<p>PIWI proteins belong to an evolutionary conserved protein family as the sister sub-family of ARGONAUTE (AGO) proteins. While AGO proteins are functionally well-characterized and shown to mediate small-RNA guided gene regulation, the function of PIWI proteins remain elusive. Here we pursued functional characterization of PIWI proteins by studying MILI and MIWI, two PIWI proteins in the mouse.</p><p>We first show that both MIWI and MILI co-immunoprecipitate with a novel class of non-coding small RNAs from the post-natal mouse testis extract, which are named Piwi-interacting RNAs (piRNAs). Our cloning efforts identified thousands of different piRNA sequences, mostly derived from intergenic regions. Interestingly, both MILI and MIWI piRNAs correspond to the same regions on the genome and differ primarily in length. We propose piRNAs in the adult testis are produced by the processing of long, single stranded RNA precursors, based on the observation that piRNAs originate in clusters from a number of sites on the genome in a head-to-tail homology. In support, we bioinformatically predicted putative promoters, and yeast one hybrid analysis on two such regions found out that they interact with Krueppel C2H2 type zinc finger transcription factors. We did not observe the features of the "ping-pong" mechanism in their biogenesis: Both MILI and MIWI piRNAs are biased for 5` Uracil without an Adenine bias on the 10th nucleotide position, and do not significantly consist of sequences complementary to each other along their first 10nt. Moreover, MILI piRNAs are not down-regulated in Miwi-/- testis. These results indicate that the post-natal testicular piRNAs are produced independent of the ping-pong mechanism. </p><p>Although piRNAs are highly complex, PAGE and in situ analyses showed that piRNAs are germ cell-specific with predominant expression in spermatocytes and round spermatids, suggestive of a meiotic function. Correspondingly, we found that Miwi-/-; Mili-/- mice undergo only male infertility with terminal spermatogenic arrest during meiosis. piRNAs show a nucleo-cytoplasmic distribution, with enrichment in the chromatoid and dense bodies, two male germ cell-specific structures. The dense body has been implicated in synapsis and in the heterochromatinization of the sex chromosomes during male meiosis, a process known as meiotic sex chromosome inactivation (MSCI). Our histological analysis on Miwi-/-; Mili-/- testes showed that, while the overall synapsis is not affected, the sex chromosomes retain the euchromatin marker acetyl-H4K16 and lacks the heterochromatin marker H3K9-dimethyl. These observations indicate that murine PIWI proteins are necessary for MSCI. Moreover, we identified piRNA production from the X chromosome before MSCI, and propose PIWI proteins utilize piRNAs to target and silence unpaired chromosomal regions during meiosis.</p> / Dissertation

Hybrid male sterility (HMS) is typically the first mechanism fortifying reproductive isolation resulting from genomic incompatibilities. Three interspecies feline breeds derived from domestic cat crosses to wild cat species (Asian leopard cat and African serval) manifest HMS through several generations of backcrossing before eventually regaining fertility. This work utilized 199 hybrid individuals with varying fertilities in a genome wide association study (GWAS) comprising 63,000 genome wide SNPs. Leveraging these results with whole-testis transcriptome sequencing and quantitative real-time PCR data facilitated the comparison of transcripts in sterile and fertile hybrids. This dissertation describes four loci with highly significant and fifty with moderately significant association to sterility within each individual hybrid domestic breed and combinations of breeds. These associations help identify epistatic targets for hybrid incompatibility contributing to sterility. Comparative QTL mapping between pairs of species provides a framework to describe the accumulation of clade-specific reproductive isolating loci. Detailed exploration of gene misregulation between domestic and hybrid individuals, as well as between littermate hybrids of varying fertilities outlines a pattern of expression consistent with a meiotic sex-chromosome inactivation failure in early generations and apoptotic failure in later hybrid generations. Combining comparative genomic association and transcriptomic characterization among hybrid felids of varying divergence, new insight is gained into the mechanisms of mammalian reproductive isolation.

Charles University in Prague Faculty of Science Ph.D. study program: Molecular and Cellular Biology, Genetics and Virology Abstract Genetics and genomics of hybrid sterility Mgr. Tanmoy Bhattacharyya Supervisor: Prof. MUDr. Jiří Forejt, DrSc. Praha 2013 Abstract Male-limited hybrid sterility restricts gene flow between the related species, an important pre- requisite of speciation. The F1 hybrid males of PWD/Ph female (Mus m. musculus subspecies) and C57BL/6J or B6 male (Mus m. domesticus) are azoospermic and sterile (PB6F1), while the hybrids from the reciprocal (B6PF1) cross are semi fertile. A disproportionately large effect of the X chromosome (Chr) on hybrid male sterility is a widespread phenomenon accompanying the origin of new species. In the present study, we mapped two phenotypically distinct hybrid sterility loci Hstx1 and Hstx2 to a common 4.7 Mb region on Chr. X. Analysis of meiotic prophase I of PB6F1 sterile males revealed meiotic block at mid-late pachynema and the TUNEL assay showed apoptosis of arrested spermatocytes. In sterile males over 95% of pachytene spermatocytes showed one or more unsynapsed autosomes visualized by anti SYCP1, HORMAD2 and SYCP3 antibodies. The phosphorylated form of H2AFX histone, normally restricted only to XY chromosome containing sex body decorated unsynapsed...

Theory predicts that sexually antagonistic mutations will be over- or under-represented on the X and Z chromosomes, depending on the average dominance coefficient of the mutations. However, as little is known about the dominance coefficients for new mutations, the effect of sexually antagonistic selection is difficult to predict. To elucidate the role of sexually antagonistic selection in the evolution of Z chromosome gene content in chicken, we analyzed publicly available microarray data from several somatic tissues as well as somatic and germ cells of the ovary. We found that the Z chromosome is enriched for genes showing preferential expression in ovarian somatic cells, but not for genes with preferential expression in primary oocytes or non-sex-specific somatic tissues. Our results suggest that sexual antagonism leads to higher abundance of female-benefit alleles on the Z chromosome. No bias towards Z-linkage of oocyte-enriched genes can be explained by lower intensity of sexually antagonistic selection in ovarian germ cells compared to ovarian somatic cells. An alternative explanation would be that meiotic Z chromosome inactivation hinders accumulation of oocyte-expressed genes on the Z chromosome. Our results are consistent with findings in mammals and indicate that recessive rather than dominant...