The Catsper1 promoter has bidirectional transcriptional activity in GC-1 spg spermatogonia cells. (a) Structure of the Catsper1 promoter constructs. Each construct was named according to the length of the promoter region inserted in either direction upstream of the Photinus luciferase gene (Luc). (b) Transcriptional activity in GC-1 spg spermatogonia cells. Transcriptional activity is expressed as a fold-increase of luciferase activity over the pGL3-Basic (empty vector), to which a value of 1 was assigned. The results represent the average activity of Photinus firefly luciferase normalized to the activity of Renilla reniformis luciferase as an internal transfection control. Bar graphs represent the means ± SD, n = 3. Statistical significance was evaluated with one-way ANOVA: *P < 0.05, **P < 0.01, ***P ≤ 0.001.

In silico analysis of the 10.4 kb region upstream of the mouse Catsper1 gene and determination of the divergent gene TSSs. (a) The new putative Catsper1 divergent gene and its structure were predicted using different web servers (see Material and Methods). Catsper1 TSS is indicated. The new gene putative TSSs predicted at −121 and −289 bp are indicated. Predicted poly(A) and Kozak sequences, as well as exons and matches to EST 1–4, are indicated. LTR, simple repeats, Alu, B4, B2, and L1 repeat sequences are also indicated. (b) TSSs of the new gene Catsper1au. TSS were determined by 5′-RACE using a nested PCR as outlined in Materials and Methods. Two Catsper1au TSSs were confirmed as indicated by the sequencing of the 300 and 350 bp bands obtained in the second reaction of the nested PCR with primers AP1-AS322 (boxed in lane 1). The distances between Catsper1au TSS1 and TSS2 (labelled as TSS1-Cau and TSS2-Cau) from Catsper1 TSS-C were 224 and 291 bp, respectively. (c) Schematic representation of the Catsper1 bidirectional promoter in testis. Bent arrows indicate TSS-C (+1 bp) and TSS1-Cau (−225) and TSS2-Cau (−292). The arrows represent the direction of gene transcription. The predicted cis-acting elements in the 5′ upstream region of Catsper1au (transcriptional factors) are boxed; the TATA box (−216 bp) is bolded. The 5′ Catsper1au sequence, determined using 5′-RACE, is shown in italics.

Expression profile of Catsper1au in male and female mouse organs. A cDNA panel was prepared from different organs and cells to amplify the Catsper1au 5′ region through PCR based on the sequences obtained using 5′-RACE. A product of 496 bp was produced using oligonucleotides AS-306REV and NG-FORW292. (a) Catsper1au expression from different adult mouse organs. S: smooth; Sm. Int.: small intestine. (b) Catsper1au expression in adult male and female gonads and organs. F: Female. M: Male. (c) Catsper1au expression profile at different stages of male mouse development. E: embryo; d.p.c: days post-coitum; n.b: newborn. W: week. A product of 93 bp was amplified for Catsper1 in 4-week and adult testis. Actb was analysed as an internal expression control and to verify the absence of genomic DNA. The negative control (NC) is shown in the last lane. (d–g) Catsper1au RNA expression levels analysed using qRT-PCR in different tissues. Expression levels were normalized with respect to Actb mRNA. Values are expressed as fold-variations of each tissue type relative to adult testis (to which a value of 1 was assigned). Statistical significance was evaluated using one-way ANOVA: a > b. (h) Catsper1 expression relative to Catsper1au. Catsper1 was expressed as fold-changes in the expression of Catsper1au (***P < 0.001; paired t test). The results are expressed as the means ± S.E.M., n = 3 for each tissue.

Sequence and characteristics of Catsper1au. The full length of the Catsper1au transcript (1402 bp) was determined by 5′/3′-RACE, cDNA cloning into pCG5 vector and sequencing. (a) A Kozak sequence (−699 bp) and the poly (A) signal sequence (−1,614 bp) are bolded. Bent arrows indicate TSS-C (+1 bp), TSS1-Cau (−225) and TSS2-Cau (−292). The 1402 bp transcribed region is indicated by the two bolded arrows from −225 to −1626 bp. (b) Expression of the 1.4 kb Catsper1au RNA in mouse adult testis. Total RNA from adult testis and heart was examined by Northern blot analysis using a digoxigenin-UTP-labelled riboprobe for Catsper1au (left panel). Ethidium bromide staining of ribosomal RNA (right panel). (c) The in silico analysis in all of the six reading frames did not reveal long open reading frame (ORF). (c) Predicted secondary structure of Catsper1au transcript. (d) Catsper1au transcript is not translated. A rabbit reticulocyte lysate system was used to express Catsper1au from pSG5 in vitro. Positive control, a construct coding 61-kDa luciferase (lane 1); pSdC1, a pSG5 derivative containing the 1.4 Kb RNA gene downstream of T7 promoter (lane 2); negative control, empty vector (pSG5) (lane 3). (e) Overexpression of Catsper1au RNA by RT-PCR in mouse germ cells. Actb was analysed as an internal expression control to verify the absence of genomic DNA.

The lncRNA Catsper1au displays a nuclear localization and is expressed in germ and somatic cells from testis. (a) Catsper1au expression in cell lines and testicular germ cells at different stages of spermatogenesis. (b) Nuclear and cytoplasmic expression of Catsper1au RNA in testis, MSC1, and GC-1 spg cells. Rnu1a1 was used as a control to verify the quality of fractions. (c) Electrophoretic analysis of RNA fractions in a 1.2% agarose gel. (d) Catsper1au is not expressed in sperm cells. Actb was analysed as an internal expression control and to verify the absence of genomic DNA. ps: pachytene spermatocyte; rs: round spermatid; es: elongated spermatid; sp: sperm cell; gc: germ cells.

Catsper1au is located in vivo in the nucleus of spermatogenic and somatic cells from the seminiferous tubule. (a) Catsper1au RNA was analysed in the mouse testis by FISH using antisense RNA probe derived from Catsper1au. (b) Sense Catsper1au RNA was included as a negative control. Antisense (c) and sense (d) Actb RNA probes were also included as controls. Dashed line, basal border of seminiferous tubule; VI and VIII, stages in the cycle of the seminiferous epithelium; SC: Sertoli cell; sg: spermatogonia cell; ps: pachytene spermatocyte; rs: round spermatid; es: elongated spermatid; sp: sperm cell. Blood vessel: bv; lc: Leydig cell.