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Background Improved genome-editing via oviductal nucleic acids delivery (i-GONAD) is a new technology that facilitates in situ genome-editing of mammalian zygotes exiting the oviductal lumen. The i-GONAD technology has been developed for use in mice, rats, and hamsters; however, oligonucleotide (ODN)-based knock-in (KI) is more inefficient in rats than mice. To improve the efficiency of i-GONAD in rats we examined KI efficiency using three guide RNAs (gRNA), crRNA1, crRNA2 and crRNA3. These gRNAs recognize different portions of the target locus, but also overlap each other in the target locus. We also examined the effects of commercially available KI -enhancing drugs (including SCR7, L755,507, RS-1, and HDR enhancer) on i-GONAD-mediated KI efficiency. Results The KI efficiency in rat fetuses generated after i-GONAD with crRNA2 and single-stranded ODN was significantly higher (24%) than crRNA1 (5%; p < 0.05) or crRNA3 (0%; p < 0.01). The KI efficiency of i-GONAD with triple gRNAs was 11%. These findings suggest that KI efficiency largely depends on the type of gRNA used. Furthermore, the KI efficiency drugs, SCR7, L755,507 and HDR enhancer, all of which are known to enhance KI efficiency, increased KI efficiency using the i-GONAD with crRNA1 protocol. In contrast, only L755,507 (15 μM) increased KI efficiency using the i-GONAD with crRNA2 protocol. None of them were significantly different. Conclusions We attempted to improve the KI efficiency of i-GONAD in rats. We demonstrated that the choice of gRNA is important for determining KI efficiency and insertion and deletion rates. Some drugs (e.g. SCR7, L755,507 and HDR enhancer) that are known to increase KI efficiency in culture cells were found to be effective in i-GONAD in rats, but their effects were limited.

It is unknown whether endocrine-disrupting chemicals (EDCs) with estrogenic activities have any modifying effects on uterine carcinogenesis. In our previous study, we established a uterine-carcinogenesis model that is useful for detecting tumor-modifying effects of EDCs by the administration of N-ethyl-N-nitrosourea (ENU) to female heterozygous p53-deficient CBA mice [p53 (+/-) mice]. To investigate the effects of ethinylestradiol (EE) and methoxychlor (MXC) on development of ENU-induced uterine tumors, female p53 (+/-) mice and their wild-type littermates [p53 (+/+) mice] received an intraperitoneal injection of 120 mg/kg body weight (bw) of ENU, followed, in Group 1, by no further treatment; in Group 2, by a diet containing 1 ppm EE; in Group 3, by a diet containing 5 ppm EE for 4 weeks and 2.5 ppm EE thereafter; and in Group 4, by a diet containing 2000 ppm MXC for 26 weeks. Uterine proliferative lesions that were induced were composed of both endometrial-stromal and epithelial-cell types. Endometrial stromal sarcomas were induced in p53 (+/-) mice of Groups 1 to 4, and the incidence (87%) in Group 3 was significantly increased compared to Group 1 (47%). Atypical hyperplasias (clear-cell type) of the endometrial gland in p53 (+/-) mice were seen at incidences of 0, 14, 60, and 0% in Groups 1, 2, 3, and 4, respectively, while their incidence in p53 (+/+) mice was 0, 7, 53, and 0%, respectively, with a significant difference between Groups 1 and 3 in both cases. One p53 (+/-) mouse in Group 3 also had an adenocarcinoma consisting of clear cells, and the PCNA labeling indices of the clear-cell atypical hyperplasias, and this endometrial adenocarcinoma, were higher than those of glandular hyperplasias. The present study suggests that 2.5 ppm EE, but not MXC, exerts tumor-promoting effects on stromal and epithelial proliferative lesions of the uteri in p53 (+/-) mice initiated with ENU.

Premenstrual syndrome (PMS) is triggered by hormonal events ensuing after ovulation. The symptoms can begin in the early, mid, or late luteal phase and are not associated with defined concentrations of any specific gonadal or non-gonadal hormone. Women with PMS experience affective or somatic symptoms that cause severe dysfunction in social or occupational realms. Although evidence for a hormonal abnormality has not been established, the symptoms of the premenopausal disorders are related to the production of progesterone by the ovary. The progesterone metabolites may bind to a neurosteroid-binding site on the membrane of the neurotransmitters. Thus, ovulation suppression is an area of focus for diagnostic and treatment options. Many treatment studies have focused on suppression of ovulation with gonadotropin-releasing hormone analogs (GnRHa), high doses of transdermal estrogen, and bilateral oophorectomy all have positive evidence as treatment options for prevention of PMS. However, because of these limitations and their substantial intensive care, these do not appear to be appropriate methods for conventional treatment of PMS. Serotonergic antidepressants, selective serotonin reuptake inhibitors, are well-established, highly effective, and first-line pharmacologic therapy.