| Buffalo is an important large livestock in southern China,with high adaptability and disease resistance,tolerance to high temperature and humidity,rough feeding and easy rearing,long service life and rich nutrition of buffalo milk.However,buffalo have poor reproductive performance,the number of primordial follicles before puberty in female buffalo before puberty（10,000-20,000）is less than that in cow and cattle（100,000-200,000）,and the rate of follicular atresia is higher.Therefore,elucidating the molecular mechanism of follicular atresia in buffalo can provide theoretical support for tapping buffalo follicular resources,reversing the fate of follicular atresia and improving buffalo reproductive efficiency.It has been demonstrated that the abnormal state of granulosa cells directly affects the follicular development process,and non-coding RNAs have a decisive role in regulating various biological functions of follicular granulosa cells.Accordingly in this study,whole transcriptome analysis of granulosa cells isolated from healthy and atretic follicles of Chinese buffalo was performed using high-throughput sequencing,with the aim of obtaining various types of differential RNA molecules,constructing an interplay regulatory network,and exploring the molecular mechanisms of non-coding RNAs regulating the physiological functions of granulosa cells,as well as providing important support for in-depth studies on the development and atresia processes of buffalo follicles.1.Buffalo follicles were divided into healthy follicles（bright yellow with abundant capillary distribution）and atretic follicles（grayish-white surface without capillary distribution）according to their appearance,and then the structure of the cumulus-oocytes complex,the apoptosis rate of granulosa cells and the hormone levels in the follicular fluid were further observed and analyzed.The results revealed that the structure of the cumulus-oocytes complex was tight and intact in healthy follicles,and the oocyte zona pellucida was clearly defined,whereas the atretic follicles had severe internal flocculation,with loss of the cumulus structure,exposed oocytes and broken zona pellucida.The apoptosis rate of granulosa cells in healthy follicles was 4.93±1.59%,which was significantly lower than that in atretic follicles（21.31±1.40%,P<0.05）.The concentrations of estradiol and progesterone in follicular fluid of healthy follicles were 126.21±9.28 pg·m L-1 and21.88±1.78 ng·m L-1,with a ratio of 5.90×10-3.Compared with76.63±1.09 pg·m L-1of estradiol and 53.43±2.68 ng·m L-1 of progesterone in the follicular fluid of atretic follicles,with a ratio of1.45×10-3.The results indicated that there were significant differences in the concentration of steroid hormones between healthy follicles and atretic follicles（P<0.05）.Granulosa cells were collected from two types of follicles and whole transcriptome analysis was performed using second-generation high-throughput sequencing.The results showed that there were 3225 differentially expressed m RNAs,of which 2058were up-regulated and 1167 were down-regulated.A total of 2446mature mi RNAs（1173 known mi RNAs,1273 novel mi RNAs）were identified,and 183 mi RNAs were significantly different,of which159 were up-regulated and 24 were down-regulated.GO annotation results showed that the target genes of differential m RNA and mi RNA were annotated to cellular process,biological regulation,single organism process,metabolic process,binding,catalytic activity,and molecular sensor activity.KEGG gene function is significantly enriched in ECM receptor interaction,focal adhesion pathway,PI3K-Akt,thyroid hormone,TGF-β,m TOR and estrogen signal pathways.At the same time,a total of 3488 known lnc RNAs and 1344 novel predicted lnc RNAs were identified.Difference analysis showed that 122 differentially expressed lnc RNAs were up-regulated and 87 were down-regulated.A total of 6443 circ RNAs were identified,most of which were 100-700 bp in length,64%of circ RNAs were formed by exon looping of coding genes,58 of 112differential circ RNAs were significantly up-regulated and 54 were down-regulated.Based on the ce RNA theory,we constructed a non-coding RNA interaction regulatory network using 1458 m RNAs,135 mi RNAs,134 lnc RNAs and 47 circ RNAs,which further revealed the regulatory role of these non-coding RNAs in buffalo follicular granulosa cells.Subsequently,lnc RNA-4040/mi R-709/HIF-1αand circ RNA-1781/mi R-212/VDR were selected for further study.The result indicated that there was a ce RNA regulatory relationship between lnc RNA-4040/mi R-709/HIF-1αin buffalo granulosa cells（P<0.05）.Overexpression of lnc RNA-4040 can significantly promote cell proliferation and inhibit apoptosis,while mi R-709 could inhibit granulosa cell proliferation and promote apoptosis by down-regulating HIF-1α（P<0.05）.Co-transfection experiments showed that lnc RNA-4040 reversed the pro-apoptotic effect of mi R-709 on granulosa cells and attenuated the inhibitory effect on HIF-1α（P<0.05）,indicating that lnc RNA-4040/mi R-709/HIF-1αaxis can regulate the proliferation and apoptosis of granulosa cells.Similarly,we found a ce RNA regulatory relationship also between circ RNA-1781/mi R-212/VDR,and overexpression of circ RNA-1781 can promote the synthesis of estrogen（P<0.05）.Mi R-212 could reduce the synthesis of estrogen and progesterone by down-regulating the expression of VDR（P<0.05）.Co-transfection experiments showed that mi R-212 can reduce the increase of VDR expression and estrogen synthesis caused by overexpression of circ RNA-178（P<0.05）1,indicating that the circ RNA-1781/mi R-212/VDR axis can regulate the synthesis of estrogen in granulosa cells.The above results suggest that atresia of buffalo follicles is accompanied by an increase in the proportion of granulosa cell apoptosis and a decrease in the ratio of follicular fluid estradiol to progesterone concentration.More mi RNAs（159）than lnc RNAs（122）and circ RNAs（58）were down-regulated during follicular atresia,suggesting that mi RNAs may play a dominant role.Lnc RNA-4040 can act as a ce RNA for mi R-709 to up-regulate the expression of HIF-1α,which in turn promotes the proliferation of buffalo granulosa cells and inhibits their apoptosis.Circ RNA-1781could act as a ce RNA for mi R-212 to upregulate the expression of VDR and thus promote estradiol synthesis in granulosa cells. |
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