Minor splicing plays an important role in vertebrate development. Zrsr1 and Zrsr2 paralog genes have essential roles in alternative splicing, mainly participating in the recognition of minor (U12) introns. To further explore their roles during early embryo development, we produced Zrsr1^mu and Zrsr2^mu mutant mice, containing truncating mutations within the second zinc finger domain. Both homozygous mutant mice were viable with a normal lifespan. When we crossed a homozygous Zrsr2^mu/mu female with Zrsr1^mu/mu male, the double heterozygotes were non-viable, giving rise to embryos that stopped developing mainly between the 2- and 4-cell stages, just after zygotic gene activation. RNA-seq analysis of Zrsr1/2^mu 2-cell embryos showed altered gene and isoform expression of thousands of genes enriched in gene ontology terms and biological pathways related to ribosome, RNA transport, spliceosome, and essential zygotic gene activation steps. Alternative splicing was analyzed, showing a significant increase in intron retention in both U2 and U12 intron-containing genes related to cell cycle and mitotic nuclear division. Remarkably, both Zrsr1 and Zrsr2 were required for the conversion of mouse-induced pluripotent stem cells into 2C-like cells. According to our results, Zrsr1 or Zrsr2 are necessary for ZGA and both are indispensable for the conversion of induced pluripotent stem cells into 2C-like cells.

Polycystic ovarian syndrome (PCOS) is the main cause of female infertility. It is a multifactorial disorder with varying clinical manifestations including metabolic/endocrine abnormalities, hyperandrogenism, and ovarian cysts, among other conditions. D-Chiro-inositol (DCI) is the main treatment available for PCOS in humans. To address some of the mechanisms of this complex disorder and its treatment, this study examines the effect of DCI on reproduction during the development of different PCOS-associated phenotypes in aged females and two mouse models of PCOS. Aged females (8 months old) were treated or not (control) with DCI for 2 months. PCOS models were generated by treatment with dihydrotestosterone (DHT) on Days 16, 17, and 18 of gestation, or by testosterone propionate (TP) treatment on the first day of life. At two months of age, PCOS mice were treated with DCI for 2 months and their reproductive parameters analyzed. No effects of DCI treatment were produced on body weight or ovary/body weight ratio. However, treatment reduced the number of follicles with an atretic cyst-like appearance and improved embryo development in the PCOS models, and also increased implantation rates in both aged and PCOS mice. DCI modified the expression of genes related to oocyte quality, oxidative stress, and luteal sufficiency in cumulus-oocyte complexes (COCs) obtained from the aged and PCOS models. Further, the phosphorylation of AKT, a main metabolic sensor activated by insulin in the liver, was enhanced only in the DHT group, which was the only PCOS model showing glucose intolerance and AKT dephosphorylation. The effect of DCI in the TP model seemed mediated by its influence on oxidative stress and follicle insufficiency. Our results indicate that DCI works in preclinical models of PCOS and offer insight into its mechanism of action when used to treat this infertility-associated syndrome.