Circular RNA (circRNA) plays a key role in the development and progression of several diseases; however, its role in intrauterine adhesions (IUAs) is not well understood. This study aims to investigate the expression profiles and potential role of circRNA in IUA. RNA-sequencing was performed to screen for abnormally expressed circRNAs in TGF-β1-induced IUA endometrial stromal cell (ESC) model (IUA group) and an SMAD3 inhibitor, SIS3-treated IUA ESC model (SIS3 group). Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analyses were performed to uncover the key functions and pathways. Interaction networks were constructed and analyzed based on the competing endogenous RNA hypothesis of circRNA. CircRNAs were validated by Sanger sequencing and quantitative polymerase chain reaction (qPCR). Cell proliferation and apoptosis were measured using MTS and flow cytometry, respectively. The protein and mRNA expression levels of fibrosis-related proteins were measured using western blotting and reverse transcription–qPCR, respectively. A total of 66 circRNAs were differentially expressed between the IUA and SIS3 groups. CircPlekha7 was identified as one of the significantly upregulated circRNAs in the SIS3 group. Overexpression of circPlekha7 enhanced apoptosis, decreased the viability of ESCs, and suppressed the expression of α-SMA, collagen I, and SMAD3 in ESCs; whereas knockdown of circPlekha7 exhibited opposite results. Altogether, the results indicate that circPlekha7 plays an anti-fibrotic role in IUA and may serve as a promising prognostic biomarker for patients with IUA. Therefore, overexpression of circPlekha7 could be a potential treatment strategy for IUA.

Porcine somatic cell nuclear transfer (SCNT) is currently inefficient, as 1–3.95% of reconstructed embryos survive to term; inadequate or erroneous epigenetic reprogramming of the specialized donor somatic nucleus could be a primary reason. Therefore, a locus-specific analysis of DNA methylation dynamics in embryogenesis and the DNA methylation status of gametes and donor cells used for SCNT were conducted in the following developmentally important gene loci: POU5F1, NANOG, SOX2, H19, IGF2, IGF2R, XIST; and the retrotransposon LINE-1. There were significant epigenetic differences between the gametes and the somatic donor cells. Three gamete-specific differentially methylated regions (DMRs) in POU5F1, XIST, and LINE-1 were identified. A delayed demethylation process at POU5F1 and LINE-1 loci occurred after three successive cleavages, compared to the in vitro fertilized (IVF) embryos. Although cloned embryos could undergo de-methylation and re-methylation dynamics at the DMRs of imprinted genes (H19, IGF2R, and XIST), the re-methylation process was compromised, unlike in fertilized embryos. LINE-1 loci are widely dispersed across the whole genome, and LINE-1 DMR might be a potential porcine nuclear reprogramming epi-marker. Data from observations in our present and previous studies, and two published articles were pooled to produce a schematic diagram of locus-specific, DNA methylation dynamics of cloned and IVF embryos during porcine early embryogenesis. This also indicated aberrant DNA methylation reprogramming events, including inadequate DNA demethylation and insufficient re-methylation in cloned embryos. Further research should focus on mechanisms underlying demethylation during the early cleavage of embryos and de novo DNA methylation at the blastocyst stage.

Cluster of differentiation (CD) 9 and CD81 are closely-related members of the tetraspanin family that consist of four-transmembrane domain proteins. Cd9 and Cd81 are highly expressed in breast cancer cells; however, their expression in healthy mammary glands is unclear. In this study, we performed quantitative real-time PCR to analyze the expression levels of Cd9 and Cd81. Histological techniques were employed to identify Cd9- and Cd81-expressing cells in rat mammary glands during pregnancy and lactation. It was observed that Cd9 and Cd81 were expressed in the mammary glands, and their expression levels correlated with mammary gland development. To identify cells expressing Cd9 and Cd81 in the mammary glands, we performed double immunohistochemical staining for CD9 and CD81, prolactin receptor long form, estrogen receptor alpha, or Ki67. The results showed that CD9 and CD81 were co-expressed in proliferating mammary epithelial cells. Next, we attempted to isolate CD9-positive epithelial cells from the mammary gland using pluriBead cell-separation technology based on antibody-mediated binding of cells to beads of different sizes, followed by isolation using sieves with different mesh sizes. We successfully isolated CD9-positive epithelial cells with 96.8% purity. In addition, we observed that small-interfering RNAs against Cd9 and Cd81 inhibited estrogen-induced proliferation of CD9-positive mammary epithelial cells. Our current findings may provide novel insights into the proliferation of mammary epithelial cells during pregnancy and lactation as well as in pathological processes associated with breast cancer.

We examined the effects of human chorionic gonadotropin (hCG) treatment on Day 5 (Day 0 = day of artificial insemination: AI) and intravaginal progesterone device (IVPD) treatment from Day 5 to 19 on the conception and detection rates of return to estrus (re-estrus) in lactating dairy cows. A total of 306 cows from a commercial dairy farm were divided into the following three groups on Day 5: non-treatment group (n = 128), untreated; hCG group (n = 71), 3,000 IU hCG was administered (intramuscularly); IVPD group (n = 107), IVPD was inserted into the vagina from Day 5 to 19. Re-estrus detection was performed up to Day 25. Pregnancy was diagnosed by rectal palpation between Day 50 and 60. There was an interaction between treatment and AI number (P < 0.01) on the conception rate of first-AI. For cows with more than three AIs, the IVPD treatment (66.7%) was more effective than the non-treatment (23.1%) (P < 0.05). The re-estrus detection rate was significantly (P < 0.05) higher in the IVPD group (60.7%) than that in the non-treatment group (41.4%) and tended (P < 0.1) to be higher than that in the hCG group (37.8%). Our results suggested that the conception rate can be improved by IVPD treatment, especially in cows with more than three AIs. In addition, IVPD treatment can induce higher estrus expression up to 25 days after AI in non-pregnant cows.

Cotton rats (Sigmodon hispidus, CRs) are commonly used as animal models in biomedical research. However, the reproductive characteristics and ovarian development in the CRs has not been widely investigated. We have previously shown that female CRs, in particular, show several unique phenotypes associated with the urogenital system, such as chronic kidney disease and pyometra. Our investigation revealed unique morphologies in CR ovaries, particularly in oocytes. Cotton rat ovaries at 6–8 weeks of age were obtained from the Hokkaido Institute of Public Health, and their sections analyzed by light microscopy and transmission electron microscopy. Although the general histology and folliculogenesis of CR ovaries were similar to those of other experimental rodents, multi-oocyte follicles (MOFs) and double nucleated oocytes (DNOs) were also observed. Although MOFs were found at all stages of follicular development, a greater frequency of MOFs was observed in the primary and secondary stages. However, DNOs tended to be frequently observed in primordial follicles. Almost all MOF oocytes and a few DNOs possessed a clear zona pellucida, expressed DEAD (Asp-Glu-Ala-Asp) box polypeptide 4 and Forkhead box protein 2, a representative marker of oocytes and follicular epithelial cells. Thus, our investigations revealed the unique phenotypes of the CR ovary. As MOFs and DNOs are occasionally observed in human patients with infertility, the CR would be a useful animal model to study for gaining a better understanding of folliculogenesis and oocytogenesis, as well as their abnormalities in humans and other animals.

We examined the effect of ploidy on mitochondrial DNA (mtDNA) copy number in embryos and the amount of cell-free mitochondrial and nucleic DNA content (cf-mtDNA and cf-nDNA) in spent culture medium (SCM). Oocytes collected from the ovaries were matured, activated, incubated in medium containing cycloheximide (CHX) or CHX and cytochalasin B (CB) for 4.5 h to produce haploid or diploid embryos (H-group and D-group embryos). These embryos were cultured for 7 days, and the blastocysts and SCM were examined. The amount of mtDNA and nDNA was determined by real-time PCR. The rate of development to the blastocyst stage was higher for the D-group than for the H-group. Moreover, D-group blastocysts had less mtDNA compared to the H-group blastocysts. After activation, the mitochondrial content was constant before the blastocyst stage in D-group embryos, but increased earlier in H-group embryos. The amount of cf-mtDNA in the SCM of D-group blastocysts was greater than that of H-group blastocysts. However, when the cf-mtDNA in the SCM of 2 cell-stage embryos (day 2 post-activation) was examined, the amount of cf-mtDNA was greater in the H-group than in the D-group embryos. When D-group embryos were cultured for 7 days, a significant correlation was observed between the total cell number of blastocysts and cf-nDNA content in the SCM. Hence, although careful consideration is needed regarding the time point for evaluating mtDNA content in the embryos and SCM, this study demonstrates that mtDNA in the embryos and SCM was affected by the ploidy of the embryos.

This study investigated the effect of aging on mitochondria in granulosa cells (GCs) collected from the antral follicles of young and aged cows (25–50 months and over 140 months in age, respectively). When GCs were cultured under 20% O₂ for 4 days, mitochondrial DNA copy number (Mt-number), determined by real-time PCR, increased throughout the culture period, and the extent of increase was greater in the GCs of young cows than in those of old cows. In a second experiment, GCs were cultured under 20% O₂ for 24 h. Protein levels of TOMM20 and TFAM in GCs were lower in aged cows than in young cows, and the amount of reactive oxygen species and the mitochondrial membrane potential were higher, whereas ATP content and proliferation activity were lower, respectively. Glucose consumption and lactate production were higher in the GCs of aged cows than in those of young cows. When GCs were cultured under 5% or 20% O₂ for 24 h, low O₂ decreased ATP content and increased glucose consumption in GCs of both age groups compared with high O₂; however, low O₂ decreased the Mt-number only in the GCs of young cows. In conclusion, we show that aging affects mitochondrial quantity, function, and response to differential O₂ tensions in GCs.

Carnosic acid (CA), a natural catechol rosin diterpene, is used as an additive in animal feeds and human foods. However, the effects of CA on mammalian reproductive processes, especially early embryonic development, are unclear. In this study, we added CA to parthenogenetically activated porcine embryos in an in vitro culture medium to explore the influence of CA on apoptosis, proliferation, blastocyst formation, reactive oxygen species (ROS) levels, glutathione (GSH) levels, mitochondrial membrane potential, and embryonic development-related gene expression. The results showed that supplementation with 10 μM CA during in vitro culture significantly improved the cleavage rates, blastocyst formation rates, hatching rates, and total numbers of cells of parthenogenetically activated porcine embryos compared with no supplementation. More importantly, supplementation with CA also improved GSH levels and mitochondrial membrane potential, reduced natural ROS levels in blastomeres, upregulated Nanog, Sox2, Gata4, Cox2, Itga5, and Rictor expression, and downregulated Birc5 and Caspase3 expression. These results suggest that CA can improve early porcine embryonic development by regulating oxidative stress. This study elucidates the effects of CA on early embryonic development and their potential mechanisms, and provides new applications for improving the quality of in vitro-developed embryos.

It is important to understand ovarian physiology when developing an artificial insemination (AI) protocol. Brown bears (Ursus arctos) have a breeding season from May to July, although the type of estrus (polyestrus or monoestrus) is still contested. The present study aimed to define the ovarian dynamics, including follicular waves and ovulatory follicle size, and estrus type in brown bears. Six brown bears were used for ovarian ultrasonography; four were observed between April and October (before the start and after the end of the breeding season) and two in June (breeding season). In addition, we attempted to induce ovulation by administering a gonadotropin releasing hormone (GnRH) agonist. We observed follicular development in April in four bears, but follicles did not develop to greater than 6.0 mm in diameter until May. Thereafter, a group of follicles developed to more than 6.0 mm and grew as dominant follicles, except in one bear. After ovulation and subsequent corpus luteum (CL) formation, the follicular waves disappeared. Furthermore, in three bears treated with GnRH, follicles between 8.2 to 11.2 mm in diameter at the time of treatment ovulated and formed CLs. In two bears, follicles between 5.8 to 8.8 mm ovulated spontaneously within the observation interval. Our results suggest that brown bears may be monoestrous animals. Therefore, AI can only be performed once during the breeding season. Our results also suggest that dominant follicles larger than 8.0 mm are a suitable size for inducing ovulation.

A phosphorylated prolactin is a kind of modified prolactin, which is produced through phosphorylation of native prolactin (PRL) by p21-activated kinase 2 (PAK2) in secretory granules in lactotrophs. Phosphorylated prolactin is involved in the regulation of the estrous cycle and in apoptosis in cancer cells, which seem to have important physiological and pathological roles, respectively. In previous research, it has been reported that estrogen induced the phosphorylation of prolactin in the mouse pituitary gland. However, the relationship between estrogen and PAK2 in the production of phosphorylated PRL has not been clarified yet. In order to examine whether PAK2 is involved in PRL phosphorylation by estrogen, we analyzed PAK2 protein levels in mice and phosphorylated prolactin levels in mouse pituitary cells by western blot analysis. The ratio of phosphorylated PAK2/total PAK2 was increased in estrogen implanted mice, but PAK2 protein and gene expression levels were decreased. In addition, the ratio of phosphorylated prolactin/non-phosphorylated prolactin was decreased in primary pituitary cells with introduced siPAK2. These findings suggest that estrogen could induce the phosphorylation of PRL through PAK2 activation. Therefore, this study contributes to better understanding of the mechanism of phosphorylated PRL production in physiological and pathological conditions associated with estrogen.

Kisspeptin has an indispensable role in gonadotropin-releasing hormone/gonadotropin secretion in mammals. In rodents, kisspeptin neurons are located in distinct brain regions, namely the anteroventral periventricular nucleus-periventricular nucleus continuum (AVPV/PeN), arcuate nucleus (ARC), and medial amygdala (MeA). Among them, the physiological role of AVPV/PeN kisspeptin neurons in males has not been clarified yet. The present study aims to investigate the acute effects of the olfactory and/or mating stimulus with a female rat on hypothalamic and MeA Kiss1 mRNA expression, plasma luteinizing hormone (LH) and testosterone levels in male rats. Intact male rats were exposed to the following stimuli: exposure to clean bedding; exposure to female-soiled bedding as a female-olfactory stimulus; exposure to female-soiled bedding and mating stimulus with a female rat. The mating stimulus significantly increased the number of the AVPV/PeN Kiss1 mRNA-expressing cells in males within 5 minutes after the exposure, and significantly increased LH and testosterone levels, followed by an increase in male sexual behavior. Whereas, the males exposed to female-soiled bedding showed a moderate increase in LH levels and no significant change in testosterone levels and the number of the AVPV/PeN Kiss1 mRNA-expressing cells. Importantly, none of the stimuli affected the number of Kiss1 mRNA-expressing cells in the ARC and MeA. These results suggest that the mating-induced increase in AVPV/PeN Kiss1 mRNA expression may be, at least partly, involved in stimulating LH and testosterone release, and might consequently ensure male mating behavior. This study would be the first report suggesting that the AVPV/PeN kisspeptin neurons in males may play a physiological role in ensuring male reproductive performance.

We investigated the effects of long-term repeated treatments with a sustainable gonadotropin-releasing hormone (GnRH) antagonist, degarelix acetate, on testicular hormonal secretion, size, ultrasound images, histology and spermatogenesis in goats to assess its efficacy as a chemical castration method. Male Shiba goats (3–6 months of age) were treated subcutaneously with degarelix acetate every 4 weeks for 24 weeks. Plasma testosterone and insulin-like peptide 3 concentrations decreased (P < 0.05) within 2 days after the first treatment and remained low until 29 weeks (P < 0.05). Scrotal circumference and testicular pixel intensity were lower from 2–6 months and from 1–6 months, respectively, compared to the pretreatment values (P < 0.05). The testis and epididymis weights were lower at 24 weeks compared to those in untreated goats (P < 0.05). There were no sperm in the seminiferous tubules of testicular tissue sections or in homogenates of the epididymis at 24 weeks. These results suggest that repeated treatment with degarelix acetate is an effective chemical castration method for goats.

We evaluated the relationship between plasma anti-Müllerian hormone (AMH) concentrations in Japanese Black (JB) heifers at 7−10 months of age and the number of embryos recovered after superovulation treatment in selected ovum pick-up donors, concomitantly with changes in their AMH concentrations before and after parturition. Plasma AMH concentrations in heifers were positively correlated with the total number of follicles (r = 0.647, P < 0.01) and embryos (r = 0.681, P < 0.01) recovered from the animals postpartum, when selected as donor cows, but did not correlate with the total number of transferable embryos. No difference was observed between the plasma AMH concentration at the heifer period and the postpartum period. Additionally, serum AMH concentrations of heifers weakly correlated with the number of follicles and embryos recovered by virgin flush after superovulation treatment at 13−15 months of age. Therefore, a single blood AMH concentration measurement may accelerate intensive JB cattle breeding.

Autoimmune orchitis is a condition related to cellular immunity. A disease model involving transfer of T lymphocytes activated by known antigens would be useful for defining pathogenical molecules. Since no method for activating rat T cells using specific antigens is available, we started the study to develop the method. T cells were collected from draining lymph nodes of immunized rats, then co-cultured with syngeneic splenocytes as antigen-presenting cells (APC) in antigen-supplemented medium (= stimulation). The cells were then incubated in medium without antigens and APC (= resting). Repetitive stimulation and resting increased the number of the T cells more than 100-fold. The antigen-specific activation was demonstrated by cell proliferation assay and ELISA assay for interferon gamma. Flow cytometry revealed that > 95% of the cells expressed tumor necrosis factor alpha, a cytokine responsible for autoimmune orchitis. The present method will provide a new procedure to evaluate antigenicity of sperm molecules.