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. 2019 Jul 19;20(14):3543.
doi: 10.3390/ijms20143543.

Sex-Dimorphic Behavioral Alterations and Altered Neurogenesis in U12 Intron Splicing-Defective Zrsr1 Mutant Mice

Francisco Alén  1 , Isabel Gómez-Redondo  2 , Patricia Rivera  3 , Juan Suárez  3 , Priscila Ramos-Ibeas  2 , Eva Pericuesta  2 , Raul Fernández-González  2 , Serafín Perez-Cerezales  2 , Keiko Horiuchi  4 , Laura Orio  1 , Fernando Rodriguez de Fonseca  1   3 , Alfonso Gutiérrez-Adán  5
Affiliations

Sex-Dimorphic Behavioral Alterations and Altered Neurogenesis in U12 Intron Splicing-Defective Zrsr1 Mutant Mice

Francisco Alén et al. Int J Mol Sci. .

Abstract

Mutant mice with respect to the splicing factor Zrsr1 present altered spermatogenesis and infertility. To investigate whether Zrsr1 is involved in the homeostatic control that the hypothalamus exerts over reproductive functions, we first analyzed both differential gene and isoform expression and alternative splicing alterations in Zrsr1 mutant (Zrsr1mu) hypothalamus; second, we analyzed the spontaneous and social behavior of Zrsr1mu mice; and third, we analyzed adult cell proliferation and survival in the Zrsr1mu hypothalamus. The Zrsr1mu hypothalamus showed altered expression of genes and isoforms related to the glutathione metabolic process, synaptonemal complex assembly, mRNA transport, and altered splicing events involving the enrichment of U12-type intron retention (IR). Furthermore, increased IR in U12-containing genes related with the prolactin, progesterone, and gonadotropin-releasing hormone (GnRH) reproductive signaling pathway was observed. This was associated with a hyperactive phenotype in both males and females, with an anxious phenotype in females, and with increased social interaction in males, instead of the classical aggressive behavior. In addition, Zrsr1mu females but not males exhibited reduced cell proliferation in both the hypothalamus and the subventricular zone. Overall, these results suggest that Zrsr1 expression and function are relevant to organization of the hypothalamic cell network controlling behavior.

Keywords: U12 introns; Zrsr1; behavior; cell proliferation; hypothalamus; neurogenesis; social interaction.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design, execution, interpretation, or writing of the study.

Figures

Figure 1
Figure 1
ZRSR1 expression in HEK293 murine cell lines and in mouse hypothalami. Numbers of differentially expressed genes (DEG) and differentially expressed isoforms (DEI). Significantly enriched Gene Ontology (GO) terms (p < 0.05) in DEGs. (A) Western blot analysis of ZRSR1 protein expression. Lanes 1 and 3 correspond to the murine cell lines expressing mZrsr1 and mZrsr1 truncated protein, respectively. Expression of ZRSR1 in wild-type (WT) mouse hypothalamus and Zrsr1mu mouse hypothalamus is shown in lanes 2 and 4. Actin was used as the loading control (lower panel). (B) Venn diagram showing the correspondence between up- and down-regulated genes and isoforms in the comparison between Zrsr1mu and WT mouse hypothalami. (C) Significant GO terms in DEGs in the pairwise comparison.
Figure 2
Figure 2
RNA-seq analysis of differential alternative splicing (AS) in Zrsr1mu mouse hypothalamus. (A) Distribution of the categories of AS events detected in the analysis. 3SS, alternative 3′ splice sites; A5SS, alternative 5′ splice sites; ES, exon skipping; IR, intron retention; MIC, alternative microexon. (B) Enrichment of the different categories of AS in our analysis in relation with the total number of events present in the vast-tools database. (C) Distribution of differentially retained introns that correspond to U12 introns (left) and U2 introns located in a U12-intron-containing gene (right) detected in our analysis. (D) GO terms associated to biological processes and KEGG pathways enriched in the U12-containing genes detected as differentially spliced.
Figure 3
Figure 3
Performance in the open field test. Panel (A) represents total distance travelled, panel (B) represents average speed of movement of male and female wild-type (control) and Zrsr1mu mice. Panel (C) represents anxiety in the elevated plus maze of male and female wild-type and Zrsr1mu mice. Data are means ± SEM of at least 8 animals per group. * p < 0.05, ** p < 0.01, **** p < 0.001, 2-way ANOVA.
Figure 4
Figure 4
Performance in a social interaction test of male wild-type and Zrsr1mu mice. Panels: (A) body care activity, (B) exploratory digging, (C) non-social exploration of the field, and (D) time spent in social exploration. Data are means ± SEM of at least 8 animals per group. *** p < 0.005.
Figure 5
Figure 5
Newborn cell survival (A) and neural stem cell proliferation (B) respectively assessed by the number of BrdU- and IdU-immunoreactive (+) cells in the subventricular zone (SVZ) of male and female wild-type and Zrsr1mu mice. Low- and high-resolution photomicrographs of representative images showing BrdU+ cells (C–F) and IdU+ cells (G–J). Arrowheads indicate labeled nuclei. Bars represent the mean ± SEM (n = 7–8/group). Tukey (A) or simple effect analysis (B): * p < 0.05, ** p < 0.01 female vs. control male and # p < 0.05 ## p < 0.01 wild-type vs. Zrsr1mu. Str, striatum; lv, lateral ventricle.
Figure 6
Figure 6
Newborn cell survival (A) and neural stem cell proliferation (B) respectively assessed by the number of BrdU- and IdU-immunoreactive (+) cells in the hypothalami of male and female wild-type and Zrsr1mu mice. Low- and high-resolution photomicrographs of representative images showing BrdU+ cells (C–F) and IdU+ cells (G–J). Arrowheads indicate labeled nuclei. Bars represent the mean ± SEM (n = 7–8/group). Tukey (A) or simple effect analysis (B): * p < 0.05 female vs. control male and # p < 0.05 wild-type vs. Zrsr1mu. ARC, arcuate nucleus; VMH, ventromedial hypothalamus; ME, median eminence.
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