Interaction between NANOS2 and the CCR4-NOT deadenylation complex is essential for male germ cell development in mouse

Nanos is one of the evolutionarily conserved proteins implicated in germ cell development and we have previously shown that it interacts with the CCR4-NOT deadenylation complex leading to the suppression of specific RNAs. However, the molecular mechanism and physiological significance of this interaction have remained elusive. In our present study, we identify CNOT1, a component of the CCR4-NOT deadenylation complex, as a direct factor mediating the interaction with NANOS2. We find that the first 10 amino acids (AAs) of NANOS2 are required for this binding. We further observe that a NANOS2 mutant lacking these first 10 AAs (NANOS2-ΔN10) fails to rescue defects in the Nanos2-null mouse. Our current data thus indicate that the interaction with the CCR4-NOT deadenylation complex is essential for NANOS2 function. In addition, we further demonstrate that NANOS2-ΔN10 can associate with specific mRNAs as well as wild-type NANOS2, suggesting the existence of other NANOS2-associated factor(s) that determine the specificity of RNA-binding independently of the CCR4-NOT deadenylation complex.     

Methylation dynamics of repetitive DNA elements in the mouse germ cell lineage

Repetitive DNA elements account for a substantial fraction of the mammalian genome. Many are subject to DNA methylation, which is known to undergo dynamic change during mouse germ cell development. We found that repeat sequences of three different classes retain high levels of methylation at E12.5, when methylation is erased from many single-copy genes. Maximal demethylation of repeats was seen later in development and at different times in male and female germ cells. At none of the time points examined (E12.5, E15.5, and E17.5) did we see complete demethylation, suggesting that methylation patterns on repeats may be passed on from one generation to the next. In male germ cells, we observed a de novo methylation event resulting in complete methylation of all the repeats in the interval between E15.5 and E17.5, which was not seen in females. These results suggest that repeat sequences undergo coordinate changes in methylation during germ cell development and give further insights into germ cell reprogramming in mice.     

Temporally controlled site-specific mutagenesis in the germ cell lineage of the mouse testis

We have obtained a PrP-Cre-ER(T) transgenic mouse line (28.8) that selectively expresses in testis the tamoxifen-inducible Cre-ER(T) recombinase under the control of a mouse Prion protein (PrP) promoter-containing genomic fragment. Cre-ER(T) is expressed in spermatogonia and spermatocytes, but not in Sertoli and Leydig cells. We also established reporter PrP-L-EGFP-L transgenic mice harboring a LoxP-flanked enhanced green fluorescent protein (EGFP) Cre reporter cassette under the control of the same PrP promoter-containing genomic fragment that exhibits prominent EGFP expression in brain and testis. Using the PrP-L-EGFP-L as well as other Cre-reporter mice, we demonstrate that tamoxifen administration efficiently and selectively induces Cre-mediated recombination in the germ cell lineage. The established PrP-Cre-ER(T) line should provide a valuable tool for studying functions of germ cell-expressed genes involved in spermatogenesis.     

Chromatin organization in the male germ line of Drosophila hydei

The chromatin organization in developing germ cells of Drosophila hydei males was studied with the highly sensitive DNA stain DAPI (4, 6-diamidino-2-phenylindole dichloride). The prophase of meiosis I is characterized by decondensed chromosomes and only late during this stage do they condense rapidly. The sex chromosomes show allocycly. During postmeiotic development the final condensation of chromatin is preceded by a cycle of condensation and subsequent decondensation. Meiotic chromosomes were studied in more detail after orcein staining. Pairing sites of the sex chromosomes could be localized in the distal end of the heterochromatic arm of the X chromosome and distally in both arms of the Y chromosome. The various heterochromatic parts of the genome condense differentially in meiosis. Chromatin reorganization was studied cytochemically with antibodies raised against histones H1 and H2A of D. melanogaster. The core histone H2A is present in spermatid nuclei until the late elongation stage. However, histone H1 is not found in the chromatin later than the early primary spermatocyte stage. Thus, chromatin reorganization during spermatogenesis in D. hydei is complex. The process is discussed with regard to possible functions.     

Nanos3 maintains the germ cell lineage in the mouse by suppressing both Bax-dependent and -independent apoptotic pathways

Cell death in the germ line is controlled by both positive and negative mechanisms that maintain the appropriate number of germ cells and that prevent the possible formation of germ cell tumors. In the mouse embryo, Steel/c-Kit signaling is required to prevent migrating primordial germ cells (PGCs) from undergoing Bax-dependent apoptosis. In our current study, we show that migrating PGCs also undergo apoptosis in Nanos3-null embryos. We assessed whether the Bax-dependent apoptotic pathway is responsible for this cell death by knocking out the Bax gene together with the Nanos3 gene. Differing from Steel-null embryos, however, the Bax elimination did not completely rescue PGC apoptosis in Nanos3-null embryos, and only a portion of the PGCs survived in the double knockout embryo. We further established a mouse line, Nanos3-Cre-pA, to undertake lineage analysis and our results indicate that most of the Nanos3-null PGCs die rather than differentiate into somatic cells, irrespective of the presence or absence of Bax. In addition, a small number of surviving PGCs in Nanos3/Bax-null mice are maintained and differentiate as male and female germ cells in the adult gonads. Our findings thus suggest that heterogeneity exists in the PGC populations and that Nanos3 maintains the germ cell lineage by suppressing both Bax-dependent and Bax-independent apoptotic pathways.     

Establishment of the germ cell lineage in mammals

The germ cell lineage in mice becomes lineage-restricted about 7.2 days postcoitum. Its progenitors have migrated from the proximal region of the epiblast. Cells from the distal region of the epiblast will also give rise to germ cells, if they are transplanted to the proximal region at the appropriate time. Cells in this region are subject to a predisposing signal from the adjacent extra-embryonic ectoderm. It appears that this and other signals determine the emergence of germ cells: unlike in some other organisms, this event is not predetermined.     

The Dr-nanos gene is essential for germ cell specification in the planarian Dugesia ryukyuensis

Homologs of nanos are required for the formation and maintenance of germline stem cell (GSC) systems and for gametogenesis in many metazoans. Planarians can change their reproductive mode seasonally, alternating between asexual and sexual reproduction; they develop and maintain their somatic stem cells (SSCs) and GCSs from pluripotent stem cells known as neoblasts. We isolated a nanos homolog, Dr-nanos, from the expressed sequence tags (ESTs) of the sexualized form of Dugesia ryukyuensis. We examined the expression of Dr-nanos in asexual and sexualized planarians by in situ hybridization and analyzed its function using RNA interference (RNAi) together with a planarian sexualization assay. A nanos homolog, Dr-nanos, was identified in the planarian D. ryukyuensis. Dr-nanos expression was observed in the ovarian primordial cells of the asexual worms. This expression increased in proportion to sexualization and was localized in the early germline cells of the ovaries and testes. In X-ray-irradiated worms, the expression of Dr-nanos decreased to a large extent, indicating that Dr-nanos is expressed in some subpopulations of stem cells, especially in GSCs. During the sexualization process, worms in which Dr-nanos was knocked down by RNAi exhibited decreased numbers of oogonia in the ovaries and failed to develop testes, whereas the somatic sexual organs were not affected. We conclude that Dr-nanos is essential for the development of germ cells in the ovaries and testes and may have a function in the early stages of germ cell specification, but not in the development of somatic sexual organs.     

Smad5 is required for mouse primordial germ cell development

Smad5, together with Smad1 and Smad8, have been implicated as downstream signal mediators for several bone morphogenetic proteins (BMPs). Recent studies have shown that primordial germ cells (PGCs) are absent or greatly reduced in Bmp4 or Bmp8b mutant mice. To define the role of Smad5 in PGC development, we examined PGC number in Smad5 mutant mice by Oct4 whole-mount in situ hybridization and alkaline phosphatase staining. We found ectopic PGC-like cells in the amnion of some Smad5 mutant mice, however, the total number of PGCs was greatly reduced or completely absent in Smad5 mutant embryos, similar to Bmp4 or Bmp8b mutant embryos. Therefore, Smad5 is an important factor involved in PGC generation and localization.     

The proto-oncogene Ret is required for male foetal germ cell survival

The spermatogenic and oogenic lineages originate from bipotential primordial germ cells in response to signalling in the foetal testis or ovary, respectively. The signals required for male germ cell commitment and their entry into mitotic arrest remain largely unknown. Recent data show that the ligand GDNF is up regulated in the foetal testis indicating that it may be involved in male germ cell development. In this study genetic analysis of GDNF-RET signalling shows that RET is required for germ cell survival. Affected germ cells in Ret-/- mice lose expression of key germ cell markers, abnormally express cell cycle markers and undergo apoptosis. Surprisingly, a similar phenotype was not detected in Gdnf-/- mice indicating that either redundancy with a Gdnf related gene might compensate for its loss, or that RET operates in a GDNF independent manner in mouse foetal germ cells. Either way, this study identifies the proto-oncogene RET as a novel component of the foetal male germ cell development pathway.     

Testis-specific sulfoglycolipid, seminolipid, is essential for germ cell function in spermatogenesis

More than 90% of the glycolipid in mammalian testis consists of a unique sulfated glyceroglycolipid, seminolipid. The sulfation of the molecule is catalyzed by a Golgi membrane-associated sulfotransferase, cerebroside sulfotransferase (CST). Disruption of the Cst gene in mice results in male infertility due to the arrest of spermatogenesis prior to the metaphase of the first meiosis. However, the issue of which side of the cell function-germ cells or Sertoli cells-is deteriorated in this mutant mouse remains unknown. Our findings show that the defect is in the germ cell side, as evidenced by a transplantation analysis, in which wild-type spermatogonia expressing the green fluorescent protein were injected into the seminiferous tubules of CST-null testis. The transplanted GFP-positive cells generated colonies and spermatogenesis proceeded over meiosis in the mutant testis. The findings also clearly show that the seminolipid is expressed on the plasma membranes of spermatogonia, spermatocytes, spermatids, and spermatozoa, as evidenced by the immunostaining of wild-type testes using an anti-sulfogalactolipid antibody, Sulph-1 in comparison with CST-null testes as a negative control, and that seminolipid appears as early as day 8 of age, when Type B spermatogonia emerge.     

Male germ cell specific sulfogalactoglycerolipid is recognized and degraded by mycoplasmas associated with male infertility

Sulfogalactosylglycerolipid (SGG) is the major mammalian male germ cell glycolipid and has been implicated in sperm/egg binding. Mycoplasma pulmonis, a species of Mollicutes, is associated with male infertility in rodents. Purified SGG incubated in the presence of M. pulmonis was enzymatically degraded by both desulfation and deacylation. Desulfation occurred primarily at alkaline pH, and deacylation also increased with increased pH, indicating that these represent novel enzymatic activities. Digestion was facilitated, but not dependent on, the presence of detergent. Rat spermatozoa exposed to M. pulmonis showed a reduction in SGG content which was particularly marked for cauda (mature) spermatozoa. With the aid of tlc overlay binding procedure, intact M. pulmonis were found to bind specifically to sulfated glycolipids and thus SGG may provide the cell membrane receptor for this organism. The topology of mycoplasma binding to rat sperm was consistent with the known topology of sperm SGG. The reduced binding (and subsequent digestion) of caput spermatozoan SGG correlates with the membrane colocalization of SGG and its endogenous binding protein at this stage. Separation of SGG and its binding protein during epididymal sperm maturation appears to facilitate M. pulmonis binding to and digestion of cauda sperm SGG. The binding and degradation of the sperm SGG by M. pulmonis may play a role in the induction of infertility which follows infection with these organisms by interfering in sperm/egg receptor recognition.     

A male contraceptive targeting germ cell adhesion

Throughout spermatogenesis, developing germ cells remain attached to Sertoli cells via testis-specific anchoring junctions. If adhesion between these cell types is compromised, germ cells detach from the seminiferous epithelium and infertility often results. Previously, we reported that Adjudin is capable of inducing germ cell loss from the epithelium. In a small subset of animals, however, oral administration of Adjudin (50 mg per kg body weight (b.w.) for 29 d) resulted in adverse effects such as liver inflammation and muscle atrophy. Here, we report a novel approach in which Adjudin is specifically targeted to the testis by conjugating Adjudin to a recombinant follicle-stimulating hormone (FSH) mutant, which serves as its 'carrier'. Using this approach, infertility was induced in adult rats when 0.5 microg Adjudin per kg b.w. was administered intraperitoneally, which was similar to results when 50 mg per kg b.w. was given orally. This represents a substantial increase in Adjudin's selectivity and efficacy as a male contraceptive.     

c-Flip(L) is expressed in undifferentiated mouse male germ cells

Apoptosis represents a fundamental process during fetal/post-natal testis development. Therefore pro- and anti-apoptotic proteins are essential to regulate testis physiology. c-Flip(L) is a known inhibitor of caspase 8/10 activity; in this study its perinatal expression in mouse male germ cells was investigated. In testis sections and seminiferous tubule whole mount c-Flip(L) was found to be expressed in undifferentiated spermatogonia and to co-localize with germ stem cells markers. In vivo investigations in the vitamin-A deficient mouse, lacking differentiated germ cells, confirmed c-Flip(L) expression in undifferentiated spermatogonia. Further analyses showed Fas expression but no significant caspase 8/10 activity when c-Flip(L) was highly expressed. Altogether these data suggest that c-Flip may control the survival rate of undifferentiated spermatogonia.