| The silver fox and the blue fox belong to different genera, and the hybrid males HBS(blue fox male × silver fox female hybrids) and HSB(silver fox female × blue fox male hybrids) are fully or partially sterile, cannot produce normal sperm. For the past few years, transcriptomic analysis has been employed in distant hybrid male sterility of horse and donkey, or cattle and yak to unravel key genes regulating spermatogenesis.In this study, the silver fox, the blue fox, HBS and HSB during the reparative mating period and the breeding season were used to elucidate potential reasons as to why the fox hybrid males cannot reproduce. To accomplish this goal, the differences in serum hormone concentrations and testicular, and epididymal anatomy among silver foxes, blue foxes, and the hybrids were analyzed. Moreover, the testicular transcriptome database of silver foxes, blue foxes, and the hyb rids of the reparative mating period and breeding season was established. To find the key genes regulating spermatogenesis, two experiments were conducted. First, the differentially expressed genes of two different development periods of the same species were analyzed. The second study was conducted to analyze the differentially expressed genes of four kinds of fox in the same period. Taking together, these data revealed physiological differences and molecular mechanism of fox hybrid male sterility.In the present study, semen volume per ejaculate had no significant difference among silver foxes, blue foxes, and the hybrids(P > 0.05). Large numbers of sperm were found in the semen of blue foxes and silver foxes, but none in that of the hybrids, indicating t hat spermatogenesis in the hybrids was arrested.Germ cell meioses in the hybrids were arrested in the stage of primary spermatocyte transiting to secondary spermatocyte. Low concentration of testosterone and estradiol, as well as high concentration of FSH, LH and prolactin, caused incomplete development of testis and seminiferous tubule in the hybrids during the reparative mating period. Low concentration of testosterone or high concentration of LH and prolactin can inhibit the completion of spermatogenesis, leading to infertility.Based on high-throughput sequencing and sequence assembls of testicular tissue of silver foxes, blue foxes, and the hybrids during the reparative mating period and the breeding season, 175.36 G Clean bases, 1142703819 transcript and 673146243 Unigenes were obtained, and a relatively comprehensive fox testicular transcriptome database was established. By annotating with NR, NT and SwissProt database, 76243, 276899 and 56851 Unigenes were annotated, respectively. Through analyzing the differentially expressed genes of two different development periods of the same species and of four kinds of fox in the same period, 151 genes were found to regulate spermatogenesis. Among them, 26 genes(ASPM, BAG6, BRDT, CCNA1, DDX4, GGN, GGNBP2, GMCL1, HSF2, HSF2 BP, KHDRBS3, MAEL, MAK, MEIG1, MLH1, MNS1, NDRG3, PACRG, PAFAH1B1, PGAM2, RAD51 C, SCMH1, SOX30, SUN5, TDRD9 and TEX15) probably played key roles in the stage of primary spermatocyte transiting to secondary spermatocyte. In addition, KATNAL1 gene specifically expressed in testis sertoli cells, and ACSBG2 gene specifically expressed in leydig cells. To further validate the result, 18 genes were selected for q PCR determination, and their expression patterns were found to be coserved with transcriptome data, suggesting that the transcriptome sequencing results basically reflected the gene expression level in testis of silver fox, blue fox, and the hybrid.In conclusion, germ cell meioses in the hybrids HBS and HSB were arrested in the stage of primary spermatocyte transiting to secondary spermatocyte, and 28 genes were found as the key genes for further study of hybrid male sterility mechanism of silver and blue fox. However, specific functions of the 28 genes in fox spermatogenesis need to be clarified further. The present study lays the foundation for systematic research of molecular mechanisms of fox hybrid male sterility. |
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