Haploid Gene Expression Research Articles

The effect of phytosulfokine alpha on haploid embryogenesis and gene expression of Brassica napus microspore cultures.

Microspore embryogenesis (ME) is the most powerful tool for creating homozygous lines in plant breeding and molecular biology research. It is still based mainly on the reprogramming of microspores by temperature, osmotic and/or nutrient stress. New compounds are being sought that could increase the efficiency of microspore embryogenesis or even induce the formation of haploid embryos from recalcitrant genotypes. Among these, the mitogenic factor phytosulfokine alpha (PSK-α) is promising due to its broad spectrum of activity in vivo and in vitro. The aim of our study was to investigate the effect of PSK-α on haploid embryogenesis from microspores of oilseed rape (Brassica napus L., DH4079), one of the most important oil crops and a model plant for studying the molecular mechanisms controlling embryo formation. We tested different concentrations (0, 0.01, 0.1 and 1 µM) of the peptide and evaluated its effect on microspore viability and embryo regeneration after four weeks of culture. Our results showed a positive correlation between addition of PSK-α and cultured microspore viability and a positive effect also on the number of developed embryos. The analysis of transcriptomes across three time points (day 0, 2 and 4) with or without PSK-α supplementation (15 RNA libraries in total) unveiled differentially expressed genes pivotal in cell division, microspore embryogenesis, and subsequent regeneration. PCA grouped transcriptomes by RNA sampling time, with the first two principal components explaining 56.8% variability. On day 2 with PSK, 45 genes (15 up- and 30 down-regulated) were differentially expressed when PSK-α was added and their number increased to 304 by day 4 (30 up- and 274 down-regulated). PSK, PSKR, and PSI gene expression analysis revealed dynamic patterns, with PSK2 displaying the highest increase and overall expression during microspore culture at days 2 and 4. Despite some variations, only PSK1 showed significant differential expression upon PSK-α addition. Of 16 ME-related molecular markers, 3 and 15 exhibited significant differential expression in PSK-supplemented cultures at days 2 and 4, respectively. Embryo-specific markers predominantly expressed after 4 days of culture, with higher expression in medium without PSK, while on day 0, numerous sporophyte-specific markers were highly expressed.

From azoospermia to macrozoospermia, a phenotypic continuum due to mutations in the ZMYND15 gene.

Thanks to tremendous advances in sequencing technologies and in particular to whole exome sequencing (WES), many genes have now been linked to severe sperm defects. A precise genetic diagnosis is obtained for a minority of patients and only for the most severe defects like azoospermia or macrozoospermia which is very often due to defects in the aurora kinase C (AURKC) gene. Here, we studied a subject with a severe oligozoospermia and a phenotypic diagnosis of macrozoospermia. AURKC analysis did not reveal any deleterious variant. WES was then initiated which permitted to identify a homozygous loss of function variant in the zinc finger MYND-type containing 15 (ZMYND15) gene. ZMYND15 has been described to serve as a switch for haploid gene expression, and mice devoid of ZMYND15 were shown to be sterile due to nonobstructive azoospermia (NOA). In man, ZMYND15 has been associated with NOA and severe oligozoospermia. We confirm here that the presence of a bi-allelic ZMYND15 variant induces a severe oligozoospermia. In addition, we show that severe oligozoospermia can be associated macrozoospermia, and that a phenotypic misdiagnosis is possible, potentially delaying the genetic diagnosis. In conclusion, genetic defects in ZMYND15 can induce complete NOA or severe oligozoospermia associated with a very severe teratozoospermia. In our experience, severe oligozoospermia is often associated with severe teratozoospermia and can sometimes be misinterpreted as macrozoospermia or globozoospermia. In these instances, specific AURKC or dpy-19 like 2 (DPY19L2) diagnosis is usually negative and we recommend the direct use of a pan-genomic techniques such as WES.

Large structural variations in the haplotype-resolved African cassava genome.

SummaryCassava (Manihot esculenta Crantz, 2n = 36) is a global food security crop. It has a highly heterozygous genome, high genetic load, and genotype‐dependent asynchronous flowering. It is typically propagated by stem cuttings and any genetic variation between haplotypes, including large structural variations, is preserved by such clonal propagation. Traditional genome assembly approaches generate a collapsed haplotype representation of the genome. In highly heterozygous plants, this results in artifacts and an oversimplification of heterozygous regions. We used a combination of Pacific Biosciences (PacBio), Illumina, and Hi‐C to resolve each haplotype of the genome of a farmer‐preferred cassava line, TME7 (Oko‐iyawo). PacBio reads were assembled using the FALCON suite. Phase switch errors were corrected using FALCON‐Phase and Hi‐C read data. The ultralong‐range information from Hi‐C sequencing was also used for scaffolding. Comparison of the two phases revealed >5000 large haplotype‐specific structural variants affecting over 8 Mb, including insertions and deletions spanning thousands of base pairs. The potential of these variants to affect allele‐specific expression was further explored. RNA‐sequencing data from 11 different tissue types were mapped against the scaffolded haploid assembly and gene expression data are incorporated into our existing easy‐to‐use web‐based interface to facilitate use by the broader plant science community. These two assemblies provide an excellent means to study the effects of heterozygosity, haplotype‐specific structural variation, gene hemizygosity, and allele‐specific gene expression contributing to important agricultural traits and further our understanding of the genetics and domestication of cassava.

Histone hyperacetylation and DNA methylation interplay during murine spermatogenesis.

Male germ cell development is a critical period during which epigenetic patterns are established and maintained. The progression from diploid spermatogonia to haploid spermatozoa involves the incorporation of testis-specific histone variants, mitotic and meiotic divisions, haploid gene expression, histone-protamine transitions and massive epigenetic reprogramming. Understanding the protein players and the epigenetic mark network involved in the setting of the epigenetic programme in spermatogenesis is an exciting new clue in the field of reproductive biology with translational outcomes. As information in the existing literature regarding cross-talk between DNA methylation and histone hyperacetylation in the advanced stages of murine spermatogenesis is still scarce and controversial we have investigated the effect of a DNA-methyltransferase inhibitor, 5-aza-2'-deoxycytidine, at the cytological and molecular level (by transmission electron microscopy, immunocytochemistry and immunoprecipitation methods). Our results revealed an important role for regulation of DNA methylation in controlling histone hyperacetylation and chromatin remodelling during spermatogenesis.

Pollen competition is the mechanism underlying a variety of evolutionary phenomena in dioecious plants.

It has long been known that more pollen grains often arrive on stigmas than there are ovules to fertilize, resulting in pollen competition. Moreover, this competition among pollen grains (gametophytes) depends, in part, on their extensive haploid gene expression. Here I review how this leads to a variety of phenomena in dioecious plants of interest to evolutionary biologists. For example, pollen competition can lead to extreme female-biased sex ratios. In addition, gene expression by individual pollen grains can slow mutation accumulation and degeneration of the Y chromosome. Lastly, I review work on how the haploid selection resulting from pollen competition has been proposed to influence which alleles are linked to the Y chromosome, and some recent empirical evidence in support of this theory.

Genetic variation for resistance to high temperature stress of mature sperm - a study in Drosophila.

Genetic variation for resistance to heat stress has been found for a number of life-history components in Drosophila species. For male and female fertility (or sterility), stress resistance of the parents is confounded with stress resistance of the haploid gametes. Many genes are known to influence male fertility in Drosophila melanogaster. Some may carry temperature sensitive alleles that reduce fertility through effects on mature sperm when exposed to heat stress. In this study, sperm from each of 320 males were either not heat shocked (control) or exposed to a heat shock (36.9°C for 2 hours) either in the male testes or in the female reproductive tract. We did not detect any temperature sensitive sterility alleles. These results are relevant in relation to haploid gene expression and the findings of considerable amounts of mRNA in mature sperm, potentially important for sperm function and fertilization.

TRF2 is recruited to the pre-initiation complex as a testis-specific subunit of TFIIA/ALF to promote haploid cell gene expression.

Mammalian genomes encode two genes related to the TATA-box binding protein (TBP), TBP-related factors 2 and 3 (TRF2 and TRF3). Male Trf2−/− mice are sterile and characterized by arrested spermatogenesis at the transition from late haploid spermatids to early elongating spermatids. Despite this characterization, the molecular function of murine Trf2 remains poorly characterized and no direct evidence exists to show that it acts as a bona fide chromatin-bound transcription factor. We show here that Trf2 forms a stable complex with TFIIA or the testis expressed paralogue ALF chaperoned in the cytoplasm by heat shock proteins. We demonstrate for the first time that Trf2 is recruited to active haploid cell promoters together with Tbp, Taf7l and RNA polymerase II. RNA-seq analysis identifies a set of genes activated in haploid spermatids during the first wave of spermatogenesis whose expression is down-regulated by Trf2 inactivation. We therefore propose that Trf2 is recruited to the preinitiation complex as a testis-specific subunit of TFIIA/ALF that cooperates with Tbp and Taf7l to promote haploid cell gene expression.

A decade of pollen transcriptomics.

Key message Overview of pollen transcriptome studies. Pollen development is driven by gene expression, and knowledge of the molecular events underlying this process has undergone a quantum leap in the last decade through studies of the transcriptome. Here, we outline historical evidence for male haploid gene expression and review the wealth of pollen transcriptome data now available. Knowledge of the transcriptional capacity of pollen has progressed from genetic studies to the direct analysis of RNA and from gene-by-gene studies to analyses on a genomic scale. Microarray and/or RNA-seq data can now be accessed for all phases and cell types of developing pollen encompassing 10 different angiosperms. These growing resources have accelerated research and will undoubtedly inspire new directions and the application of system-based research into the mechanisms that govern the development, function and evolution of angiosperm pollen.Electronic supplementary materialThe online version of this article (doi:10.1007/s00497-015-0261-7) contains supplementary material, which is available to authorized users.

Inbreeding depression in male gametic performance.

One key objective in evolutionary ecology is to understand the magnitude of inbreeding depression expressed across sex-specific components of fitness. One major component of male fitness is fertilization success, which depends on male gametic performance (sperm and pollen performance in animals and plants, respectively). Inbreeding depression in male gametic performance could create sex-specific inbreeding depression in fitness, increase the benefit of inbreeding avoidance and reduce the efficacy of artificial insemination and pollination. However, there has been no assessment of the degree to which inbreeding generally depresses male gametic performance and hence post-copulatory or post-pollination fertilization success. Because inbreeding depression is understood to be a property of diploid entities, it is not clear what degree of inbreeding depression in haploid gametic performance should be expected. Here, we first summarize how inbreeding depression in male gametic performance could potentially arise through gene expression in associated diploid cells and/or reduced genetic diversity among haploid gametes. We then review published studies that estimate the magnitude of inbreeding depression in traits measuring components of sperm or pollen quantity, quality and competitiveness. Across 51 published studies covering 183 study traits, the grand mean inbreeding load was approximately one haploid lethal equivalent, suggesting that inbreeding depresses male gametic performance across diverse systems and traits. However, there was an almost complete lack of explicit estimates from wild populations. Future studies should quantify inbreeding depression in systematic sets of gametic traits under naturally competitive and noncompetitive conditions and quantify the degree to which gamete phenotypes and performance reflect haploid vs. diploid gene expression.

Histone crotonylation specifically marks the haploid male germ cell gene expression program

The haploid male germ cell differentiation program controls essential steps of male gametogenesis and relies partly on a significant number of sex chromosome-linked genes. These genes need to escape chromosome-wide transcriptional repression of sex chromosomes, which occurs during meiosis and is largely maintained in post-meiotic cells. A newly discovered histone lysine modification, crotonylation (Kcr), marks X/Y-linked genes that are active in post-meiotic male germ cells. Histone Kcr, by conferring resistance to transcriptional repressors, could be a dominant element in maintaining these genes active in the globally repressive environment of haploid cell sex chromosomes. Furthermore, the same mark was found associated with post-meiotically activated genes on autosomes. Histone Kcr therefore appears to be an indicator of the male haploid cell gene expression program and a notable element of genome programming in the post-meiotic phases of spermatogenesis.

PLOIDALLY ANTAGONISTIC SELECTION MAINTAINS STABLE GENETIC POLYMORPHISM

Understanding the maintenance of genetic variation in the face of selection remains a key issue in evolutionary biology. One potential mechanism for the maintenance of genetic variation is opposing selection during the diploid and haploid stages of biphasic life cycles universal among eukaryotic sexual organisms. If haploid and diploid gene expression both occur, selection can act in each phase, potentially in opposing directions. In addition, sex-specific selection during haploid phases is likely simply because male and female gametophytes/gametes tend to have contrasting life histories. We explored the potential for the maintenance of a stable polymorphism under ploidally antagonistic as well as sex-specific selection. Furthermore, we examined the role of the chromosomal location of alleles (autosomal or sex-linked). Our analyses show that the most permissible conditions for the maintenance of polymorphism occur under negative ploidy-by-sex interactions, where stronger selection for an allele in female than male diploids is coupled with weaker selection against the allele in female than male haploids. Such ploidy-by-sex interactions also promote allele frequency differences between the sexes. With constant fitness, ploidally antagonistic selection can maintain stable polymorphisms for autosomal and X-linked genes but not for Y-linked genes. We discuss the implications of our results and outline a number of biological settings where the scenarios modeled may apply.

Zmynd15 Encodes a Histone Deacetylase-dependent Transcriptional Repressor Essential for Spermiogenesis and Male Fertility

Spermatogenesis is a complex process through which male germ line stem cells undergo a multi-step differentiation program and sequentially become spermatogonia, spermatocytes, spermatids, and eventually spermatozoa. In this process, transcription factors act as switches that precisely regulate the expression of genes that in turn control the developmental program of male germ cells. Transcription factors identified to be essential for normal haploid gene expression all display transcription-activating effects and thus serve as the "on" switch for haploid gene expression. Here, we report that ZMYND15 acts as a histone deacetylase-dependent transcriptional repressor and controls normal temporal expression of haploid cell genes during spermiogenesis. Inactivation of Zmynd15 results in early activation of transcription of numerous important haploid genes including Prm1, Tnp1, Spem1, and Catpser3; depletion of late spermatids; and male infertility. ZMYND15 represents the first transcriptional repressor identified to be essential for sperm production and male fertility.

Functional transformation of the chromatoid body in mouse spermatids requires testis-specific serine/threonine kinases

The cytoplasmic chromatoid body (CB) organizes mRNA metabolism and small regulatory RNA pathways, in relation to haploid gene expression, in mammalian round spermatids. However, little is known about functions and fate of the CB at later steps of spermatogenesis, when elongating spermatids undergo chromatin compaction and transcriptional silencing. In mouse elongating spermatids, we detected accumulation of the testis-specific serine/threonine kinases TSSK1 and TSSK2, and the substrate TSKS, in a ring-shaped structure around the base of the flagellum and in a cytoplasmic satellite, both corresponding to structures described to originate from the CB. At later steps of spermatid differentiation, the ring is found at the caudal end of the newly formed mitochondrial sheath. Targeted deletion of the tandemly arranged genes Tssk1 and Tssk2 in mouse resulted in male infertility, with loss of the CB-derived ring structure, and with elongating spermatids possessing a collapsed mitochondrial sheath. These results reveal TSSK1- and TSSK2-dependent functions of a transformed CB in post-meiotic cytodifferentiation of spermatids.

Sperm length is not influenced by haploid gene expression in the flies Drosophila melanogaster and Scathophaga stercoraria

Recent theoretical models have postulated a role for haploid-diploid conflict and for kin selection favouring sperm cooperation and altruism in the diversification and specialization of sperm form. A critical assumption of these models-that haploid gene expression contributes to variation in sperm form-has never been demonstrated and remains contentious. By quantifying within-male variation in sperm length using crosses between males and females from populations that had been subjected to divergent experimental selection, we demonstrate that haploid gene expression does not contribute to variation in sperm length in both Drosophila melanogaster and Scathophaga stercoraria. This finding casts doubt on the importance of haploid-diploid conflict and kin selection as evolutionary influences of sperm phenotypes.

Male gametophyte development: a molecular perspective

Pollen grains represent the highly reduced haploid male gametophyte generation in flowering plants, consisting of just two or three cells when released from the anthers. Their role is to deliver twin sperm cells to the embryo sac to undergo fusion with the egg and central cell. This double fertilization event along with the functional specialization of the male gametophyte, are considered to be key innovations in the evolutionary success of flowering plants. This review encompasses important recent advances in our understanding of the molecular mechanisms controlling male gametophyte development. A brief overview of pollen development is presented, followed by a discussion of genome-wide transcriptomic studies of haploid gene expression. The progress achieved through genetic analysis of landmark events of male gametogenesis is discussed, with a focus on sperm cell production, and an emerging model of the regulatory network governing male germline development is presented. The review concludes with a perspective of the impact these data will have on future research strategies to further develop our understanding of the gametophytic control of pollen development.

Sperm competition and sperm cooperation: the potential role of diploid and haploid expression

Sperm competition is a powerful selective force driving the evolution of sperm shape and function. Recent findings suggest that sperm cooperation is a potential evolutionary response to sperm competition. Sperm cooperation may enhance the performance of the ejaculate increasing a male's chance to outcompete rival males in competition for fertilisation. Whether and how sperm cooperation may evolve is the focal point of this review. The relative importance of haploid and diploid gene expression for the evolution of sperm cooperation and the potential conflict of interest between (i) haploid sperm and diploid male and (ii) among sibling sperm, since sibling sperm only share an average of 50% of their genes in a diploid organism, are discussed. Furthermore, sperm cooperation is defined and the literature for empirical evidence of sperm cooperation is reviewed in light of the author's definitions.

Male germ cell gene expression.

Formation of the male gamete occurs in sequential mitotic, meiotic, and postmeiotic phases. Many germ cell-specific transcripts are produced during this process. Their expression is developmentally regulated and stage specific. Some of these transcripts are product of genes that are male germ cell-specific homologs of genes expressed in somatic cells, while some are expressed from unique genes unlike any others in the genome. Others are alternate transcripts derived from the same gene as transcripts in somatic cells but differing from them in size and/or overall sequence. They are generated during gene expression by using promoters and transcription factors that activate transcription at different start sites upstream or downstream of the usual site, by incorporation of alternate exons, by germ cell-specific splicing events, and by using alternate initiation sites for polyadenylation. Male germ cell development consists of an assortment of unique processes, including meiosis, genetic recombination, haploid gene expression, formation of the acrosome and flagellum, and remodeling and condensation of the chromatin. These processes are intricate, highly ordered, and require novel gene products and a precise and well-coordinated program of gene expression to occur. The regulation of gene expression in male germ cells occurs at three levels: intrinsic, interactive, and extrinsic. A highly conserved genetic program "intrinsic" to germ cells determines the sequence of events that underlies germ cell development. This has been underscored by recent studies showing that meiosis involves many genes that have been conserved during evolution from yeast to man. During meiosis and other processes unique to germ cells, the intrinsic program determines which genes are utilized and when they are expressed. In the postmeiotic phase, it coordinates the expression of genes whose products are responsible for constructing the sperm. The process of spermatogenesis occurs in overlapping waves, with cohorts of germ cells developing in synchrony. The intrinsic program operating within a particular germ cell requires information from and provides information to neighboring cells to achieve this coordination. Sertoli cells are crucial for this "interactive" process as well as for providing essential support for germ cell proliferation and progression through the phases of development. The interactive level of regulation is dependent on "extrinsic" influences, primarily testosterone and follicle-stimulating hormone (FSH). Studies during the last 4 years have established that FSH is not essential for germ cell development but instead serves an important supportive role for this process. While testosterone is essential for maintenance of spermatogenesis, it acts on Sertoli cells and peritubular cells and has indirect effects on germ cells. The extrinsic and interactive processes are extremely important for establishing and maintaining an optimum environment within which gametogenesis occurs. Nevertheless, an intrinsic evolutionarily conserved genetic program regulates male germ cell gene expression and development.

Molecular Cloning of Human Testis mRNA Specifically Expressed in Haploid Germ Cells, Having Structural Homology with the A-Kinase Anchoring Proteins

We have cloned a human testis specific gene (hi), which encodes for a protein having regional homologies to the domain of A-kinase anchoring proteins. Open reading frame encodes a protein of 860 amino acids. The predicted protein has a molecular weight 95.8 kD and contains 32 cysteine residues and 34 potential phosphorylation sites. The deduced protein analysis revealed an eukaryotic secretory signal with a potential cleavage site. Northern blot analysis detected a single transcript of approximately 3.0 kb in testis and not in any other somatic tissues. Expression of hi transcript was observed only in round spermatids indicating post meiotic haploid gene expression.

Models for male infertility: the t haplotypes.

The t haplotypes are variant alleles of genes in the proximal region of mouse Chromosome 17, linked together by four inversions. While females carrying two t haplotypes are fertile, males are sterile. Their spermatozoa exhibit severe motility defects and are unable to penetrate zona pellucida-free oocytes. Spermatozoa from males carrying one t haplotype (t/+) exhibit mild motility deficits and a delay in penetration of the zona-free oocyte. The inversions of the t haplotypes contain several genes that cause or contribute to male sterility, at least some of which can be identified by analysis of mice carrying Mus spretus-Mus domesticus recombinant Chromosomes 17. The t haplotypes specify a number of sperm biochemical abnormalities, but these have not been related directly to defects in fertilization. In t/+ males, spermatozoa not bearing the t haplotype are defective in fertilization compared with t-bearing spermatozoa. The mechanism causing this is likely to involve haploid gene expression confined to the t-bearing spermatids. Since many genes situated in the region of the t haplotypes have human homologues, an understanding of t haplotype sterility in mice is expected to contribute significantly to our knowledge of the genetic basis for human sperm dysfunction.

Models for male infertility: the t haplotypes

The t haplotypes are variant alleles of genes in the proximal region of mouse Chromosome 17, linked together by four inversions. While females carrying two t haplotypes are fertile, males are sterile. Their spermatozoa exhibit severe motility defects and are unable to penetrate zona pellucida-free oocytes. Spermatozoa from males carrying one t haplotype (t/+) exhibit mild motility deficits and a delay in penetration of the zona-free oocyte. The inversions of the t haplotypes contain several genes that cause or contribute to male sterility, at least some of which can be identified by analysis of mice carrying Mus spretus-Mus domesticus recombinant Chromosomes 17. The t haplotypes specify a number of sperm biochemical abnormalities, but these have not been related directly to defects in fertilization. In t/+ males, spermatozoa not bearing the t haplotype are defective in fertilization compared with t-bearing spermatozoa. The mechanism causing this is likely to involve haploid gene expression confined to the t-bearing spermatids. Since many genes situated in the region of the t haplotypes have human homologues, an understanding of t haplotype sterility in mice is expected to contribute significantly to our knowledge of the genetic basis for human sperm dysfunction.