CircSry regulates spermatogenesis by enhancing γH2AX expression via sponging miR-138-5p

Sry on the Y chromosome is the master switch in sex determination in mammals. It has been well established that Sry encodes a transcription factor that is transiently expressed in somatic cells of male gonad, inducing a series of events that lead to the formation of testes. In the testis of adult mice, Sry is expressed as a circular RNA (circRNA) transcript, a type of noncoding RNA that forms a covalently linked continuous loop. However, the physiological function of this Sry circRNA (circSry) remains unknown since its discovery in 1993. Here we show that circSry is mainly expressed in the spermatocytes, but not in mature sperms and Sertoli cells. Loss of circSry led to the reduction of sperm number and the defect of germ cell development. The expression of γH2AX was decreased and failure of XY body formation was noted in circSry KO germ cells. Further study demonstrates that circSry regulates H2AX mRNA indirectly in pachytene spermatocytes through sponging miR-138-5p. Our study demonstrates that, in addition to its well-known sex-determination function, Sry also plays important role in spermatogenesis as a circRNA.


39
Long non-coding RNAs are relatively abundant in the mammalian transcriptome (1), and play 40 important roles in gene regulation in development and reproduction (2). Circular RNA (circRNA) is 41 a unique type of non-coding RNAs generated through back-splicing to form a covalently linked 3 Sry is best known as the sex-determination gene on Y chromosome. In mouse, Sry is 51 expressed as a transcription factor from 10.5 to 12.5 post-coitum(dpc)in the genital ridge 52 somatic cells, initiating testis development (16). Introduction of Sry locus into the female mouse 53 embryo switches the sex to male, while the targeted mutation in male embryos leads to complete 54 male-to-female sex reversal (17)(18)(19). Moreover, mutation of SRY causes a range of sex-disorder 55 development with profound effects in human (20). Recently, a cryptic second exon of mouse Sry 56 hidden in the palindromic sequence was identified and this two-exon Sry transcript plays primary 57 role in sex determination (21). Sry is also expressed in adult mouse testis as a circular RNA 58 (circSry) (22-24). A linear transcript containing long inverted repeats is transcribed from a distal 59 promoter, followed by a back splicing event that covalently links an acceptor splice site at the 5' 60 end to a donor site at a downstream 3' end ( Figure 1A) (4). Although the presence of circSry in 61 the testis has long been discovered, the significance of circSry remains elusive.

62
In this study, we generated mouse models that did not express circSry, without interfering with 63 male sex determination. We found that circSry played an important role in spermatogenesis, and 64 further dissected the underlying mechanism. Our findings highlight a unique synergy between 65 Sry's male sex-determination role as a protein and its regulatory role as a circular RNA in male 66 germ cells.

71
To identify Sry transcripts in adult mouse testis, divergent primers and convergent primers were 72 designed to amplify circSry or Sry linear RNA respectively ( Figure 1A). Upon RNase R treatment, 73 circSry was still detectable by RT-PCR, while linear RNA was not ( Figure 1B). The expression of 74 circSry was abundant when random hexamer primers were used for reverse transcription, while 75 only weak signal was detected using oligo (dt) 18 primers. In comparison, the expression of Sry 76 linear RNA was barely detectable using either random hexamer primers or oligo (dt) 18 primers 77 4 ( Figure 1C). The presence of head-to-tail splicing site of circSry was verified by Sanger

116
Quantitative analyses of Sry gene in control and circSry KO gonads at E11.5. The mRNA expression level of Sry in in 117 control and circSry KO gonads was not significantly changed at E11.5. The gender of the embryos was confirmed with 118 PCR using Sry primers. Data are presented as the mean ± s.e.m; unpaired, two tailed t test, ns, not significant, p > 0.05.

119
(C)SOX9 was expressed in Sertoli cells of both control and circSry KO gonads at E12.5 and E14.5. SOX9/DDX4 double-120 staining experiment was performed with control and circSry KO embryos at E12.5 (a-f) and E14.5 (g-l). Germ cells were 121 labeled with DDX4 (green). DAPI (blue) was used to stain the nuclei. The arrowheads point to SOX9-positive Sertoli cells 122 (red). The gender of the embryos was confirmed with PCR using Sry primers. Scale bars indicate 50 μm.    To determine the function of circSry, we assessed the testes of 6 to 10 week-old males and 138 examined the germ cell development in circSry KO mice. The size of testes from circSry KO mice 139 was smaller than that of control (wild-type C57BL/6) mice at 8 weeks ( Figure 2D). The testis to 140 body weight ratio of circSry KO mice was comparable to that of control mice at 7 weeks, whereas 141 it became significantly lower from 8 to 10 weeks ( Figure 2E). The development of germ cells was 142 examined by MVH staining. As shown in Figure 2F, the histology of the seminiferous tubules was  tubules, but not in control mice ( Figure 4D, a to h). These results suggest that lack of circSry 230 leads to defects in meiosis.

231
Since it has been reported that loss of histone γH2AX resulted in aberrant synapsis of sex

289
Since cytoplasmic circRNAs could act as miRNA sponge to regulate gene expression indirectly,

302
Next, we performed RNA-RNA pull down experiment to examine the interaction between miR-303 138-5p and circSry in 293T cell line that highly expressed circSry ( Figure 5 figure supplement 1B).

316
Transfection of luciferase reporter containing H2AX sequence together with miR-138-5p mimics 317 showed significantly reduced luciferase signal, while co-transfection of circSry rescued the 318 decrease of luciferase signal ( Figure 5G).

319
Taken together, these results show that circSry regulates the expression of H2AX mRNA at

363
CircSry was also expressed in the mouse brain during embryonic stage and its expression was 364 diminished after birth (36). Whether circSry plays a role in mouse brain needs further 365 investigation.

366
Human SRY is also expressed in adult testis.