Hypothalamic kisspeptin neurons are master regulators of mammalian reproduction via direct stimulation of gonadotropin-releasing hormone and consequent gonadotropin release. Here, we generated novel Kiss1 (kisspeptin gene)-Cre rats and investigated the developmental changes and sex differences in visualized Kiss1 neurons of Kiss1-Cre-activated tdTomato reporter rats. First, we validated Kiss1-Cre rats by generating Kiss1-expressing cell-specific Kiss1 knockout (Kiss1-KpKO) rats, which were obtained by crossing the current Kiss1-Cre rats with Kiss1-floxed rats. The resulting male Kiss1-KpKO rats lacked Kiss1 expression in the brain and exhibited hypogonadotropic hypogonadism, similar to the hypogonadal phenotype of global Kiss1 KO rats. Histological analysis of Kiss1 neurons in Kiss1-Cre-activated tdTomato reporter rats revealed that tdTomato signals in the anteroventral periventricular nucleus (AVPV) and arcuate nucleus (ARC) were not affected by estrogen, and that tdTomato signals in the ARC, AVPV, and medial amygdala (MeA) were sexually dimorphic. Notably, neonatal AVPV tdTomato signals were detected only in males, but a larger number of tdTomato-expressing cells were detected in the AVPV and ARC, and a smaller number of cells in the MeA was detected in females than in males at postpuberty. These findings suggest that Kiss1-visualized rats can be used to examine the effect of estrogen feedback mechanisms on Kiss1 expression in the AVPV and ARC. Moreover, the Kiss1-Cre and Kiss1-visualized rats could be valuable tools for further detailed analyses of sexual differentiation in the brain and the physiological role of kisspeptin neurons across the brain in rats.

Although embryo transfer is widely applied in cattle, many of the transferred embryos do not result in pregnancy. To determine a new parameter for bovine embryo evaluation, we investigated the relationships between in vitro hatchability and embryo morphological parameters using optical coherence tomography (OCT) that we established recently. Bovine embryos were obtained from Japanese Black cattle by in vitro fertilization (IVF). The quality of the blastocysts was examined under an inverted microscope and confirmed as Codes 1–3 according to the IETS standards for embryo evaluation. The OCT images of the embryos were captured on Day 7 after IVF, and the embryos were cultured until Day 9 to determine their hatchability. During OCT, the embryos were irradiated with near-infrared light for a few minutes to obtain three-dimensional images. In total, 22 parameters were assessed for each of the 42 embryos, of which 25 hatched (H embryos) and 17 did not (NH embryos). The thickness of the trophectoderm (TE) and TE+zona pellucida (ZP) was lesser, and the volumes of the TE, ZP, blastocoel, and whole embryo and blastocoel diameter were greater in the H embryos than in the NH embryos. PCA identified that the increase in the blastocoel-related value along with the decrease in the thickness-related value of the TE and/or ZP could be indicators for evaluating the hatchability of bovine IVF embryos. These results support the idea that OCT-captured structural data of blastocyst-stage embryos can be used as a potential model to predict the quality of bovine embryos.

Intrauterine extracellular vesicles (EVs) are involved in establishing proper conceptus-endometrial communication, which is essential for conceptus implantation and subsequent successful placentation. Despite several studies on intrauterine EVs, the composition and quantitative changes in conceptus and endometrial EVs, as well as the effects of intrauterine EVs on endometrial epithelial cells (EECs) during the peri-implantation period, have not been well characterized. To elucidate global changes in proteins in EVs extracted from uterine flushings (UFs) during the pre-implantation (P17), just-implantation (P20), and post-implantation (P22) periods, the datasets of the proteome iTRAQ analysis were compared among P17, P20, and P22 EVs. These analyses revealed that the composition and function of proteins in the EVs changed dramatically during peri-implantation in cattle. Notably, intrauterine P17 EVs affected the high expression of “Developmental Biology” and “morphogenesis of an endothelium” compared with those in P20 and P22 EVs. Furthermore, P20 EVs had the functions of the high expression of “mitochondrial calcium ion homeostasis” and “Viral mRNA Translation” compared with those in P17 EVs. Transcripts extracted from EECs treated with P17, P20, or P22 EVs were subjected to RNA-seq analysis. These analyses identified 60 transcripts in EECs commonly induced by intrauterine EVs recovered from P17, P20, and P22, a large number of which were associated with “type I interferon signaling pathway”. Collectively, these findings reveal the presence and multiple functions of EVs that are potentially implicated in facilitating conceptus implantation into the uterine epithelium during the peri-implantation period.

We investigated the effects of differences in milk production during early pregnancy on placental characteristics at full term, calf birth weights, and their metabolic status. Thirty-four Holstein cows were categorized into three groups (Low, n = 9; Middle, n = 16; High, n = 9) based on the quartile of average daily 4% fat-corrected milk production during early pregnancy. The High group showed higher milk component production than the other groups (P < 0.05) during early and mid-pregnancy. Although most placental characteristics did not differ significantly among the groups, cows in the High group had larger individual cotyledons and fewer medium-sized cotyledons than those in the Low group (P < 0.05). Plasma amino acid concentrations of calves in the Low and High groups were significantly higher than those of calves in the Middle group, although calf birth weights were similar among the groups. Furthermore, cows in the Low group had longer dry periods than those in the High (P = 0.004) and Middle (P = 0.058) groups. This suggests that cows in the Low group may have provided more amino acids to the fetus because of low lactation and long dry periods. Conversely, cows in the High group required more energy for lactation during early pregnancy, which can reduce nutrient availability to the placenta and fetus; however, increasing individual cotyledonary sizes during late pregnancy may ensure that the same amounts of amino acids as those in cows in the Low group are supplied to the fetus, recovering the birth weights.

A high temperature-humidity index during summer has deleterious effects on mitochondrial function, reducing oocyte developmental competence. 5-Aminolevulinic acid (5-ALA) and sodium ferrous citrate (SFC) are both known to support mitochondrial function and have strong anti-oxidant and anti-apoptotic activities. This study aimed to determine the mechanism of action of 5-ALA/SFC on oocyte quality. Bovine oocytes were collected from medium-sized follicles during summer (July–September, temperature-humidity index:76.6), cultured with 0, 1, 2, 4, and 8 µM 5-ALA with SFC at a molar ratio of 1:0.125, fertilized, and cultured for 10 days. The addition of 8/1 µM 5-ALA/SFC had a deleterious effect on oocyte cleavage rate in comparison with control oocytes, but did not affect the blastocyst rate, while 1/0.125 µM 5-ALA/SFC had a significantly higher increase in blastocyst rate than 8/1 µM 5-ALA/SFC. The addition of 1/0.125 and 2/0.25 µM 5-ALA/SFC improved oocyte quality by increasing the mitochondrial distribution pattern and metaphase-II oocytes, reducing reactive oxygen species and upregulating nuclear factor erythroid-2-related factor 2, heme oxygenase-1, and superoxide dismutase-1 in oocytes, and nuclear factor erythroid-2-related factor 2 and mitochondrial transcription factor A in cumulus cells. These results indicate that 1/0.125 and 2/0.25 µM 5-ALA/SFC may support oocyte quality and developmental competence and provide anti-oxidant actions in cumulus-oocyte complexes.

The signals of the transforming growth factor β (TGF-β) superfamily play a critical role in follicular development in mammals. ACVR1B/TGFBR1/ACVR1C receptors mediate the signaling of several TGF-β superfamily ligands in granulosa cells. Although the requirement for ACVR1B/TGFBR1/ACVR1C receptor signaling in follicular development has been confirmed using mutant mouse models, the detailed roles of the signaling in granulosa cell and oocyte development have not been clearly defined. In the present study, we examined the requirement for ACVR1B/TGFBR1/ACVR1C receptor signaling in granulosa cells using an in vitro growth culture of oocyte-granulosa cell complexes (OGCs) and SB431542, a potent inhibitor of the receptor signaling. Although cumulus-oocyte complexes isolated from the control OGCs were able to undergo cumulus expansion, those isolated from OGCs grown with the inhibitor were not competent, even in the presence of in vivo-grown oocytes. The diameter of the oocytes in the SB431542-treated OGCs was comparable with that of the control; however, these oocytes were not competent for complete meiotic maturation or preimplantation development. Therefore, ACVR1B/TGFBR1/ACVR1C receptor signaling is not required for oocytes to increase their volume but is essential for the normal development of cumulus cells and oocyte developmental competence.

Progesterone (P) is a well-known enhancer of hyperactivation which is associated with the success of in vitro fertilization (IVF). In this study, we examined whether P-enhanced hyperactivation affected IVF success in rats. When rat spermatozoa were exposed to 10, 20, and 40 ng/ml P, 20 ng/ml P enhanced hyperactivation via the membrane progesterone receptor. In addition, the enhancement of hyperactivation by 20 ng/ml P was regulated by phospholipase C, transmembrane adenylate cyclase, and protein kinase A. However, 20 ng/ml P did not affect IVF success. These results suggest that 20 ng/ml P enhances rat spermatozoal hyperactivation through non-genomic pathways. Because the concentration of P changes during the estrous cycle, it seems that rat spermatozoa are hyperactivated in response to the oviductal environment. However, the effect of 20 ng/ml P does not seem to fully capacitate spermatozoa.