The gamete fusion process is defective in eggs of Cd9-deficient mice

The cell-surface molecule Cd9, a member of the transmembrane-4 superfamily, interacts with the integrin family and other membrane proteins. and is postulated to participate in cell migration and adhesion. Expression of Cd9 enhances membrane fusion between muscle cells and promotes viral infection in some cells. Fertilization also involves membrane fusion, between gametes. In mammals, the sperm binds to microvilli on the egg surface, and sperm-egg membrane fusion first occurs around the equatorial region of the sperm head12. The fused membrane is then disrupted, and the sperm nucleus as well as the cytoplasm is incorporated into the egg. Cd9 is expressed on the plasma membrane of the mouse egg, and an anti-Cd9 monoclonal antibody inhibits sperm-egg surface interactions. We generated Cd9 mice and found that homozygous mutant females were infertile. Sperm-egg binding was normal, but sperm-egg fusion was almost entirely inhibited in eggs from Cd9 females. Intracellular Ca2 oscillations, which signal fertilization, were absent in almost all mutant eggs; in rare cases, a response occurred after a long time period. In normal animals, Cd9 molecules were expressed on the egg microvilli and became densely concentrated at the sperm attachment site. Thus, our results show that Cd9 is important in the gamete fusion process at fertilization.     

Genomic and functional evolution of the Drosophila melanogaster sperm proteome

In addition to delivering a haploid genome to the egg, sperm have additional critical functions, including egg activation, origination of the zygote centrosome and delivery of paternal factors. Despite this, existing knowledge of the molecular basis of sperm form and function is limited. We used whole-sperm mass spectrometry to identify 381 proteins of the Drosophila melanogaster sperm proteome (DmSP). This approach identified mitochondrial, metabolic and cytoskeletal proteins, in addition to several new functional categories. We also observed nonrandom genomic clustering of sperm genes and underrepresentation on the X chromosome. Identification of widespread functional constraint on the proteome indicates that sexual selection has had a limited role in the overall evolution of D. melanogaster sperm. The relevance of the DmSP to the study of mammalian sperm function and fertilization mechanisms is demonstrated by the identification of substantial homology between the DmSP and proteins of the mouse axoneme accessory structure.     

Epigenetic asymmetry of imprinted genes in plant gametes

Plant imprinted genes show parent-of-origin expression in seed endosperm, but little is known about the nature of parental imprints in gametes before fertilization. We show here that single differentially methylated regions (DMRs) correlate with allele-specific expression of two maternally expressed genes in the seed and that one DMR is differentially methylated between gametes. Thus, plants seem to have developed similar strategies as mammals to epigenetically mark imprinted genes.     

Haploinsufficiency of protamine-1 or -2 causes infertility in mice

Protamines are the major DNA-binding proteins in the nucleus of sperm in most vertebrates and package the DNA in a volume less than 5% of a somatic cell nucleus. Many mammals have one protamine, but a few species, including humans and mice, have two. Here we use gene targeting to determine if the second protamine provides redundancy to an essential process, or if both protamines are necessary. We disrupted the coding sequence of one allele of either Prm1 or Prm2 in embryonic stem (ES) cells derived from 129-strain mice, and injected them into blastocysts from C57BL/6-strain mice. Male chimeras produced 129-genotype sperm with disrupted Prm1 or Prm2 alleles, but failed to sire offspring carrying the 129 genome. We also found that a decrease in the amount of either protamine disrupts nuclear formation, processing of protamine-2 and normal sperm function. Our studies show that both protamines are essential and that haploinsufficiency caused by a mutation in one allele of Prm1 or Prm2 prevents genetic transmission of both mutant and wild-type alleles.     

Rate of molecular evolution of the seminal protein gene SEMG2 correlates with levels of female promiscuity

Postcopulatory sperm competition is a key aspect of sexual selection and is believed to drive the rapid evolution of both reproductive physiology and reproduction-related genes. It is well-established that mating behavior determines the intensity of sperm competition, with polyandry (i.e., female promiscuity) leading to fiercer sperm competition than monandry. Studies in mammals, particularly primates, showed that, owing to greater sperm competition, polyandrous taxa generally have physiological traits that make them better adapted for fertilization than monandrous species, including bigger testes, larger seminal vesicles, higher sperm counts, richer mitochondrial loading in sperm and more prominent semen coagulation. Here, we show that the degree of polyandry can also impact the dynamics of molecular evolution. Specifically, we show that the evolution of SEMG2, the gene encoding semenogelin II, a main structural component of semen coagulum, is accelerated in polyandrous primates relative to monandrous primates. Our study showcases the intimate relationship between sexual selection and the molecular evolution of reproductive genes.     

Fine-scale recombination patterns differ between chimpanzees and humans

Recombination rates seem to vary extensively along the human genome. Pedigree analysis suggests that rates vary by an order of magnitude when measured at the megabase scale, and at a finer scale, sperm typing studies point to the existence of recombination hotspots. These are short regions (1-2 kb) in which recombination rates are 10-1,000 times higher than the background rate. Less is known about how recombination rates change over time. Here we determined to what degree recombination rates are conserved among closely related species by estimating recombination rates from 14 Mb of linkage disequilibrium data in central chimpanzee and human populations. The results suggest that recombination hotspots are not conserved between the two species and that recombination rates in larger (50 kb) genomic regions are only weakly conserved. Therefore, the recombination landscape has changed markedly between the two species.     

An abundance of X-linked genes expressed in spermatogonia

Spermatogonia are the self-renewing, mitotic germ cells of the testis from which sperm arise by means of the differentiation pathway known as spermatogenesis. By contrast with hematopoietic and other mammalian stem-cell populations, which have been subjects of intense molecular genetic investigation, spermatogonia have remained largely unexplored at the molecular level. Here we describe a systematic search for genes expressed in mouse spermatogonia, but not in somatic tissues. We identified 25 genes (19 of which are novel) that are expressed in only male germ cells. Of the 25 genes, 3 are Y-linked and 10 are X-linked. If these genes had been distributed randomly in the genome, one would have expected zero to two of the genes to be X-linked. Our findings indicate that the X chromosome has a predominant role in pre-meiotic stages of mammalian spermatogenesis. We hypothesize that the X chromosome acquired this prominent role in male germ-cell development as it evolved from an ordinary, unspecialized autosome.     

Mort cellulaire programmée et embryogenèse

The investigation of programmed cell death (PCD) events in embryogenesis is almost as ancient as cell theory. Firstly, PCD is massively involved in the elimination of anatomical structures in the embryo which are no longer present in the adult: non functional anlagen of structures inherited from the evolutionary story of the species; structures used in one sex and not in the other; or structures functional in the embryo, but whose function is taken over by other organs in the adult. Apart from the complete disappearance of such tissues or cell types, more localized PCD events accompany most of the morphogenetic movements which shape organs; cavitation, folding and closing of epithelial sheets, sculpturing of mesenchymal spaces. Finally, cell populations which functionally interact - typically neurons, glial cells and muscle fibers, or the various leukocyte species of the immune system - regulate their respective numbers by PCD. PCD events in embryogenesis involve the same processes of cellular suicide as those which contribute to homeostasis in the adult. Localized PCD associated with organogenesis usually follows the standard apoptotic scheme, while the massive PCDs involved in the elimination of whole tissues usually involve a distinct form of PCD, in which the cells destroy themselves by autophagy. In the embryo, depending on the local context, the same biochemical signals may induce proliferation, differentiation, or PCD in the cells that receive them. A given molecular signal often acts on the genesis of an organ by having at least two of these effects, on distinct cell populations within the concerned anlage.     

An azoospermic man with a de novo point mutation in the Y-chromosomal gene USP9Y

In humans, deletion of any one of three Y-chromosomal regions- AZFa, AZFb or AZFc-disrupts spermatogenesis, causing infertility in otherwise healthy men. Although candidate genes have been identified in all three regions, no case of spermatogenic failure has been traced to a point mutation in a Y-linked gene, or to a deletion of a single Y-linked gene. We sequenced the AZFa region of the Y chromosome and identified two functional genes previously described: USP9Y (also known as DFFRY) and DBY (refs 7,8). Screening of the two genes in 576 infertile and 96 fertile men revealed several sequence variants, most of which appear to be heritable and of little functional consequence. We found one de novo mutation in USP9Y: a 4-bp deletion in a splice-donor site, causing an exon to be skipped and protein truncation. This mutation was present in a man with nonobstructive azoospermia (that is, no sperm was detected in semen), but absent in his fertile brother, suggesting that the USP9Y mutation caused spermatogenic failure. We also identified a single-gene deletion associated with spermatogenic failure, again involving USP9Y, by re-analysing a published study.     

The t complex-encoded GTPase-activating protein Tagap1 acts as a transmission ratio distorter in mice

Transmission ratio distortion in the mouse is caused by several t-complex distorters (Tcds) acting in trans on the t-complex responder (Tcr). Tcds additively affect the flagellar movement of all spermatozoa derived from t/+ males; sperm carrying Tcr are rescued, resulting in an advantage for t sperm in fertilization. Here we show that Tagap1, a GTPase-activating protein, can act as a distorter. Tagap1 maps to the Tcd1 interval and has four t loci, which encode altered proteins including a C-terminally truncated form. Overexpression of wild-type Tagap1 in sperm cells phenocopied Tcd function, whereas a loss-of-function Tagap1 allele reduced the transmission rate of the t6 haplotype. The combined data strongly suggest that the t loci of Tagap1 produce Tcd1a. Our results unravel the molecular nature of a Tcd and demonstrate the importance of small G proteins in transmission ratio distortion in the mouse.     

Human recombination hot spots hidden in regions of strong marker association

The fine-scale distribution of meiotic recombination events in the human genome can be inferred from patterns of haplotype diversity in human populations but directly studied only by high-resolution sperm typing. Both approaches indicate that crossovers are heavily clustered into narrow recombination hot spots. But our direct understanding of hot-spot properties and distributions is largely limited to sperm typing in the major histocompatibility complex (MHC). We now describe the analysis of an unremarkable 206-kb region on human chromosome 1, which identified localized regions of linkage disequilibrium breakdown that mark the locations of sperm crossover hot spots. The distribution, intensity and morphology of these hot spots are markedly similar to those in the MHC. But we also accidentally detected additional hot spots in regions of strong association. Coalescent analysis of genotype data detected most of the hot spots but showed significant differences between sperm crossover frequencies and historical recombination rates. This raises the possibility that some hot spots, particularly those in regions of strong association, may have evolved very recently and not left their full imprint on haplotype diversity. These results suggest that hot spots could be very abundant and possibly fluid features of the human genome.     

Inactivation of the Wip1 phosphatase inhibits mammary tumorigenesis through p38 MAPK-mediated activation of the p16(Ink4a)-p19(Arf) pathway

Modulation of tumor suppressor activities may provide new opportunities for cancer therapy. Here we show that disruption of the gene Ppm1d encoding Wip1 phosphatase activated the p53 and p16 (also called Ink4a)-p19 (also called ARF) pathways through p38 MAPK signaling and suppressed in vitro transformation of mouse embryo fibroblasts (MEFs) by oncogenes. Disruption of the gene Cdkn2a (encoding p16 and p19), but not of Trp53 (encoding p53), reconstituted cell transformation in Ppm1d-null MEFs. In vivo, deletion of Ppm1d in mice bearing mouse mammary tumor virus (MMTV) promoter-driven oncogenes Erbb2 (also called c-neu) or Hras1 impaired mammary carcinogenesis, whereas reduced expression of p16 and p19 by methylation-induced silencing or inactivation of p38 MAPK correlated with tumor appearance. We conclude that inactivation or depletion of the Wip1 phosphatase with resultant p38 MAPK activation suppresses tumor appearance by modulating the Cdkn2a tumor-suppressor locus.     

Age distribution of human gene families shows significant roles of both large- and small-scale duplications in vertebrate evolution

The zebrafish embryo is transparent and can tolerate absence of blood flow because its oxygen is delivered by diffusion rather than by the cardiovascular system. It is therefore possible to attribute cardiac failure directly to particular genes by ruling out the possibility that it is due to a secondary effect of hypoxia. We focus here on pickwickm171 (pikm171), a recessive lethal mutation discovered in a large-scale genetic screen. There are three other alleles in the pik complementation group with this phenotype (pikm242, pikm740, pikm186; ref. 3) and one allele (pikmVO62H) with additional skeletal paralysis. The pik heart develops normally but is poorly contractile from the first beat. Aside from the edema that inevitably accompanies cardiac dysfunction, development is normal during the first three days. We show by positional cloning that the 'causative' mutation is in an alternatively-spliced exon of the gene (ttn) encoding Titin. Titin is the biggest known protein and spans the half-sarcomere from Z-disc to M-line in heart and skeletal muscle. It has been proposed to provide a scaffold for the assembly of thick and thin filaments and to provide elastic recoil engendered by stretch during diastole. We found that nascent myofibrils form in pik mutants, but normal sarcomeres are absent. Mutant cells transplanted to wildtype hearts remain thin and bulge outwards as individual cell aneurysms without affecting nearby wildtype cardiomyocytes, indicating that the contractile deficiency is cell-autonomous. Absence of Titin function thus results in blockage of sarcomere assembly and causes a functional disorder resembling human dilated cardiomyopathies, one form of which is described in another paper in this issue.     

Imprinting along the Kcnq1 domain on mouse chromosome 7 involves repressive histone methylation and recruitment of Polycomb group complexes

Imprinted genes are clustered in domains, and their allelic repression is mediated by imprinting control regions. These imprinting control regions are marked by DNA methylation, which is essential to maintain imprinting in the embryo. To explore how imprinting is regulated in placenta, we studied the Kcnq1 domain on mouse distal chromosome 7. This large domain is controlled by an intronic imprinting control region and comprises multiple genes that are imprinted in placenta, without the involvement of promoter DNA methylation. We found that the paternal repression along the domain involves acquisition of trimethylation at Lys27 and dimethylation at Lys9 of histone H3. Eed-Ezh2 Polycomb complexes are recruited to the paternal chromosome and potentially regulate its repressive histone methylation. Studies on embryonic stem cells and early embryos support our proposal that chromatin repression is established early in development and is maintained in the placenta. In the embryo, however, imprinting is stably maintained only at genes that have promoter DNA methylation. These data underscore the importance of histone methylation in placental imprinting and identify mechanistic similarities with X-chromosome inactivation in extraembryonic tissues, suggesting that the two epigenetic mechanisms are evolutionarily linked.     

Dynamics of the p53-Mdm2 feedback loop in individual cells

The tumor suppressor p53, one of the most intensely investigated proteins, is usually studied by experiments that are averaged over cell populations, potentially masking the dynamic behavior in individual cells. We present a system for following, in individual living cells, the dynamics of p53 and its negative regulator Mdm2 (refs. 1,4-7): this system uses functional p53-CFP and Mdm2-YFP fusion proteins and time-lapse fluorescence microscopy. We found that p53 was expressed in a series of discrete pulses after DNA damage. Genetically identical cells had different numbers of pulses: zero, one, two or more. The mean height and duration of each pulse were fixed and did not depend on the amount of DNA damage. The mean number of pulses, however, increased with DNA damage. This approach can be used to study other signaling systems and suggests that the p53-Mdm2 feedback loop generates a 'digital' clock that releases well-timed quanta of p53 until damage is repaired or the cell dies.     

CCDC39 is required for assembly of inner dynein arms and the dynein regulatory complex and for normal ciliary motility in humans and dogs

Primary ciliary dyskinesia (PCD) is an inherited disorder characterized by recurrent infections of the upper and lower respiratory tract, reduced fertility in males and situs inversus in about 50% of affected individuals (Kartagener syndrome). It is caused by motility defects in the respiratory cilia that are responsible for airway clearance, the flagella that propel sperm cells and the nodal monocilia that determine left-right asymmetry. Recessive mutations that cause PCD have been identified in genes encoding components of the outer dynein arms, radial spokes and cytoplasmic pre-assembly factors of axonemal dyneins, but these mutations account for only about 50% of cases of PCD. We exploited the unique properties of dog populations to positionally clone a new PCD gene, CCDC39. We found that loss-of-function mutations in the human ortholog underlie a substantial fraction of PCD cases with axonemal disorganization and abnormal ciliary beating. Functional analyses indicated that CCDC39 localizes to ciliary axonemes and is essential for assembly of inner dynein arms and the dynein regulatory complex.     

Ras-MAPK signaling promotes trophectoderm formation from embryonic stem cells and mouse embryos

In blastocyst chimeras, embryonic stem (ES) cells contribute to embryonic tissues but not extraembryonic trophectoderm. Conditional activation of HRas1(Q61L) in ES cells in vitro induces the trophectoderm marker Cdx2 and enables derivation of trophoblast stem (TS) cell lines that, when injected into blastocysts, chimerize placental tissues. Erk2, the downstream effector of Ras-mitogen-activated protein kinase (MAPK) signaling, is asymmetrically expressed in the apical membranes of the 8-cell-stage embryo just before morula compaction. Inhibition of MAPK signaling in cultured mouse embryos compromises Cdx2 expression, delays blastocyst development and reduces trophectoderm outgrowth from embryo explants. These data show that ectopic Ras activation can divert ES cells toward extraembryonic trophoblastic fates and implicate Ras-MAPK signaling in promoting trophectoderm formation from mouse embryos.