Journal of Reproduction and Development最新文献

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.

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 simultaneouslydouble-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.

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 hours 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.

In mammals, secretion of tonic (pulsatile) gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) is often suppressed during lactation. Suppression of GnRH/LH pulses in lactating dams is assumed to be caused by suckling stimuli and a chronic negative energy balance due to milk production. The present study aimed to investigate whether the central enkephalin-δ opioid receptor (DOR) signaling mediated the suppression of LH secretion by acute suckling stimuli and/or chronic negative energy balance due to milk production in rats during late lactation when dams were under a heavy energy demand. On postpartum day 16, the number of Penk (enkephalin mRNA)-expressing cells in the arcuate nucleus was significantly higher in lactating rats than in non-lactating control rats. Pulsatile LH secretion was suppressed in rats with chronic suckling or acute 1-h suckling stimuli 6 h after pup removal on day 16 of lactation. Central DOR antagonism significantly increased the mean LH concentrations and the baseline of LH pulses in rats with chronic suckling but not with acute suckling stimuli on day 16 of lactation. Besides, central κ opioid receptor (KOR) antagonism increased the amplitude of LH pulses in rats with the acute suckling stimuli on day 16 of lactation. These results suggest that central DOR signaling mediates the suppression of LH secretion caused by a negative energy balance in rats receiving chronic suckling during late lactation. On the other hand, central KOR signaling likely mediates acute suckling stimuli-induced suppression of LH secretion in rats during late lactation.

The adenohypophysis is composed of the anterior and intermediate lobes (AL and IL, respectively), and secretes hormones that play an important role in reproduction. CD9- and SOX2-double (CD9/SOX2) positive cells located in the marginal cell layer (MCL) facing the Rathke’s cleft in the AL and IL form the primary stem cell niche in the adult adenohypophysis of rats. In this study, we successfully obtained 3-dimensional (3D) cell aggregates that closely resembled the primary niche of MCL in vivo. After incubation in a Matrigel containing several growth factors, approximately 20% of the cells in the CD9/SOX2-positive cell aggregates were differentiated into hormone-producing cells. The cell aggregates generated in this study may provide insight into the regulation of the pituitary stem/progenitor cell niche and the turnover of hormone-producing cells.

Understanding how stress hormones induce apoptosis in oviductal epithelial cells (OECs) and mural granulosa cells (MGCs) can reveal the mechanisms by which female stress impairs embryonic development and oocyte competence. A recent study showed that tissue plasminogen activator (tPA) ameliorates corticosterone-induced apoptosis in MGCs and OECs by acting on its receptors low-density lipoprotein receptor-related protein 1 (LRP1) and Annexin A2 (ANXA2), respectively. However, whether tPA is involved in corticotropin-releasing hormone (CRH)-induced apoptosis and whether it uses the same or different receptors to inhibit apoptosis induced by different hormones in the same cell type remains unknown. This study showed that CRH triggered apoptosis in both OECs and MGCs and significantly downregulated tPA expression. Moreover, tPA inhibits CRH-induced apoptosis by acting on ANXA2 in both OECs and MGCs. While ANXA2 inhibits apoptosis via phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling, LRP1 reduces apoptosis via mitogen-activated protein kinase (MAPK) signaling. Thus, tPA used the same receptor to inhibit CRH-induced apoptosis in both OECs and MGCs, however used different receptors to inhibit corticosterone-induced apoptosis in MGCs and OECs. These data helps understand the mechanism by which female stress impairs embryo/oocyte competence and proapoptotic factors trigger apoptosis in different cell types.

For the intracytoplasmic sperm injection (ICSI) procedure in pigs, an electrical pulse (EP) has been used as an effective method for oocyte stimulation, but unlike sperm, EP is unable to induce Ca²⁺ oscillations. In this study, we investigated the effects of generating artificial Ca²⁺ oscillations with phospholipase Cζ (PLCζ) mRNA, a candidate sperm factor, on fertilization, embryonic development, and gene expression after ICSI. Firstly, the concentration of PLCζ mRNA of a fixed volume (1.0 pl) that would induce a pattern of Ca²⁺ rise similar to that of in vitro fertilized (IVF) sperm was examined and determined to be 300 ng/μl. Secondly, the effects of oocyte stimulation methods on fertilization and embryonic development were investigated. ICSI-oocytes were activated by EP (EP group) or by PLCζ mRNA (PLCζ group). Furthermore, IVF-oocytes (IVF group) and ICSI-oocytes with and without an injection of buffer (buffer and untreated groups, respectively) were used as controls. It was found that the rates of normal fertilization in the PLCζ and EP groups were significantly higher than those in the buffer and untreated groups. The blastocyst formation rates did not differ among the groups. The embryo quality in the EP group was inferior to those in the PLCζ and IVF groups. Additionally, the expression level of a proapoptosis-related gene (Caspase-3) in the EP group was significantly higher than those in the PLCζ and IVF groups. Our data suggest that oocyte activation by PLCζ mRNA has the effect of improving embryo quality.

Intracytoplasmic sperm injection (ICSI) is clinically used to treat obstructive/nonobstructive azoospermia. This study compared the efficacy of ICSI with cauda epididymal and testicular sperm in Wistar (WI) and Brown-Norway (BN) rats. The transfer of ICSI oocytes with cryopreserved epididymal and testicular WI sperm resulted in offspring production of 26.2% and 3.7%-4.7%, respectively (P < 0.05). Treatments for artificial oocyte activation (AOA) and acrosome removal improved pronuclear formation in BN-ICSI oocytes; however, only AOA treatment was effective in producing offspring (3.7%-6.5%). In the case of ICSI with testicular sperm (TESE-ICSI), one offspring (0.6%) was derived from the BN-TESE-ICSI oocytes. The application of AOA or a hypo-osmotic sperm suspension did not improve the production of TESE-ICSI offspring. Thus, outbred WI rat offspring can be produced by using ICSI and less efficiently by using TESE-ICSI. Challenges in producing offspring by using ICSI/TESE-ICSI in inbred BN strain require further investigation.

This study aimed to develop a method to evaluate the quality of bovine in vitro fertilized (IVF) embryos based on gene expression profiling via whole-transcriptome amplification. The expression of 11 developmentally important genes in individual bovine in vivo-derived (IVD) and IVF embryos were examined. Gene expression profiling was conducted by classifying the expression level of each gene in individual embryos as low, medium, or high. The IVF group had a higher (P < 0.01) proportion of embryos with low expression of SOX2, NANOG, and FGF4. In addition, a correlation analysis between the expression levels of each gene in individual embryos demonstrated that the relationship between gene expression differed with respect to IVD and IVF embryos. Our results suggest that the expression profiling of developmentally important genes using IVD embryos as normal controls could be a useful indicator for evaluating the quality of bovine IVF embryos.

The present study examined whether male resveratrol intake affected mitochondrial DNA copy number (mt-cn) and telomere length (TL) in blastocysts fathered by young and aged male mice. C57BL/6N male mice supplied with water or water containing 0.1 mM resveratrol were used for embryo production at 14-23 and 48-58 weeks of age. Two-cell-stage embryos were collected from the oviducts of superovulated female mice (8-15 weeks old) and cultured for 3 days until the blastocyst stage. Mt-cn and TL levels were measured by real-time polymerase chain reaction. Resveratrol intake did not affect body weight or water consumption. Resveratrol intake increased the expression levels of SIRT1 in the liver, the antioxidative ability of serum, and extended TL in the heart, whereas there was no significant difference in mt-cn in the heart or TL in sperm. The rate of blastocyst development was significantly lower in aged male mice than in younger mice, and resveratrol intake increased the total number of blastocysts derived from both young and aged males. Resveratrol intake did not affect mt-cn or TL in blastomeres of blastocyst-stage embryos derived from young mice, but significantly increased both mt-cn and TL in blastomeres of blastocysts derived from aged fathers. In conclusion, resveratrol intake increased mt-cn and TL levels in blastocysts derived from aged male mice.