| Follicular development and ovulation in the mammalian ovary are characterized by interdependence between oocytes and neighboring somatic cells,both of which play a crucial role in the process.During the process of follicular formation,the oocytes and their surrounding cumulus cells join together to create a complex known as the cumulus-oocyte complexes(COCs).Due to their mutual reliance,the investigation of COCs in different species has been a prominent area of research in recent decades.The treatment of 4 to8-week-old lambs with follicle-stimulating hormone(FSH)has been shown through both domestic and international studies to promote the development of ovarian follicles,resulting in a substantial yield of oocytes.However,the ability of oocytes to develop in vitro is low,which affects the comprehensive application of this technology.Objective: The research focused on Chinese Merino(Xinjiang Type)lambs and adult ewes.Transcriptome sequencing was conducted on oocytes and cumulus cells of lambs and adult ewes after in vitro maturation(IVM).Furthermore,functional verification of the CSTB gene in the oocytes and NPY gene in the cumulus cells was performed.Our research augmented the gene expression profile information of lamb and adult sheep oocytes and cumulus cells,serving as a foundation for elucidating the molecular mechanism of sheep oocyte development and maturation.Method: Experiment 1: FSH treatment was administered to Chinese Merino(Xinjiang Type)lamb and adult ewes,and COCs were collected surgically and IVM was performed.After 24 hours of IVM,oocytes and cumulus cells(n=3)were collected respectively,and transcriptome sequencing was carried out through c DNA library construction.Through differential expression analysis,the differentially expressed genes in oocytes and cumulus cells between lambs and adult ewes were compared,and the GO and KEGG databases were used for functional annotation and enrichment analysis of differentially expressed genes,and the protein interaction analysis was carried out using online database STRING 11.0.Experiment 2: Immunohistochemistry,qPCR,WB were used to analyze the expression level and localization of the differential gene Cystatin B(CSTB)in the oocytes of lamb and adult ewes in the heart,liver,spleen,lung,kidney and other tissues of ovine,follicles of different diameters,various follicular cells and ovarian structures.Recombinant CSTB protein was obtained by prokaryotic expression and its inhibitory activity was detected;The effects of CSTB on the maturation rate,cleavage rate and blastocyst rate of sheep oocytes were analyzed by supplementing recombinant CSTB in in-vitro maturation system and microinjection of siRNA.The effects of CSTB on the ROS,mitochondrial membrane potential and ATP levels of oocytes were detected,and the level of autophagy associated protein LC3A/LC3 B in oocytes was detected with immunofluorescence method.Experiment 3: The expression level and localization of neuropeptide Y(NPY),a differential gene screened from cumulus cells of lamb and adult ewes,were analyzed in the heart,liver,spleen,lung,kidney and other tissues of ovine,follicles of different diameters,various follicular cells,ovarian structures and primary cultured cumulus cells by immunohistochemistry,qPCR,WB methods;EdU and TUNEL staining,qPCR and WB were used to analyze the effects of NPY on the proliferation and apoptosis of sheep cumulus cells;Add NPY receptor inhibitor to determine the receptor pathway of NPY affecting cumulus cell proliferation;To study the effect of adding NPY in in-vitro maturation culture on the maturation rate,cleavage rate and blastocyst rate of sheep oocytes.Results: Experiment 1:(1)The study revealed that lambs and adult sheep oocytes displayed differential expression of 50 genes.In comparison to adult sheep,11 genes were up-regulated and 39 genes were down-regulated in lambs oocytes.Notably,certain genes such as CUL1 and TRIM17 were up-regulated multiple times,while others including SHFM1 and CSTB were significantly down-regulated.The results of the GO annotation revealed that the differentially expressed genes were primarily involved in biological processes,cellular components,and molecular functions related to hormone secretion,reproduction,extracellular matrix,and antioxidant activity.The KEGG pathway analysis revealed that the differentially down-regulated genes were significantly enriched in oxidative phosphorylation,oocyte meiosis,and the TGF-β signaling pathway.(2)A total of 191 differentially expressed genes were identified in cumulus cells.Among lamb oocytes,72 genes were found to be up-regulated while 119 genes were down-regulated relative to adult sheep.Notably,the expression of NPY,MAP1LC3 C,MGP,and PAGE4 genes was significantly suppressed,whereas that of H1F0,MARCKS,and APOA1 genes was significantly enhanced.A multitude of genes were identified through KEGG analysis as being enriched in the endocytosis,chemokine signal pathway,and PI3K-Akt signal pathway.Additionally,there were no commonly differentially expressed genes observed in oocytes and cumulus cells.Experiment 2:(1)the CSTB gene is expressed in the heart,liver,and uterus of sheep,with the uterus displaying the highest level of expression.Furthermore,the CSTB protein is expressed in primary and secondary follicles,corpus luteum,oocytes,cumulus cells,granulosa cells,and membrane cells,expressed in oocytes.(2)The recombinant CSTB protein was produced and evaluated for its ability to inhibit cathepsin B using the cathepsin B(CTSB)activity detection kit.The findings demonstrated that the recombinant protein displayed a highly significant inhibitory effect on cathepsin B(P<0.01).(3)Addition of recombinant CSTB in vitro maturation medium can significantly improve the maturation rate,cleavage rate and blastocyst rate of small follicular oocytes in a dose-dependent manner(P<0.05),The effect of adding100 ng/μL concentration is the best,close to the blastocyst rate of large follicular oocytes;However,after microinjection of siCSTB at GV stage,the maturation rate,cleavage rate and blastocyst rate decreased significantly(P<0.05),which was the first time that the recombinant protein of endogenous cathepsin inhibitor Cystatin B(CSTB)could affect the development ability of sheep embryos in vitro.(4)Additional examination was conducted on the levels of reactive oxygen species(ROS),mitochondrial membrane potential,adenosine triphosphate(ATP)content,and autophagic activity of oocytes.The findings indicated that CSTB enhanced the in vitro developmental potential of sheep embryos by diminishing the ROS level of sheep oocytes,elevating membrane potential,and augmenting ATP content and autophagic activity.Experiment 3:(1)The NPY are universally expressed in all tissues in mRNA and protein levels,with a comparatively heightened expression in the ovary and oviduct.Significantly,the expression level in cumulus and granulosa cells is notably greater than that in membrane cells and oocytes.The immunohistochemical findings revealed the presence of NPY protein in primary,primary and secondary follicles,corpus luteum,oocytes,cumulus cells,granulosa cells,and membrane cells.The cellular immunofluorescence assessment demonstrated that NPY protein was primarily expressed in the cytoplasm and cell membrane of sheep CCs.(2)The results of EdU demonstrated that the addition of 50 nM NPY to CCs significantly enhanced their proliferation(P<0.01),leading to an increase in the expression of genes related to proliferation such as PCNA and Cyclin D1(P<0.01).However,when NPY was interfered with,cell proliferation decreased significantly(P<0.01),and the expression of PCNA,Cyclin D1,and other genes also decreased.Similarly,TUNEL results showed that 50 nM NPY had no effect on cell apoptosis,and the percentage of apoptotic cells increased significantly after interference(P<0.01),accompanied by a significant increase in the expression level of genes such as Caspase 3 and Bax/Bcl-2(P<0.01).(3)In the meantime,inhibitors targeting NPY receptors NPY1 R,NPY2R,and NPY5 R were included.The addition of NPY5 R inhibitors led to the suppression of CC proliferation(P<0.05),while the inclusion of NPY1 R and NPY2 R inhibitors did not affect cell proliferation(P>0.05),as demonstrated by EdU and qPCR analyses.(4)Significant improvements in the maturation rate,cleavage rate and blastocyst rate of sheep small follicle oocytes can be achieved by adding 50 nM NPY to the in vitro maturation medium(P<0.05).Conclusions:(1)The findings indicated that there existed genes that were differentially expressed in the oocytes and cumulus cells of both lamb and adult ewes.(2)The activity of cathepsin B can be significantly inhibited by recombinant CSTB protein.(3)The CSTB protein has the ability to enhance the maturation rate,cleavage rate and blastocyst rate of small follicle oocytes in sheep by diminishing the level of ROS in oocytes,elevating membrane potential,augmenting ATP content,and promoting autophagic activity.(4)The combination of NPY with NPY5 R promotes the proliferation of sheep CCs and up-regulates the expression of proliferation-related genes.(5)The rate of maturation,cleavage and blastocyst in small follicle oocytes of sheep can be significantly enhanced by NPY by promoting cumulus cell proliferation. |
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