Hybrid Male Sterility Research Articles

Different complex regulatory phenotypes underlie hybrid male sterility in divergent rodent crosses.

Hybrid incompatibilities are a critical component of species barriers and may arise due to negative interactions between divergent regulatory elements in parental species. We used a comparative approach to identify common themes in the regulatory phenotypes associated with hybrid male sterility in two divergent rodent crosses, dwarf hamsters and house mice. We investigated three potential characteristic gene expression phenotypes in hybrids including the propensity of transgressive differentially expressed genes towards over or underexpression, the influence of developmental stage on patterns of misexpression, and the role of the sex chromosomes on misexpression phenotypes. In contrast to near pervasive overexpression in hybrid house mice, we found that misexpression in hybrid dwarf hamsters was dependent on developmental stage. In both house mouse and dwarf hamster hybrids, however, misexpression increased with the progression of spermatogenesis, although to varying extents and with potentially different consequences. In both systems, we detected sex-chromosome specific overexpression in stages of spermatogenesis where inactivated X chromosome expression was expected, but the hybrid overexpression phenotypes were fundamentally different. Importantly, misexpression phenotypes support the presence of multiple developmental blocks to spermatogenesis in dwarf hamster hybrids, including a potential role of meiotic stalling or breakdown early in spermatogenesis. Collectively, we demonstrate that while there are some similarities in hybrid expression phenotypes of house mice and dwarf hamsters, there are also clear differences that point towards unique mechanisms underlying hybrid male sterility. Our results highlight the potential of comparative approaches in helping to understand the causes and consequences of disrupted gene expression in speciation.

Identification of a novel hybrid sterility locus S67 between temperate japonica subgroup and basmati subgroup in Oryza sativa L

Asian cultivated rice (Oryza sativa L.) is the most important cultivated species in the AA genome species of the genus Oryza. basmati is a special and famous subgroup in Asian cultivated rice, and temperate japonica is one of the most important cultivated subgroup, too. However, hybrid sterility hinders the introgression of favorable traits and the utilization of hybrid vigour between the two subgroups. The genetic basis of intraspecific hybrid sterility between temperate japonica and basmati remained elusive. In this study, a novel hybrid sterility locus S67 was identified, which caused hybrid male sterility in hybrids between the temperate japonica rice variety Dianjingyou 1 (DJY1) and the basmati rice variety Dom-sufid. Initial mapping with BC1F1, BC4F1, BC4F2 populations and DNA markers located S67 between RM5362 and K1-40.6 on the long arm of chromosome 1. Genetic analysis confirmed that S67 caused a transmission advantage for the temperate japonica rice S67-te allele in the hybrid offsprings. This result not only fills the gap in the research on hybrid sterility between basmati and temperate japonica, but also lays a good foundation for the systematic study of the genetic nature of hybrid sterility between basmati and other subgroups, as well as the full exploration and utilization of this subgroup through the creation of wide or specific compatibility lines to overcome hybrid sterility. In addition, this result can also help us broaden our understanding of genetic differentiation within Asian cultivated rice and hybrid sterility between inter-subgroups.

Different complex regulatory phenotypes underlie hybrid male sterility in divergent rodent crosses.

Hybrid incompatibilities are a critical component of species barriers and may arise due to negative interactions between divergent regulatory elements in parental species. We used a comparative approach to identify common themes in the regulatory phenotypes associated with hybrid male sterility in two divergent rodent crosses, dwarf hamsters and house mice. We investigated three potential characteristic gene expression phenotypes in hybrids including the propensity of transgressive differentially expressed genes towards over or underexpression, the influence of developmental stage on patterns of misexpression, and the role of the sex chromosomes on misexpression phenotypes. In contrast to near pervasive overexpression in hybrid house mice, we found that misexpression in hybrid dwarf hamsters was dependent on developmental stage. In both house mouse and dwarf hamster hybrids, however, misexpression increased with the progression of spermatogenesis, although to varying extents and with potentially different consequences. In both systems, we detected sex-chromosome specific overexpression in stages of spermatogenesis where inactivated X chromosome expression was expected, but the hybrid overexpression phenotypes were fundamentally different. Importantly, misexpression phenotypes support the presence of multiple developmental blocks to spermatogenesis in dwarf hamster hybrids, including a potential role of meiotic stalling or breakdown early in spermatogenesis. Collectively, we demonstrate that while there are some similarities in hybrid expression phenotypes of house mice and dwarf hamsters, there are also clear differences that point towards unique mechanisms underlying hybrid male sterility. Our results highlight the potential of comparative approaches in helping to understand the causes and consequences of disrupted gene expression in speciation.

Sharp decline in male fertility in F2 hybrids of the female-heterogametic silk moth Bombyx.

Sexual selection drives rapid evolution of morphological, physiological, and behavioral traits, especially in males, and it may also drive the rapid evolution of hybrid male sterility. Indeed, the faster male theory of speciation was once viewed as a major cause of Haldane's rule in male-heterogametic XY taxa, but is increasingly being replaced by the genetic conflict hypothesis partly because it cannot explain the faster evolution of hybrid female sterility in female-heterogametic ZW taxa. The theory nonetheless predicts that there should be more genes for hybrid male sterility than for hybrid female sterility even in such taxa, but this remains untested. Thus, finding evidence for the faster male theory of reproductive isolation beyond the F1 generation in ZW systems still represents a challenge to studying the impact of sexual selection. In this study, we examined F2 hybrids between the domesticated silkworm Bombyx mori and the wild silk moth Bombyx mandarina, which have ZW sex determination. We found that although only females showed reduced fertility in the F1 generation, the F2 hybrid males had a significant reduction in fertility compared with the parental and F1 males. Importantly, 27% of the F2 males and 15% of the F2 females were completely sterile, suggesting the presence of recessive incompatibilities causing male sterility in female-heterogametic taxa.

Transformation of meiotic drive into hybrid sterility in Drosophila.

Hybrid male sterility is one of the fastest evolving intrinsic reproductive barriers between recently isolated populations. A leading explanation for the evolution of hybrid male sterility involves genomic conflicts with meiotic drivers in the male germline. There are, however, few examples directly linking meiotic drive to hybrid sterility. In this study, we report that the Sex-Ratio chromosome of Drosophila pseudoobscura, which causes X-chromosome drive within the USA subspecies, causes near-complete male sterility when it is moved into the genetic background of the Bogota subspecies. In addition, we show that this new form of sterility is genetically distinct from the sterility of F1 hybrid males in crosses between USA males and Bogota females. Our observations provide a tractable study system where noncryptic drive within species is transformed into strong hybrid sterility between very young subspecies.

Overdrive is essential for targeted sperm elimination by Segregation Distorter.

Intra-genomic conflict driven by selfish chromosomes is a powerful force that shapes the evolution of genomes and species. In the male germline, many selfish chromosomes bias transmission in their own favor by eliminating spermatids bearing the competing homologous chromosomes. However, the mechanisms of targeted gamete elimination remain mysterious. Here, we show that Overdrive (Ovd), a gene required for both segregation distortion and male sterility in Drosophila pseudoobscura hybrids, is broadly conserved in Dipteran insects but dispensable for viability and fertility. In D. melanogaster, Ovd is required for targeted Responder spermatid elimination after the histone-to-protamine transition in the classical Segregation Distorter system. We propose that Ovd functions as a general spermatid quality checkpoint that is hijacked by independent selfish chromosomes to eliminate competing gametes.

Identification and genetic analysis of a pervasive 'needle-eye' sperm phenotype in Drosophila sterile hybrid males.

Interspecies hybrid sterility has been extensively studied, especially in the genus Drosophila. Hybrid sterility is more often found in the heterogametic (XY or ZW) sex, a trend called Haldane's rule. Although this phenomenon is pervasive, identification of a common genetic mechanism remains elusive, with modest support found for a range of potential theories. Here, we identify a single precise morphological phenotype, which we call 'needle-eye sperm', that is associated with hybrid sterility in three separate species pairs that span the Drosophila genus. The nature of the phenotype indicates a common point of meiotic failure in sterile hybrid males. We used 10 generations of backcross selection paired with whole-genome pooled sequencing to genetically map the regions underlying the needle-eye (NE) sperm phenotype. Surprisingly, the sterility phenotype was present in ~50% of males even after 10 generations of backcrossing, and only a single region of the X chromosome was associated with sterility in one direction of backcross. Owing to the common phenotype among sterile male hybrids, and the strong effect of individual loci, further exploration of these findings may identify a universal mechanism for the evolution of hybrid sterility.

Genome-wide sequence divergence of satellite DNA could underlie meiotic failure in male hybrids of bighead catfish and North African catfish (Clarias, Clariidae)

Hybrid sterility, a hallmark of postzygotic isolation, arises from parental genome divergence disrupting meiosis. While chromosomal incompatibility is often implicated, the underlying mechanisms remain unclear. This study investigated meiotic behavior and genome-wide divergence in bighead catfish (C. macrocephalus), North African catfish (C. gariepinus), and their sterile male hybrids (important in aquaculture). Repetitive DNA analysis using bioinformatics and cytogenetics revealed significant divergence in satellite DNA (satDNA) families between parental species. Notably, one hybrid exhibited successful meiosis and spermatozoa production, suggesting potential variation in sterility expression. Our findings suggest that genome-wide satDNA divergence, rather than chromosome number differences, likely contributes to meiotic failure and male sterility in these catfish hybrids.

MRNA-Seq of testis and liver tissues reveals a testis-specific gene and alternative splicing associated with hybrid male sterility in dzo.

Alternative splicing (AS) plays an important role in the co-transcription and post-transcriptional regulation of gene expression during mammalian spermatogenesis. The dzo is the male F1 offspring of an interspecific hybrid between a domestic bull (Bos taurus ♂) and a yak (Bos grunniens ♀) which exhibits male sterility. This study aimed to identify the testis-specific genes and AS associated with hybrid male sterility in dzo. The iDEP90 program and rMATS software were used to identify the differentially expressed genes (DEG) and differential alternative splicing genes (DSG) based on RNA-seq data from the liver (n = 9) and testis (n = 6) tissues of domestic cattle, yak, and dzo. Splicing factors (SF) were obtained from the AmiGO2 and the NCBI databases, and Pearson correlation analysis was performed on the differentially expressed SFs and DSGs. We focused on the testis-specific DEGs and DSGs between dzo and cattle and yak. Among the top 3,000 genes with the most significant variations between these 15 samples, a large number of genes showed testis-specific expression involved with spermatogenesis. Cluster analysis showed that the expression levels of these testis-specific genes were dysregulated during mitosis with a burst downregulation during the pachynema spermatocyte stage. The occurrence of AS events in the testis was about 2.5 fold greater than in the liver, with exon skipping being the major AS event (81.89% to 82.73%). A total of 74 DSGs were specifically expressed in the testis and were significantly enriched during meiosis I, synapsis, and in the piRNA biosynthesis pathways. Notably, STAG3 and DDX4 were of the exon skipping type, and DMC1 was a mutually exclusive exon. A total of 36 SFs were significantly different in dzo testis, compared with cattle and yak. DDX4, SUGP1, and EFTUD2 were potential SFs leading to abnormal AS of testis-specific genes in dzo. These results show that AS of testis-specific genes can affect synapsis and the piRNA biosynthetic processes in dzo, which may be important factors associated with hybrid male sterility in dzo.

A comprehensive evolutionary scenario for the origin and neofunctionalization of the Drosophila speciation gene Odysseus (OdsH).

Odysseus (OdsH) was the first speciation gene described in Drosophila related to hybrid sterility in offspring of mating between Drosophila mauritiana and Drosophila simulans. Its origin is attributed to the duplication of the gene unc-4 in the subgenus Sophophora. By using a much larger sample of Drosophilidae species, we showed that contrary to what has been previously proposed, OdsH origin occurred 62 MYA. Evolutionary rates, expression, and transcription factor-binding sites of OdsH evidence that it may have rapidly experienced neofunctionalization in male sexual functions. Furthermore, the analysis of the OdsH peptide allowed the identification of mutations of D. mauritiana that could result in incompatibility in hybrids. In order to find if OdsH could be related to hybrid sterility, beyond Sophophora, we explored the expression of OdsH in Drosophila arizonae and Drosophila mojavensis, a pair of sister species with incomplete reproductive isolation. Our data indicated that OdsH expression is not atypical in their male-sterile hybrids. In conclusion, we have proposed that the origin of OdsH occurred earlier than previously proposed, followed by neofunctionalization. Our results also suggested that its role as a speciation gene might be restricted to D. mauritiana and D. simulans.

A toxin-antidote system contributes to interspecific reproductive isolation in rice

Breakdown of reproductive isolation facilitates flow of useful trait genes into crop plants from their wild relatives. Hybrid sterility, a major form of reproductive isolation exists between cultivated rice (Oryza sativa) and wild rice (O. meridionalis, Mer). Here, we report the cloning of qHMS1, a quantitative trait locus controlling hybrid male sterility between these two species. Like qHMS7, another locus we cloned previously, qHMS1 encodes a toxin-antidote system, but differs in the encoded proteins, their evolutionary origin, and action time point during pollen development. In plants heterozygous at qHMS1, ~ 50% of pollens carrying qHMS1-D (an allele from cultivated rice) are selectively killed. In plants heterozygous at both qHMS1 and qHMS7, ~ 75% pollens without co-presence of qHMS1-Mer and qHMS7-D are selectively killed, indicating that the antidotes function in a toxin-dependent manner. Our results indicate that different toxin-antidote systems provide stacked reproductive isolation for maintaining species identity and shed light on breakdown of hybrid male sterility.

Disruption of the Contents of Endogenous Hormones Cause Pollen Development Obstruction and Abortion in Male-Sterile Hybrid Lily Populations.

Lilies are well-known flowers with large anthers and a high quantity of pollen that easily contaminates clothing and tepals. The anthers need to be artificially removed, leading to production problems. Cultivating male-sterile or pollen-free lilies could solve these problems. The key period of male sterility in a specific male-sterile hybrid lily population was determined through cytological observation. The contents of hormones, soluble sugar, soluble protein, and proline were determined by high-performance liquid chromatography, tandem mass spectrometry and colorimetry. Transcriptome sequencing was used to identify the genes with altered expression. The key period of male sterility was determined to be the microspore mother and tetrad stages. The hormone contents were abnormal in the sterile line compared with the fertile line. The indole-3-acetic acid (IAA) content was higher in the sterile line than in the fertile line at all stages, while the gibberellic acid 4 (GA4) content showed the opposite result. Abscisic acid (ABA) accumulated in the sterile line in both the microspore mother and tetrad stages, and the zeatin riboside (ZR) content in the sterile line increased at the microspore mother stage but decreased at the tetrad stage. The contents of soluble sugar, soluble protein and proline were higher in the fertile line than in the sterile line. Genes involved in auxin and ABA synthesis and signalling pathways were highly expressed in the male-sterile line. Our data suggested that abnormal contents of hormones in the microspore mother and tetrad stages resulted in pollen abortion in a male-sterile hybrid lily population, which indicated that the hormone balance in specific stages plays critical functions in pollen development in lilies.

PSIV-21 Comparing the Nutritional Value and the Trade-Off Between Sugars and Starch of Male-Sterile with Non-Sterile Sorghum Hybrids

Abstract The objective of this study was to assess the effect male-sterile versus non-sterile sorghum hybrids on nutritional value with emphasis on carbohydrates. The experiment was conducted on a dairy farm in West Texas under center pivot irrigation. Two male-sterile (Non-BMR, F465; and BMR12, F430) and three non-sterile sorghum hybrids (non-BMR F10; BMR12, F382, and F431; Richardson Seeds) were used in a split-plot design to reduce cross-pollination. Because of westerly prevailing winds, male-sterile hybrids were allocated to the west and non-sterile hybrids to the east, plots were blocked by irrigation section, and replicated 3 times. Plots were harvested 148 days after seeding with non-sterile hybrids after the hard dough stage and male-sterile hybrids 7 weeks after boot stage and harvested with a self-propelled forage harvester with a theoretical length of cut of 12 mm and roll gap setting at 2 mm. Duplicate samples of freshly chopped forage were obtained from each plot and immediately placed into a cooler with ice until harvest was completed. Samples were then frozen until shipped to Rock River laboratory where near-infrared spectroscopy analysis was performed. In addition, water soluble carbohydrates (WSC) and NDFD at 30h were analyzed with wet chemistry methods. The statistical analysis was performed using mixed linear regression models (GLIMMIX procedure of SAS). Type (Sterile and non-Sterile), trait (BMR12 and non-BMR) and its interaction were included as predictor variables in all models. Normal distribution of residuals was assessed using Shapiro-Wilk test (P > 0.05) and visual diagnostic of residual plots. A summary of the results is presented in Table 1. WSC was 2.5-fold greater for the sterile hybrids when compared with non-sterile, whereas starch was 2.2-fold greater for non-sterile when compared with sterile hybrids (P < 0.0001). Non-fiber carbohydrates were greater for non-sterile over sterile sorghum hybrids (P < 0.0001); however, non-sterile hybrids were past the optimal stage of harvest (soft dough), which would result in poor digestibility of grain starch and protein. In addition, crude protein (CP) was less for sterile than non-sterile (8.29% and 10.2% respectively; P < 0.0001). Compared with non-sterile hybrids, male sterile hybrids DM was decreased (28.4% and 40.8%, respectively; P < 0.0001) due to its proportionally greater vegetative plant parts compared with reproductive plant parts. The trade-off between WSC and starch in male-sterile sorghum hybrids is because sugars are not translocated to the grain where starch is developed as in non-sterile hybrids. This research indicates that concerns about low sorghum starch digestibility can be overcome by using male-sterile sorghum hybrids that will, at least partially, compensate for the reduced starch yield by increasing water-soluble carbohydrates storage.

Strong postmating reproductive isolation in Mimulus section Eunanus.

Postmating reproductive isolation can help maintain species boundaries when premating barriers to reproduction are incomplete. The strength and identity of postmating reproductive barriers are highly variable among diverging species, leading to questions about their genetic basis and evolutionary drivers. These questions have been tackled in model systems but are less often addressed with broader phylogenetic resolution. In this study we analyse patterns of genetic divergence alongside direct measures of postmating reproductive barriers in an overlooked group of sympatric species within the model monkeyflower genus, Mimulus. Within this Mimulus brevipes species group, we find substantial divergence among species, including a cryptic genetic lineage. However, rampant gene discordance and ancient signals of introgression suggest a complex history of divergence. In addition, we find multiple strong postmating barriers, including postmating prezygotic isolation, hybrid seed inviability and hybrid male sterility. M. brevipes and M. fremontii have substantial but incomplete postmating isolation. For all other tested species pairs, we find essentially complete postmating isolation. Hybrid seed inviability appears linked to differences in seed size, providing a window into possible developmental mechanisms underlying this reproductive barrier. While geographic proximity and incomplete mating isolation may have allowed gene flow within this group in the distant past, strong postmating reproductive barriers today have likely played a key role in preventing ongoing introgression. By producing foundational information about reproductive isolation and genomic divergence in this understudied group, we add new diversity and phylogenetic resolution to our understanding of the mechanisms of plant speciation.

Diversifying selection in aly, bam, dj and topi genes: Association between Rapid divergence and Hybrid male sterility in Drosophila bipectinata species complex

The diversity of millions of species depends on the multiplication of species and speciation, in which reproductive isolation evolves, such as hybrid sterility: a puzzle that still remains unknown. Crosses between closely related species results in sterile hybrid males, the perturbances of spermatogenesis in the F1 hybrid males usually affect the later stages of development, arresting at the sperm individualization stage, which could be due to the rapid interspecies divergence of reproductive genes experiencing strong selective constraints between species. We annotated and analyzed the molecular evolution of 6 key spermatogenesis genes playing an important role in the sperm developmental pathway in 15 strains of Drosophila bipectinata species subcomplex, where the interspecies crosses are known to produce sterile hybrid males. We analyzed nucleotide divergence and characterized the patterns of sequence polymorphism in the protein-coding regions to validate or reject the nature of adaptive divergence using its phylogenetic histories. Except for spermatocyte arrest (sa) and twine, all 4 genes - always early(aly), bag of marbles(bam), don juan (dj) and matotopetli (topi) showed signatures of positive selection with more nonsynonymous to synonymous fixed differences between species. However, departure from neutrality in polymorphism analysis was only significant for topi, twine and aly. Consistent with our analysis proving rapid divergence of don juan(dj) and topi, hybrids involving the three species Drosophila bipectinata, Drosophila parabipectinata and Drosophila malerkotliana, have found immotile sperm and unindividualized spermatids. Bag of marbles(bam) and aly showed rapid divergence coherent to the aspermic testes abnormality found in the hybrid males from crosses involving the Drosophila pseudoananassae with the other three species. We propose that faster evolution of spermatogenesis genes due to a yet unknown underlying mechanism acting upon the germ cell line developmental pathway of these four species might be operating as a perpetual dynamo for interspecies divergence and hybrid male sterility. Our study is the first attempt to understand the molecular genetic basis of post-zygotic reproductive isolation in bipectinata species complex and lays a foundation for future detailed exploration to better understand the mechanisms involved.

Inheritance regularities of economic and valuable traits in selection of sugar beet simple cytoplasmic male sterile hybrids

Aim. To study the influence of parental components of the crossing on the manifestation of the traits “yield of root crops” and “sugar content” in the hybrid progeny for the production of simple CМS hybrids. To create high-yielding simple CМS hybrids as a parent component for the formation of experimental sugar beet hybrids on а CМS basіs. Methods. Field (experiments, phenological observations), laboratory (to determine sugar content), measuring and weighing (to determine the crop structure), statistical (mathematical processing of research results). Results. The nature of the inheritance of productivity traits for the formation of simple CМS sugar beet hybrids has been established. For different combinations of crossings, the value of the inheritance coefficients (h2) of the trait “yield of root crops” from the parent form (O-type line) to the hybrid varied within 0.11–0.71 and 0.52–0.63 from the maternal form (CМS line). The inheritance coefficients of the trait “sugar content” from the parental form were in the range of 0.46–0.53 and 0.42–0.55 from the maternal form. An intermediate type of inheritance was observed for the sugar content in root crops. The best CМS lines and O-type lines have been identified as sources of economic and valuable traits. Seven combining-capable simple CМS hybrids with a high level of basic productivity were created for further use as a maternal component in the selection of sugar beet hybrids on а CМS basіs. Conclusions. A significant influence of the maternal component genotype on the level of yield of root crops of simple CМS hybrids has been established. A regularity has been revealed that the higher the total combining ability of CМS lines, the lower the degree of transfer of the trait “yield of root crops” from the parental form (O-type line) to the hybrid. There is no “mother plant” effect on sugar content in root crops, and an intermediate type of inheritance was observed in hybrids. The use of simple CМS hybrids with an increased level of basic productivity as a maternal component will significantly increase the efficiency of the breeding process for creating competitive sugar beet hybrids on а CМS basіs.

Genomic Architecture of Hybrid Male Sterility in a Species Without Sex Chromosomes (Tigriopus californicus, Copepoda: Harpacticoida).

Sterility among hybrids is one of the most prevalent forms of reproductive isolation delineating species boundaries and is expressed disproportionately in heterogametic XY males. While hybrid male sterility (HMS) due to the "large X effect" is a well-recognized mechanism of reproductive isolation, it is less clear how HMS manifests in species that lack heteromorphic sex chromosomes. We evaluated differences in allele frequencies at approximately 460,000 SNPs between fertile and sterile F2 interpopulation male hybrids to characterize the genomic architecture of HMS in a species without sex chromosomes (Tigriopus californicus). We tested associations between HMS and mitochondrial-nuclear and/or nuclear-nuclear signatures of incompatibility. Genomic regions associated with HMS were concentrated on a single chromosome with the same primary 2-Mbp regions identified in one pair of reciprocal crosses. Gene Ontology analysis revealed that annotations associated with spermatogenesis were the most overrepresented within the implicated region, with nine protein-coding genes connected with this process found in the quantitative trait locus of chromosome 2. Our results indicate that a narrow genomic region was associated with the sterility of male hybrids in T. californicus and suggest that incompatibilities among select nuclear loci may replace the large X effect when sex chromosomes are absent.

Whole-Mount Fluorescence In Situ Hybridization to Study Spermatogenesis in the Anopheles Mosquito.

Spermatogenesis is a complex biological process during which diploid cells undergo successive mitotic and meiotic division followed by large structural changes to form haploid spermatozoa. Besides the biological aspect, studying spermatogenesis is of paramount importance for understanding and developing genetic technologies such as gene drive and synthetic sex ratio distorters, which, by altering Mendelian inheritance and the sperm sex ratio, respectively, could be used to control pest insect populations. These technologies have proven to be very promising in lab settings and could potentially be used to control wild populations of Anopheles mosquitoes, which are vectors of malaria. Due to the simplicity of the testis anatomy and their medical importance, Anopheles gambiae, a major malaria vector in sub-Saharan Africa, represents a good cytological model for studying spermatogenesis. This protocol describes how whole-mount fluorescence in situ hybridization (WFISH) can be used to study the dramatic changes in cell nuclear structure through spermatogenesis using fluorescent probes that specifically stain the X and Y chromosomes. FISH usually requires the disruption of the reproductive organs to expose mitotic or meiotic chromosomes and allow the staining of specific genomic regions with fluorescent probes. WFISH enables the preservation of the native cytological structure of the testis, coupled with a good level of signal detection from fluorescent probes targeting repetitive DNA sequences. This allows researchers to follow changes in the chromosomal behavior of cells undergoing meiosis along the structure of the organ, where each phase of the process can clearly be distinguished. This technique could be particularly useful for studying chromosome meiotic pairing and investigating the cytological phenotypes associated with, for example, synthetic sex ratio distorters, hybrid male sterility, and the knock-out of genes involved in spermatogenesis.

Chromosome Asynapsis Is the Main Cause of Male Sterility in the Interspecies Hybrids of East Asian Voles (Alexandromys, Rodentia, Arvicolinae).

Closely related mammalian species often have differences in chromosome number and morphology, but there is still a debate about how these differences relate to reproductive isolation. To study the role of chromosome rearrangements in speciation, we used the gray voles in the Alexandromys genus as a model. These voles have a high level of chromosome polymorphism and substantial karyotypic divergence. We investigated testis histology and meiotic chromosome behavior in the captive-bred colonies of Alexandromys maximowiczii, Alexandromys mujanensis, two chromosome races of Alexandromys evoronensis, and their interracial and interspecies hybrids, to explore the relationship between karyotypic differences and male hybrid sterility. We found that the seminiferous tubules of the males of the parental species and the interracial hybrids, which were simple heterozygotes for one or more chromosome rearrangements, contained germ cells at all stages of spermatogenesis, indicating their potential fertility. Their meiotic cells displayed orderly chromosome synapsis and recombination. In contrast, all interspecies male hybrids, which were complex heterozygotes for a series of chromosome rearrangements, showed signs of complete sterility. Their spermatogenesis was mainly arrested at the zygotene- or pachytene-like stages due to the formation of complex multivalent chains, which caused extended chromosome asynapsis. The asynapsis led to the silencing of unsynapsed chromatin. We suggest that chromosome asynapsis is the main cause of meiotic arrest and male sterility in the interspecies hybrids of East Asian voles.

Comparative proteomic analysis identifies differentially expressed proteins associated with meiotic arrest in cattle-yak hybrids.

Cattle-yak, the interspecific hybrid between yak and taurine cattle, exhibits male-specific sterility. Massive loss of spermatogenic cells, especially spermatocytes, results in azoospermia in these animals. Currently, the mechanisms underlying meiosis block and defects in spermatocyte development remain elusive. The present study was designed to investigate the differences in the protein composition of spermatocytes isolated from 12-month-old yak and cattle-yak testes. Histological analysis confirmed that spermatocytes were the most advanced germ cells in the testes of yak and cattle-yak at this developmental stage. Comparative proteomic analysis identified a total of 452 differentially abundant proteins (DAPs) in the fluorescence-activated cell sorting (FACS) isolated spermatocytes from cattle-yak and yak. A total of 291 proteins were only present in yak spermatocytes. Gene Ontology analysis revealed that the downregulated DAPs were mostly enriched in the cellular response to DNA damage stimulus and double-strand break (DSB) repair via break-induced replication, while the proteins specific for yak were related to cell division and cycle, spermatogenesis, and negative regulation of the extrinsic apoptotic signaling pathway. Ultimately, these DAPs were related to the critical process for spermatocyte meiotic events, including DSBs, homologous recombination, synapsis, crossover formation, and germ cell apoptosis. The database composed of proteins associated with spermatogenesis, including KPNA2, HTATSF1, TRIP12, STIP1, LZTFL1, LARP7, MTCH2, STK31, ROMO1, CDK5AP2, DNMT1, RBM44 and CHRAC1 is the focus of further research on male hybrid sterility. In total, these results provide insight into the molecular mechanisms underlying failed meiotic processes and male infertility in cattle-yak. This article is protected by copyright. All rights reserved.