich secretory protein specifically expressed in male haploid germ cells. Although likely to be involved in gamete interaction, evidence for a functional role of CRISP2 in fertilization still remains poor. In the present study, we used a mouse model to examine the subcellular localization of CRISP2 in sperm and its involvement in the different stages of fertilization. Results from indirect immunofluorescence and protein extraction experiments indicated that mouse CRISP2 is an intraacrosomal component that remains associated with sperm after capacitation and the acrosome reaction (AR). In vitro fertilization assays using zona pellucida-intact mouse eggs showed that an antibody against the protein significantly decreased the percentage of penetrated eggs, with a coincident accumulation of perivitelline sperm. The failure to inhibit zona pellucida penetration excludes a detrimental effect of the antibody on sperm motility or the AR, supporting a specific participation of CRISP2 at the sperm-egg fusion step. In agreement with this evidence, recombinant mouse CRISP2 (recCRISP2) specifically bound to the fusogenic area of mouse eggs, as previously reported for rat CRISP1, an epididymal protein involved in gamete fusion. In vitro competition investigations showed that incubation of mouse zona-free eggs with a fixed concentration of recCRISP2 and increasing amounts of rat CRISP1 reduced the binding of recCRISP2 to the egg, suggesting that the proteins interact with common complementary sites on the egg surface. Our findings indicate that testicular CRISP2, as observed for epididymal CRISP1, is involved in sperm-egg fusion through its binding to complementary sites on the egg surface, supporting the idea of functional cooperation between homologous molecules to ensure the success of fertilization.

Isolation of male germ stem cell-like cells from testicular tissue of nonobstructive azoospermic patients and differentiation into haploid male germ cells in vitro

Isolation of male germ stem cell-like cells from testicular tissue of nonobstructive azoospermic patients and differentiation into haploid male germ cells in vitro

The 193-Base Pair Gsg2 (Haspin) Promoter Region Regulates Germ Cell-Specific Expression Bidirectionally and Synchronously

Haspin is a unique protein kinase expressed predominantly in haploid male germ cells. The genomic structure of haspin (Gsg2) has revealed it to be intronless, and the entire transcription unit is in an intron of the integrin alphaE (Itgae) gene. Transcription occurs from a bidirectional promoter that also generates an alternatively spliced integrin alphaE-derived mRNA (Aed). In mice, the testis-specific alternative splicing of Aed is expressed bidirectionally downstream from the Gsg2 transcription initiation site, and a segment consisting of 26 bp transcribes both genomic DNA strands between Gsg2 and the Aed transcription initiation sites. To investigate the mechanisms for this unique gene regulation, we cloned and characterized the Gsg2 promoter region. The 193-bp genomic fragment from the 5' end of the Gsg2 and Aed genes, fused with EGFP and DsRed genes, drove the expression of both proteins in haploid germ cells of transgenic mice. This promoter element contained only a GC-rich sequence, and not the previously reported DNA sequences known to bind various transcription factors--with the exception of E2F1, TCFAP2A1 (AP2), and SP1. Here, we show that the 193-bp DNA sequence is sufficient for the specific, bidirectional, and synchronous expression in germ cells in the testis. We also demonstrate the existence of germ cell nuclear factors specifically bound to the promoter sequence. This activity may be regulated by binding to the promoter sequence with germ cell-specific nuclear complex(es) without regulation via DNA methylation.

Changes in intranuclear chromatin architecture induce bipolar nuclear localization of histone variant H1T2 in male haploid spermatids

Spermiogenesis entails a major biochemical and morphological restructuring of the germ cell packing the DNA into the condensed spermatid nucleus. H1T2 is a histone H1 variant selectively and transiently expressed in male haploid germ cells during spermiogenesis that specifically localizes to a chromatin domain at the apical pole under the acrosome. We explored the mechanisms determining polar localization of H1T2 in spermatids. In acrosome-deficient round spermatids of hrb −/− and gopc −/− mice, H1T2 localization is not altered, indicating that proper acrosome development is not required for specifying nuclear polarity. In contrast, in late round spermatids from trf2 −/− or hmgb2 −/− mice, a bipolar H1T2 localization was observed revealing that polarity is modified by loss of proteins specifying chromatin architecture. Our results show that intranuclear chromatin organization is critical for correct polar localization of H1T2 and that H1T2 can be a useful molecular marker revealing chromatin disorganization in spermatids.

FGFR-1 signaling is involved in spermiogenesis and sperm capacitation

Cloning of the fibroblast growth factor receptor (FGFR) adaptor Snt-2 cDNA and the identification of FGFR-1 protein in association with sperm tails, suggested that FGFR-1 signaling was involved in either sperm tail development or function. This hypothesis was tested by the creation of transgenic mice that specifically expressed a dominant-negative variant of FGFR-1 in male haploid germ cells. Mating of transgenic mice showed a significant reduction in pups per litter compared with wild-type littermates. Further analysis demonstrated that this subfertility was driven by a combination of reduced daily sperm output and a severely compromised ability of those sperm that were produced to undergo capacitation prior to fertilization. An analysis of key signal transduction proteins indicated that FGFR-1 is functional on wild-type sperm and probably signals via the phosphatidylinositol 3-kinase pathway. FGFR-1 activation also resulted in the downstream suppression of mitogen activated protein kinase signaling. These data demonstrate the FGFR-1 is required for quantitatively and qualitatively normal spermatogenesis and has a key role in the regulation of the global tyrosine phosphorylation events associated with sperm capacitation.

Different expression and activity of the α1 and α4 isoforms of the Na,K-ATPase during rat male germ cell ontogeny

Two catalytic isoforms of the Na,K-ATPase, alpha1 and alpha4, are present in testis. While alpha1 is ubiquitously expressed in tissues, alpha4 predominates in male germ cells. Each isoform has distinct enzymatic properties and appears to play specific roles. To gain insight into the relevance of the Na,K-ATPase alpha isoforms in male germ cell biology, we have studied the expression and activity of alpha1 and alpha4 during spermatogenesis and epididymal maturation. This was explored in rat testes at different ages, in isolated spermatogenic cells and in spermatozoa from the caput and caudal regions of the epididymis. Our results show that alpha1 and alpha4 undergo differential regulation during development. Whereas alpha1 exhibits only modest changes, alpha4 increases with gamete differentiation. The most drastic changes for alpha4 take place in spermatocytes at the mRNA level, and with the transition of round spermatids into spermatozoa for expression and activity of the protein. No further changes are detected during transit of spermatozoa through the epididymis. In addition, the cellular distribution of alpha4 is modified with development, being diffusely expressed at the plasma membrane and intracellular compartments of immature cells, finally to localize to the midregion of the spermatozoon flagellum. In contrast, the alpha1 isoform is evenly present along the plasma membrane of the developing and mature gametes. In conclusion, the Na,K-ATPase alpha1 and alpha4 isoforms are functional in diploid, meiotic and haploid male germ cells, alpha4 being significantly upregulated during spermatogenesis. These results support the importance of alpha4 in male gamete differentiation and function.

Isolation of male germ stem cell-like cells from testicular tissue of non-obstructive azoospermic patients and differentiation into haploid male germ cells in vitro

The purpose of this study was to establish the culture conditions required to isolate, identify and expand male germ stem cell-like cells (GSC-LC) from the testicular tissue of patients with non-obstructive azoospermia (NOA). Testicular tissues obtained from patients (two with maturation arrest (MA, n = 2) and Sertoli cell-only syndrome (SCOS, n = 11) were dissociated and plated into gelatin-coated dishes. After 2-4 weeks, cultures from both MA patients (100%) and four SCOS patients (36.3%) exhibited multicellular colonies, which proliferated successfully until passage 10. GSC-LC in the colonies displayed alkaline phosphatase activity, as well as Oct-4 and integrin b1 expression after every passage. After the fifth passage, GSC-LC were differentiated by encapsulation in calcium alginate and further cultivation. At 2 and 6 weeks, cells expressed c-Kit, Scp3, testis-specific histone protein 2B (TH2B), and transition protein (TP)-1. Fluorescence in situ hybridization additionally disclosed a few tetraploid and haploid cells at 6 weeks. Human oocytes were activated in the absence of artificial activation and cleaved after the injection of presumptive spermatids. Our novel culture system may be useful for diagnosing the existence of germ cells and facilitating the treatment of NOA patients.

MBD3L2 Interacts with MBD3 and Components of the NuRD Complex and Can Oppose MBD2-MeCP1-mediated Methylation Silencing

MBD2 and MBD3 are two proteins that contain methyl-CpG binding domains and have a transcriptional repression function. Both proteins are components of a large CpG-methylated DNA binding complex named MeCP1, which consists of the nucleosome remodeling and histone deacetylase complex Mi2-NuRD and MBD2. MBD3L2 (methyl-CpG-binding protein 3-like 2) is a protein with substantial homology to MBD2 and MBD3, but it lacks the methyl-CpG-binding domain. Unlike MBD3L1, which is specifically expressed in haploid male germ cells, MBD3L2 expression is more widespread. MBD3L2 interacts with MBD3 in vitro and in vivo, co-localizes with MBD3 but not MBD2, and does not localize to methyl-CpG-rich regions in the nucleus. In glutathione S-transferase pull-down assays, MBD3L2 is found associated with several known components of the Mi2-NuRD complex, including HDAC1, HDAC2, MTA1, MBD3, p66, RbAp46, and RbAp48. Gel shift experiments with nuclear extracts and a CpG-methylated DNA probe indicate that recombinant MBD3L2 can displace a form of the MeCP1 complex from methylated DNA. MBD3L2 acts as a transcriptional repressor when tethered to a GAL4-DNA binding domain. Repression by GAL4-MBD3L2 is relieved by MBD2 and vice versa, and repression by MBD2 from a methylated promoter is relieved by MBD3L2. The data are consistent with a role of MBD3L2 as a transcriptional modulator that can interchange with MBD2 as an MBD3-interacting component of the NuRD complex. Thus, MBD3L2 has the potential to recruit the MeCP1 complex away from methylated DNA and reactivate transcription.

Polar nuclear localization of H1T2, a histone H1 variant, required for spermatid elongation and DNA condensation during spermiogenesis

Spermiogenesis entails a major biochemical and morphological restructuring of the germ cell involving replacement of the somatic histones by protamines packing the DNA into the condensed spermatid nucleus and elimination of the cytoplasm during the elongation phase. We describe H1T2, an histone H1 variant selectively and transiently expressed in male haploid germ cells during spermiogenesis. In round and elongating spermatids, H1T2 specifically localizes to a chromatin domain at the apical pole, revealing a polarity in the spermatid nucleus. Inactivation by homologous recombination shows that H1T2 is critical for spermiogenesis as male H1t2(-/-) mice have greatly reduced fertility. Analysis of spermiogenesis in H1t2 mutant mice shows delayed nuclear condensation and aberrant elongation. As a result, mutant spermatids are characterized by the presence of residual cytoplasm, acrosome detachment, and fragmented DNA. Hence, H1T2 is a protein required for proper cell restructuring and DNA condensation during the elongation phase of spermiogenesis.

322 DIFFERENTIAL DEVELOPMENT OF RABBIT EMBRYOS FOLLOWING MICROINSEMINATION USING SPERM AND SPERMATIDS

Microinsemination is a technique that delivers male germ cells directly into the ooplasm. The efficiency of fertilization and subsequent embryo development after microinsemination varies with species and the male germ cells used. This study examined the developmental ability of rabbit embryos in vitro and in vivo following microinsemination using haploid male germ cells at different stages. First, we injected rabbit spermatozoa, elongated spermatids, and round spermatids into mouse oocytes to assess their oocyte-activating capacity. Mouse oocytes are a good experimental model for assessing the oocyte-activating capacity of male germ cells from different species. The majority of mouse oocytes were activated irrespective of the stage of rabbit male germ cells injected (77, 61, and 73% for spermatozoa, elongated spermatids, and round spermatids, respectively). By contrast, these male germ cells activated homologous rabbit oocytes at rates of 100, 59, and 29%, respectively. After 120 h in culture, 69, 55, and 13% of these activated rabbit oocytes (pronuclear eggs) developed into blastocysts, respectively. The rate of embryo development into blastocysts following round spermatid injection was significantly improved when oocytes were activated by an electric pulse shortly before microinsemination. The total number of cells was counted in embryos that reached the morula/blastocyst stages in culture using nuclear-staining with propidium iodide. The average cell number of embryos derived from elongated (89 ± 41; mean ± SD) or round spermatid (98 ± 34) injection was significantly lower than that of control embryos (in vivo fertilization) (211 ± 44) (P < 0.01). After 24 h in culture, some four- to eight-cell-stage embryos were transferred into the oviducts of pseudopregnant females. Normal pups were born from embryos involving sperm (4 offspring/16 transfers; 25%) and elongated spermatid (3/26; 12%) injection, but none from those involving round spermatid injection (0/68). These findings indicate that rabbit male germ cells acquire the ability to activate oocytes and to support subsequent embryo development as they undergo spermiogenesis. Immaturity of the nuclear genome or difficulty in coordinating the behavior of the male and female chromosomes might compromise embryo development.

The role of histones in chromatin remodelling during mammalian spermiogenesis.

One of the most dramatic chromatin remodelling processes takes place during mammalian spermatogenesis. Indeed, during the postmeiotic maturation of male haploid germ cells, or spermiogenesis, histones are replaced by small basic proteins, which in mammals are transition proteins and protamines. However, nothing is known of the mechanisms controlling the process of histone replacement. Two hints from the literature could help to shed light on the underlying molecular events: one is the massive synthesis of histone variants, including testis-specific members, and the second is a stage specific post-translational modification of histones. A new testis-specific 'histone code' can therefore be generated combining both histone variants and histone post-translational modifications. This review will detail these two phenomena and discuss possible functional significance of the global chromatin alterations occurring prior to histone replacement during spermiogenesis.

Transcription in Haploid Male Germ Cells

Major modifications in chromatin organization occur in spermatid nuclei, resulting in a high degree of DNA packaging within the spermatozoon head. However, before arrest of transcription during midspermiogenesis, high levels of mRNA are found in round spermatids. Some transcripts are the product of genes expressed ubiquitously, whereas some are generated from male germ cell-specific gene homologs of somatic cell genes. Others are transcript variants derived from genes with expression regulated in a testis-specific fashion. The haploid genome of spermatids also initiates the transcription of testis-specific genes. Various general transcription factors, distinct promoter elements, and specific transcription factors are involved in transcriptional regulation. After meiosis, spermatids are genetically but not phenotypically different, because of transcript and protein sharing through cytoplasmic bridges connecting spermatids of the same generation. Interestingly, different types of mRNAs accumulate in the sperm cell nucleus, raising the question of their origin and of a possible role after fertilization.

Transcriptional control in male germ cells: general factor TFIIA participates in CREM-dependent gene activation.

Regulation of gene expression in haploid male germ cells follows a number of specific rules that differ from somatic cells. In this physiological context, transcriptional control mediated by the activator CREM (cAMP-responsive element modulator) represents an established paradigm. In somatic cells activation by CREM requires its phosphorylation at a unique regulatory site (Ser117) and subsequent interaction with the ubiquitous coactivator CBP (cAMP response element binding protein-binding protein). In testis, CREM transcriptional activity is controlled through interaction with a tissue-specific partner, ACT (activator of CREM in testis), which confers a powerful, phosphorylation-independent activation capacity. In addition to specialized transcription factors and coactivators, a variety of general factors of the basal transcriptional machinery, and their distinct tissue-specific isoforms, are highly expressed in testis, supporting the general notion that testis-specific gene expression requires specialized mechanisms. Here, we describe that CREM interacts with transcription factor IIA (TFIIA), a general transcription factor that stimulates RNA polymerase II-directed transcription. This association was identified by a two-hybrid screen, using a testis-derived cDNA library, and confirmed by coimmunoprecipitation. The interaction is restricted to the activator isoforms of CREM and does not require Ser117. Importantly, CREM does not interact with TFIIAtau-ALF, a testis-specific TFIIA homolog. CREM and TFIIA are expressed in a spatially and temporally coordinated fashion during the differentiation program of germ cells. The two proteins also colocalize intracellularly in spermatocyte and spermatid cells. These findings contribute to the understanding of the highly specialized rules of transcriptional regulation in haploid germ cells.