lncRNA 1700009J07Rik Impaired Male Fertility by Interfering with Sexual Behaviors in Mice

Abstract

Long non-coding (lnc) RNAs exhibit tissue-specific expression characteristics and have been shown to be involved in the regulation of various biological processes. The testis is one of the organs with the most abundant lncRNAs. However, the functions of many testis-specific or -enriched lncRNAs in male fertility remain undisclosed. In this study, we screened lncRNA 1700009J07Rik (07Rik) to investigate its roles in spermatogenesis and male fertility using knockout (KO) mice. We found that 07Rik mainly acted as an intact lncRNA rather than a small protein, being highly expressed in various spermatogenic cells, which suggests its potential involvement in spermatogenesis. Unexpectedly, the deletion of 07Rik did not impact spermatogenesis or sperm functions. Intriguingly, two-thirds of the male KO were infertile, which was ascribed to the lack of sexual behaviors rather than abnormalities in spermatogenesis or sperm functions. Further results reveal that, compared with wild-type mice, free testosterone content in serum was significantly reduced in the KO infertile (KO-I) mice, whereas it was remarkably elevated in the testes. Correspondingly, Hsd3b2, a key gene that promotes testosterone synthesis, was dramatically upregulated. Cyp19a1 and Cyp11b1, which are responsible for testosterone metabolism, were downregulated in the testes. In addition, the expression of sex hormone-binding globulin was observably elevated in the testes of 07Rik KO-I mice, which might partially explain the decrease in testosterone in the serum. These results suggest that disruptions in testosterone synthesis and metabolism might contribute to the loss of libido in 07Rik KO-I mice. Our findings expand the understanding of lncRNA function and provide novel insights into the role of lncRNAs in male fertility, particularly in relation to hormonal turnover disorders that mediate sexual behavior defects.

Keywords: 1700009J07Rik; lncRNA; male infertility; sexual behavior; spermatogenesis; testosterone.

Figure 1

Expression profiles and characteristics of lncRNA 1700009J07Rik (07Rik). (A) Expression levels of 07Rik in various tissues. (B) 07Rik expression levels in the testes at different development times and (C) in different spermatogenetic cells. (D) Overexpression levels of different 07Rik transcripts in 293T cells. Spermatogenic cells were sorted using flow cytometry. Total RNA was extracted from each sample using a MiniBEST Universal RNA Extraction Kit and gene expression levels were detected by RT-PCR or qPCR. Gapdh was used as a loading control to normalize the gene expression levels in different samples. (E) Validation of protein-encoding abilities of different transcripts of 07Rik by immunofluorescence (magnification, 200×). (F) The expression levels of 07Rik CDS encoded a small protein in 293T cell lines. Different 07Rik sequences with the flag tag were constructed into a Eukaryotic expression vector and then transfected into 293T cells. Immunofluorescence staining and Western blot with a Flag antibody was used to verify the expression of the presumed fusion protein. The tubulin protein was used as a loading control. All data are means ± SEM (n = 3). p values are calculated using the Tukey–Kramer test. * p < 0.05, ** p < 0.01, compared with the vector control group. Spg, spermatogonia; PL, preleptotene spermatocytes; L, leptotene spermatocytes; Z, zygotene spermatocytes; P, pachytene spermatocytes; D, diplotene spermatocytes; MII, meiosis II spermatocytes; RS, round spermatids; ES, elongated spermatids; Sperm, spermatozoa; OE, Overexpression; FL, full length.

Figure 2

The effect of 1700009J07Rik (07Rik) deletion on the development and morphology of testis and epididymis, and sperm parameters. (A) Development and histomorphology of the testis in adult knockout (KO) and wild-type (WT) male mice. (B) Development and morphology of the epididymis. Histopathological analyses of the testis and epididymis were performed by periodic acid-Schiff + hematoxylin and hematoxylin + eosin staining, respectively. Representative images from stages I–III, IV–V, VI–VIII and IX–IIX of the spermatogenesis progress and caput epididymis are shown. (C–L) Evaluation of sperm motility parameters in KO and WT mice. Sperm were collected from the caudal epididymis and we analyzed sperm count and motility on a computer assisted sperm analysis system. (C) Concentration, (D) total motility, (E) progressive motility, (F) ALH (amplitude of lateral head displacement), (G) beat frequency, (H) VAP (average path velocity), (I) VSL (straight-line velocity), (J) VCL (curvilinear velocity), (K) LIN (linearity), and (L) straightness were recorded, respectively. All data are means ± SEM (n > 3). p values were calculated using multiple comparison with Tukey–Kramer test. ** p < 0.01, compared with the group of WT.

Figure 3

The knockout (KO) of 1700009J07Rik (07Rik) did not affect sperm function but impaired male fertility by obstructing sexual behaviors. (A) External morphological comparison of the testis and epididymis in KO and wild-type (WT) mice. (B) Contrast of testes weight. (C) Ratio of testicular weight to body weight. (D) Pregnancy and (E) litter size between WT and KO mice. (F) The representative pictures of two-cell embryos developed from WT and KO mice with infertility (KO-I) sperm by in vitro fertilization (magnification, 100×). (G) Statistical analysis of the two-cell formation rate from (F). (H) Analysis of in vivo fertilization and mating behaviors in the 07Rik KO male mice. Representative pictures of two-cell embryos developed from WT, KO mice with fertility (KO-F) and KO-I sperm (magnification, 100×). (I) The formation rate of two-cell embryos and mating behavior recording. Videos of sexual acts of male mice were recorded over a long period of time (12 h) after being caged with 2 female mice in estrus with hormone stimulation. The ampulla of the fallopian tube was dissected the next day and the development of two-cell embryos was statistically analyzed. All data are means ± SEM (n = 10–15). p values were calculated using a non-paired “Student-t” test or multiple comparison with Tukey–Kramer test. * p < 0.05, ** p < 0.01, *** p < 0.001, compared with the WT group.

Figure 4

The evaluation of sexual behaviors in the 1700009J07Rik (07Rik) knockout (KO) mice. (A,B) Duration and number of sniffing behaviors. (C,D) Duration and number of mating behaviors. (E,F) Duration and number of mount behaviors. The experimental male mice were caged 1:2 with ICR female mice in estrus stimulated by artificial hormones PMSG and HCG in advance. Sexual behaviors were video-recorded and the above parameters were analyzed in detail for each male mice after a 4 h caging period. All data are means ± SEM (n = 4). p values were calculated via multiple comparison with Tukey–Kramer test. * p < 0.05, ** p < 0.01, *** p < 0.001, compared with the WT group. PMSG, pregnant mare serum gonadotropin; HCG, human chorionic gonadotropin.

Figure 5

1700009J07Rik (07Rik) excision indirectly disturbed testosterone synthesis and metabolism. After RNA isolation, RNA-sequencing was performed to detect the differentially expressed genes (DEGs) in 07Rik knockout (KO) mouse testes. The functions and pathways affected by DEGs were analyzed by GO and KEGG enrichment, respectively. (A) The number of DEGs in the testes after 07Rik cancellation. (B) Biological processes significantly influenced by DEGs. (C) KEGG pathways markedly disturbed by DEGs. The contents of free testosterone in the serum (D) and testes (E). Free testosterone concentration was detected using a commercial Elisa kit. (F) Hsd3b2, (G) Cyp19a1, (H) Cyp11b1, and (I) Gnrh1 transcript expression levels in KO and wild-type (WT) male mouse testes. Gene expressions were determined by qPCR assay. Gapdh was used as a loading control to normalize the gene expression levels. All data are means ± SEM (n = 3–10). p values were calculated using multiple comparison with Tukey–Kramer test. * p < 0.05, ** p < 0.01, *** p < 0.001, compared with the WT group. Testo: testosterone.