Journal of Reproduction and Development最新文献

Apoptosis is an essential physiological process involved in embryonic development, immune responses, and tissue homeostasis. Despite many studies on pro-apoptotic genes, few reports have directly compared the lethality-inducing potential between them under comparable conditions. In this study, we evaluated the lethality-inducing potential of three representative pro-apoptotic genes, Bax, Casp3, and Casp9, in mouse early embryos under defined conditions using the doxycycline (Dox)-inducible tetracycline-regulated gene expression system in combination with the PiggyBac transposon system. All genes were transcriptionally induced by Dox, and Bax showed the strongest lethal effect, followed by Casp9, while Casp3 did not show any effects. Notably, Bax expression severely impaired blastocyst formation and led to the intense accumulation of the DNA damage marker γH2AX, along with a pronounced increase in the apoptotic cells. These findings suggest that introducing upstream apoptotic regulators leads to the more efficient and widespread activation of the apoptotic cascade. Overall, this study is expected to contribute to a deeper understanding of apoptotic mechanisms and future advancements in regenerative medicine, reproductive engineering, and cancer research.

During spermatogenesis, chromatin remodeling regulated by histone modification is essential for spermatogenic cell development, and multiple epigenetic regulators are involved in this process. Recent studies reported that ATAD2 was a newly discovered cancer/testis factor that functioned in chromatin remodeling in somatic cells by binding acetylated histone. However, the physiological role of ATAD2 in spermatogenesis is largely unknown. In this study, we characterized the expression pattern of ATAD2 in mouse testes and found that the highly expressed ATAD2 in the gonads was lowly expressed in meiotic spermatocytes with distinct localization in nucleus, but highly expressed in round spermatids. By generating Atad2 knockout (KO) mice using CRISPR/Cas9 technology, we revealed that ATAD2 deletion leads to failure of DSB repair and chromosome synapsis in spermatocytes and impairs spermiogenesis in spermatids. Atad2 KO mice were subfertile, as characterized by reduced sperm count, impaired motility, and abnormal morphology. RNA-Seq analysis showed that hundreds of genes were dysregulated in Atad2-KO round spermatids. As revealed by GSEA analysis, the gene set related to spermatid development was downregulated, while gene sets related to chromatin binding and positive and negative DNA-templated transcription were upregulated. In conclusion, our results indicate that ATAD2 contributes to meiotic progression and participates in spermiogenesis by regulating RNA transcription in spermatids.

The cauda epididymis protects and stores mature sperm in mammals. Recently, comprehensive transcriptomic and proteomic analyses have been conducted to understand its molecular functions; however, fundamental information on its metabolites has not been reported. In this study, we optimized a system for the comprehensive metabolic analysis of the cauda epididymis in mature and juvenile mice. This system identified 116 and 92 metabolites in mature and juvenile mice, respectively. Comparative analysis revealed that 44 and 13 metabolites were upregulated and downregulated, respectively, in the cauda epididymis of mature and juvenile mice. Based on the identified metabolites, 34 metabolic and unique pathways (mature: four pathways and juvenile: one pathway) were determined. In conclusion, the levels of certain metabolites in the cauda epididymis differed between mature and juvenile mice. These results contribute to understanding of the unique functions of the cauda epididymis based on dynamic changes in metabolites.

Although it is well-known that sperm tyrosine-phosphorylated proteins increase in a cyclic AMP (cAMP)-dependent manner during capacitation, it remains unclear whether or not this reaction is involved directly in sperm fertilization-related events. This study aimed to examine the effects of pharmacological suppression of cAMP-dependent increases in tyrosine-phosphorylated proteins on the induction of full-type hyperactivation and penetration into eggs in boar spermatozoa. Ejaculated spermatozoa were treated with a cAMP analog (cBiMPS) and subsequently used for Western blotting, flagellar beating assays, and egg penetration assays. Marked increases in tyrosine-phosphorylated proteins and full-type hyperactivation were observed in spermatozoa treated with cBiMPS in the presence of CaCl₂. These spermatozoa effectively penetrated eggs. A spleen tyrosine kinase (SYK) inhibitor OXSI2 suppressed large cBiMPS-dependent increases in sperm tyrosine-phosphorylated proteins, in contrast to the ineffectiveness of SU6656 (a SRC family kinase inhibitor) and dasatinib (a c-ABL inhibitor). However, this suppression was not associated with changes in the state of full-type hyperactivation or the outcomes of egg penetration assays conducted in insemination medium without OXSI2. Conversely, adding OXSI2 to both the cBiMPS treatment medium and the sperm insemination medium suppressed increases in sperm tyrosine-phosphorylated proteins during insemination and significantly decreased the average number of spermatozoa that penetrated eggs. These results suggest that high SYK activation and large cAMP-dependent increases in tyrosine-phosphorylated proteins are not absolutely necessary for inducing full-type hyperactivation and penetration into eggs in boar spermatozoa in vitro, but may influence sperm functions associated with the outcomes of egg penetration assays.

Sixteen multiparous Santa Inês ewes underwent transrectal ovarian ultrasonography from days 6 to 17 (D6-17; D0 = ovulation). On D6, ewes received a single subcutaneous injection of Firmagon^® at a low dose (LD; n = 5; 215 µg/kg body weight [b.w.]) or a high dose (HD; n = 6; 235 µg/kg b.w.); controls (C; n = 5) received saline. Ovulations were detected in 100% of controls, but only in 20% of LD and 17% of HD animals. Mean daily numbers of large antral follicles (> 4.49 mm) decreased (P < 0.05) between D6 and D7 in HD group. Compared with controls, LD ewes had higher (P < 0.05) ovarian artery pulsatility index (PI; D16-17) and resistive index (RI; D17), but lower flow-propagation velocity (FPV; D17), whereas HD ewes exhibited higher PI (D15) and RI (D17), along with lower FPV (D8 and D15) and end-diastolic velocity (D17). Firmagon^® treatment was associated with a reduced number of large antral follicles and alterations in ovarian blood perfusion.

Gonadotropin-releasing hormone (GnRH) tightly regulates the synthesis and the release of gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), while the intracellular molecular mechanisms following GnRH signal for the regulation of transcription remains incompletely understood. In this study, we used primary culture of rat anterior pituitary cells to investigate the role of NR4A transcription factors (NR4A1, NR4A2, and NR4A3) in GnRH regulation of gonadotropin secretion. GnRH agonist stimulation rapidly and transiently increased Nr4a1, Nr4a2, and Nr4a3 expression within one hour, accompanied by a time-dependent increase in Fshb mRNA levels and the secretion of both FSH and LH. The knockdown of each Nr4a gene using siRNA significantly reduced Fshb expression under GnRH stimulation. Nr4a1 knockdown caused the most pronounced decrease in FSH secretion. Although Lhb and Cga mRNA levels were largely unaffected, LH secretion was consistently reduced following NR4A knockdown. These findings suggest that NR4A transcription factors act downstream of GnRH signaling to promote Fshb transcription and facilitate gonadotropin secretion, thereby modulating GnRH-dependent control of FSH and LH secretion.

Three-dimensional (3D) culture systems provide physiologically relevant models that better replicate in vivo cell architecture and function compared to the conventional two-dimensional cultures. In reproductive biology, 3D models of endometrial cells have emerged as powerful tools to investigate uterine physiology, embryo-maternal interactions, and implantation processes. Gel-based cultures using natural extracellular matrices such as collagen or Matrigel enable endometrial epithelial and stromal cells to form gland-like or fibroblast-like structures while maintaining hormone responsiveness and secretory activity. In addition, spheroid cultures recapitulate cell polarity, paracrine signaling, and tissue remodeling, offering insights into implantation biology and endometrial pathologies such as endometriosis. Our studies have demonstrated that bovine endometrial epithelial cells embedded in Matrigel exhibit enhanced glandular gene expression and that engineered hetero-spheroids provide stable models for studying interferon-induced matrix remodeling. Furthermore, co-culture of cultured endometrial explants with rat hatched blastocyst facilitates in vitro analysis of embryo attachment and uterine receptivity. Advances in organoid and microfluidic platforms extend these models by enabling long-term culture and dynamic hormonal regulation. Collectively, 3D culture approaches bridge the gap between in vitro experimentation and in vivo physiology, offering translational applications in reproductive medicine, livestock fertility management, and drug discovery.

Tubal abnormalities were evaluated on hysterosalpingo-contrast sonography (HyCoSy) using sodium alginate solution as a contrast agent in 18 repeat breeder cattle. Clear contrast enhancement was observed from the uterine horn to the tubal infundibulum in the patent cases, and the percentage of cases with unilateral or bilateral passage disorder was 64.7%. The post-examination artificial insemination conception rate when ovulatory follicles were present on the patent side was 64.3%, which was significantly higher than 12.5% seen when ovulatory follicles were present on the side affected by passage disorder (P < 0.05). The polymorphonuclear leukocyte percentage in the recovered contrast agent was significantly higher on the passage disorder side than on the patent side, whereas there was no significant difference in C-reactive protein concentrations between the patent and passage disorder sides. The present method can be used for diagnosing tubal abnormalities, and chronic inflammatory changes may be related to tubal passage disorder.

The conventional ovum pick-up method requires oocytes to be transported from local farms to the laboratory, where they undergo nuclear maturation. However, atmospheric conditions for oocyte transportation differ from those for normal oocyte maturation in vitro. In this study, we examined the effects of conventional and modified oocyte transport conditions on oocyte quality and subsequent embryonic development. Cumulus-oocyte complexes were collected from slaughterhouse-derived bovine ovaries and cultured in few drops of medium on plastic plates in a CO₂-incubator (Control), in plastic tubes containing medium (C-T) in air, or in tubes containing gellan gum and medium (MC-T) in air. C-T conditions reduced mitochondrial functionality (mitochondrial membrane potential and adenosine triphosphate), lipid content, and DNA methylation but increased mitochondrial DNA copy number and phosphorylated AMP-activated protein kinase (P-AMPK) levels compared to those in control oocytes. Furthermore, RNA sequencing analysis of blastocysts derived from these oocytes revealed that C-T conditions affected mitophagy- and AMPK-signaling-related genes. However, MC-T conditions attenuated these C-T-associated changes. In conclusion, conventional C-T conditions affect oocyte metabolism and alter embryo quality, whereas the use of gellan gum as a substrate ameliorates such adverse effects. The oocyte transportation system is inadequate for embryonic production and can induce epigenetic changes. Modifying these conditions with gellan gum is a useful counter-measure.

Correlative light-electron microscopy (CLEM) combines fluorescence microscopy and scanning electron microscopy (SEM) to achieve nanoscale resolution while highlighting regions of interest identified by fluorescence microscopy. CLEM is becoming increasingly important in life sciences but traditionally requires highly dried samples to withstand the high vacuum of SEM. The NanoSuit method, which mimics native extracellular substances, was developed to address this limitation by encasing samples in a thin, vacuum-proof membrane, allowing SEM observation of live or wet multicellular organisms. While previous NanoSuit CLEM studies focused on formalin-fixed paraffin-embedded sections and cultured cells, cryosections had not yet been explored. In this study, NanoSuit CLEM with diluted NanoSuit solution was applied to cryosections of bovine anterior pituitary tissue. Secretory granules in gonadotrophs, which constitute less than 12% of anterior pituitary cells, were successfully visualized. However, other organelles remained unobserved due to fixation conditions. Therefore, NanoSuit CLEM enabled visualization of the ultrastructure of important cells in cryosections, even from large animals.