Absence of an intron splicing silencer in porcine Smn1 intron 7 confers immunity to the exon skipping mutation in human SMN2.

Spinal Muscular Atrophy is caused by homozygous loss of SMN1. All patients retain at least one copy of SMN2 which produces an identical protein but at lower levels due to a silent mutation in exon 7 which results in predominant exclusion of the exon. Therapies targeting the splicing of SMN2 exon 7 have been in development for several years, and their efficacy has been measured using either in vitro cellular assays or in vivo small animal models such as mice. In this study we evaluated the potential for constructing a mini-pig animal model by introducing minimal changes in the endogenous porcine Smn1 gene to maintain the native genomic structure and regulation. We found that while a Smn2-like mutation can be introduced in the porcine Smn1 gene and can diminish the function of the ESE, it would not recapitulate the splicing pattern seen in human SMN2 due to absence of a functional ISS immediately downstream of exon 7. We investigated the ISS region and show here that the porcine ISS is inactive due to disruption of a proximal hnRNP A1 binding site, while a distal hnRNP A1 binding site remains functional but is unable to maintain the functionality of the ISS as a whole.

The effect of hormonal estrus induction on maternal effect and apoptosis-related genes expression in porcine cumulus-oocyte complexes.

BACKGROUND: The effect of hormonal estrus induction on maternal effect (MATER - maternal antigen that embryo requires, ZAR-1 - zygote arrest 1, and BMP15 - bone morphogenetic protein 15) and apoptosis-related genes expression (BCL-2 and BAX) in porcine cumulus-oocyte complexes (COCs) and selected follicular parameters was investigated in this study. METHODS: Gilts were divided into three groups: (I) with natural estrus; (II) stimulated with PMSG/hCG; and (III) with PMSG/hCG + PGF2alpha. Analysis of maternal effect and apoptosis-related transcripts expression in COCs, and progesterone synthesis pathway genes expression (P450scc and 3betaHSD) in granulosa cells was performed by qPCR. BMP15 protein expression in follicular fluid (FF) was analyzed by western blot. Oocyte nuclear maturation was assessed by aceto-orcein staining. Progesterone (P4) and estradiol (E2) concentrations in FF and serum were measured by ELISA. Data were analyzed with the one-way ANOVA and Bonferroni post-test or Kruskal-Wallis test and Dunns post-test. RESULTS: The highest expression of MATER, ZAR-1, and BMP15 genes was found in COCs recovered from gilts treated with PMSG/hCG when compared to PMSG/hCG + PGF2alpha-stimulated or non-stimulated gilts. Hormonal treatment did not affect the BMP15 protein expression in FF, but increased the expression of genes participating in P4 synthesis in granulosa cells. The higher percentage of immature oocytes was found in PMSG/hCG-treated when compared to the non-stimulated gilts. The expression of BCL-2 and BAX mRNA, and BCL-2/BAX mRNA ratio was significantly higher in COCs derived from PMSG/hCG-treated when compared to PMSG/hCG + PGF2alpha-treated or non-stimulated subjects. The level of P4 in serum was similar in animals from all experimental groups, while its concentration in FF was greater in gilts subjected to PMSG/hCG treatment than in PMSG/hCG + PGF2alpha-stimulated and non-stimulated gilts. The concentration of E2 did not differ in the serum or FF between the control group and the hormonally stimulated groups. CONCLUSIONS: Hormonal induction of estrus affected maternal effect gene transcripts levels in COCs and and oocyte nuclear maturation. The inclusion of PGF2alpha into the stimulation protocol enabled maintaining of physiological concentration of P4 in FF. Additionally, both hormonal treatments seem to be beneficial for apoptosis prevention through increasing BCL-2/BAX transcript ratio.

Effects of seminal plasma and the presence of a conceptus on regulation of lymphocyte-cytokine network in porcine endometrium.

Infusion of seminal plasma in the uterus is known to elicit an instant inflammatory response in the porcine uterus, but whether or not it prepares a uterine immunological response to the presence of conceptuses is not well understood. Seminal plasma induced long-term modulatory effects and conceptus-induced immune changes in leukocyte populations were measured by flow cytometry and mRNAs for various cytokines by quantitative reverse-transcriptase PCR in porcine endometrium collected on Days 6 and 13 from cycling and pregnant animals or from animals given seminal plasma infusions. Seminal plasma infusion induced long-term modulatory effects, resulting in significantly more endometrial FoxP3-positive T-regulatory and T-helper cells 6 days after infusion as compared to cycling and pregnant animals. The number of T-cytotoxic and T-null cells did not change between the studied groups. The early molecular effects of seminal plasma were not observed at 13-days post-infusion, although animals on Day 13 of pregnancy did show significantly more T-cells (of any type investigated). Seminal plasma also showed a delayed effect on cytokine expression, specifically exhibiting a significant increase in interleukin 10 (IL10) and a decrease in granulocyte macrophage colony-stimulating factor (GMCSF) gene expression on Day 13 as compared to Day 6 of cycling or pregnant gilts. The results indicate a delayed regulatory effect of seminal plasma on immune responses in the porcine uterus, which are similar to immune changes generated by implanting conceptuses.