Journal of Reproduction and Development最新文献

Anti-Müllerian hormone (AMH) is a marker for predicting embryo production in cows subjected to superovulation; however, it remains to be established as a reliable predictor of reproductive performance. We hypothesized that the serum AMH concentration of donors affects the ovarian response to follicle-stimulating hormone (FSH) treatment during superovulation. Herein, we retrospectively investigated the association between different FSH doses and AMH concentrations in donor Japanese Black cows in a superovulation program and analyzed the number of total and transferable embryos recovered. The number of transferable embryos recovered from donors with high AMH levels was significantly higher than that recovered from donors with low AMH levels. Additionally, it increased further with a reduction in the FSH dose. These results illustrate that the AMH concentration is a useful marker for predicting embryo production after superovulation, and donors with high AMH levels produce more transferable embryos at low FSH doses than at high doses.

Progesterone (P₄)-impregnated controlled internal drug-releasing device for goats and sheep (CIDR-G) is used in the estrus synchronization protocol; however, it is not commercially available in Japan. In this study, we investigated whether a modification of CIDR-B, designed for cattle, could be used as a P₄ release device for synchronizing estrus in goats. An in vitro analysis showed that the dissolution rate was not significantly different between CIDR-G and modified CIDR-B (CIDR-M). The administration of CIDR-G or CIDR-M to dairy goats resulted in plasma P₄ concentrations being maintained at the functional luteal phase levels (>2 ng/ml) in both groups for 12 days after administration. However, P₄ concentrations at 2, 4, and 8 days after the administration of CIDR-G were significantly higher than those after the administration of CIDR-M. These results suggest that the CIDR-M examined in this study can maintain P₄ levels for 12 days after its administration as a P₄ release device.

Oocyte developmental competence declines in women aged 35 and older resulting in many women resorting to IVF. The present study determined whether adding Granulocyte-macrophage colony-stimulating factor (GM-CSF) during in vitro oocyte maturation (IVM) could improve oocyte developmental competence in a mouse model of advanced maternal age. Oocytes from 12-14 month C57BL6 J × CBA mice were treated with 10 ng/ml of GM-CSF during IVM, and embryo development, mitochondrial activity, spindle formation and chromosomal alignment were examined. The addition of GM-CSF tended to increase fertilisation rates (76.19 vs. 82.03%; P = 0.07) but did not affect cumulus expansion compared with control. The addition of GM-CSF also increased blastocysts rates (51.10 vs. 61.52%; P < 0.01) and the number of good quality blastocysts (33.31 vs. 44.13%; P < 0.05) present at 96 h of culture as well as inner cell mass (12.64 vs. 15.62; P < 0.01) and total cell number (42.98 vs. 48.78; P < 0.05). GM-CSF treatment also increased mitochondrial membrane potential two to three fold in the outer (2.86 vs. 0.97; P < 0.001), intermediate (3.25 vs. 0.89; P < 0.001) and peri nuclear areas (3.62 vs. 1.08; P < 0.001). GM-CSF treatment did not influence spindle formation or chromosomal alignment. Together our results indicate that the addition of GM-CSF during IVM may improve oocyte quality in women of advanced maternal age.

Selenoprotein P (SeP) is synthesized in the liver and plays a vital role in maintaining selenium homeostasis via transport throughout the body. Previous studies have shown that SeP-deficient mice have severely reduced expression of selenoproteins essential for testicular function, leading to male infertility. We previously reported that the high expression of Ccdc152 in hepatocytes acts as a lncRNA, suppressing SeP expression in the liver. Ccdc152 reduces SeP translation by binding to SeP mRNA and decreasing its interaction with SECIS-binding protein 2. Although Ccdc152 is highly expressed in testes, its function remains unclear. Therefore, this study aimed to elucidate the role of Ccdc152 in the testes. Using the CRISPR/Cas9 system, we generated mice lacking all exons of Ccdc152 and found that SeP expression levels in the liver and plasma, as well as overall selenium homeostasis, remained unchanged. No significant differences were observed in the expression of glutathione peroxidase 1/4 or level of selenium in the testes. Subsequent investigation of the impact on male reproductive function revealed no abnormalities in sperm motility or Mendelian ratios of the offspring. However, a slight decrease in testicular weight and an increased rate of sperm malformations in the epididymis were observed. RNA-seq and pathway analyses identified the reduced expression of multiple genes related to kinesin and reproductive pathways. Based on these findings, Ccdc152 may not be essential for male reproductive function, but it may enhance reproductive capabilities by maintaining the expression of genes necessary for reproduction.

Prepubertal animals are often preferred as sources of oocytes for assisted reproductive technologies (ARTs) in laboratory mice, but the normality and developmental competence of these oocytes remain controversial. This study systematically examined in vitro fertilization competence, embryo development, and fetal development after embryo transfer (ET) using oocytes from C57BL/6J mice aged 3 to 10 weeks. Oocytes were collected from superovulated females, fertilized, and cultured in vitro for 96 h or transferred at 2-cell stage to recipient females. Additionally, fetal development was compared between unfrozen and frozen-thawed in vitro-fertilized 2-cell embryos across different age groups. The number of ovulated oocytes per animal decreased with age, while the percentage of morphologically normal oocytes was highest in 3-week-old mice (99%) compared to older ages (70-86%, P < 0.05). Although fertilization percentages were consistently high (≥ 97%), blastocyst development in vitro, the nuclear counts of blastocysts and fetal development after ET were lowest for embryos from 3-week-old mice. Development of frozen-thawed embryos to fetuses was significantly reduced compared to unfrozen embryos in all age groups, except for those from 10-week-old mice. These findings suggest that oocytes from prepubertal mice, particularly from 3-week-old mice, are less developmentally competent than those from older mice. Therefore, the age of animals for oocyte source should be carefully considered based on the specific requirements of the research or ART applications.

Ovulation disorders are a major cause of low pregnancy rates and infertility in humans and livestock. Kisspeptin neurons located in the anteroventral periventricular nucleus (AVPV) are responsible for the generation of the gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) surge and the consequent ovulation in female rodents. The present study aimed to examine whether purinergic neurons are direct upstream stimulators of AVPV kisspeptin neurons that trigger the GnRH/LH surge and consequent ovulation in Kiss1-Cre rats. We specifically knocked down the mRNA expression of the P2rx2 purinergic receptor in AVPV kisspeptin neurons by administering an adeno-associated virus (AAV) vector containing Cre-dependent P2rx2 short hairpin RNA (shRNA) into the AVPV region of ovariectomized (OVX) Kiss1-Cre rats treated with a proestrus level of estradiol-17β (OVX + high E2) or ovary-intact Kiss1-Cre rats. The E2-induced afternoon LH surge was significantly suppressed by AVPV kisspeptin neuron-specific knockdown of P2rx2 in OVX + high E2 Kiss1-Cre rats compared with scrambled shRNA-treated control OVX + high E2 Kiss1-Cre rats. Furthermore, the specific knockdown of P2rx2 in AVPV kisspeptin neurons largely disrupted the estrous cycle, spontaneous LH surge, and ovulation in ovary-intact Kiss1-Cre rats. These findings suggest that purinergic neurons directly stimulate AVPV kisspeptin neurons via P2X2 receptors (P2RX2) to induce the GnRH/LH surge and consequent ovulation in female rats.

The size of the ovarian reserve, an indicator of the number of primordial follicles, varies widely among individuals, and declines with age. However, the association between the ovarian reserve and fertility remains unclear. Therefore, in the present study, we analyzed the relationship between plasma concentrations of anti-Müllerian hormone (AMH), a marker of ovarian reserve, and reproductive outcomes in Japanese Black cattle. AMH level quartiles were positively associated with pregnancy following artificial insemination (AI), and the median number of days to pregnancy in Q4 (13 days, 95% confidence interval [CI] = 7-18 days) was significantly shorter than that in Q1 (21 days, 95% CI = 15-46 days). The odds ratio for the predicted pregnancy rate by logistic regression analysis in Q4 (4.06, 95% CI = 1.54-10.67) was also significantly higher than that in Q1. Plasma AMH concentrations were significantly higher in summer (June-August) than in winter (December-February). Furthermore, a strong correlation (r = 0.829, P < 0.001) was observed between plasma AMH concentrations at 2 and 14 months of age. Calves with plasma AMH concentrations of > 700 pg/ml at 2 months old showed a transient increase and maximum AMH concentration within 5 months of birth. Overall, the results of this study indicate that the plasma AMH concentration serves as a predictive marker for the probability of conception following AI in Japanese Black cattle. The current findings contribute to the reliable assessment of AMH production and the early prediction of reproductive performance in sexually mature heifers.

Granulosa cells (GCs) in secondary follicles differentiate into cumulus cells (CCs) and mural granulosa cells (MGCs) in the antral follicle. Only CCs maintain direct connections with oocytes through transzonal projections (TZPs) and support oocyte growth. Here, we examined whether granulosa cells (GCs) from secondary follicles and MGCs from early and late antral follicles were able to reconstruct complexes with TZP-free denuded oocytes (DOs) and regenerate TZPs. Furthermore, to confirm that the regenerated TZPs were functional, the development of the reconstructed complexes and oocyte growth in the complexes were evaluated. After coculture, GCs and MGCs from early antral follicles reconstructed the complexes with DOs and regenerated TZPs. Furthermore, the oocytes in the integrally reconstructed complexes grew fully and acquired meiotic competence, suggesting that the regenerated TZPs were functional. In contrast, MGCs from the late antral follicles lost their ability to elongate TZPs. As the ability to regenerate TZPs differed among cells, we analyzed the transcriptomes of GCs, CCs, and MGCs collected from follicles of different sizes. The characteristics of TZP generation coincided with the transcriptome changes in two directions: from GCs to CCs and MGCs. In conclusion, until the early antral follicle stage, bovine GCs, CCs, and MGCs have common characteristics to elongate TZPs and form antrum-like structures that support oocyte growth in vitro. Furthermore, as the follicle develops, MGCs lose the ability to elongate TZPs.

The developmental activation of the corpus luteum (CL) structurally and functionally is critical for the temporally regulated establishment, maintenance, and termination of pregnancy in rats. In this study, we have investigated the possible involvement of autophagy in the regulation of the CL during pregnancy in rats. The expression ratio of microtubule-associated protein light chain 3 (LC3)-II/-I, a widely used indicator of autophagic activity, in the CL remained relatively stable until day 15 of pregnancy. Subsequently, it progressively increased until day 21, and then declined until day 3 postpartum. This fluctuation was closely associated with the tissue weight of the CL rather than progesterone (P4) production activity. Light and electron microscopy revealed the presence of immunoreactive LC3 aggregates and irregularly shaped autolysosome-like microstructures in the cytoplasm of luteal cells during late pregnancy. Notably, a bolus intrabursal injection of the autophagy inhibitor bafilomycin A1 on day 15 of pregnancy resulted in a significant reduction in luteal cell size and disrupted the normal alteration of circulating P4 levels. Consequently, treatment with this inhibitor increased the likelihood of the varied timing (both advanced and delayed) of delivery and led to reduced body weight in neonates when compared with the vehicle-treated control group. Our findings suggest that autophagy in the rat CL contributes to luteal tissue growth, influences P4 production, and thereby fine-tunes the regulation of gestation length in rats.

Heifer growth and milk production in lactating cows may diminish the nutrient supply to the fetus. This study aimed to analyze the characteristics of the nutrient supply to the fetus in primiparous and multiparous cows. We investigated maternal, umbilical cord, and calf blood glucose and amino acid levels, as well as placental development in 28 primiparous (PP) and 30 multiparous (MP) Holstein cows. Although the total cotyledonary weight and surface area showed no significant differences, the MP group exhibited larger individual cotyledons (P < 0.01) and fewer medium-sized cotyledons (P < 0.05). Within the PP group, total cotyledonary weight and surface area positively correlated with blood glucose (r = 0.71-0.77; P < 0.01) and total essential amino acid (r = 0.55; P < 0.05) concentrations in the umbilical veins. However, no significant correlation was observed in the MP group. Blood glucose and amino acid concentrations in the umbilical vein, umbilical artery, and calf were significantly lower in the MP group (P < 0.05), although no difference was observed in the dams between the groups. In conclusion, the nutrient status of primiparous cows can alter fetal nutrient supply. Moreover, multiparous cows have larger individual cotyledons as an adaptive response to increased milk production during pregnancy. However, this adaptive response in multiparous cows did not completely restore nutrient supply to the fetus to the same extent as that in primiparous cows. Therefore, the nutritional management of multiparous cows during pregnancy must be reconsidered.