Expression dynamics, relationships, and transcriptional regulations of diverse transcripts in mouse spermatogenic cells

<p>Among all tissues of the metazoa, the transcritpome of testis displays the highest diversity and specificity. However, its composition and dynamics during spermatogenesis have not been fully understood. Here, we have identified 20,639 message RNAs (mRNAs), 7,168 long non-coding RNAs (lncRNAs) and 15,101 circular RNAs (circRNAs) in mouse spermatogenic cells, and found many of them were specifically expressed in testes. lncRNAs are significantly more testis-specific than mRNAs. At all stages, mRNAs are generally more abundant than lncRNAs, and linear transcripts are more abundant than circRNAs. We showed that the productions of circRNAs and piRNAs were highly regulated instead of random processes. Based on the results of a small-scale functional screening experiment using cultured mouse spermatogonial stem cells, many evolutionarily conserved lncRNAs are likely to play roles in spermatogenesis. Typical classes of transcription factor binding sites are enriched in the promoters of testis-specific m/lncRNA genes. Target genes of CREM and RFX2, 2 key TFs for spermatogenesis, were further validated by using ChIP-chip assays and RNA-seq on RFX2-knockout spermatogenic cells. Our results contribute to the current understanding of the transcriptomic complexity of spermatogenic cells and provide a valuable resource from which many candidate genes may be selected for further functional studies.</p

Assessment of the effects of SLXL1 antisera on <i>in vitro</i> fertilization, motility and acrosome reaction of spermatozoa.

<p>(A) The inhibitory effect of SLXL1 antisera on <i>in vitro</i> fertilization (IVF) rate. Successful fertilization was indicated by zygote cleavage. Spermatozoa were treated with PBS, preimmune serum, anti-SLXL1^1–155aa, anti-SLXL1^1–155aa+SLXL1, anti-SLXL1^65–155aa and anti-SLX^95–212aa before <i>in vitro</i> fertilization assays were performed. (B). Assessment of effect of anti-SLXL1^1–155aa on the motility of spermatozoa. (C). Assessment of effect of anti-SLXL1^1–155aa on the progressive movement of spermatozoa. (D). Assessment of effect of anti-SLXL1^1–155aa on the acrosome reaction of spermatozoa. **Denotes group that is significantly different (p<0.01) from the control groups.</p

The inhibitory effect of SLXL1 antiserum on <i>in vitro</i> zona pellucida binding/penetration of spermatozoa.

<p>(A). Bright (left panel) and fluorescent (right panel) images of eggs and spermatozoa incubated with HTF, control, anti-SLXL1^1–155aa (antiserum) and anti-SLXL1^1–155aa plus SLXL1 (Antiserum+SLXL1). (B) Statistical analysis of binding/penetration of spermatozoa to oocytes. The number of bound spermatozoa on each egg was evaluated. *Denotes significantly different groups compared with control group (p<0.05).</p

mRNA and protein expression of Slxl1 in mouse tissues.

<p>(A). Positions of primers used in RT-PCRs. (B) Slxl1 mRNA was exclusively expressed in mouse testes indicated by RT-PCR analysis of multiple tissues; Slxl1 transcript was first detected at 14 days post partum (dpp) in postnatal mouse testes. (C) From 10.5 dpp to 22.5 dpp, Slxl1 transcript level increased gradually as detected by RT-PCR using the ad and ac primer sets. (D) One protein of about 25 kDa in adult testis was detected in Western blotting, however, the protein was not detected in the spermatozoa using anti-SLX^95–212aa antibody. Using anti-SLXL1^1–155aa, two proteins of about 25 kDa and 18 kDa were detected in testes, while only the 18 kDa one was detected in spermatozoa. Only the 18 kDa protein was detected in either testis or spermatozoa using anti-SLXL1^65–155aa. (E–F) The expression of SLX and SLXL1 in mouse tissues and testes of different days post partum were detected using anti-SLXL1^1–155aa (E) and anti-SLXL1^65–155aa (F). mRNA and protein expression of GAPDH were used as internal controls for RT-PCR and Western Blot, respectively.</p

Co-immunoprecipitation and co-localization of SLXL1 and DKKL1.

<p>(A). Coimmunoprecipitation of DKKL1-FLAG with SLXL1-GFP expressed in 293T cells. (B) Coimmunoprecipitation of DKKL1 with SLXL1 from testis lysates. Protein lysates were analyzed by standard Western blotting with anti-FLAG, anti-GFP, anti-SLXL1 and anti-DKKL1 antisera, respectively. (C) Co-localization of SLXL1 (red) and DKKL1 (green) in seminiferous tubules. The nuclei were stained with DAPI (blue).</p