Gene-knockout mice lacking ACRBP, a proacrosin-binding protein localized in the acrosome of sperm, have been shown to exhibit male subfertility, owing to abnormal formation of the acrosome. In this study, to elucidate the mechanism contributing to the subfertility phenotype, we examined the behavior of ACRBP-deficient mouse sperm in the female reproductive tract. When sperm that had migrated into the uterus and oviduct after mating were counted, the number of ACRBP-deficient sperm was noticeably smaller in the oviduct of mice post mating. However, ACRBP-deficient sperm recovered from the oviduct possessed morphologically normal head shape and retained normal motility. Importantly, ACRBP-deficient sperm displayed a marked reduction in the ability to successfully gain access to unfertilized oocytes. These data suggest that male subfertility of ACRBP-deficient mice may be attributed to incompleteness of the acrosome reaction rather than impairment in sperm migration from the uterus to the oviduct.

Recently, the modification of the epigenetic status of somatic cell nuclear transfer (SCNT) embryos by treatment with histone deacetylase inhibitors (HDACis) has made it possible to alter epigenetic traits and improve the developmental competence of these embryos. In the current study, we examined the effects of an HDACi, quisinostat (JNJ), on the in vitro development of porcine cloned embryos and their epigenetic nuclear reprogramming status. SCNT embryos were cultured under various conditions, and we found that treatment with 100 nM JNJ for 24 h post activation could improve blastocyst formation rates compared to the control (P < 0.05). Therefore, this was chosen as the optimal condition and used for further investigations. To explore the effects of JNJ on the nuclear reprogramming of early stage embryos and how it improved cloning efficiency, immunofluorescence staining and quantitative real-time PCR were performed. From the pseudo-pronuclear to 2-cell stages, the levels of acetylation of histone 3 at lysine 9 (AcH3K9) and acetylation of histone 4 at lysine 12 (AcH4K12) increased, and global DNA methylation levels revealed by anti-5-methylcytosine (5-mC) antibody staining were decreased in the JNJ-treated group compared to the control (P < 0.05). However, JNJ treatment failed to alter AcH3K9, AcH4K12, or 5-mC levels at the 4-cell embryo stage. Moreover, JNJ treatment significantly upregulated the expression of the development-related genes OCT4, SOX2, and NANOG, and reduced the expression of genes related to DNA methylation (DNMT1, DNMT3a, and DNMT3b) and histone acetylation (HDAC1, HDAC2, and HDAC3). Together, these results suggest that treatment of SCNT embryos with JNJ could promote their developmental competence by altering epigenetic nuclear reprogramming events.

This study aimed to investigate the effect of resveratrol supplementation in maturation medium on the developmental ability and bioenergetic\oxidative status of prepubertal goat oocytes selected by brilliant cresyl blue (BCB). Oocytes collected from slaughterhouse-derived ovaries were selected by 13 µM BCB staining and classified as grown BCB+ and growing BCB- oocytes. All oocytes were matured in vitro in our conventional maturation medium and supplemented with 1 µM (BCB+R and BCB-R) and without (Control groups: BCB+C and BCB-C) resveratrol. After 24 h, IVM-oocytes were fertilized with fresh semen and presumptive zygotes were in vitro cultured for 8 days. Oocytes were assessed for blastocyst development and quality, mitochondrial activity and distribution, and levels of GSH, ROS, and ATP. BCB+R (28.3%) oocytes matured with resveratrol presented significantly higher blastocyst development than BCB+C (13.0%) and BCB- groups (BCB-R: 8.3% and BCB-C: 4.7%). Resveratrol improved blastocyst development of BCB-R oocytes at the same rate as BCB+C oocytes. No differences were observed in blastocyst quality among groups. GSH levels were significantly higher in resveratrol groups (BCB+R: 36554.6; BCB-R: 34946.7 pixels/oocyte) than in control groups (BCB+C: 27624.0; BCB-C: 27655.4 pixels/oocyte). No differences were found in mitochondrial activity, ROS level, and ATP content among the groups. Resveratrol-treated oocytes had a higher proportion of clustered active mitochondria in both BCB groups (BCB+R: 73.07%; BCB-R: 79.16%) than control groups (BCB+C: 19.35%; BCB-C: 40%). In conclusion, resveratrol increased blastocyst production from oocytes of prepubertal goats, particularly in better quality oocytes (BCB+).

About 10% of male infertile patients show abnormalities in spermatogenesis. The microdeletion of azoospermia factor a (AZFa) region of the Y chromosome is thought to be a cause of spermatogenic failure. However, candidate gene responsible for the spermatogenic failure in AZFa deleted patients has not been elucidated yet. Using mice, we explored the function of Ddx3y, a strong candidate gene in the Azfa region, and Ddx3x, a Ddx3y paralog on the X chromosome, in spermatogenesis. We first generated Ddx3y KO male mice using CRISPR/Cas9 and found that the Ddx3y KO male mice show normal spermatogenesis, produce morphologically normal spermatozoa, and sire healthy offspring. Because Ddx3x KO males were embryonic lethal, we next generated chimeric mice, which contain Ddx3x and Ddx3y double KO (dKO) germ cells, and found that the dKO germ cells can differentiate into spermatozoa and transmit their mutant alleles to offspring by normal mating. We conclude that Ddx3x and Ddx3y are dispensable for spermatogenesis at least in mice. Unlike human, mice have an additional Ddx3y paralog D1pas1, that has been reported to be essential for spermatogenesis. These findings suggest that human and mouse DDX3 related proteins have distinct differences in their functions.

Hindbrain ependymocytes are postulated to have a glucose-sensing role in regulating gonadal functions. Previous studies have suggested that malnutrition-induced suppression of gonadotropin secretion is mediated by noradrenergic inputs from the A2 region in the solitary tract nucleus to the paraventricular nucleus (PVN), and by corticotropin-releasing hormone (CRH) release in the hypothalamus. However, no morphological evidence to indicate the neural pathway from the hindbrain ependymocytes to hypothalamic kisspeptin neurons, a center for reproductive function in mammals, currently exists. The present study aimed to examine the existence of a neuronal pathway from the hindbrain ependymocytes to kisspeptin neurons in the arcuate nucleus (ARC) and anteroventral periventricular nucleus (AVPV). To determine this, wheat-germ agglutinin (WGA), a trans-synaptic tracer, was injected into the fourth ventricle (4V) in heterozygous Kiss1-tandem dimer Tomato (tdTomato) rats, where kisspeptin neurons were visualized by tdTomato fluorescence. 48 h after the WGA injection, brain sections were taken from the forebrain, midbrain and hindbrain and subjected to double immunohistochemistry for WGA and dopamine β-hydroxylase (DBH) or CRH. WGA immunoreactivities were found in vimentin-immunopositive ependymocytes of the 4V and the central canal (CC), but not in the third ventricle. The WGA immunoreactivities were detected in some tdTomato-expressing cells in the ARC and AVPV, DBH-immunopositive cells in the A1–A7 noradrenergic nuclei, and CRH-immunopositive cells in the PVN. These results suggest that the hindbrain ependymocytes have neuronal connections with the kisspeptin neurons, most probably via hindbrain noradrenergic and CRH neurons to relay low energetic signals for regulation of reproduction.

Recently, gradual decline in human sperm production has become a serious worldwide concern because it leads to increased rates of infertility. Endocrine disrupters, lifestyle changes, and varicocele, all of which elevate testicular temperature, are thought to be the main causes of this decline. The present study aimed to determine whether the dietary phytochemicals Angelica keiskei (Ashitaba) powder (57.5 mg/kg) and its functional component, xanthoangelol (3 mg/kg), can prevent heat stress-induced impairment in sperm density and quality in mice. Sperm parameters were analyzed 28 days after mice exposure to heat. Supplementation with Ashitaba powder completely prevented heat-induced impairment in sperm parameters, including densities of motile sperms and progressive sperms (> 25 μm/sec), and amplitude of lateral head displacement. Xanthoangelol did not exert a complete protective effect; nevertheless, it significantly prevented heat stress-induced reduction in most parameters. Both Ashitaba powder and xanthoangelol elevated the expression of the widely expressed heat shock proteins (HSPs) Hspa1a and Hsp40 and the antioxidant enzyme glutathione synthase in non-stressed testes. Ashitaba powder significantly prevented heat stress-induced reduction in the expression of Hspa1l and Hspa2, which are highly expressed in the testes and critical for fertility. Our results showed that Ashitaba powder and xanthoangelol protected testicular cells from heat stress, probably by elevating the levels of antioxidant enzymes and HSPs. Supplementation with dietary functional phytochemicals may help prevent heat stress-induced male infertility.

Previously, we reported that neurotensin (NT), which is expressed in the uterus and oviduct, enhanced bovine sperm capacitation and acrosome reactions. As NT mRNA expression in bovine oviducts increases dramatically in the follicular phase, we hypothesized that NT modulates fertilization and subsequent conception in cattle. The objective of this study was to evaluate the effect of NT on embryo development and blastocyst quality. The rate of embryo cleavage was significantly increased by the addition of NT to the fertilization medium. Furthermore, the total number of cells and numbers of cells in the inner cell mass of blastocysts were significantly increased by NT during in vitro fertilization (IVF). These results suggested that NT enhanced the efficiency of early bovine embryo development and blastocyst quality. The expression of NT receptors (NTRs) in sperm, testes, oocytes, and cumulus cells was evaluated to determine whether NT acted via NTRs in sperm alone or in both male and female reproductive cells during IVF. Immunocytochemistry and reverse transcription polymerase chain reaction revealed that NTR1 and NTR2 were expressed in sperm and testes, but not in oocytes and cumulus cells. We propose that NT selectively acts upon sperm via NTR1 and NTR2 during IVF to improve the cleavage rate and quality of blastocysts, which are important determinants of sperm quality for successful conception. This research supports our hypothesis that NT acts as a key modulator of fertilization and conception in cattle. Further studies are necessary to apply our findings to the industrial framework of bovine reproduction.

The mitochondrial sheath is composed of mitochondria that coil tightly around the midpiece of sperm flagellum. These mitochondria are recruited from the cytoplasm to the flagellum late in spermatogenesis. Initially, recruited mitochondria are spherical-shaped but then elongate laterally to become crescent-like in shape. Subsequently, crescent-like mitochondria elongate continuously to coil tightly around the flagellum. Recently, disorganization of the mitochondrial sheath was reported in Glycerol kinase 2 (Gk2) disrupted mice. To analyze the disorganization of the mitochondrial sheath further, we generated Gk2-deficient mice using the CRISPR/Cas9 system and observed sperm mitochondria in testis using a freeze-fracture method with scanning electron microscopy. Gk2-disrupted spermatids show abnormal localization of crescent-like mitochondria, in spite of the initial proper alignment of spherical mitochondria around the flagellum, which causes abnormal mitochondrial sheath formation leading to exposure of the outer dense fibers. These results indicate that GK2 is essential for proper arrangement of crescent-like mitochondria to form the mitochondrial sheath during mouse spermatogenesis.

Lycium barbarum polysaccharide (LBP) exhibits multiple pharmacological and biological effects, including displaying antioxidant and cytoprotective properties. The current study investigated the effects of LBP-supplemented culture medium on mitochondrial distribution, mitochondrial membrane potential (MMP), adenosine triphosphate (ATP) production, mitochondrial deoxyribonucleic acid (mtDNA) copy number, reactive oxygen species (ROS) accumulation, and development of previously-cryopreserved murine two-cell embryos. Results indicate that LBP enhances development of such embryos, and that potential mechanisms include: (1) mitochondrial function enhancement via altering mitochondrial distribution and increasing MMP, ATP production, mtDNA copy number, and expression of genes involved in mitochondrial biogenesis and energy metabolism (NAD-dependent deacetyltransferase sirtuin-1 (SIRT1) and phosphorylated adenosine monophosphate-activated protein kinase (pAMPK)); (2) down-regulation of ROS generation and enhanced expression of the antioxidant genes glutathione peroxidase 4 (GPX4) and superoxide dismutase 1 (SOD1), thereby increasing embryo oxidative stress tolerance; and (3) increased expression of B-cell lymphoma-2 (BCL2), a critical gene for cell survival and embryo development. These results demonstrate that LBP improves development of previously-cryopreserved murine two-cell embryos via restoration of mitochondrial function and down-regulated generation of ROS.

Follicular fluid contains cell-free DNA (cfDNA), which may serve as a useful biomarker of oocyte ability. The present study evaluates whether nuclear and mitochondrial cfDNAs in conditioned oocyte growth medium determine the quality of oocytes cultured in vitro. Oocyte and granulosa cell complexes (OGCs) derived from early antral follicles of gilt ovaries were cultured for 14 days and the amount of cfDNA and lactate concentration in the conditioned culture medium were measured and compared to evaluate oocyte maturation ability. The amount of nuclear cfDNA, but not mitochondrial cfDNA, strongly correlated with the number of dead cells in OGCs. Furthermore, low mitochondrial cfDNA content and high lactate concentration in the medium was associated with high maturation ability of oocytes cultured in vitro. In conclusion, the amounts of nuclear and mitochondrial cfDNAs differentially reflect the conditions of OGCs, and low mitochondrial cfDNA, low glucose content, and high lactate concentration in the medium are associated with the proper maturation of oocytes.

We examined the effect of the timing of removing cumulus cells surrounding porcine oocytes from small follicles (SFs, < 3 mm in diameter) and medium follicles (MFs; 3–6 mm in diameter) on the meiotic and developmental competence of the oocytes. Cumulus-oocyte complexes (COCs) were collected from SFs and MFs, and the oocytes were denuded at 0, 20, and 44 h after the start of in vitro maturation (IVM), and the meiotic progression of the oocytes was assessed at the end of the IVM period. The incidence of mature oocytes was significantly affected by both the origin of the COCs and the time when the oocytes were denuded. Although the percentage of mature oocytes was always higher when the COCs were collected from MFs than that when the COCs were collected from SFs, the maturation rate was significantly higher when the oocytes were denuded at 20 h than when they were denuded at 44 h after the start of IVM. When the mature oocytes were activated electrically, the developmental competence of the oocytes denuded at 20 and 44 h to reach the blastocyst stage did not differ, whereas the competence of the MF-derived oocytes was significantly higher than that of SF-derived oocytes. When the intracellular cAMP and cGMP levels in SF-derived oocytes were examined at 24 h of IVM, the levels of both were significantly decreased only in the oocytes denuded at 20 h. In conclusion, denuding oocytes at 20 h of IVM caused a significant reduction in ooplasmic cAMP and cGMP levels and increased the meiotic competence of the oocytes without any reduction in blastocyst formation, even in the case of SF-derived oocytes.

We examined whether the use of in vivo-matured oocytes, collected by ovum pick-up (OPU) from superstimulated Japanese Black cows, can improve the productivity and quality of in vitro produced embryos. The cows were superstimulated by treatment with progesterone, GnRH, FSH and prostaglandin F2α according to a standardized protocol. The resulting in vivo-matured oocytes were collected by OPU and used subsequently for the other experiments. The immature oocytes from cows in the non-stimulated group were collected by OPU and then subjected to maturation in vitro. We found that the rate of normally distributed cortical granules of the matured oocyte cytoplasm in the superstimulated group was significantly higher than that in the non-stimulated group. The normal cleavage rate (i.e., production of embryos with two equal blastomeres without fragmentation) and freezable blastocyst rate were significantly higher in the superstimulated group than in the non-stimulated group. Among the transferable blastocysts, the ratio of embryos from normal cleavage was also significantly higher in the superstimulated group than in the non-stimulated group. For in vivo-matured oocytes, it was observed that the pregnancy rates were significantly higher when normally cleaved embryos were used for transfer. Taken together, these results suggest that high-quality embryos with respect to developmental kinetics can be efficiently produced with the use of in vivo-matured oocytes collected by OPU from superstimulated Japanese Black cows.

In a previous study on monovular cows, follicles revealed a mean antral (follicular fluid) temperature 1.54°C cooler than rectal temperatures in ovulating cows, whereas no such temperature differences were detected in non-ovulating cows. The present study adds to our previous work, this time considering 24 bi-ovular cows (one follicle per ovary). In order to increase the number of pre-ovulatory follicles failing to ovulate, this study was performed under heat-stress conditions. Follicular temperatures of the ovulating follicles (n = 31) were 0.93°C significantly cooler (P < 0.0001) than rectal temperatures, whereas no significant differences in temperature were found in non-ovulating follicles (n = 17). Eight cows became pregnant. The results of the present study indicate that, similar to those in monovular cows, pre-ovulatory follicles in bi-ovular cows were cooler than deep rectal temperatures and those temperature gradients were not found in follicles showing ovulation failure.