The reproductive characteristics and ovarian development in cotton rats (Sigmodon hispidus, CRs) are unclear, although CRs are commonly used as animal models in biomedical research. We previously reported that young (6–8 weeks) CRs showed multi-oocyte follicles (MOFs) and double nucleated oocytes (DNOs) in different stages of follicles. The developmental changes in neonatal CR ovaries were investigated in the present study and were compared with our findings in previous studies of unique phenotypes, particularly in oocytes. CR ovaries at postnatal days (PND) 0, 4, and 7 were obtained from the Hokkaido Institute of Public Health. Samples were analyzed by light and transmission electron microscopy. The general histology and folliculogenesis in CR ovaries were similar to those in other experimental rodents. However, DNOs were observed in all age categories and were frequently observed in primordial follicles, whereas MOFs started to develop from PND4 with greater frequency in primary follicles. Almost all developing follicles expressed DEAD (Asp-Glu-Ala-Asp) box polypeptide 4 and forkhead box L2, which are representative markers of oocytes and follicular epithelial cells, respectively. Ki-67 staining demonstrated the proliferative activity of granulosa cells, but not of oocytes, in follicles. Moreover, rapid folliculogenesis of CR due to a small number of apoptotic oocytes was suggested, based on results of the terminal deoxynucleotidyl transferase dUTP nick end labeling assay, confirming the formation of DNOs or MOFs. These findings clarify the development of unique phenotypes of neonatal CR ovaries and support it as a useful model to better understand folliculogenesis and oocytogenesis as well as their abnormalities in humans and other animals.

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Cotton rats are one of the experimental rodents used for testing different infectious and non-infectious diseases, including gastrointestinal tract pathology. However, their intestinal morphological characteristics are still poorly understood. Here, we clarified the anatomical and histological characteristics of the cecum and ascending colon (AC) of young (1-3-month old), adult (4-6-month old), and old (10-12-month old) cotton rats. The large intestine (LI) in cotton rats is composed of the cecum, AC, transverse and descending colons, and rectum, and is similar to that of other mammals. The AC begins with a double or triple spiral loop-like flexure (SLLF) and ends with a coupled horseshoe-like flexure (HSLF). A single longitudinal mucosal fold (SLMF) was found at the beginning of the AC along the mesentery line and developed with age. Furthermore, the SLMF contained several lymphatic nodules (LNs), indicating their role in digestive and immunological functions. Small and large protuberant LNs were found in the cecum and SLLF, respectively, whereas thin and flat LNs were observed in the HSLF and transverse colon, respectively. Regarding sex-related differences, adult females had a significantly longer AC with a higher number of SLLFs compared to males. The SLMF length and LN number were also longer and higher, respectively, in adult females compared to adult males. These are crucial findings, indicating the presence of sex-related differences in the morphology of the LI in cotton rats, and ours is the first study to discover a sex difference in the mammalian LI lining. Our study clarified the unique morphology of the LI in cotton rats, which could serve as the principal model for elucidating species-specific digestive tract functions and gastrointestinal disorders.

Most mammalian ovarian follicles contain only a single oocyte having a single nucleus. However, two or more oocytes and nuclei are observed within one follicle and one oocyte, respectively, in several species, including cotton rat (CR, Sigmodon hispidus). The present study compared ovarian histology, focusing on folliculogenesis, between two inbred CR strains, HIS/Hiph and HIS/Mz. At 4 weeks of age, ovarian sections from both the strains were analyzed histologically. Multi-oocyte follicles (MOFs) and double-nucleated oocytes (DNOs) were observed in all stages of developing follicles in HIS/Hiph, whereas HIS/Mz had MOFs up to secondary stages and lacked DNOs. The estimated total follicles in HIS/Mz were almost half that of HIS/Hiph, but interstitial cells were well developed in HIS/Mz. Furthermore, immunostaining revealed no clear strain differences in the appearance of oocytes positive for Ki67, PCNA, and p63 in MOF or DNOs; no cell death was observed in these oocytes. Ultrastructural analysis revealed more abundant mitochondrial clouds in oocytes of HIS/Hiph than HIS/Mz. Thus, we clarified the strain differences in the CR ovary. These findings indicate that early events during folliculogenesis affect the unique ovarian phenotypes found in CRs, including MOFs or DNOs, and their strain differences.