The RNA-binding protein Tsunagi interacts with Mago Nashi to establish polarity and localize oskar mRNA during Drosophila oogenesis

In Drosophila melanogaster, formation of the axes and the primordial germ cells is regulated by interactions between the germ line-derived oocyte and the surrounding somatic follicle cells. This reciprocal signaling results in the asymmetric localization of mRNAs and proteins critical for these oogenic processes. Mago Nashi protein interprets the posterior follicle cell-to-oocyte signal to establish the major axes and to determine the fate of the primordial germ cells. Using the yeast two-hybrid system we have identified an RNA-binding protein, Tsunagi, that interacts with Mago Nashi protein. The proteins coimmunoprecipitate and colocalize, indicating that they form a complex in vivo. Immunolocalization reveals that Tsunagi protein is localized within the posterior oocyte cytoplasm during stages 1-5 and 8-9, and that this localization is dependent on wild-type mago nashi function. When tsunagi function is removed from the germ line, egg chambers develop in which the oocyte nucleus fails to migrate, oskar mRNA is not localized within the posterior pole, and dorsal-ventral pattern abnormalities are observed. These results show that a Mago Nashi-Tsunagi protein complex is required for interpreting the posterior follicle cell-to-oocyte signal to define the major body axes and to localize components necessary for determination of the primordial germ cells.     

The adaptor-like protein ROG-1 is required for activation of the Ras-MAP kinase pathway and meiotic cell cycle progression in Caenorhabditis elegans

The Ras-MAP kinase pathway regulates varieties of fundamental cellular events. In Caenorhabditis elegans, this pathway is required for oocyte development; however, the nature of its up-stream regulators has remained elusive. Here, we identified a C. elegans gene, rog-1, which encodes the only protein having the IRS-type phosphotyrosine-binding (PTB) domain in the worms. ROG-1 has no obvious domain structure aside from the PTB domain, suggesting that it could serve as an adaptor down-stream of protein-tyrosine kinases (PTKs). RNA interference (RNAi)-mediated down-regulation of rog-1 mRNA significantly decreased brood size. rog-1(tm1031) truncation mutants showed a severe disruption in progression of developing oocytes from pachytene to diakinesis, as was seen in worms carrying a loss-of-function mutation in the let-60 Ras or mpk-1 MAP kinase gene. Furthermore, let-60 Ras-regulated activation of MPK-1 in the gonad is undetectable in rog-1(tm1031) mutants. Conversely, a gain-of-function mutation in the let-60 Ras gene rescues the brood size reduction and germ cell abnormality in rog-1(tm1031) worms. Consistently, rog-1 is preferentially expressed in the germ cells and its expression in the gonad is essential for oocyte development. Thus, ROG-1 is a key positive regulator of the Ras-MAP kinase pathway that permits germ cells to exit from pachytene.     

Fragile X mental retardation protein modulates the fate of germline stem cells in Drosophila

Fragile X syndrome, a common form of inherited mental retardation, is caused by the loss of fragile X mental retardation protein (FMRP). FMRP, which may regulate translation in neurons, associates not only with specific mRNAs and microRNAs (miRNA), but also with components of the miRNA pathway, including Dicer and Argonaute proteins. In Drosophila, dFmr1 is also known to be involved in germ cell and oocyte specification; however, the question of whether dFmr1 is required for controlling the fate of germline stem cells (GSCs) has gone unanswered. Here we show that dFmr1 is required for both GSC maintenance and repressing differentiation. Furthermore, we demonstrate that in Drosophila ovary, dFmr1 protein interacts with Argonaute protein 1 (AGO1), a key component of the miRNA pathway. Thus dFmr1 could modulate the fate of GSCs, likely via the miRNA pathway. Our results provide the first evidence that FMRP might be involved in the regulation of adult stem cells.     

Human testicular protein TPX1/CRISP-2: localization in spermatozoa, fate after capacitation and relevance for gamete interaction

Testicular protein Tpx-1, also known as CRISP-2, is a cysteine-rich secretory protein specifically expressed in the male reproductive tract. Since the information available on the human protein is limited to the identification and expression of its gene, in this work we have studied the presence and localization of human Tpx-1 (TPX1) in sperm, its fate after capacitation and acrosome reaction (AR), and its possible involvement in gamete interaction. Indirect immunofluorescence studies revealed the absence of significant staining in live or fixed non-permeabilized sperm, in contrast to a clear labelling in the acrosomal region of permeabilized sperm. These results, together with complementary evidence from protein extraction procedures strongly support that TPX1 would be mainly an intra-acrosomal protein in fresh sperm. After in vitro capacitation and ionophore-induced AR, TPX1 remained associated with the equatorial segment of the acrosome. The lack of differences in the electrophoretic mobility of TPX1 before and after capacitation and AR indicates that the protein would not undergo proteolytical modifications during these processes. The possible involvement of TPX1 in gamete interaction was evaluated by the hamster oocyte penetration test. The presence of anti-TPX1 during gamete co-incubation produced a significant and dose-dependent inhibition in the percentage of penetrated zona-free hamster oocytes without affecting sperm motility, the AR or sperm binding to the oolema. Together, these results indicate that human TPX1 would be a component of the sperm acrosome that remains associated with sperm after capacitation and AR, and is relevant for sperm-oocyte interaction.     

The testis anion transporter TAT1 (SLC26A8) physically and functionally interacts with the cystic fibrosis transmembrane conductance regulator channel: a potential role during sperm capacitation

The Slc26 gene family encodes several conserved anion transporters implicated in human genetic disorders, including Pendred syndrome, diastrophic dysplasia and congenital chloride diarrhea. We previously characterized the TAT1 (testis anion transporter 1; SLC26A8) protein specifically expressed in male germ cells and mature sperm and showed that in the mouse, deletion of Tat1 caused male sterility due to a lack of sperm motility, impaired sperm capacitation and structural defects of the flagella. Ca(2+), Cl(-) and HCO(3)(-) influxes trigger sperm capacitation events required for oocyte fertilization; these events include the intracellular rise of cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA)-dependent protein phosphorylation. The cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in mature sperm and has been shown to contribute to Cl(-) and HCO(3)(-) movements during capacitation. Furthermore, several members of the SLC26 family have been described to form complexes with CFTR, resulting in the reciprocal regulation of their activities. We show here that TAT1 and CFTR physically interact and that in Xenopus laevis oocytes and in CHO-K1 cells, TAT1 expression strongly stimulates CFTR activity. Consistent with this, we show that Tat1 inactivation in mouse sperm results in deregulation of the intracellular cAMP content, preventing the activation of PKA-dependent downstream phosphorylation cascades essential for sperm activation. These various results suggest that TAT1 and CFTR may form a molecular complex involved in the regulation of Cl(-) and HCO(3)(-) fluxes during sperm capacitation. In humans, mutations in CFTR and/or TAT1 may therefore be causes of asthenozoospermia and low fertilizing capacity of sperm.     

The Caenorhabditis elegans DDX-23, a homolog of yeast splicing factor PRP28, is required for the sperm-oocyte switch and differentiation of various cell types

DEAD/H-box proteins are involved in various aspects of RNA metabolism. Here we report the developmental function of a DEAD-box protein, DDX-23, in Caenorhabditis elegans, which has significant homology with the yeast splicing factor PRP28. We found by RNAi and mutant analyses that DDX-23 is essential for both embryonic and post-embryonic development, and required for differentiation of the majority of somatic tissues. When the germline function of ddx-23 was inhibited, hermaphrodite animals showed a reduced number of germ cells and failed to switch from spermatogenesis to oogenesis. These phenotypes were similar to those of the mutants of the three DEAH-box proteins (MOG-1, MOG-4, and MOG-5) whose yeast orthologs are involved in the pre-mRNA splicing pathway. We speculate that DDX-23 functions with the three MOG proteins in the same pathway to regulate tissue differentiation, robust germline proliferation, and the sperm/oocyte switch through modulations of ribonucleoprotein complexes.     

Identification of stem cells during prepubertal spermatogenesis via monitoring of nucleostemin promoter activity

The nucleostemin (NS) gene encodes a nucleolar protein found at high levels in several types of stem cells and tumor cell lines. The function of NS is unclear but it may play a critical role in S-phase entry by stem/progenitor cells. Here we characterize NS expression in murine male germ cells. Although NS protein was highly expressed in the nucleoli of all primordial germ cells, only a limited number of gonocytes showed NS expression in neonatal testes. In adult testes, NS protein was expressed at high levels in the nucleoli of spermatogonia and primary spermatocytes but at only low levels in round spermatids. To evaluate the properties of cells expressing high levels of NS, we generated transgenic reporter mice expressing green fluorescent protein (GFP) under the control of the NS promoter (NS-GFP Tg mice). In adult NS-GFP Tg testes, GFP and endogenous NS protein expression were correlated in spermatogonia and spermatocytes but GFP was also ectopically expressed in elongated spermatids and sperm. In testes of NS-GFP Tg embryos, neonates, and 10-day-old pups, however, GFP expression closely coincided with endogenous NS expression in developing germ cells. In contrast to a previous report, our results support the existence in neonatal testes of spermatogonial stem cells with long-term repopulating capacity. Furthermore, our data show that NS expression does not correlate with cell-cycle status during prepuberty, and that strong NS expression is essential for the maintenance of germline stem cell proliferation capacity. We conclude that NS is a marker of undifferentiated status in the germ cell lineage during prepubertal spermatogenesis.     

The paternal methylation imprint of the mouse H19 locus is acquired in the gonocyte stage during foetal testis development

BACKGROUND: Germline-specific differential DNA methylation that persists through fertilization and embryonic development is thought to be the 'imprint' distinguishing the parental alleles of imprinted genes. If such methylation is to work as the imprinting mechanism, however, it has to be reprogrammed following each passage through the germline. Previous studies on maternally methylated genes have shown that their methylation imprints are first erased in primordial germ cells (PGCs) and then re-established during oocyte growth. RESULTS: We have examined the timing of the reprogramming of the paternal methylation imprint of the mouse H19 gene during germ cell development. In both male and female PGCs, the paternal allele is partially methylated whereas the maternal allele is unmethylated. This partial methylation is completely erased in the female germline by entry into meiosis, establishing the oocyte methylation pattern. In the male germline, both alleles become methylated, mainly during the gonocyte stage, establishing the sperm methylation pattern. CONCLUSION: The paternal methylation imprint of H19 is established in the male germline and erased in the female germline at specific developmental stages. The identification of the timings of the methylation and demethylation should help to identify and characterize the biochemical basis of the reprogramming of imprinting.     

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

BACKGROUND: 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. METHODS: We examined expression of the CTAs MAGE-A1, GAGE and NY-ESO-1 in fetal gonads by single and double immunohistochemical staining. RESULTS: 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. CONCLUSIONS: 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.     

MSP domain protein-1 from Ascaris suum and its possible role in the regulation of major sperm protein-based crawling motility

Nematode sperm utilize a crawling motility based on a nematode sperm-specific cytoskeletal protein called the major sperm protein (MSP). Although MSP has no similarity to actin in sequence or structure, the motility mediated by these two proteins is nearly indistinguishable at a phenotypic level. As with the traditional actin cytoskeleton, the central component of MSP-based motility (MSP) interacts with accessory proteins that regulate polymerization and depolymerization and play a key role in cell motility. A bioinformatics approach has led to the identification of proteins with enhanced expression in the Ascaris suum male germ line, including five new proteins each containing an MSP domain. One of these MSP domain proteins (As-MDP-1) contains an MSP domain in the C-terminus and a N-terminal extension rich in prolines and alanines. The 15.6 kDa As-MDP-1 was shown to be >90% identical at the amino acid level to members of a small family of Ascaris proteins that have been shown to bind to the MSP cytoskeleton and to negatively regulate MSP fiber growth. Further, it was demonstrated that As-MDP-1 is the smallest member of the MFP1 triplet of negative regulators of MSP cytoskeleton formation. Antibodies were used to detect the presence of As-MDP-1 along the entire length of the MSP fibers suggesting that As-MDP-1 binds directly to the higher order forms of MSP. This protein has orthologues in other nematode species, is present in Ascaris in at least six allelic forms, and is likely to form multimers.     

Stem cell based therapeutical approach of male infertility by teratocarcinoma derived germ cells

Infertility affects 13-18% of couples and growing evidence from clinical and epidemiological studies suggests an increasing incidence of male reproductive problems. There is a male factor involved in up to half of all infertile couples. The pathogenesis of male infertility can be reflected by defective spermatogenesis due to failure in germ cell proliferation and differentiation. We report here in vitro generation of a germ cell line (SSC1) from the pluripotent teratocarcinoma cells by a novel promoter-based sequential selection strategy and show that the SSC1 cell line form mature seminiferous tubule structures, and support spermatogenesis after transplantation into recipient testes. To select differentiated germ cell population, we generated a fusion construct (Stra8-EGFP) harbouring the 1.4 kb promoter region of germ line specific gene Stra8 and coding region of enhanced green fluorescence protein. This region was sufficient to direct gene expression to the germinal stem cells in testis of transgenic mice. The purified cells expressed the known molecular markers of spermatogonia Rbm, cyclin A2, Tex18, Stra8 and Dazl and the beta1- and alpha6-integrins characteristic of the stem cell fraction. This cell line undergoes meiosis and can develop into sperm when transplanted into germ cell depleted testicular tubules. Sperm were viable and functional, as shown by fertilization after intra-cytoplasmic injection into mouse oocytes. This approach provides the basis that is essential for studying the development and differentiation of male germ line stem cell, as well as for developing new approaches to reproductive engineering and infertility treatment.     

Localization of a specific germ cell marker, DAZL1, in testicular germ cell neoplasias

DAZ-like 1( DAZL1) is a germ cell-specific protein expressed in both male and female gonads. The DAZL1 gene, which maps to chromosome 3 in humans, is an autosomal homologue to the Deleted in AZoospermia ( DAZ) gene(s) located on the Y chromosome. We studied the expression of DAZL1 by means of immunohistochemistry in order to determine its distribution among testicular germ cell neoplasias and among the intratubular lesions in their vicinity. Our results demonstrated that expression of DAZL1 protein was consistently observed in scattered cells in all intratubular germ cell neoplasias (IGCN) of the unclassified type, as well as in some of the intratubular seminomas. Foci of DAZL1 immunopositive cells were detected in pure seminomas, while single immunopositive cells were dispersed in the seminomatous component of mixed germ cell neoplasias. All the nonseminomatous components were negative for DAZL1 expression. These findings demonstrate an antigenic heterogeneity of seminoma cells. The localization of a specific germ cell protein, DAZL1, in the putative ontogenic progenitor, IGCN, and in their putative derivative, seminoma, provides further support for the hypothesis that IGCN is the precursor of germ cell neoplasias.