The Dobzhansky-Muller model of incompatibilities explains reproductive isolation between species by incorrect epistatic interactions. Although the mechanisms of speciation are of great interest, no incompatibility has been characterized at the gene level in mammals. The Hybrid sterility 1 gene (Hst1) participates in the arrest of meiosis in F₁ males of certain strains from two Mus musculus subspecies, e.g., PWD from M. m. musculus and C57BL/6J (henceforth B6) from M. m. domesticus. Hst1 has been identified as a meiotic PR-domain gene (Prdm9) encoding histone 3 methyltransferase in the male offspring of PWD females and B6 males, (PWD×B6)F₁. To characterize the incompatibilities underlying hybrid sterility, we phenotyped reproductive and meiotic markers in males with altered copy numbers of Prdm9. A partial rescue of fertility was observed upon removal of the B6 allele of Prdm9 from the azoospermic (PWD×B6)F₁ hybrids, whereas removing one of the two Prdm9 copies in PWD or B6 background had no effect on male reproduction. Incompatibility(ies) not involving Prdm9^B6 also acts in the (PWD×B6)F₁ hybrids, since the correction of hybrid sterility by Prdm9^B6 deletion was not complete. Additions and subtractions of Prdm9 copies, as well as allelic replacements, improved meiotic progression and fecundity also in the progeny-producing reciprocal (B6×PWD)F₁ males. Moreover, an increased dosage of Prdm9 and reciprocal cross enhanced fertility of other sperm-carrying male hybrids, (PWD×B6-C3H.Prdm9)F₁, harboring another Prdm9 allele of M. m. domesticus origin. The levels of Prdm9 mRNA isoforms were similar in the prepubertal testes of all types of F₁ hybrids of PWD with B6 and B6-C3H.Prdm9 despite their different prospective fertility, but decreased to 53% after removal of Prdm9^B6. Therefore, the Prdm9^B6 allele probably takes part in posttranscriptional dominant-negative hybrid interaction(s) absent in the parental strains. Disturbed gametogenesis in the progeny of two fertile parental forms is called hybrid sterility; it is an important part of reproductive barriers between species. The Dobzhansky-Muller model of incompatibilities explains reproductive isolation between species by incorrect interactions between genes. Hybrid sterility 1 (Hst1) is one of the genes causing meiotic arrest in F₁ male hybrids between certain Mus musculus musculus (e.g., the PWD strain) and M. m. domesticus (C57BL/6J etc.) mice. Hst1, the first mammalian candidate for a speciation gene, was identified as a meiotic PR/SET-domain gene, Prdm9, but the mechanism causing sterility has remained unknown. While the F₁ male offspring of C57BL/6J males and PWD females produce no sperm, the males from the reciprocal cross using PWD males and C57BL/6J females yield progeny. Here we show that the meiotic progress and fertility of hybrid males from both F₁ crosses improved by removal as well as overexpression of the C57BL/6J allele of Prdm9, suggesting that Prdm9 interactions not present in the parental species (incompatibilities) play a role in hybrid sterility. Furthermore, the Prdm9 dosage also controlled fecundity in other F₁ hybrids, indicating that this gene is an important regulator of mouse hybrid fertility.

A hallmark of meiosis is the rearrangement of parental alleles to ensure genetic diversity in the gametes. These chromosome rearrangements are mediated by the repair of programmed DNA double-strand breaks (DSBs) as genetic crossovers between parental homologs. In mice, humans, and many other mammals, meiotic DSBs occur primarily at hotspots, determined by sequence-specific binding of the PRDM9 protein. Without PRDM9, meiotic DSBs occur near gene promoters and other functional sites. Studies in a limited number of mouse strains showed that functional PRDM9 is required to complete meiosis, but despite its apparent importance, Prdm9 has been repeatedly lost across many animal lineages. Both the reason for mouse sterility in the absence of PRDM9 and the mechanism by which Prdm9 can be lost remain unclear. Here, we explore whether mice can tolerate the loss of Prdm9. By generating Prdm9 functional knockouts in an array of genetic backgrounds, we observe a wide range of fertility phenotypes and ultimately demonstrate that PRDM9 is not required for completion of male meiosis. Although DSBs still form at a common subset of functional sites in all mice lacking PRDM9, meiotic outcomes differ substantially. We speculate that DSBs at functional sites are difficult to repair as a crossover and that by increasing the efficiency of crossover formation at these sites, genetic modifiers of recombination rates can allow for meiotic progression. This model implies that species with a sufficiently high recombination rate may lose Prdm9 yet remain fertile.

PR-domain 9 (Prdm9) is the first hybrid sterility gene identified in mammals. The incompatibility between Prdm9 from Mus musculus domesticus (Mmd; the B6 strain) and the Hstx2 region of chromosome (Chr) X from M. m. musculus (Mmm; the PWD strain) participates in the complete meiotic arrest of mouse intersubspecific (PWD×B6)F1 hybrid males. Other studies suggest that also semisterile intersubspecific hybrids are relevant for mouse speciation, but the genes responsible remain unknown. To investigate the causes of this semisterility, we analyzed the role of Prdm9 and Chr X in hybrids resulting from the crosses of PWK, another Mmm-derived inbred strain. We demonstrate that Prdm9 and Chr X control the partial meiotic arrest and reduced sperm count in (PWK×B6)F1 males. Asynapsis of heterosubspecific chromosomes and semisterility were partially suppressed by removal of the B6 allele of Prdm9. Polymorphisms between PWK and PWD on Chr X but not in the Prdm9 region were responsible for the modification of the outcome of Prdm9 - Chr X F1 hybrid incompatibility. Furthermore, (PWK×B6)F1 hybrid males displayed delayed fertility dependent on the Prdm9 incompatibility. While the Drosophila hybrid sterility gene Overdrive causes both delayed fertility and increased transmission of its own chromosome to the offspring, the segregation of Chr X and the Prdm9 region from the mouse (PWK×B6)F1 males was normal. Our results indicate extended functional consequences of Prdm9 - Chr X intersubspecific incompatibility on the fertility of hybrids and should influence the design of fertility analyses in hybrid zones and of laboratory crosses between Mmm and Mmd strains.

Background: Vertebrate meiotic recombination events are concentrated in regions (hotspots) that display open chromatin marks, such as trimethylation of lysines 4 and 36 of histone 3 (H3K4me3 and H3K36me3). Mouse and human PRDM9 proteins catalyze H3K4me3 and H3K36me3 and determine hotspot positions, whereas other vertebrates lacking PRDM9 recombine in regions with chromatin already opened for another function, such as gene promoters. While these other vertebrate species lacking PRDM9 remain fertile, inactivation of the mousePrdm9gene, which shifts the hotspots to the functional regions (including promoters), typically causes gross fertility reduction; and the reasons for these species differences are not clear.Results: We introducedPrdm9deletions into theRattus norvegicusgenome and generated the first rat genome-wide maps of recombination-initiating double-strand break hotspots. Rat strains carrying the same wild-typePrdm9allele shared 88% hotspots but strains with differentPrdm9alleles only 3%. AfterPrdm9deletion, rat hotspots relocated to functional regions, about 40% to positions corresponding toPrdm9-independent mouse hotspots, including promoters. Despite the hotspot relocation and decreased fertility,Prdm9-deficient rats of the SHR/OlaIpcv strain produced healthy offspring. The percentage of normal pachytene spermatocytes in SHR-Prdm9mutants was almost double than in the PWD male mouse oligospermic sterile mutants. We previously found a correlation between the crossover rate and sperm presence in mousePrdm9mutants. The crossover rate of SHR is more similar to sperm-carrying mutant mice, but it did not fully explain the fertility of the SHR mutants. Besides mild meiotic arrests at rat tubular stages IV (mid-pachytene) and XIV (metaphase), we also detected postmeiotic apoptosis of round spermatids. We found delayed meiosis and age-dependent fertility in both sexes of the SHR mutants.Conclusions: We hypothesize that the relative increased fertility of rat versus mousePrdm9mutants could be ascribed to extended duration of meiotic prophase I. While rat PRDM9 shapes meiotic recombination landscapes, it is unnecessary for recombination. We suggest that PRDM9 has additional roles in spermatogenesis and speciation—spermatid development and reproductive age—that may help to explain male-specific hybrid sterility.

Reduced fertility of male mouse hybrids relative to their parents, or hybrid sterility, is governed by the hybrid sterility 1 (Hst1) locus. Rescue experiments with transgenes carrying sequences within or near Hst1 manifested that Hst1 contains the gene encoding meiosis-specific histone methyltransferase PRDM9. The Prdm9 gene is responsible for partial meiotic arrest, testicular atrophy, and low sperm count in (C57BL/6J x PWD)F1 mouse hybrids. Here we report that these male hybrids suffer an additional reproductive disadvantage, decreased sperm quality, which is (i) further exacerbated by the introduction of long transgene(s) carrying sequences from Hst1 with incomplete Prdm9 into their genome, and (ii) controlled by the Prdm9 dosage. These transgenic male hybrids displayed the features of severe oligoasthenoteratozoospermia (OAT), a human infertility syndrome characterized by a low number of spermatozoa with poor motility and morphological abnormalities. Analysis of spermiogenesis in these mice revealed acrosome detachment, aberrant elongation and condensation of the nucleus. As a result, the transgenic sperm had acrosome malformations, abnormal chromatin packaging, and fragmented DNA with elevated base oxidation, revealed by using multiple methods. Heterozygosity for one null Prdm9 allele improved meiotic progression and sperm quality of both non- and transgenic hybrids. Our results indicate that genomic analysis of OAT patients should include consideration of allelic variants in PRDM9, and our transgenic models can serve as tools to understand the diverse molecular processes that, when perturbed, can cause this disease.

PRDM9 is a protein with histone-3-methyltransferase activity, which specifies the sites of meiotic recombination in mammals. Deficiency of the Prdm9 gene in the laboratory mouse results in complete arrest of the meiotic prophase of both sexes. Moreover, the combination of certain PRDM9 alleles from different mouse subspecies causes hybrid sterility, e.g., the male-specific meiotic arrest found in the (PWD/Ph × C57BL/6J)F1 animals. The fertility of all these mice can be rescued using a Prdm9 -containing transgene. Here we characterized a transgene made from the clone RP24-346I22 that was expected to encompass the entire Prdm9 gene. Both (PWD/Ph × C57BL/6J)F1 intersubspecific hybrid males and Prdm9 -deficient laboratory mice of both sexes carrying this transgene remained sterile, suggesting that Prdm9 inactivation occurred in the Tg(RP24-346I22) transgenics. Indeed, comparative qRT-PCR analysis of testicular RNAs from transgene-positive versus negative animals revealed similar expression levels of Prdm9 mRNAs from the exons encoding the C-terminal part of the protein but elevated expression from the regions coding for the N-terminus of PRDM9, indicating that the transgenic carries a new null Prdm9 allele. Two naturally occurring alternative Prdm9 mRNA isoforms were overexpressed in Tg(RP24-346I22), one formed via splicing to a 3’-terminal exon consisting of short interspersed element B2 and one isoform including an alternative internal exon of 28 base pairs. However, the overexpression of these alternative transcripts was apparently insufficient for Prdm9 function or for increasing the fertility of the hybrid males.