Male Haploid Research Articles

Stimulation of DNA repair by the spermatidal TP1 protein.

The chromatin remodeling process that takes place during spermiogenesis in mammals is characterized by a transient increase in DNA single-strand breaks (SSB). The mammalian transition proteins (TPs) are expressed at a high level at mid-spermiogenesis steps coincident with chromatin remodeling and could be involved in the repair of these lesions since SSB are no longer detected in terminally differentiated spermatids. We report that TP1 can stimulate the repair of SSB in vitro and demonstrate that in vivo repair of UV-induced DNA lesions is enhanced in mammalian cells stably expressing TP1. These results suggest that, aside from its role in DNA compaction, this major transition protein may contribute to the yet unidentified enzymatic activity responsible for the repair of SSB at mid-spermiogenesis steps. These results also suggest that the TP1 proteins have the potential to participate in the repair process following genotoxic insults and therefore may play an active role in the maintenance of the integrity of the male haploid genome during spermiogenesis.

In-vitro development of mouse zygotes following reconstruction by sequential transfer of germinal vesicles and haploid pronuclei.

We evaluated whether mouse oocytes reconstructed by germinal vesicle (GV) transfer can develop to blastocyst stage. The oocytes were artificially activated with sequential treatment of A23187 and anisomycin; fertilization was then established by transfer or exchange of pronuclei with those of zygotes fertilized in vivo. Type 1 zygotes were constructed by placing the male haploid pronucleus from a zygote into the cytoplasm of an oocyte that underwent GV transfer, in-vitro maturation and activation; for type 2 zygotes, the female pronucleus was removed from a zygote and replaced with the female pronucleus of an oocyte subjected to GV transfer, in-vitro maturation and activation. Karyotypes of activated oocytes and type 2 zygotes were also subjected to analysis. When cultured in human tubal fluid (HTF) medium, reconstructed oocytes matured and, following artificial activation, consistently developed a pronucleus with a haploid karyotype; the activation rate for this medium was two- to three-fold higher than that of oocytes cultured in M199 (87% versus 30% respectively). Following transfer of a male pronucleus, only 47% of the type 1 zygotes developed to morula or blastocyst stage and embryo morphology was poor. In contrast, 73% of the type 2 zygotes developed to morula or blastocyst stage, many even hatching, with few morphological anomalies. Normal karyotypes were observed in 88% of the type 2 zygotes analysed. These observations demonstrate that the nucleus of a mouse oocyte subjected to sequential nuclear transfer at GV and pronucleus stages is, nonetheless, capable of maturing meiotically, activating normally and supporting embryonic development to hatching blastocyst stage. In contrast, the developmental potential of the cytoplasm of such oocytes appears to be compromised by these procedures.

Genesis of a novel human sequence from the protamine PRM1 gene

The members of the male haploid expressed protamine 1 (PRM1)→protamine 2 (PRM2)→transition protein 2 (TNP2) locus exist as a single, coordinately expressed genic domain. Previous analysis has revealed that the genes within the human PRM1→PRM2→TNP2 domain are inter-related, as they share significant sequence similarity at both the nucleotide and amino acid levels. Analysis described here supports the view that a fourth candidate coding region, gene4/Prm3, was derived from PRM1 during the genesis of the PRM1→PRM2→TNP2 domain. In some species, gene4 has diverged to a great extent, which can limit its expression.

Extended analysis of the region encompassing the PRM1→PRM2→TNP2 domain: Genomic organization, evolution and gene identification

The human male haploid expressed protamine 1 (PRM1)-->protamine 2 (PRM2)-->transition protein 2 (TNP2) locus comprises a coordinately regulated multigenic domain. This region of 16p13.13 has been used as a model to address how the organization of genes and genic domains within the human genome may influence tissue specific gene expression. Toward this goal, we have completed an extensive computational and biological analysis of the region encompassing the PRM1-->PRM2-->TNP2 domain. These analyses have revealed the likely genesis of this domain. Interestingly, the SOCS-1 gene and an hnRNPC-class pseudogene lies just 3' of this domain. Regions of nuclear matrix attachment also mark these newly identified genes.

Genetic testing for male infertility: a postulated role for mutations in sperm nuclear matrix attachment regions.

Numerous reports have suggested that disturbances in nuclear condensation may result in male infertility. This notion has been supported by the observation of infertile individuals with a decrease or absence of the male sperm-specific chromatin packaging protamine proteins. To date, no correlation between the absence of protamine proteins and a mutation within the coding regions of the protamine genes has been documented. To address this issue, PCR-based mutation scanning analysis has been performed across the human male haploid expressed PRM1-->PRM2-->TNP2 domain in several oligozoospermic infertile individuals. This analysis identified a candidate mutation in a region of contact with the sperm nuclear matrix from 2 of 5 affected individuals. This is the first report of a mutation scan covering the entire PRM1-->PRM2-->TNP2 locus in affected individuals.

Evolutionary origin of expandable G-C-rich triplet repeat DNA sequences

A model explaining properties exhibited by fragile-X DNA systems arises from observations that time-dependent base substitutions are expressed at G-C sites but not at A-T sites (Biochem. Genet. 32:383, 1994). [CGG]n sequences are classified as most sensitive to evolutionary base substitution processes involving time-dependent populating of G-C sites with enol-imine states having enhanced stability. Increased density of these states in oocyte DNA would introduce a ground-state collapse double-helix of reduced energy that would inhibit strand separation by the replicase. Evolutionarily altered G' in CG'G triplets allows CG'G to be transcribed as CTG, an initiation codon. And this will cause reinitiation of DNA synthesis, thereby adding additional CGG units to the collapsed double helix. This situation would not occur in slower-evolving male haploid DNA that replicates frequently.

Karyotypes of S and L type rotifers Brachionus plicatilis O.F. Müller

A comparative study on the karyotypes of S and L types of the rotifer Brachionus plicatilis O.F. Müller was carried out to clarify their cytological differences. Embryos, stained with alcoholic hydrochloric acid-carmine, were made into squash chromosome preparations. Mitotic chromosomes of S type prometaphase cells and L type metaphase cells were studied. The two karyotypes differed in chromosome number and morphology. The female diploid embryo of the S type had 25 chromosomes (2 n = 25), whereas that of L type had 22 chromosomes (2 n = 22). The chromosome numbers of male haploid embryos of S and L types were n = 12 and 11, respectively. Differences in karyotypes suggest that S and L types are so differentiated they should be considered different species. The possible evolutionary steps of chromosomes changes leading to the two types are discussed.

GENIC VARIATION IN MALE HAPLOIDS UNDER DETERMINISTIC SELECTION

Genic variation in male haploids and male diploids was compared assuming constant fitnesses (derived from computer-generated random numbers) and infinite population size. Several models were studied, differing by the fitness correlation between the sexes (r(s)) and genotypes (r(g)), and by the intensity of selection as measured by the coefficient of variation (CV) of the fitness distribution. Genic variation was quantified using the proportion of polymorphic loci, P, the gene diversity at polymorphic loci, H(p), and the gene diversity over all loci, H(a). The two genetic systems were compared via variation ratios: variation in male haploidy/variation in male diploidy.-P and H(a) were markedly lower for male-haploids than for male diploids, the variation ratios declining with increasing r(s), r(g) and CV, but the two genetic systems were similar for H(p). Except for male diploids with r(s) = 1, the two sexes had different equilibrium gene frequencies but the sample sizes required to detect such differences reliably were larger than usually possible in surveys of natural populations.-Data from natural populations fit the above trends qualitatively, but the variation ratios are much lower than those from our analyses, except that for H(p), which is higher when Drosophila is excluded. Also, the frequency distribution of most common alleles from electrophoretic data has a deficiency of intermediate frequencies compared to that from the computer-generated sets of fitnesses, possibly reflecting either the influence of stochastic processes shifting frequencies away from equilibrium or the involvement of alleles under selection-mutation balance.--While electrophoretic data suggest that Drosophila has unusually high levels of genic variation, unusually low levels of genic variation in male haploids compared with male diploids are not strongly indicated. However, if further data confirm male haploids as having low levels of genic variation, likely explanations are that the bulk of electrophoretically detected variation involves fixed-fitness balancing selection, selection-mutation balance involving slightly deleterious recessive alleles, new favorable male haploid alleles moving more rapidly to fixation than under male diploidy and thus carrying linked loci to fixation faster, or some combination of these possible factors.

A Fundamental Theorem of Natural Selection for Sex Linkage or Arrhenotoky

Equation (11) is a general equation for the change in geometric mean fitness at a male haploid or sex-linked locus when the selection intensity is small. The rate of change in fitness depends on the additive genetic variance in fitness, which is defined as the variance of the additive genotypic values when the additive model of gene action assumes no dominance in females and complete dosage compensation in males. If the relative fitnesses of genotypes are constant, then under slow selection the increment in fitness is equal to the additive variance if either (1) the population is in random mating or (2) there is no dominance in females and complete dosage compensation in males. If both conditions hold, then a male haploid population evolves one-third faster than the corresponding diploid population and one-third slower than the corresponding haploid population. In computer-simulated populations undergoing random mating, the additive variance is a good approximation to actual fitness changes over a wide ra...

XI.—The Cytology of Pharaoh's Ant,Monomorium pharaonis(L.)

SynopsisPharaoh's Ant,Monomorium pharaonis(L.), is arrhenotokously parthenogenetic, conforming essentially to the pattern of reproduction characteristic of Hymenoptera generally. The female is diploid, 2n= 22, the male haploid,n= 11, in certain somatic cells and in the gonia, though polyploidy exists in some larval tissues. The 11 discrete chromosomes of the male cells comprise a single genome, in which are distinctive only one large clubbed chromosome and at least one of intermediate size. Spermatogenesis is characterized by a first abortive division and a second equational division, resulting in two spermatozoa being derived from each haploid spermatogonium. Spermateleosis is described in some detail. Certain peculiar features are discussed, viz. the cytoplasmic buds appearing during spermatogenesis; unequal second spermatocyte divisions; the single chromosome sometimes observed as lying apart from the other chromosomes during first spermatocyte metaphase; the division and grouping of polar body chromosomes. For reasons given, several recent conclusions of Whelden and Haskins (1953) regarding the cytology of ants are regarded as mistaken.