Long non-coding RNA Loc105611671 promotes the proliferation of ovarian granulosa cells and steroid hormone production upregulation of CDC42.

Granulosa cells (GCs) are essential for follicular development, and long non-coding RNAs (LncRNAs) are known to support the maintenance of this process and hormone synthesis in mammals. Nevertheless, the regulatory roles of these lncRNAs within sheep follicular GCs remain largely unexplored. This study delved into the influence of a Loc105611671, on the proliferation and steroid hormone synthesis of sheep ovarian GCs and the associated target genes in vitro. Cell Counting Kit-8 (CCK-8) gain-of-function experiments indicated that overexpression of Loc105611671 significantly boosted GCs proliferation, along with estrogen (E2) and progesterone (P4) levels. Further mechanistic scrutiny revealed that Loc105611671 is primarily localized within the cytoplasm of ovarian granulosa cells and engages in molecular interplay with CDC42. This interaction results in the upregulation of CDC42 protein expression. Moreover, it was discerned that increased CDC42 levels contribute to augmented proliferation of follicular granulosa cells and the secretion of E2 and P4. Experiments involving co-transfection elucidated that the concurrent overexpression of CDC42 and Loc105611671 acted synergistically to potentiate these effects. These findings provide insights into the molecular underpinnings of fecundity in ovine species and may inform future strategies for enhancing reproductive outcomes.

Comprehensive Analysis of Differentially Expressed CircRNAs in the Ovaries of Low- and High-Fertility Sheep.

CircRNAs are essential in regulating follicle growth and development and the female reproductive system at multiple levels. However, the molecular mechanism by which circRNAs regulate reproduction in sheep is unclear and requires further exploration. In this study, RNA sequencing was performed to reveal the circRNA expression profiles in the ovaries of Cele black sheep and Hetian sheep during estrus. Analysis of the number of circRNAs in their host genes revealed that 5031 genes could produce 20,835 circRNAs. Among the differentially expressed circRNAs (DEcircRNA), 75 were upregulated, and 105 were downregulated. Functional enrichment analysis showed that the host genes of DEcircRNA were involved in several pathways, including the MAPK and Hippo signaling pathway. In addition, we constructed a subnetwork of competitive endogenous RNA (ceRNA) containing 4 mRNAs, 4 microRNAs (miRNAs), and 10 circRNAs, potentially related to follicle development. Functional circRNAs (e.g., novel_circ_0003851, novel_circ_0015526, novel_circ_0008117) were found to act as ceRNAs for follicle growth and development-related mRNAs (CUEDC1, KPNB1, ZFPM2) by sponging functional miRNAs (miR-29a, miR-29b, miR-17-5p). Finally, through an RNA pull-down assay, oar-miR-125b was selected and confirmed as the target miRNA of novel-circ-0041512. We analyzed the overall expression of circRNAs in sheep ovaries. Further, we explored the potential mechanisms underlying the circRNA functions, providing a theoretical basis for the genetic progress of reproductive traits in sheep.

Identification of hub genes associated with follicle development in multiple births sheep by WGCNA.

Sheep exhibit a distinct estrous cycle that includes four different phases: proestrus, estrus, late estrus, and luteal phase. As the estrous cycle repeats, follicular development regularly alternates. We thus investigated ovarian transcriptome data from each of the four phases using weighted gene co-expression network analysis (WGCNA) to identify modules, pathways, and genes essential to follicle growth and development. We clustered mRNA and long non-coding RNA (lncRNA) into different modules by WGCNA, and calculated correlation coefficients between genes and Stages of the estrous cycle. Co-expression of the black module (cor = 0.81, P<0.001) and the yellow module (cor = 0.61, P<0.04) was found to be critical for follicle growth and development. A total of 2066 genes comprising the black and yellow modules was used for functional enrichment. The results reveal that these genes are mainly enriched in Cell cycle, PI3K-Akt signaling pathway, Oocyte meiosis, Apoptosis, and other important signaling pathways. We also identified seven hub genes (BUB1B, MAD2L1, ASPM, HSD3B1, WDHD1, CENPA, and MXI1) that may play a role in follicle development. Our study may provide several important new markers allowing in depth exploration of the genetic basis for multiparous reproduction in sheep.

RNAi as a tool to control the sex ratio of mouse offspring by interrupting Zfx/Zfy genes in the testis.

The objective of this study was to explore a novel method to alter the sex-ratio balance of mouse offspring by silencing the paralogous genes Zfx/Zfy (Zinc finger X/Y-chromosomal transcription factor gene) during spermatogenesis. Four recombined vectors PRZ1, PRZ2, PRZ3, and PRZ4 (RNAi-Ready-pSIREN-RetroQ-ZsGreen) were constructed for interrupting the Zfx gene. Additionally, a recombined vector Psilencer/Zfy-shRNA was constructed for interrupting the Zfy gene. Male mice were randomly divided into 8 groups, with 20 animals per group. Five groups of mice were injected with PRZ1, PRZ2, PRZ3, PRZ4, and Psilencer/Zfy-shRNA vectors, respectively. The three control groups were injected with an equal volume of physiological saline, empty RNAi-Ready-pSIREN-RetroQ-ZsGreen vector, and empty Psilencer/Zfy-shRNA vector, respectively. All groups were injected every 7 days for a total of four injections. Fourteen days after the fourth injection, 10 male mice from each group were mated individually with 10 females. Testicular tissue of 10 male mice in each group was collected, and the expression level of Zfx/Zfy mRNA was determined by qRT-PCR. Results showed that, compared with the empty RNAi-Ready-pSIREN-RetroQ-ZsGreen vector and the physiological saline group, expression of Zfx mRNA decreased significantly after injection of PRZ1 (p < 0.01), PRZ3 (p < 0.01), and PRZ4 (p < 0.01), and 78.75 ± 7.50% of the offspring were male in PRZ4 group, significantly higher than the offspring derived from the empty RNAi-Ready-pSIREN-RetroQ-ZsGreen vector and physiological saline group (p < 0.01). In the PRZ1 group, the expression of Zfx mRNA was also significantly lower (p < 0.01), but the male rate of offspring was not different (p > 0.05). Conversely, the expression of Zfy mRNA decreased significantly after injection of Psilencer/Zfy-shRNA (p < 0.01) and 31.00 ± 11.00% of the offspring were male, significantly lower than in the physiological saline group (p < 0.01). In conclusion, our findings show that RNAi-mediated disruption of Zfx/Zfy in mouse testis affected X/Y spermatogenesis. Additionally, results suggest that the paralogous genes Zfx/Zfy play an important role in the process of X and Y sperm development. The individual interference of Zfx/Zfy may predict the outcome of X and Y haploid sperms. Presented herein is an advanced method developed to control mouse X/Y spermatogenesis and sex ratio of offspring.

Identification of SNPs within the sheep PROP1 gene and their effects on wool traits.

Regarding mutations of PROP1 (Prophet of POU1F1) gene significantly associating with combined pituitary hormone deficiency (CPHD) in human patients and animals, PROP1 gene is a novel important candidate gene for detecting genetic variation and growth, reproduction, metabolism traits selection and breeding. The aim of this study was to detect PROP1 gene mutation of the exon 1-3 and its association with wool traits in 345 Chinese Merino sheep. In this study, on the basis of PCR-SSCP and DNA sequencing methods, ten novel SNPs within the sheep PROP1 gene, namely, AY533708: g.45A>G resulting in Glu15Glu, g.1198A>G, g.1341G>C resulting in Arg63Ser, g.1389G>A resulting in Ala79Ala, g.1402C>T resulting in Leu84Leu, g.1424A>G resulting in Asn91Ser, g.1522C>T, g.1556A>T, g.1574T>C, g.2430C>G were reported. In addition, association analysis showed that three genotypes of P4 fragment were significantly associated with fiber diameter in the analyzed population (P=0.044). These results strongly suggested that polymorphisms of the PROP1 gene could be a useful molecular marker for sheep breeding and genetics through marker-assisted selection (MAS).