Journal of Reproduction and Development Advance Publications

Previous studies have shown that grafted neonatal chicken testicular tissue can develop and produce functional sperm; however, it was unclear whether regenerative processes or proportional growth caused the re-appearance of spermatogenic tissue. We dissociated testicular tissues, performed subcutaneous auto-transplantation of the re-aggregated cells to castrated cockerels, and monitored the post-surgery development of these transplanted aggregates. We found that these transplanted cell aggregates experienced compensatory growth in the form of a 300-fold increase in size, rather than the 30-fold increase observed in normal testis development. Further, these dissociated testicular cell aggregates restored seminiferous tubule structure and were able to produce testosterone and motile sperm. Therefore, we concluded that the dissociated testicular cells from 11-week-old cockerels retained a strong regenerative potential, as they exhibited compensatory growth, restored destroyed structure, and sustained spermatogenesis.

In mammals, germ cells originate outside of the developing gonads and follow a unique migration pattern through the embryonic tissue toward the genital ridges. Many studies have attempted to identify critical receptors and factors involved in germ cell migration. However, relatively few reports exist on germ cell receptors and chemokines that are involved in germ cell migration in avian species. In the present study, we investigated the specific migratory function of C-X-C chemokine receptor type 4 (CXCR4) in chicken primordial germ cells (PGCs). We induced loss-of-function via a frameshift mutation in the CXCR4 gene in chicken PGCs using clustered regularly interspaced short palindromic repeat-CRISPR-associated protein 9 (CRISPR/Cas9) genome editing. The migratory capacity of CXCR4 knockout PGCs was significantly reduced in vivo after transplantation into recipient embryos. However, CXCR4-expressing somatic cell lines, such as chicken DT40 and DF1, failed to migrate into the developing gonads, suggesting that another key factor(s) is necessary for targeting and settlement of PGCs into the genital ridges. In conclusion, we show that CXCR4 plays a critical role in the migration of chicken germ cells.

Intrauterine growth retardation (IUGR) impairs fetal intestinal development, and is associated with high perinatal morbidity and mortality. However, the mechanism underlying this intestinal injury is largely unknown. We aimed to investigate this mechanism through analysis of intestinal autophagy and related signaling pathways in a rat model of IUGR. Normal weight (NW) and IUGR fetuses were obtained from primiparous rats via ad libitum food intake and 50% food restriction, respectively. Maternal serum parameters, fetal body weight, organ weights, and fetal blood glucose were determined. Intestinal apoptosis, autophagy, and the mechanistic target of rapamycin (mTOR) signaling pathway were analyzed. The results indicated that maternal 50% food restriction reduced maternal serum glucose, bilirubin, and total cholesterol and produced IUGR fetuses, which had decreased body weight; blood glucose; and weights of the small intestine, stomach, spleen, pancreas, and kidney. Decreased Bcl-2 and increased Casp9 mRNA expression was observed in IUGR fetal intestines. Analysis of intestinal autophagy showed that the mRNA expression of WIPI1, MAP1LC3B, Atg5, and Atg14 was also increased, while the protein levels of p62 were decreased in IUGR fetuses. Compared to NW fetuses, IUGR fetuses showed decreased mTOR protein levels and enhanced mRNA expression of ULK1 and Beclin1 in the small intestine. In summary, the results indicated that maternal 50% food restriction on gestational days 10–21 reduced maternal serum glucose, bilirubin, and total cholesterol contents, and produced IUGR fetuses that had low blood glucose and reduced small intestine weight. Intestinal injury of IUGR fetuses caused by maternal food restriction might be due to enhanced apoptosis and autophagy via the mTOR signaling pathway.

Transcriptional activity is repressed due to the packaging of sperm chromatins during spermiogenesis. The detection of numerous transcripts in sperm, however, raises the question whether transcriptional events exist in sperm, which has been the central focus of the recent studies. To summarize the transcriptional activity during spermiogenesis and in sperm, we reviewed the documents on transcript differences during spermiogenesis, in sperm with differential motility, before and after capacitation and cryopreservation. This will lay a theoretical foundation for studying the mechanism(s) of gene expression in sperm, and would be invaluable in making better use of animal sires and developing reproductive control technologies.

In embryo transfer experiments in mice, pseudopregnant females as recipients are prepared by sterile mating with vasectomized males. Because only females at the proestrus stage accept males, such females are selected from a stock of animals based on the appearance of their external genital tract. Therefore, the efficiency of preparing pseudopregnant females largely depends on the size of female colonies and the skill of the operators who select females for sterile mating. In this study, we examined whether the efficiency of preparing pseudopregnant females could be improved by applying an estrous cycle synchronization method by progesterone (P4) pretreatment, which significantly enhances the superovulation outcome in mice. We confirmed that after two daily injections of P4 (designated Days 1 and 2) in randomly selected females, the estrous cycles of most females (about 85%) were synchronized at metestrus on Day 3. When P4-treated females were paired with vasectomized males for 4 days (Days 4–8), a vaginal plug was found in 63% (20/32) of the females on Day 7. After the transfer of vitrified-warmed embryos into their oviducts, 52% (73/140) of the embryos successfully developed into offspring, the rate being comparable to that of the conventional embryo transfer procedure. Similarly, 77% (24/31) of females became pregnant by fertile mating with intact males for 3 days, which allowed the scheduled preparation of foster mothers. Thus, our estrous cycle synchronization method may omit the conventional experience-based process of visually observing the vagina to choose females for embryo transfer. Furthermore, it is expected that the size of female stocks for recipients can be reduced to less than 20%, which could be a great advantage for facilities/laboratories undertaking mouse-assisted reproductive technology.

The present study was conducted to establish haploid embryonic stem (ES) cell lines using fluorescent marker-carrying rats. In the first series, 7 ES cell lines were established from 26 androgenetic haploid blastocysts. However, only 1 ES cell line (ahES-2) was found to contain haploid cells (1n = 20 + X) by fluorescence-activated cell sorting (FACS) and karyotypic analyses. No chimeras were detected among the 10 fetuses and 41 offspring derived from blastocyst injection with the FACS-purified haploid cells. In the second series, 2 ES cell lines containing haploid cells (13% in phES-1 and 1% in phES-2) were established from 2 parthenogenetic haploid blastocysts. Only the phES-2 cell population was purified by repeated FACS to obtain 33% haploid cells. Following blastocyst injection with the FACS-purified haploid cells, no chimera was observed among the 11 fetuses; however, 1 chimeric male was found among the 47 offspring. Although haploid rat ES cell lines can be established from both blastocyst sources, FACS purification may be necessary for maintenance and chimera production.

Oocyte cryopreservation is the technique of choice for the long-term storage of female gametes. However, it induces an irreversible loss of oocyte viability and function. We examined the effects of vitrification and a Rho-associated coiled-coil containing protein kinase 1 (ROCK1) inhibitor (ROCKi) on the meiotic and developmental competence of feline oocytes. We examined the expression of LIM kinase (LIMK) 1 and 2, with and without ROCKi treatment. Cumulus oocyte complexes (COCs) were matured in vitro with 0, 10, 20, and 40 µM ROCKi. The oocytes were subsequently assessed for maturation rate and embryo development following in vitro fertilization. We repeated the COC experiment, but vitrified and warmed the COCs prior to culture. We detected LIMK1 and LIMK2 expression in feline oocytes, which could be downregulated by ROCKi treatment. The ROCKi at 10 µM affected neither meiotic nor developmental competence (P > 0.05, versus control). However, high concentrations of ROCKi during maturation induced meiotic arrest at metaphase I. Appropriate concentrations of ROCKi significantly improved the normal fertilization rate of vitrifiedwarmed oocytes (49.4 ± 3.4%) compared with that of the control (42.8 ± 8.6%, P < 0.05). The ROCKi also significantly improved the embryo cleavage rate (36.1 ± 3.8%) as compared with the non-treated control (27.4 ± 2.5%, P < 0.05). Thus, this study revealed that the main mediators of the ROCK cascade (LIM kinases) are expressed in feline oocytes. The ROCKi (10 µM) did not compromise the meiotic or developmental competence of feline oocytes. In addition, 10 µM ROCKi improved the cytoplasmic maturation of vitrified–warmed oocytes as indicated by their fertilization competence.

Recent studies demonstrated that G-protein-coupled receptor 30 (GPR30) on the plasma membrane of gonadotroph cells mediates picomolar, but not nanomolar, levels of estradiol (E2) to rapidly suppress gonadotropin-releasing hormone (GnRH)-induced luteinizing hormone (LH) secretion in the anterior pituitary (AP). While estrone (E1) and estriol (E3) are considered “weak” estrogens that exert suppressive effects through estrogen receptors α and β, it is conceivable that they also strongly suppress GnRH-induced LH secretion via GPR30. Both E1 and E3 are likely present within the blood at picomolar or nanomolar concentrations, indicating that such concentrations are sufficient to suppress GnRH-induced LH secretion. To evaluate this possibility, bovine AP cells were cultured under steroid-free conditions and then incubated with various concentrations (0.01 pM to 10 nM) of E2, E1, or E3, prior to stimulation with GnRH. Notably, GnRH-induced LH secretion from AP cells was inhibited by 1–100 pM E2, 1–10 pM E1, and 1–100 pM E3. GnRH-induced LH secretion from AP cells was not inhibited by lower (0.01–0.1 pM) or higher (1–10 nM) concentrations of E2, E1, and E3. These suppressive effects were inhibited by pre-treatment of AP cells with the GPR30 antagonist G36, but not with the estrogen receptor alpha antagonist. Treatment with E1 or E3 also yielded decreased cytoplasmic cAMP levels in cultured AP cells pre-treated with dopamine and phosphodiesterase inhibitors. Therefore, these results suggest that GPR30 mediates the suppressive effects of E1, E3, and E2 on GnRH-induced LH secretion from bovine AP.

The mammalian oocyte undergoes an asymmetric division during meiotic maturation, producing a small polar body and a haploid gamete. This process involves the dynamics of actin filaments, and the guanosine triphosphatase (GTPase) protein superfamily is a major regulator of actin assembly. In the present study, the small GTPase CDC42 was shown to participate in the meiotic maturation of porcine oocytes. Immunofluorescent staining showed that CDC42 was mainly localized at the periphery of the oocytes, and accumulated with microtubules. Deactivation of CDC42 protein activity with the effective inhibitor ML141 caused a decrease in actin distribution in the cortex, which resulted in a failure of polar body extrusion. Moreover, western blot analysis revealed that besides the Cdc42-N-WASP pathway previously reported in mouse oocytes, the expression of ROCK and p-cofilin, two molecules involved in actin dynamics, was also decreased after CDC42 inhibition during porcine oocyte maturation. Thus, our study demonstrates that CDC42 is an indispensable protein during porcine oocyte meiosis, and CDC42 may interact with N-WASP, ROCK, and cofilin in the assembly of actin filaments during porcine oocyte maturation.

Preimplantation genomic selection based on single nucleotide polymorphism (SNP) genotypes is expected to accelerate genetic improvement in cattle. However, genome-wide genotyping at the early embryonic stage has several limitations, such as the technical difficulty of embryonic biopsy and low accuracy of genotyping resulting from a limited number of biopsied cells. After hatching from the zona pellucida, the morphology of the bovine embryo changes from spherical to filamentous, in a process known as elongation. The bovine nonsurgical elongating conceptus transfer technique was recently developed and applied for sexing without requiring specialized skills for biopsy. In order to develop a bovine preimplantation genomic selection system combined with the elongating conceptus transfer technique, we examined the accuracy of genotyping by SNP chip analysis using the DNA from elongating conceptuses (Experiment 1) and optimal cryopreservation methods for elongating conceptuses (Experiment 2). In Experiment 1, the call rates of SNP chip analysis following whole genome amplification in biopsied cells from two elongating conceptuses were 95.14% and 99.32%, which were sufficient for estimating genomic breeding value. In Experiment 2, the rates of dead cells in elongating conceptuses cryopreserved by slow freezing were comparable to those in fresh elongating conceptuses. In addition, we obtained healthy calves by the transfer of elongating conceptuses cryopreserved by slow freezing. Our findings indicate that the elongating conceptus transfer technology enables preimplantation genomic selection in cattle based on SNP chip analysis. Further studies on the optimization of cryopreservation methods for elongating conceptuses are required for practical application of the selection system.

DNA repair protein RAD51 homolog 1 (RAD51) plays a central role in homologous recombination (HR) repair of DNA breaks. HR depends on the formation of a RAD51 recombinase filament that facilitates strand invasion. However, the role of RAD51 during porcine oocyte maturation is unknown. The objective of this study was to investigate the expression and function of RAD51 during porcine oocyte maturation in vitro. RAD51 was mainly localized to the nucleus at the germinal vesicle (GV) stage, and was widely distributed in the cytoplasm between the GV breakdown (GVBD) and metaphase II stage. DNA damage induced by etoposide was accompanied by the formation of RAD51 foci that were colocalized with γH2AX. Inhibition of RAD51 increased DNA damage and induced metaphase I arrest along with spindle defects, chromosomal misalignment, and abnormal spindle assembly checkpoint (SAC) activity. Inhibition of RAD51 also increased ROS levels and led to an abnormal mitochondrial distribution. Our results indicate that RAD51 plays a critical role in maintaining chromosome integrity and mitochondrial activity during porcine oocyte maturation.

Sperm sorting by flow cytometry is a useful technology in the bovine industry, but the conception rates after artificial insemination using sex-sorted sperm are lower than when using the un-sorted sperm. In this study, we have investigated the causes for these low conception rates. We have focused on changes caused by flow cytometry to the glycocalyx, which forms the outermost surface of the sperm membrane. We have also evaluated the effects of capacitation on the glycocalyx since capacitation involves a redistribution of the sperm membrane that is vital for successful fertilization and conception. Lectin histochemistry was used to visualize the structure of the sperm glycocalyx. Lectin-staining sites were examined in non-treated sperm, sex-sorted sperm, and capacitated sperm. We have detected six different staining patterns related to different labeling regions of the sperm. Phaseolus vulgaris–erythroagglutinin (PHA-E) lectin-staining patterns of non-treated sperm were very different from those observed for sex-sorted sperm or capacitated sperm, suggesting that both, sex sorting by flow cytometry and the capacitation process affected the glycocalyx structures in the sperm. In addition, the total tyrosine-phosphorylation level in sex-sorted sperm was significantly higher than that in the non-treated sperm. Therefore, we concluded that the unexpected capacitation of bovine sperm during flow cytometry is associated with changes in the glycocalyx. Since premature capacitation leads to low conception rates, this unexpected capacitation could be a cause of low conception rates after artificial insemination using sex-sorted sperm.

Geminin plays a critical role in cell cycle regulation by regulating DNA replication and serves as a transcriptional molecular switch that directs cell fate decisions. Spermatogonia lacking Geminin disappear during the initial wave of mitotic proliferation, while geminin is not required for meiotic progression of spermatocytes. It is unclear whether geminin plays a role in pre-meiotic DNA replication in later-stage spermatogonia and their subsequent differentiation. Here, we selectively disrupted Geminin in the male germline using the Stra8-Cre/loxP conditional knockout system. Geminin-deficient mice showed atrophic testes and infertility, concomitant with impaired spermatogenesis and reduced sperm motility. The number of undifferentiated spermatogonia and spermatocytes was significantly reduced; the pachytene stage was impaired most severely. Expression of cell proliferation-associated genes was reduced in Gmnn^fl/Δ; Stra8-Cre testes compared to in controls. Increased DNA damage, decreased Cdt1, and increased phosphorylation of Chk1/Chk2 were observed in Geminin-deficient germ cells. These results suggest that geminin plays important roles in pre-meiotic DNA replication and subsequent spermatogenesis.

Thyroid hormones (THs) are essential for the correct development of nearly every structure in the body from the very early stages of development, yet the embryonic thyroid gland is not functional at these stages. To clarify the roles of the egg yolk as a source of THs, the TH content in the yolk and the expression of TH regulator genes in the yolk sac membrane were evaluated throughout the 21-day incubation period of chicken embryos. The yolk TH content (22.3 ng triiodothyronine and 654.7 ng thyroxine per total yolk on day 4 of incubation) decreased almost linearly along with development. Real-time PCR revealed gene expression of transthyretin, a principal TH distributor in the chicken, and of a TH-inactivating iodothyronine deiodinase (DIO3), until the second week of incubation when the embryonic pituitary-thyroid axis is generally thought to start functioning. The TH-activating deiodinase (DIO2) and transmembrane transporter of thyroxine (SLCO1C1) genes were expressed in the last week of incubation, which coincided with a marked increase of circulating thyroxine and a reduction in the yolk sac weight. DIO1, which can remove iodine from inactive THs, was expressed throughout the incubation period. It is assumed that the chicken yolk sac inactivates THs contained abundantly in the yolk and supplies the hormones to the developing embryo in appropriate concentrations until the second week of incubation, while THs may be activated in the yolk sac membrane in the last week of incubation. Additionally, the yolk sac could serve as a source of iodine for the embryo.

The success of implantation is an interactive process between the blastocyst and the uterus. Synchronized development of embryos with uterine differentiation to a receptive state is necessary to complete pregnancy. The period of uterine receptivity for implantation is limited and referred to as the “implantation window”, which is regulated by ovarian steroid hormones. Implantation process is complicated due to the many signaling molecules in the hierarchical mechanisms with the embryo–uterine dialogue. The mouse is widely used in animal research, and is uniquely suited for reproductive studies, i.e., having a large litter size and brief estrous cycles. This review first describes why the mouse is the preferred model for implantation studies, focusing on uterine morphology and physiological traits, and then highlights the knowledge on uterine receptivity and the hormonal regulation of blastocyst implantation in mice. Our recent study revealed that selective proteolysis in the activated blastocyst is associated with the completion of blastocyst implantation after embryo transfer. Furthermore, in the context of blastocyst implantation in the mouse, this review discusses the window of uterine receptivity, hormonal regulation, uterine vascular permeability and angiogenesis, the delayed-implantation mouse model, morphogens, adhesion molecules, crosslinker proteins, extracellular matrix, and matricellular proteins. A better understanding of uterine and blastocyst biology during the peri-implantation period should facilitate further development of reproductive technology.

Resveratrol is a potent activator of NAD-dependent deacetyltransferase sirtuin-1 (SIRT1) and affects lipid metabolism and ATP generation in somatic cells. In the present study, the effects of supplementing culture medium with resveratrol on lipid metabolism, ATP generation, and cryosensitivity of bovine in vitro produced embryos were investigated. Bovine early cleaved-stage embryos were cultured in medium containing 0 or 0.5 µM resveratrol for 1 or 5 days. Resveratrol treatment for both 1 day and 5 days increased the expression levels of SIRT1 and phosphorylated AMP-activated protein kinase (pAMPK) in the embryos. Furthermore, resveratrol treatment was effective to increase ATP generation and reduce lipid content of the embryos. The effects of resveratrol treatment were diminished by the SIRT1 inhibitor “EX527”, and the reduced lipid content was reversed by treatment with etomoxir (a potent inhibitor of beta-oxidation). Blastocysts developed after resveratrol treatment showed low levels reactive oxygen species and increased cryotolerance. These results demonstrate that resveratrol improves in vitro development of bovine embryos, while reducing cytoplasmic lipid content through activation of beta-oxidation, thereby effective for production of bovine blastocysts with enhanced cryotolerance.

Although the laboratory rabbit has long contributed to many paradigmatic studies in biology and medicine, it is often considered to be a “classical animal model” because in the last 30 years, the laboratory mouse has been more often used, thanks to the availability of embryonic stem cells that have allowed the generation of gene knockout (KO) animals. However, recent genome-editing strategies have changed this unrivaled condition; so far, more than 10 mammalian species have been added to the list of KO animals. Among them, the rabbit has distinct advantages for application of genome-editing systems, such as easy application of superovulation, consistency with fertile natural mating, well-optimized embryo manipulation techniques, and the short gestation period. The rabbit has now returned to the stage of advanced biomedical research.