A gene catalogue of the euchromatic male-specific region of the horse Y chromosome: comparison with human and other mammals

Abstract

Studies of the Y chromosome in primates, rodents and carnivores provide compelling evidence that the male specific region of Y (MSY) contains functional genes, many of which have specialized roles in spermatogenesis and male-fertility. Little similarity, however, has been found between the gene content and sequence of MSY in different species. This hinders the discovery of species-specific male fertility genes and limits our understanding about MSY evolution in mammals. Here, a detailed MSY gene catalogue was developed for the horse--an odd-toed ungulate. Using direct cDNA selection from horse testis, and sequence analysis of Y-specific BAC clones, 37 horse MSY genes/transcripts were identified. The genes were mapped to the MSY BAC contig map, characterized for copy number, analyzed for transcriptional profiles by RT-PCR, examined for the presence of ORFs, and compared to other mammalian orthologs. We demonstrate that the horse MSY harbors 20 X-degenerate genes with known orthologs in other eutherian species. The remaining 17 genes are acquired or novel and have so far been identified only in the horse or donkey Y chromosomes. Notably, 3 transcripts were found in the heterochromatic part of the Y. We show that despite substantial differences between the sequence, gene content and organization of horse and other mammalian Y chromosomes, the functions of MSY genes are predominantly related to testis and spermatogenesis. Altogether, 10 multicopy genes with testis-specific expression were identified in the horse MSY, and considered likely candidate genes for stallion fertility. The findings establish an important foundation for the study of Y-linked genetic factors governing fertility in stallions, and improve our knowledge about the evolutionary processes that have shaped Y chromosomes in different mammalian lineages.

Figure 1. Fluorescence in situ hybridization (FISH) using ECAY probes.

a. painting-like signal with flow-sorted Y probe on ECAY eu- and heterochromatin; b. painting-like signal with a probe comprising of 33 MSY BAC clones on ECAY euchromatin; c. and d. distinct signals with UBE1Y cDNA in metaphase and interphase Y chromosome; e. and f. painting-like signals with ZNF33bY cDNA on ECAY and ECAXq21 heterochromatin in metaphase and interphase. Scale bar, 1 µm.

Figure 2. A gene map of the horse male-specific region on Y (MSY).

A G-banded ideogram (ISCNH 1997) of the horse Y chromosome (ECAY) is shown at the top. Positive G-bands are black, negative G-bands are white and the centromere is shown in grey color. Horizontal lines below the ideogram demarcate the span of the euchromatic and male specific regions of Y. A schematic of ECAY contig map is at the bottom. White blocks with Roman numerals demarcate the currently available five BAC contigs. Heterochromatic region is shown at the far left and the pseudoautosomal region at the far right. The approximate locations of the 37 MSY genes are shown at the top of the map. Markers in bold were mapped in this study. Arrowheads with Ycen and Ytel show the directions towards the centromere and telomere, respectively.

Figure 3. The results of RT-PCR (30 cycles) showing the expression of horse MSY genes in nine adult tissues.

a. Testis-specific expression. b. Intermediate expression; c. Ubiquitous expression (ACTB was used as an internal control). Lanes: M - molecular markers (100 bp ladder, New England Biolabs); 1 - brain, 2 - kidney, 3 - heart, 4 – skeletal muscle, 5 - liver, 6 - lung, 7 - spleen, 8 - seminal vesicle, 9 - testis, 10 - no mRNA control, 11 - no RT control, 12 - no genomic DNA control, 13 - male genomic DNA control, 14 - female genomic DNA control.

Figure 4. The results of RT-PCR (30 cycles) showing the expression of horse MSY genes in donkey testis.

1 - horse testis (positive control); 2–3 – testis of two different donkeys; 4 - no mRNA control; 5 - male horse genomic DNA control; 6 - male donkey genomic DNA control; M - molecular markers (100 bp ladder, New England Biolabs). ACTB was used as an internal control.