Embryonic stem (ES) cells, derived from the inner cell mass of a blastocyst, are believed to pluripotent cells and give rise to embryonic, but not extraembryonic, tissues. In mice, totipotent 2-cell stage embryo-like (2-cell-like) cells, which are identified by reactivation of murine endogenous retrovirus with leucin transfer RNA primer (MuERV-L), arise at a very few frequencies in ES cell cultures. Here, we found that a lipid droplet forms during the transition from ES cells to 2-cell-like cells, and we propose that 2-cell-like cells utilize a unique energy storage and production pathway.

The structure of microtubules is essential for the fertilizing ability of spermatozoa. Acetylation of α-tubulin plays an important role in flagellar elongation and spermatozoa motility. Previous reports have suggested that alpha-tubulin N-acetyltransferase 1 (ATAT1) is the main acetyltransferase involved in the acetylation of α-tubulin. Although ATAT1 is reported to express in the testis, no information is available regarding its expression in elongated spermatids, epididymis, and mature spermatozoa. Hence, it remains unclear whether ATAT1 is involved in spermatozoa maturation and capacitation. Therefore, we evaluated the expression of ATAT1 in the mouse male reproductive system using immunostaining and western blotting. Our results showed that ATAT1 was expressed in spermatids during spermiogenesis in mouse testes, but its expression varied according to the seminiferous tubule stage. We observed ATAT1 in the cytoplasm of round spermatids, the flagella of elongated spermatids, and in the cytoplasm of step 16 spermatids, just before its release into the lumen. In addition, ATAT1 was expressed in epithelial cells of the epididymis. In spermatozoa of the cauda epididymis, ATAT1 expression was primarily observed in the midpiece of the spermatozoa. The localization of ATAT1 protein in the male germline was observed during spermiogenesis as well as during spermatozoa maturation. Our results suggest that ATAT1 may be involved in the formation of flagella and in the acetylation process, which has attracted attention in recent years regarding male infertility.

Cluster of differentiation (CD) 9 and CD81 are closely-related members of the tetraspanin family that consist of four-transmembrane domain proteins. Cd9 and Cd81 are highly expressed in breast cancer cells; however, their expression in healthy mammary glands is unclear. In this study, we performed quantitative real-time PCR to analyze the expression levels of Cd9 and Cd81. Histological techniques were employed to identify Cd9- and Cd81-expressing cells in rat mammary glands during pregnancy and lactation. It was observed that Cd9 and Cd81 were expressed in the mammary glands, and their expression levels correlated with mammary gland development. To identify cells expressing Cd9 and Cd81 in the mammary glands, we performed double immunohistochemical staining for CD9 and CD81, prolactin receptor long form, estrogen receptor alpha, or Ki67. The results showed that CD9 and CD81 were co-expressed in proliferating mammary epithelial cells. Next, we attempted to isolate CD9-positive epithelial cells from the mammary gland using pluriBead cell-separation technology based on antibody-mediated binding of cells to beads of different sizes, followed by isolation using sieves with different mesh sizes. We successfully isolated CD9-positive epithelial cells with 96.8% purity. In addition, we observed that small-interfering RNAs against Cd9 and Cd81 inhibited estrogen-induced proliferation of CD9-positive mammary epithelial cells. Our current findings may provide novel insights into the proliferation of mammary epithelial cells during pregnancy and lactation as well as in pathological processes associated with breast cancer.

Maintaining genomic integrity in mammalian early embryos, which are deficient in DNA damage repair, is critical for normal preimplantation and subsequent development. Abnormalities in DNA damage repair in preimplantation embryos can cause not only developmental arrest, but also diseases such as congenital disorders and cancers. Histone H4 lysine 20 monomethylation (H4K20me1) is involved in DNA damage repair and regulation of gene expression. However, little is known about the role of H4K20me1 during mouse preimplantation development. In this study, we revealed that H4K20me1 mediated by SETD8 is involved in maintaining genomic integrity. H4K20me1 was present throughout preimplantation development. In addition, reduction in the level of H4K20me1 by inhibition of SETD8 activity or a dominant-negative mutant of histone H4 resulted in developmental arrest at the S/G2 phase and excessive accumulation of DNA double-strand breaks. Together, our results suggest that H4K20me1, a type of epigenetic modification, is associated with the maintenance of genomic integrity and is essential for preimplantation development. A better understanding of the mechanisms involved in maintaining genome integrity during preimplantation development could contribute to advances in reproductive medicine and technology.

The spermatogonial stem cell (SSC) population in testis is small, and the lack of SSC markers has severely handicapped research on these cells. During our attempt to identify genes involved in SSC aging, we found that CD2 is expressed in cultured SSCs. Flow cytometric analysis and spermatogonial transplantation experiments showed that CD2 is expressed in SSCs from mature adult mouse testes. Cultured SSCs transfected with short hairpin RNAs (shRNAs) against CD2 proliferated poorly and showed an increased frequency of apoptosis. Moreover, functional analysis of transfected cells revealed impairment of SSC activity. Fluorescence activated cell sorting and spermatogonial transplantation experiments showed that CD2 is expressed not only in mouse but also in rat SSCs. The results indicate that CD2 is a novel SSC surface marker conserved between mouse and rat SSCs.

We determined the length of time within which frozen-thawed semen delivered a high conception rate in present-day lactating dairy cows. The cows utilized were a total 100 milking Holstein-Friesian cows kept in tie-stall style farms. We carried out artificial insemination (AI) during the periovulatory period at a predicted time based on ovulation, and checked ovulation at 6-h intervals after AI. The period from AI to ovulation ranged from 48 h (i.e., 48 h before ovulation) to -12 h (i.e., 12 h after ovulation). High conception rates averaging 63.0% were obtained by carrying out AI > 6–30 h before ovulation, significantly higher than the conception rates of 30.0% (P < 0.05) and 26.9% (P < 0.01) from AI carried out earlier than 30 h before ovulation and later than 6 h before ovulation, respectively. It was concluded that frozen-thawed semen delivers a conception rate of ≥ 60% for > 24–30 h after AI, and that a conception rate of ≥ 60% can be achieved by carrying out AI 6–30 h before ovulation using frozen-thawed semen.

The controlled activation of dormant primordial follicles is important for the maintenance of periodic ovulation. Previous reports have clearly identified the signaling pathway in granulosa cells and oocytes that controls the activation of primordial follicles; however, the exact cue for the in vivo activation of dormant primordial follicles is yet to be elucidated. In this study, we found that almost all activated primordial follicles made contact with blood vessels. Based on this result, we speculated that the contact between primordial follicles and blood vessels may provide a cue for the activation of dormant primordial follicles. To confirm this hypothesis, we attempted to activate dormant primordial follicles within the ovaries by inducing angiogenesis through the use of biodegradable gels containing recombinant vascular endothelial growth factor and in cultured ovarian tissues by increasing the serum concentration within the culture medium. The activation of dormant primordial follicles was promoted in both experiments, and our results indicated that an increase in the supply of the serum component, from new blood vessels formed via angiogenesis, to the dormant primordial follicles is the cue for their in vivo activation. In the ovaries, angiogenesis often occurs during every estrous cycle, and it is therefore likely that angiogenesis is the crucial event that influences the activation of primordial follicles.

Improving artificial oocyte activation is essential for assisted reproduction or animal biotechnology that can obtain healthy offspring with a high success rate. Here, we examined whether intracytoplasmic injection of equine sperm-specific phospholipase C zeta (ePLCζ) mRNA, the PLCζ with the strongest oocyte activation potential in mammals, could improve the mouse oocyte activation rate and subsequent embryonic development using inactivated spermatozoa. mRNA of mouse PLCζ (mPLCζ) or ePLCζ were injected into mouse oocytes to determine the optimal mRNA concentration to maximize the oocyte activation rate and developmental rate of parthenogenetic embryos in vitro. Full-term development was examined using NaOH-treated inactive spermatozoa using the optimal activation method. We found that the most optimal ePLCζ mRNA concentration was 0.1 ng/µl for mouse oocyte activation, which was ten times stronger than mPLCζ mRNA. The concentration did not affect parthenogenetic embryo development in vitro. Relatively normal blastocysts were obtained with the same developmental rate (52–53% or 48–51%, respectively) when inactive spermatozoa were injected into activated oocytes using ePLCζ or mPLCζ mRNA injection. However, the birth rate after embryo transfer was slightly but significantly decreased in oocytes activated by ePLCζ mRNA (24%) compared to mPLCζ mRNA (37%) or strontium treatment (40%) activation. These results suggest that the higher activation rate does not always correlate the higher birth rate, and some mechanisms might exist in the oocyte activation process that could affect the later developmental stages like full-term development.

Animal cloning technology has been developed to produce progenies genetically identical to a given donor cell. However, in nuclear transfer protocols, the recipient oocytes contribute a heritable mitochondrial genomic (mtDNA) background to the progeny. Additionally, a small amount of donor cell-derived mitochondria accompanies the transferred nucleus in the process; hence, the mtDNAs of two origins are mixed in the cytoplasm (heteroplasmy) of the reconstituted oocyte. Herein, I would like to introduce some of our previous results concerning five key considerations associated with animal cloning, including: mtDNA heteroplasmy in somatic cell nuclear transferred (SCNT) animals, the variation in the transmission of mtDNA heteroplasmy to subsequent generations SCNT cows and pigs, the influence of mtDNA sequence differences on mitochondrial proteins in SCNT cows and pigs, the effects of the introduction of mitochondria derived from somatic cells into recipient oocytes on embryonic development, and alterations of mtDNA heteroplasmy in inter/intraspecies nuclear transfer embryos.

Differentiated nuclei can be reprogrammed/remodelled to totipotency after their transfer to enucleated metaphase II (MII) oocytes. The process of reprogramming/remodelling is, however, only partially characterized. It has been shown that the oocyte nucleus (germinal vesicle – GV) components are essential for a successful remodelling of the transferred nucleus by providing the materials for pseudo-nucleus formation. However, the nucleus is a complex structure and exactly what nuclear components are required for a successful nucleus remodelling and reprogramming is unknown. Till date, the only nuclear sub-structure experimentally demonstrated to be essential is the oocyte nucleolus (nucleolus-like body, NLB). In this study, we investigated what other GV components might be necessary for the formation of normal-sized pseudo-pronuclei (PNs). Our results showed that the removal of the GV nuclear envelope with attached chromatin and chromatin-bound factors does not substantially influence the size of the remodelled nuclei in reconstructed cells and that their nuclear envelopes seem to have normal parameters. Rather than the insoluble nuclear lamina, the GV content, which is dissolved in the cytoplasm with the onset of oocyte maturation, influences the characteristics and size of transferred nuclei.

Eggs are female germ cells that are required for producing offspring through sexual reproduction. In mammals, eggs are produced in the ovary and ovulated into the oviduct. It is well known that over 99% of eggs are degenerated without ovulation, so that many studies have attempted in vitro folliculogenesis to produce many eggs in different species for a few decades. Although many methods have been developed, a success of in vitro egg production with the resultant live birth of offspring has been limited, especially in livestock animals. More recently, we have succeeded in producing live pups derived from in vitro/ex vivo egg production in mice. This review aims to introduce our recent findings with a brief history of in vitro/ex vivo culture systems for follicles and ovaries.

Recently, the demand of transferable embryos in cattle industry is increasing, and the number of embryos produced in vitro is also increasing in the world. Although oocytes are collected from individual elite cattle by ovum-pick up (OPU) and used for in vitro production (IVP) of embryos, the cattle are mono-ovulatory animal. It means that most of oocytes collected from ovaries are destined to degenerate. To improve the IVP efficiency, we should predict the developmental competence of oocytes correctly and culture them by the suitable way. In addition, in vitro production of bovine oocytes by in vitro growth (IVG) culture system will become a candidate of supply source of oocytes for IVP. If we can produce high competent oocytes by IVG, IVP efficiency will be improved and the genetic improvement of cattle will be dramatically accelerated. In the review, I introduce our researches related to oocyte morphology, the developmental competence, and the production of oocytes having high developmental competence by IVG culture.

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.

Mammalian oocyte quality degrades over time after ovulation in vitro, which can cause fatal defects such as chromosomal aneuploidy. As various oocyte manipulations employed in assisted reproductive technology are time consuming, post-ovulatory aging is a serious problem to overcome in reproductive medicine or ova research. In this study, we investigated the effects of postovulatory aging on the incidence of chromosome aneuploidy during meiosis II, with a focus on the expression of functional proteins from the spindle assembly checkpoint (SAC). Chromosome analysis was used to assess the rate of aneuploidy in in vitro aged oocytes, or in early embryos derived from aged oocytes. Immunofluorescent staining was used to detect the localization of MAD2, which is a SAC signal that monitors the correct segregation of sister chromatids. Immunoblotting was used to quantify cohesin subunits, which are adhesion factors connecting sister chromatids at the metaphase II (MII) centromere. It was shown that post-ovulatory oocyte aging inhibits MAD2 localization to the sister kinetochore. Furthermore, oocyte aging prevented cohesin subunits from being maintained or degraded at the appropriate time. These data suggest that the destabilization of SAC signaling causes sister chromatid segregation errors in MII oocytes, and consequently increases the incidence of aneuploidy in early embryos. Our findings have provided distinct evidence that the post-ovulatory aging of oocytes might also be a risk factor for aneuploidy, irrespective of maternal age.

Kisspeptin, identified as a natural ligand of GPR54 in 2001, is now considered as a master regulator of puberty and subsequent reproductive functions in mammals. Our previous studies using Kiss1 knockout (KO) rats clearly demonstrated the indispensable role of kisspeptin in gonadotropin-releasing hormone (GnRH)/gonadotropin secretion. In addition, behavioral analyses of Kiss1 KO rats revealed an organizational effect of kisspeptin on neural circuits controlling sexual behaviors. Our studies using transgenic mice carrying a region-specific Kiss1 enhancer-driven reporter gene provided a clue as to the mechanism by which estrogen regulates Kiss1 expression in hypothalamic kisspeptin neurons. Analyses of Kiss1 expression and gonadotropin secretion during the pubertal transition shed light on the mechanism triggering GnRH/gonadotropin secretion at the onset of puberty in rats. Here, we summarize data obtained from the aforementioned studies and revisit the physiological roles of kisspeptin in the mechanism underlying reproductive functions in mammals.

Sperm freeze-drying is a revolutionary technique, which has been gaining prominence in recent years. The first related significant result was Wakayama and Yanagimachi’s demonstration in 1998 of the birth of healthy mouse offspring by Intracytoplasmic Sperm Injection (ICSI), using epididymal freeze-dried spermatozoa. Mouse, rat, and hamster models were the first small mammals born from lyophilized epididymal spermatozoa, whereas most other studies in this field used ejaculated spermatozoa. In this work, we applied this technique to ram epididymal spermatozoa, checking the correlation between DNA integrity and embryo development following ICSI. To do this, epididymal sperm from four rams was lyophilized in a trehalose, glucose, KCl, HEPES, and Trolox media. To evaluate DNA damage and fragmentation after rehydration, samples were processed for Sperm Chromatin Dispersion test (SCD), Two-Tailed Comet Assay, and were used for ICSI. Ram #2 had a higher rate of spermatozoa with intact DNA compared with rams #1, #3, and #4 (28% vs. 3.8%, 2.8%, and 5%, respectively) and the lowest rate of Single-Strand Breaks (SSBs) (70% vs. 95.9%, 92.6%, and 93% respectively). Ram #3 had a higher level of Double-Strand Breaks (DSBs) compared to Ram #1 (4.6% vs. 0.33%, respectively). Embryo development to the blastocyst stage following ICSI was only reached from rams whose sperm had higher level of intact DNA – Rams #2 and #4 (6%, 5/147 and 6.3%, 4/64, respectively). Definitively, the impact of sperm DNA damage on embryonic development depends on the balance between sperm DNA fragmentation extent, fragmentation type (SSBs or DSBs), and the oocyte’s repair capacity.

Oog1, an oocyte-specific gene that encodes a protein of 425 amino acids, is present in five copies on mouse chromosomes 4 and 12. In mouse oocytes, Oog1 mRNA expression begins at embryonic day 15.5 and almost disappears by the late two-cell stage. Meanwhile, OOG1 protein is detectable in oocytes in ovarian cysts and disappears by the four-cell stage; the protein is transported to the nucleus in late one-cell to early two-cell stage embryos. In this study, we examined the role of Oog1 during oogenesis in mice. Oog1 RNAi-transgenic mice were generated by expressing double-stranded hairpin Oog1 RNA, which is processed into siRNAs targeting Oog1 mRNA. Quantitative RT-PCR revealed that the amount of Oog1 mRNA was dramatically reduced in oocytes obtained from Oog1-knockdown mice, whereas the abundance of spermatogenesis-associated transcripts (Klhl10, Tekt2, Tdrd6, and Tnp2) was increased in Oog1 knockdown ovaries. Tdrd6 is involved in the formation of the chromatoid body, Tnp2 contributes to the formation of sperm heads, Tekt2 is required for the formation of ciliary and flagellar microtubules, and Klhl10 plays a key role in the elongated sperm differentiation. These results indicate that Oog1 down-regulates the expression of spermatogenesis-associated genes in female germ cells, allowing them to develop normally into oocytes.

This study was conducted to investigate whether the treatment of dog to pig interspecies somatic cell nuclear transfer (iSCNT) embryos with a histone deacetylase inhibitor, to improve nuclear reprogramming, can be applied to dog SCNT embryos. The dog to pig iSCNT embryos were cultured in fresh porcine zygote medium-5 (PZM-5) with 0, 1, or 10 µM suberoylanilide hydroxamic acid (SAHA) for 6 h, then transferred to PZM-5 without SAHA. Although there were no significant differences in cleavage rates, the rates of 5-8-cell stage embryo development were significantly higher in the 10 µM group (19.5 ± 0.8%) compared to the 0 µM groups (13.4 ± 0.8%). Acetylation of H3K9 was also significantly higher in embryos beyond the 4-cell stage in the 10 µM group compared to the 0 or 1 µM groups. Treatment with 10 µM SAHA for 6 h was chosen for application to dog SCNT. Dog cloned embryos with 0 or 10 µM SAHA were transferred to recipients. However, there were no significant differences in pregnancy and delivery rates between the two groups. Therefore, it can be concluded that although porcine oocytes support nuclear reprogramming of dog fibroblasts, treatment with a histone deacetylase inhibitor that supports nuclear reprogramming in dog to pig iSCNT embryos was not sufficient for reprogramming in dog SCNT embryos.

Macromolecular crowded culture medium formed by addition of polyvinylpyrrolidone (PVP; molecular weight = 360 000), positively influences the viability, growth, and development of bovine oocytes. Owing to its apparently various effects, uncovering the specific mechanisms of crowding responsible for these outcomes is important. The present study was conducted to determine the effects of crowding on oocytes with a particular focus on the intimacy of contacts between oocyte and cumulus/granulosa cells. Growing mouse oocyte-granulosa cell complexes were cultured for 10 days in a modified α-minimum essential medium, supplemented with PVP at a concentration of 0%, 1%, 2%, or 3% (w/v). Although the complexes developed in all groups, 2% and 3% PVP medium induced a substantial morphological modification, and a larger proportion of oocytes associated with cumulus cells survived in 3% PVP medium than in the 0% or 1% PVP medium. No significant difference was found in the frequencies of polar body extrusion (78–88%) and blastocyst formation (approximately 40%) after in vitro fertilization among the experimental groups. Confocal laser scanning microscopy indicated a higher number of transzonal processes reaching the oocyte from cumulus cells in 2% PVP medium than in 0% PVP medium. Transmission electron microscopy depicted close adhesion of the oocyte with cumulus cells in 2% PVP medium —bearing a resemblance to their in vivo counterparts— and loose adhesion in 0% PVP medium. In conclusion, we found that a mechanism for the action of crowded conditions involves the strengthening of contacts and communication between oocytes and companion cumulus/granulosa cells.

This study was aimed at evaluating the effects of multi-layered cumulus cells (MCCs) during vitrification and in vitro fertilization (IVF) of mature bovine oocytes and embryogenesis after IVF. The rates of cleavage and blastocyst formation were higher in vitrified and fertilized oocytes with MCCs than in denuded oocytes (P < 0.05), but were comparable to the rates in fresh oocytes with MCCs or without (denuded). When the MCC-enclosed oocytes were denuded before IVF, blastocyst formation rate reduced compared with that in vitrified oocytes with MCCs (P < 0.05). This suggested that the MCCs surrounding the mature bovine oocytes play important roles during cryopreservation: protecting them against freezing and promoting their survival and development post IVF, thereby increasing the success rates of IVF and embryonic development. Herein, we showed for the first time that calves could be produced using only 14–19 vitrified mature oocytes with MCCs from the ovaries of individual cows post slaughter.