Journal of Reproduction and Development最新文献

The mammalian X chromosome exhibits enrichment in genes associated with germ cell development. Previously, we generated a rat model of Becker muscular dystrophy (BMD) characterized by an in-frame mutation in the dystrophin gene, situated on the X chromosome and responsible for encoding a protein crucial for muscle integrity. Male BMD rats are infertile owing to the absence of normal spermatids in the epididymis. Within the seminiferous tubules of BMD rats, elongated spermatids displayed abnormal morphology. To elucidate the cause of infertility, we identified a putative gene containing an open reading frame situated in the intronic region between exons 6 and 7 of the dystrophin gene, specifically deleted in male BMD rats. This identified gene, along with its encoded protein, exhibited specific detection within the testes, exclusively localized in round to elongated spermatids during spermiogenesis. Consequently, we designated the encoded protein as dystrophin-locus-derived testis-specific protein (DTSP). Given the absence of DTSP in the testes of BMD rats, we hypothesized that the loss of DTSP contributes to the infertility observed in male BMD rats.

Embryonic transfer of bovine blastocysts produced using in vitro fertilization (IVF) is widely used, although the challenge of compromised conception rates remains. Using bovine oviduct epithelial cells (BOEC) to improve embryo culture conditions has attracted attention, particularly since the recent discovery of extracellular vesicles from BOEC. The selection of embryos for transfer has also been the subject of various studies, and a set of evaluation criteria to predict pregnancy success has been suggested, in which the embryos are judged by their kinetics and morphology at the early stages. In the present study, we established a spontaneously immortalized BOEC line (SI-BOEC) and examined the effects of conditioned medium on IVF embryos, focusing on the results of the recommended criteria. A modified KSOM (mKSOM) was used to prepare conditioned media. Presumptive zygotes were cultured in mKSOM (control), SI-BOEC-conditioned medium, mKSOM supplemented with sediment (pellet) collected after the ultracentrifugation of the conditioned medium (mKSOM/sediment), and the supernatant. A significantly higher percentage of embryos satisfied the recommended criteria when grown in the conditioned medium than in the mKSOM. A higher proportion of embryos developed into blastocysts after achieving the four criteria. A similar tendency was observed when grown in mKSOM/sediment compared to mKSOM; however, this was not observed in the supernatant. Vesicles with a size similar to that of exosomes were observed in the sediment. In conclusion, the culture medium conditioned by SI-BOEC promoted the production of bovine blastocysts that satisfied the four evaluation criteria recommended for embryo selection.

J Reprod Dev, Vol. 70, No. 4, p. 255 Table 2 have been corrected. For the bottom of Survived (%) row, the Survived value, which read "69", should be replaced with "86".

This study investigated the effects of timed artificial insemination (TAI) and equine chorionic gonadotropin (eCG) administration on lactating dairy cows under heat-stress conditions (average temperature-humidity index: 80). Timed artificial insemination was performed on the cows with (n = 57) or without (control, n = 41) supplementation with 500 IU of eCG at the day of PGF_2α treatment using the CIDR-Ovsynch protocol. GnRH was administered, and a progesterone device (CIDR) was inserted on Day −10 of the treatment protocol. The CIDR was removed on Day −3, and the cows were treated with PGF_2α. Two days later, a 2^nd GnRH injection was administered. Subsequently, AI was performed on Day 0 (16–20 h after the 2^nd GnRH injection), and pregnancy was diagnosed on Days 32 and 60. Plasma progesterone (P₄) concentrations were measured after AI. Results showed that the eCG group had a higher pregnancy per AI (P/AI) than the control group (43.9 vs. 12.2%, P = 0.002), which was also accompanied by elevated P₄ levels. Four cows in the eCG group had multiple calves, representing 7.0 and 16.0% of the group and pregnant cows, respectively. In conclusion, 500 IU of eCG combined with CIDR-Ovsynch in lactating dairy cows under severe heat stress conditions successfully improved fertility. However, the protocol may have a slight risk of multiple births.

The aim of the present study was to develop a semi-quantitative urine pregnancy test for mares based on the Cuboni reaction and to verify the reliability of this test. The urine specimens were hydrolyzed by heating in the presence of hydrochloric acid. The resulting free estrogen were extracted from the urine matrix using toluene. Sulfuric acid was added to the toluene extract and the mixture was heated again. The lower layer in the test tube containing sulfuric acid was used for fluorescence measurements with excitation at 355 nm and measurement at 535 nm. The fluorometric Cuboni test revealed that the fluorescence counts in urine samples collected after the second trimester of gestation were significantly higher than those obtained from barren mares. The levels of estrogens, including equilin, estrone and estardiol-17β exhibited a dose-dependent increase in fluorescence counts, whereas other steroids, such as progesterone, testosterone, and cortisol, did not affect fluorescence. Heat treatment of urine samples with hydrochloric acid significantly increased the fluorescence counts in those collected after the second trimester of gestation compared to non-pregnant samples, implying the presence of large amounts of conjugated estrogens in pregnant mare urine. Fluorescence counts in urine samples obtained during pregnancy showed a positive relationship with estrone concentrations as measured by enzyme immunoassay. The results of the present study showed that the fluorometric Cuboni test facilitates urine fluorescence counts depending on the urinary estrogen content and is capable of discriminating between pregnancy and non-pregnancy states beyond the second trimester of gestation in mares.

This study sought to examine the impact of negative photoperiod on the incidence of multiple ovulations and pregnancies in dairy cattle. The study population consisted of 5,373 pregnant cows in their third or greater lactation that experienced their first post-partum pregnancy after spontaneous estrus. The positive photoperiod (increasing day-length) extends from December 22 to June 21, whereas the negative photoperiod (decreasing day-length) extends from June 22 to December 21. The odds ratios (ORs) for multiple ovulations and pregnancies in cows that became pregnant during the negative photoperiod and the remaining cows that became pregnant during the positive photoperiod were 1.4 and 1.3 (P < 0.0001), respectively. The ORs for cows that became pregnant ≥ 90 days in milk and the remaining cows that became pregnant < 90 days in milk were 4.3 and 4.1 (P < 0.0001), respectively. No significant differences were detected in the monthly rates of multiple ovulations or pregnancies during positive and negative photoperiods. Thus, the present study demonstrates that the ovarian function in cows is related to changes in day-length, with decreasing day-length being associated with greater multiple ovulation and pregnancy rates. The present study also shows that positive and negative photoperiods exhibit different trends. The results of this study are consistent with a growing body of work demonstrating the effects of photoperiod patterns on the reproductive physiology of cows, with clear implications for twin pregnancy prevention.

Incorporation of bovine serum-derived albumin formulation (AlbuMAX) into a basic culture medium, MEMα, enables the completion of in vitro spermatogenesis through testicular tissue culture in mice. However, this medium was not effective in other animals. Therefore, we sought an alternative approach for in vitro spermatogenesis using a synthetic medium without AlbuMAX and aimed to identify its essential components. In addition to factors known to be important for spermatogenesis, such as retinoic acid and reproductive hormones, we found that antioxidants (vitamin E, vitamin C, and glutathione) and lysophospholipids are vital for in vitro spermatogenesis. Moreover, based on our experience with microfluidic devices (MFD), we developed an alternative approach, the PDMS-ceiling method (PC method), which involves simply covering the tissue with a flat chip made of PDMS, a silicone resin material used in MFD. The PC method, while straightforward, integrates the advantages of MFD, enabling improved and uniform oxygen and nutrient supply via tissue flattening. Furthermore, our studies underscored the significance of lowering the oxygen concentration to 10–15%. Using an integrated cultivation method based on these findings, we successfully achieved in vitro spermatogenesis in rats, which has been a long-standing challenge. Further improvements in culture conditions would pave the way for spermatogenesis completion in diverse animal species.

Communication between oocytes and the surrounding granulosa cells during follicular development is essential for complete oocyte growth. Oocytes contain lipid droplets (LDs), organelles assembled in the endoplasmic reticulum (ER) that store neutral lipids, including triglycerides and cholesterol esters. Although the LD content varies among animals, LDs stored in oocytes have been shown to play an important role in oocyte maturation and preimplantation embryonic development. However, knowledge is lacking regarding how and when LDs are initially produced in developing oocytes within follicles. In the present study, we found that LDs appeared in mouse oocytes in a specific phase during follicular development. The emergence of LDs in intrafollicular oocytes was induced within a similar time window in vitro and in vivo. Fluorescence imaging and electron microscopy revealed that LDs emerging in oocytes during the early stages of follicular growth were in close proximity to the ER. Furthermore, fatty-acid-tracking experiments have revealed that exogenous fatty acids are rapidly incorporated into oocytes, and their uptake is regulated by the interaction between oocytes and granulosa cells, likely in part through transzonal projections. In summary, our results suggest that LD synthesis observed in growing oocytes is spatiotemporally regulated and that oocyte–granulosa cell contact may be involved in LD biosynthesis during follicular development.

Insulin-like growth factor-1 (IGF-1) plays a crucial role in follicular growth and stimulates steroid hormone production in bovine follicles. Steroid hormones are synthesized through the actions of steroidogenic enzymes, specifically STAR, CYP11A1, HSD3B, and CYP19A1 in both theca cells (TCs) and granulosa cells (GCs), under the influence of gonadotropins. Particularly, estradiol 17β (E2) assumes a central role in follicular development and selection by activating estrogen receptors β (ESR2) in GCs. We assessed ESR2 mRNA expression in GCs of developing follicles and investigated the impact of IGF-1 on the mRNA expression of ESR2, CYP19A1, FSHR, and LHCGR, STAR, CYP11A1, and HSD17B in cultured GCs and TCs, respectively. Additionally, we assessed the influence of IGF-1 on androstenedione (A4), progesterone (P4), and testosterone (T) production in TCs. Small-sized follicles (< 6 mm) exhibited the highest levels of ESR2 mRNA expression, whereas medium-sized follicles (7-8 mm) displayed higher levels than large-sized follicles (≥ 9 mm) (P < 0.05). IGF-1 increased the mRNA expression of ESR2, CYP19A1, and FSHR in GCs of follicles of both sizes, except for FSHR mRNA in medium-sized follicles (P < 0.05). IGF-1 significantly elevated mRNA expression of LHCGR, STAR, CYP11A1, and CYP17B in TCs of small- and medium-sized follicles (P < 0.05). Moreover, IGF-1 augmented the production of A4 and P4 but had no impact on T production in TCs of small- and medium-sized follicles. Taken together, our findings indicate that IGF-1 upregulates steroidogenic enzymes and steroid hormone production, underscoring the crucial role of IGF-1 in follicle development and selection.

Dynamic changes in the endometrium are crucial for establishing early pregnancy in ruminants. Blastocyst elongation and implantation require hormones and nutrients to be secreted from the maternal endometrium. The fatty acid-binding protein FABP4 is a widely expressed fatty acid transport protein that promotes cell proliferation, migration, and invasion and is involved in conceptus implantation. However, the mechanism underlying the functional regulation of endometrial epithelial cells (EECs) by FABP4 during ovine peri-implantation remains unclear. We simulated hormonal changes in vitro in sheep EECs (SEECs) during the peri-implantation period and found that it elevated FABP4 expression. FABP4 inhibition significantly reduced cell migration, endoplasmic reticulum stress, and autophagy, suggesting that FABP4 regulates endometrial function in sheep. Moreover, the FABP4 inhibitor BMS309403 counteracted hormone-mediated functional changes in SEECs, and an endoplasmic reticulum stress activator and autophagy inhibitor reversed the abnormal secretion of prostaglandins induced by FABP4 inhibition. These results suggest that FABP4 affects ovine endometrial function during early gestation by regulating endoplasmic reticulum stress and autophagy in SEECs.