Expression,structure and function analysis of the sperm-oocyte fusion genes Juno and Izumo1 in sheep(Ovis aries)

Background:JUNO and IZUMO1 are the first receptor-ligand protein pairs discovered to be essential for spermoocyte fusion;their interaction is indispensable for fertilization.Methods:PCR was used to clone the full-length DNA sequence of the Juno gene in sheep.The single nucleotide polymorphism(SNP)loci of Juno were genotyped by Sequenom MassARRAY®.PCR combined with rapid amplification of cDNA Ends were used to clone the full-length cDNA sequence of Juno and Izumo1.Reverse transcriptase-PCR(RT-PCR)and real time-quantitative-PCR(RT-qPCR)were used to analyze the genes’expression in tissues of sheep,and single cell RNA-seq was used to analyze the genes’expression in oocytes,granulosa cells and follicular theca of polytocous and monotocous Small Tail Han ewes.Bioinformatics was used to analyze advanced structure and phylogeny of JUNO and IZUMO1 proteins.Results:The full-length DNA sequence of the Juno gene in sheep was cloned and nine SNPs were screened.We found a significant association between the g.848253 C>A locus of Juno and litter size of Small Tail Han sheep(P<0.05).The full-length cDNA sequence of Juno and Izumo1 genes from Small Tail Han sheep were obtained.We found a new segment of the Izumo1 CDS consisting of 35 bp,and we confirmed the Izumo1 gene has 9 exons,not 8.RT-qPCR showed that Juno and Izumo1 genes were highly expressed in ovarian and testicular tissues,respectively(P<0.01).Single cell RNA-seq showed Juno was specifically expressed in oocytes,but not in granulosa cells or follicular theca,while Izumo1 displayed little to no expression in all three cell types.There was no difference in expression of the Juno gene in oocyte and ovarian tissue in sheep with different litter sizes,indicating expression of Juno is not related to litter size traits.Bioinformatic analysis revealed the g.848253 C>A locus of Juno results in a nonconservative missense point mutation leading to a change from Phe to Leu at position 219 in the amino acid sequence.Conclusions:For the first time,this study systematically analyzed the expression,structure and function of Juno and Izumo1 genes and their encoded proteins in Small Tail Han sheep,providing the basis for future studies of the regulatory mechanisms of Juno and Izumo1 genes.

Expression of TAC1 and Its Receptors TACR1,TACR2,TACR3 Genes in Different Goat Tissues Under Different Photoperiods

This study was conducted to analyze the regulation mechanisms of TAC1,TACR1,TACR2 and TACR3 genes on reproduction of goat under different photoperiods. The expression conditions of TAC1,TACR1,TACR2 and TACR3 genes in 12 tissues( oviduct,ovary,uterus,gluteus,mesenteric fat,brain,cerebellum,medulla oblongata,hart,lung,liver,kidney) of adult Henan Huai goat under different photoperiods( short day light,Light 8 h∶ dark 16 h,and long day light,light 16 h∶ dark 8 h) were analyzed by q-PCR method. TAC1,TACR1,TACR2 and TACR3 genes were expressed in all the 12 tissues of goats,with different expression characteristics; and the expression levels of all the genes were affected by photoperiod and changing of light signal between light and dark under short photoperiod. Shifting of light signal from dark to light was more conductive to the expression of these genes in all tissues than that of light signal from light to dark.There were significant differences in the expression levels of genes between light shifting from light to dark and from dark to light when the genes were expressed at a higher level in some tissues. TAC1 and TACR2 genes were expressed at a higher level than TACR1 and TACR3 genes in the various tissues,which implied that TACR2 is a receptor given priority to combine with TAC1 when TAC1 is functional.

Assessment of Serum PRL,MLT,TSH,FSH and LH Concentrations in the Goat (Capra hircus) Exposing to Different Photoperiod Regimes

In many seasonally breeding species, photoperiod is an important factor involved in reproduction. Capra hircus breeds are normally considered short-day breeders. An experiment was carried out to evaluate whether or not photoperiodic regimens influence circulating hormone levels （prolactin, melatonin, thyrotropin stimulating hormine, luteinizing hormone,follicle stimulating hormone） of Henan huai local goat exposing to different photoperiodic regimens. The results showed that plasma levels of prolactin, melatonin, thyrotropin stimulating hormine, luteinizing hormone and follicle stimulating hormone were higher in the 8 L∶ 16 D photoperiodic regimen than that in the 16 L∶ 8 D photoperiodic regimen. Levels of those hormones decreased on day 7 and day 28 when the 16 L∶ 8 D photoperiodic regimen was used continuously. It was concluded that serum concentrations of the hormones increased in summer when short day （8 L∶ 16 D） was used and decreased when the goats were transferred from short day for 42 d to long day exposure for 28 d. We suggest that in summer, levels of hormones changing may be due to short day animals. Changing of related hormones is still determined by the mechanism of internal regulation of short day animal s exposure to light changes. Shortening light may also help increase the reproductive activity of goat and thus increase productivity.

Behavioral Observations on Introduced Hu Sheep in Henan Province,China

Hu Sheep is a local sheep breed in the plain of Taihu, China. Due to the natural conditions in Taihu and human domestication, Hu Sheep is gradually featured with high adaptability, fast growth rate, early maturity and high productivity, which make them to be a unique and rare breed of sheep. In the present study, an experiment was conducted by means of instantaneous scan sampling, focal animal sampling and all-occurrence sampling to learn about Hu Sheep's behavior and activity pattern under certain feeding management and ecological condition. The result showed that the activity time budgets for several behaviors were different between rams and ewes, with respect to feeding (rams 30.60% vs. ewes 33.23%), ruminating (33.43% vs.35.30%); sleeping (19.92% vs. 13.69%), standing (4.13% vs. 5.26%), moving (8.10% vs. 7.46%), and other behaviors (3.86% vs.5.06%). A circadian activity rhythm was shown in some behaviors; e.g., total feeding time was greater in the daytime than at night, whereas resting time showed the opposite tendency. Moving mostly occurred in the daytime, and ruminating mainly occurred at night. Additionally, feeding activity occurred 32.22 times per day in rams and 31.73 times per day in ewes, whereas ruminating activity after feeding occurred 19.83 times per day in rams and 24.34 times per day in ewes. For each food bolus,the average ruminating time was 51.22 s in rams and 49.52 s in ewes, with 68.22 bouts of rumination per day in rams and57.35 bouts of rumination in ewes. By conducting the experiment, we studied and grasped the local Hu Sheep's main behavior and regularity which are of great significance on improving the feeding management and reducing diseases.