Herein, we evaluated the effects of gonadotropin hormone-releasing hormone (GnRH) administration 84 h after medroxyprogesterone acetate (MAP) sponge removal on follicular growth, ovulation timing, and pregnancy per artificial insemination (AI) in cosynchronized postpartum Nili Ravi buffaloes. In this study, 58 Nili Ravi postpartum buffaloes (DIM = 103 ± 1.64) were randomly divided into two treatment groups (n = 29/treatment): GnRH-TAI-84 and TAI-84. All buffaloes were administered a MAP sponge for seven days. Upon MAP sponge removal, all the subjects received prostaglandin F_2α (PGF_2α) and Timed AI (TAI) was performed 84 h after sponge removal. In the GnRH-TAI-84 group, the buffaloes received GnRH alongside insemination, whereas in the TAI-84 group, the buffaloes were inseminated without GnRH administration. Follicle diameter and blood estradiol levels were measured every 6 h from 72–108 h after MAP sponge removal. The animals were checked for pregnancy using ultrasonography 40 days after AI. Animals subjected to the GnRH-TAI-84 protocol had a higher follicular growth rate and preovulatory follicle size than those in the TAI-84 group. The follicular diameter was also larger in animals that received GnRH-TAI-84 than in those that received TAI-84 90 and 96 h after MAP sponge removal. Buffaloes in the GnRH-TAI-84 group had lower estradiol concentrations at 90, 96, 102, and 108 h than those in the TAI-84 group. Ovulation in GnRH-TAI-84 buffaloes occurred 11 h earlier than that in buffaloes from the TAI-84 group. A shorter interval between AI and ovulation in GnRH-TAI-84 buffaloes (14 h vs. 25 h) led to greater pregnancies per AI (62% vs. 17%) compared to buffaloes from the TAI-84 group.

CRISPR/Cas9-based multiplex genome editing via electroporation is relatively efficient; however, lipofection is versatile because of its ease of use and low cost. Here, we aimed to determine the efficiency of lipofection in CRISPR/Cas9-based multiplex genome editing using growth hormone receptor (GHR) and glycoprotein alpha-galactosyltransferase 1 (GGTA1)-targeting guide RNAs (gRNAs) in pig zygotes. Zona pellucida-free zygotes were collected 10 h after in vitro fertilization and incubated with Cas9, gRNAs, and Lipofectamine 2000 (LP2000) for 5 h. In Experiment 1, we evaluated the mutation efficiency of gRNAs targeting either GHR or GGTA1 in zygotes transfected using LP2000 and cultured in 4-well plates. In Experiment 2, we examined the effects of the culture method on the development, mutation rate, and mutation efficiency of zygotes with simultaneously double-edited GHR and GGTA1, cultured using 4-well (group culture) and 25-well plates (individual culture). In Experiment 3, we assessed the effect of additional GHR-targeted lipofection before and after simultaneous double gRNA-targeted lipofection on the mutation efficiency of edited embryos cultured in 25-well plates. No significant differences in mutation rates were observed between the zygotes edited with either gRNA. Moreover, the formation rate of blastocysts derived from GHR and GGTA1 double-edited zygotes was significantly increased in the 25-well plate culture compared to that in the 4-well plate culture. However, mutations were only observed in GGTA1 when zygotes were transfected with both gRNAs, irrespective of the culture method used. GHR mutations were detected only in blastocysts derived from zygotes subjected to GHR-targeted lipofection before simultaneous double gRNA-targeted lipofection. Overall, our results suggest that additional lipofection before simultaneous double gRNA-targeted lipofection induces additional mutations in the zygotes.

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.

MiR-145-5p has been implicated in the development and progression of various disorders, and it is primarily recognized as a tumor suppressor in numerous cancers types. Its expression has been reported to decrease in the granulosa cells of patients with polycystic ovarian syndrome (PCOS). This study aimed to investigate whether miR-145-5p plays a role in granulosa cell proliferation and to shed light on the underlying pathological mechanisms of follicular development in patients with PCOS. Follicular fluid samples were collected from patients with PCOS and healthy individuals. The Cell Counting Kit-8 and bromodeoxyuridine assays were performed to assess KGN cell proliferation. The expression of miR-145-5p was significantly decreased in PCOS granulosa cells than in control cells, whereas the expression of SET was increased. Furthermore, miR-145-5p suppressed the proliferation of KGN cells. SET was identified as a direct target of miR-145-5p. Additionally, SET promoted the proliferation of KGN cells, and knockdown of SET counteracted the effect of the miR-145-5p inhibitor. Therefore, miR-145-5p regulates granulosa cell proliferation by targeting the SET in KGN cells; this process may be a potential pathological pathway that contributes to follicular developmental disorders in PCOS.

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 = 0.856, 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.

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.

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.

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.