Herein, we report a case of pregnancy of a female bottlenose dolphin (Tursiops truncatus) that was subjected to artificial insemination (AI) in water based on its estrous behavior using simple instruments. AI was performed on this female dolphin once or twice daily for 4 days at the detection of estrous behavior, such as floating horizontally and showing reduced responsiveness, likely indicating the appropriate timing for AI. The female was placed in supine a position in the water to position the genital slit above the water surface. A Nélaton catheter (Fr. 10, 40 cm length), with its tip modified, was inserted approximately 20 cm into the vagina through the genital slit, and 1-2 ml of fresh semen was injected. The AI procedure was performed within 1 min by two technicians. Thus, this AI method may be a new choice for artificial reproduction, as pregnancy success can be achieved with relatively less cost, less difficulty, and less invasive treatments of cetaceans.

In mammals, secretion of tonic (pulsatile) gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) is often suppressed during lactation. Suppression of GnRH/LH pulses in lactatingdams 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 opioidreceptor (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 whendams 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 inlactating 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 oflactation. 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 16of 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 suggestthat 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, centralKOR signaling likely mediates acute suckling stimuli-induced suppression of LH secretion in rats during late lactation.

Metabolic stress and subsequent hepatic dysfunction in high-producing dairy cows are associated with inflammatory diseases and declining fertility. Lipopolysaccharide (LPS)-binding protein(LBP) is produced by hepatocytes and controls the immune response, suggesting that it is involved in the pathophysiology of inflammation-related attenuation of reproductive functions duringmetabolic stress. This study investigated the effect of LBP on the inflammatory status, oocyte quality, and steroidogenesis in the follicular microenvironment of dairy cows. Using bovineovaries obtained from a slaughterhouse, follicular fluid and granulosa cells were collected from large follicles to evaluate the follicular status of metabolism, inflammation, andsteroidogenesis. Cumulus-oocyte complexes were aspirated from small follicles and subjected to in vitro embryo production. The results showed that follicular fluid LBPconcentrations were significantly higher in cows with fatty livers and hepatitis than in those with healthy livers. Follicular fluid LBP and LPS concentrations were negatively correlated,whereas LPS concentration showed a positive correlation with the concentrations of non-esterified fatty acids (NEFA) and β-hydroxybutyric acid in follicular fluid. The blastulation rate ofoocytes after in vitro fertilization was impaired in cows in which coexisting large follicles had high NEFA levels. Follicular fluid NEFA concentration was negativelycorrelated with granulosa cell expression of the estradiol (E2) synthesis-related gene (CYP19A1). Follicular fluid LBP concentration was positively correlatedwith follicular fluid E2 concentration and granulosa cell CYP19A1 expression. In conclusion, follicular fluid LBP may be associated with favorable conditions inthe follicular microenvironment, including low LPS levels and high E2 production by granulosa cells.

After pregnancy, the corpus luteum (CL) functions as a transient endocrine gland that produces progesterone, which is necessary to maintain pregnancy. To maintain constant progesteroneproduction, CLs are enriched in lipids as its precursors. Lipid droplets (LDs) are organelles that originate from the endoplasmic reticulum and store neutral lipids such as triacylglycerolsand cholesteryl esters. The size and number of LDs in a cell are regulated by LD-associated proteins that coat their surface. LD degradation is regulated by either neutral lipid hydrolases(lipolysis), selective autophagic mechanism (lipophagy), or both. Mammalian CLs are long known to be enriched in LDs, but LDs are rapidly depleted after pregnancy and reappear near the timeof delivery. In this present study, we hypothesized that LDs synthesized by luteinization are massively degraded after pregnancy. Using mCherry-HPos mice, in which LD synthesis can bevisualized in vivo, we found that LD synthesis, which was activated during luteal development, was suppressed after implantation. In CLs, LD synthesis remained low duringpregnancy, but was reactivated before and after delivery. These changes in LDs were confirmed using electron microscopy and immunostaining. Furthermore, LD degradation was mediated bylipolysis rather than lipophagy. In summary, our findings indicate that luteinization-induced LD synthesis is suppressed after pregnancy onset and that CLs are lipid-poor during pregnancybecause LDs stored during luteal development are extensively degraded by lipolysis.

The physiological functions of the mammalian epididymis are typically regulated by the testes. In addition to sex steroids secreted by testicular Leydig cells, which act on the epididymis in an endocrine manner, there is a non-sex-steroidal signaling pathway known as the lumicrine pathway. This lumicrine signaling pathway involves ligand proteins secreted from germ cells within the testicular seminiferous tubules traversing the male reproductive tract, which induce epithelial differentiation in the epididymis. These findings prompted an inquiry into whether treatments influencing testis physiology can disrupt epididymal function by interfering with testis-epididymis communication. Busulfan, an alkylating agent commonly used to deplete testicular germ cells in reproductive biology, has not been sufficiently explored because of its effects on the epididymis. This study investigated the effects of busulfan administration on the proximal epididymis using histological and transcriptomic analyses. Notably, busulfan, as opposed to the vehicle dimethyl sulfoxide (DMSO), altered the morphology of the initial segment of the epididymis, leading to a reduction in the cell height of the luminal epithelium. RNA sequencing identified 185 significantly downregulated genes in the proximal epididymis of busulfan-administered mice compared to DMSO-administered mice. Comparative transcriptome analyses revealed similarities between the epididymal transcriptome of busulfan-administered mice and lumicrine-deficient mice, such as efferent-duct-ligated W/Wv and Nell2-/- mice. However, this differed from that of bilaterally orchidectomized mice, in which both the endocrine and lumicrine signaling pathways were simultaneously ablated. Collectively, these results suggested that the harmful effects of busulfan on the proximal epididymis are secondary consequences of the ablation of testis-epididymis lumicrine signaling.

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.

Poly(A)-binding proteins (PABPs) play roles in mRNA maturation, translational activity, and decay. The functions of PABPs, especially PABPN1 and PABPC1, in somatic cells have beenwell-studied. However, little is known about the roles of PABPs in oocytes because of the unique mechanisms of mRNA metabolism in oocytes. This study focused on PABPN1L and generatedPabpn1l knockout (KO) mice using the CRISPR/Cas9 system. After mating tests, we found that Pabpn1l KO females were infertile due to the failure of theembryos to develop to the 4-cell stage. RNA-seq analysis revealed aberrant mRNA persistence in Pabpn1l KO-MII oocytes, which indicates impaired mRNA degradation during thegerminal vesicle (GV) to MII transition. We also revealed that the exogenous expression of Pabpn1l mRNA in KO-GV oocytes recovered defects of embryonic development. PABPN1Lis partly indispensable for female fertility in mice, owing to its necessity for embryonic development, which is supported by mRNA degradation during GV to MII maturation.

In this study, we evaluated the effects of pre-maturational culture (pre-IVM) on the developmental competence of bovine oocytes derived from an 8-day in vitro growth (IVG)culture system. IVG oocytes were subjected to 5 h pre-IVM before in vitro maturation, followed by in vitro fertilization (IVF). The proportion of oocytesthat progressed to the germinal vesicle breakdown stage was similar between groups with and without pre-IVM. Although metaphase II oocytes and cleavage rates after IVF were similarregardless of pre-IVM culture, the blastocyst rate was significantly higher in the group with pre-IVM (22.5%) than without pre-IVM (11.0%, P < 0.05). In conclusion, pre-IVM cultureimproved the developmental competence of bovine oocytes derived from an 8-day IVG system.

Japanese native horses, which consists of 8 breeds, are threatened with extinction. Embryo transfer (ET) is used to reproduce endangered animals in various mammalian species. We aimed toperform ET using native ponies from Kiso and Hokkaido as donors and recipients, respectively. ET operation included long-distance transport of non-cryopreserved embryos from NaganoPrefecture to Hokkaido. Embryos were transported 1500 km over 9 h in a container maintained at 22°C. After transferring two embryos to two recipients, one mare delivered a healthy live foal.These results demonstrated that reciprocal ET with long-distance transportation of fresh embryos between the isolated breeds may allow for the proliferation of Japanese native horses.