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The vitrification of zygotes is important for their use as donors for generating genome-edited mice. We previously reported the successful vitrification of mouse zygotes using carboxylated ε-poly-L-lysine (COOH-PLL). However, this vitrification solution contains fetal calf serum (FCS), which contains unknown factors and presents risks of pathogenic viral and microbial contamination. In this study, we examined whether polyvinyl alcohol (PVA) can be used as an alternative to FCS in vitrification solutions for mouse zygotes. When COOH-PLL was added to the vitrification solutions, zygotes vitrified with solutions containing 0.01% PVA (PV0.01) and those vitrified in a control solution containing FCS (75.6%) developed into blastocysts (78.4%). In addition, there were no significant differences in the ability to develop to term between the control solution (46.6%) and PV0.01 (44.1%) groups. In conclusion, we clearly demonstrated that PVA can replace FCS in our vitrification solution supplemented with COOH-PLL for mouse zygotes.

Purpose Although giant oocytes (GOs) having about twice cytoplasmic volume compared with general oocytes in mammals including the human are rarely recovered, it is thought that GOs have potentially chromosomal abnormalities. The aim of the present study was to assess chromosome numbers in chromosome‐spindle complexes (CSCs) and polar bodies of human GOs by using micromanipulation for sampling and next‐generation sequencing (NGS) for analyses of the chromosome numbers. Methods When recovered oocytes whose cytoplasm has lager than 140 µm or above, the oocytes were defined as GOs, and recovered GOs were vitrified. After warming, the CSCs, polar bodies, and enucleated cytoplasm were collected by micromanipulation from 3 GOs. The collected samples were analyzed by NGS. Results Chromosomal aneuploidy in the GOs was confirmed in all the three GOs. Comparing the CSCs with the chromosomes from polar bodies, the deletion and overlapping chromosome numbers were complementary in each GO. Conclusions The authors could collect the CSCs and the polar bodies from human GOs by micromanipulation, and then could analyze the chromosome numbers of the GOs by NGS method. As our data suggest that human GOs have chromosomal abnormalities, GOs should be excluded from clinical purpose as gamete sources for embryo transfer in the human.

It has been known that different protocols are used for embryo preservation at different stages due to different sensitivity to the physical and physiological stress caused by vitrification. In this study, we developed a common vitrification protocol using carboxlated ε-poly-l-lysine (COOH-PLL), a new cryoprotective agent for the vitrification of mouse embryos at different stages. The IVF-derived Crl:CD1(ICR) x B6D2F1/Crl pronuclear, 2-cell, 4-cell, and 8-cell, morula and blastocyst stage embryos were vitrified with 15% (v/v) ethylene glycol (EG) and 10% (w/v) COOH-PLL (E15P15) or 15% (v/v) EG and 15% (v/v) dimethyl sulfoxide (E15D15) using the minimal volume cooling method. The survival of vitrified embryos from pronuclear to blastocyst stages was equivalent between E15P15 and E15D15 groups. However, the rate of development to blastocysts was significantly lower in E15P15 than E15D15. The rates of survival and development to blastocysts were dramatically improved by a slight modification of EG and COOH-PLL concentrations (E20P10). After transferring 17 (E20P10) and 15 (E15D15) vitrified/warmed blastocysts, 8 and 7 pups were obtained (47.1% and 46.7%, respectively). Taken together, these results indicate that our vitrification protocol is appropriate for the vitrification of mouse embryos at different stages.

Although it is well known that calcium oscillations are required for fertilization in all mammalian species studied to date, recent studies also showed the ejection of zinc into the extracellular milieu in a series of coordinated events, called "zinc spark," during mammalian fertilization. These results led us to the hypothesis that a zinc ion-dependent signal is important for oocyte maturation, fertilization (activation), and further embryonic development. In this study, we evaluated the amounts and localization of intracellular zinc ions during maturation, fertilization, activation, and embryonic development in mouse oocytes. Our results show that abundant zinc ions are present in both immature and mature oocytes. After in vitro fertilization, the amounts of zinc ions were dramatically decreased at the pronuclear (PN) stage. Artificial activation by cycloheximide, SrCl

The vitrification of immature germinal vesicle (GV) oocytes is an important way to preserve genetic resources and female fertility. However, it is well known that cryopreserved GV oocytes have very poor developmental ability and that further improvement in this technique is needed. We previously reported the successful vitrification of matured mouse oocytes with enclosed cumulus cells using the calcium-free vitrification solution supplemented with ethylene glycol (EG) by the minimal volume cooling (MVC) method. In this study, we investigated whether our method is applicable to the vitrification of mouse oocytes at the GV stage (GV oocytes). Following maturation and fertilization in vitro, vitrified GV oocytes showed high survival (94.3 ± 2.0%) and maturation (94.3 ± 2.1%) rates. Although the fertilization and blastocyst rates of vitrified oocytes (fertilization: 46.6 ± 4.9% and blastocyst: 46.6 ± 3.0%) were significantly lower than those of fresh oocytes (fertilization: 73.0 ± 7.1% and blastocyst: 71.6 ± 8.0%) (P < 0.01), there were no differences in the ability to develop to term between fresh oocytes (50.0 ± 8.4%) and vitrified oocytes (37.5 ± 4.6%) (P > 0.05). In conclusion, we here show, for the first time, the efficient production of live mice derived from vitrified GV oocytes.

In mice, the conditional knockout strategy using the Cre-loxP system is useful for various types of research. The Cre mouse line with progesterone receptor promoter (PgrCre ) has been widely used to produce specific uterine gene-deficient mice, but in the Cre line, endogenous Pgr gene is replaced by Cre recombinase gene, which makes the breeding of homozygous mice (PgrCre/Cre ) difficult because they are infertile. Yang et al. (2013, https://10.1016/j.cell.2013.04.017) reported the generation of another PgriresCre mouse line that still has endogenous Pgr gene, and they inserted Cre recombinase downstream of the Pgr gene via an internal ribosome entry site (IRES). It is possible that this new PgriresCre line would be useful for uterine research as the mice can be bred as homozygotes (PgriresCre/iresCre ). Herein, we confirmed the PgriresCre mice effectively directed recombination in the female reproductive tract and was capable of genetic alteration in the endometrium that enables the studies of its uterine function. Our findings demonstrate that the new PgriresCre mouse line is also useful for the generation of uterine-specific knockout mice. The findings using PgriresCre mouse will contribute to the understanding of reproductive systems and diseases in humans and domestic animals.

During mammalian fertilization, sperm is fused with the oocyte's membrane, triggering the resumption of meiosis from the metaphase II arrest, the extrusion of the second polar body, and the exocytosis of cortical granules; these events are collectively called 'oocyte activation.' In all species studied to date, the transient rise in the cytosolic level of calcium (in particular, the repeated calcium increases called 'calcium oscillations' in mammals) is required for these events. Researchers have focused on identifying the factor(s) that can induce calcium oscillations during fertilization. Sperm‐specific phospholipase C, i.e., PLC zeta (PLCζ), is a strong candidate of the factor(s), and several research groups using different species obtained evidence that PLCζ is a sperm factor that can induce calcium oscillations during fertilization. However, postacrosomal sheath Tryptophan‐Tryptophan (WW)—domain‐binding protein (PAWP) was recently shown to have a pivotal role in inducing calcium oscillations in some species. In this review, we focus on PLCζ and PAWP as sperm factors, and we discuss this controversy: Which of these two molecules survives as a sperm factor?

STUDY QUESTION Could aromatase inhibitors (AI) be used to reduce risks of uterine endometrial cancer growth or recurrence during ovarian stimulation? SUMMARY ANSWER In a xenograft mouse model of endometrial cancer, concomitant AI administration suppressed the growth of endometrial cancer during ovarian stimulation. WHAT IS KNOWN ALREADY Recurrence and mortality rates of estrogen receptor-positive early breast cancer are reduced by long-term AI administration. Concomitant AI use for ovarian stimulation in patients with breast cancer is recommended for reducing estrogen-related potential risks. However, the efficacy of concomitant AI use for estrogen receptor-positive endometrial cancer have not been demonstrated conclusively by clinical or experimental animal studies. STUDY DESIGN, SIZE, DURATION Forty nude mice xenografted with uterine endometrial cancer cells were allocated to four groups. Group 1: no ovarian stimulation (control). Group 2: ovarian stimulation. Group 3: AI administration + ovarian stimulation. Group 4: ovariectomy and ovarian stimulation. Tumor growth was evaluated during the 6-week treatment period. PARTICIPANTS/MATERIALS, SETTING, METHODS Ishikawa 3-H-12 uterine endometrial cancer cells (estrogen and progesterone receptors-positive) were transplanted into 6-week-old BALB/cSlc-nu/nu nude mice, followed by interventions 2 weeks later. MAIN RESULTS AND THE ROLE OF CHANCE Compared to ovarian stimulation alone (Group 2), significant suppressions of tumor growth were observed in other three groups (Groups 1, 3 and 4, all at P < 0.05) and correlated with estrogen levels. AI administration had no apparent impact on embryo development. LIMITATIONS, REASONS FOR CAUTION In this study, we examined the growth of endometrial cancer tumors using one endometrial cancer cell line. Clinical endometrial cancer or hyperplasia cells can have diverse origins and AI may not be effective against other cancer cell types. WIDER IMPLICATIONS OF THE FINDINGS Concomitant AI use may provide a chance for safer childbirth by for patients with endometrial cancer or hyperplasia. STUDY FUNDING/CONPETING INTEREST(S) This study was supported by the Graduate Student Aid from the St. Marianna University School of Medicine. The authors declare no competing interests.

Human infertility has become a serious medical and social issue worldwide, especially in developed countries. Numerous assisted reproductive technologies (ARTs) (e.g., artificial insemination [AI], in vitro fertilization [IVF], and intracytoplasmic sperm injection [ICSI]) are widely used to treat infertility, but when embryos produced using these techniques are transferred, the pregnancy outcome is only approx. 20–50%, necessitating further improvement in ARTs. Many factors (including zinc) affect successful fertilization, further embryonic development, and the establishment and maintenance of pregnancy. Zinc signaling has many physiological functions in various types of cells, and it is thus speculated that zinc signaling has an important role in the abovementioned reproductive events, even in germ cells and reproductive tissues. Prior studies demonstrated that zinc deficiency results in a number of anomalies in the formation and maturation of spermatozoa, ovulation, and fertilization. During pregnancy, zinc deficiency causes spontaneous abortion, pregnancy-related toxemia, extended pregnancy or preterm birth, malformations, and retarded growth. The molecular mechanisms underlying zinc signaling in reproduction are not yet clear. In this chapter, we summarize the current knowledge about zinc signaling in reproduction—mainly in mammals—and we discuss recent advances in this field.

A major goal of testicular xenografting is to salvage germ cells from immature animals that cannot be used for reproduction and generate their offspring. In this study, we investigated whether porcine fetal testicular tissue would acquire the ability to produce sperm with full developmental competence after they had been cryopreserved and grafted into nude mice. Testicular fragments from fetuses at 35, 55 and 90 days postartificial insemination (dpi) were vitrified and stored in liquid nitrogen. Immediately after warming, testicular fragments at each fetal stage were transplanted under the back skin of castrated nude mice (Crlj:CD1- Foxn1 nu ) (35-, 55- and 90-dpi groups, respectively) (day 0 = grafting). Before grafting, the testicular fragments contained seminiferous cords consisting of only gonocytes and Sertoli cells. Histological analyses of the testicular grafts revealed that the differentiation of seminiferous tubules was largely dependent on the time after grafting, and not on donor age. On day 180 in each group, 10–20% of the total number of tubule/cord cross-sections examined had germ cells that had progressed beyond the spermatogonial stage. Fewer than 5% of tubule cross-sections contained elongated spermatids or sperm. Between days 360 and 420, tubule differentiation advanced further, until more than 45% of the tubule cross-sections contained elongated spermatids or sperm. Sperm were recovered for the first time from a single mouse in the 55-dpi group on day 180, although on days 360–420 sperm were recovered from most mice in all of the groups. Serum concentrations of inhibin and testosterone in host mice in all of the groups were higher than those in castrated mice that had received no testicular grafts. Single sperm collected from mice in each group on day 300 or later were injected into individual in vitro -matured oocytes, and these sperm-injected oocytes were transferred to the oviducts of 2 or 3 estrus-synchronized recipient gilts. None of the recipients in any of the groups produced piglets. The present results clearly indicate that porcine fetal testes during the gestational period acquire endocrine and exocrine functions after being cryopreserved and grafted into nude mice. However, the ability of xenogeneic sperm derived from fetal testis to generate piglets was not confirmed in the present study.

The objective of this study was to evaluate fertility and full-term development of rat vitrified oocytes after in vitro fertilization (IVF) with cryopreserved sperm. Oocytes with or without surrounding cumulus cells were vitrified with 30% ethylene glycol + 0.5 mol/L sucrose + 20% fetal calf serum by using the Cryotop method. The warmed oocytes were co-cultured with sperm. Although the denuded/vitrified oocytes were not fertilized, some of the oocytes vitrified with cumulus cells were fertilized (32.7%) after IVF with fresh sperm. When IVF was performed with cryopreserved sperm, vitrified or fresh oocytes with cumulus cells were fertilized (62.9% or 41.1%, respectively). In addition, to confirm the full-term development of the vitrified oocytes with surrounding cumulus cells after IVF with cryopreserved sperm, 108 vitrified oocytes with two pronuclei (2PN) were transferred into eight pseudopregnant females, and eight pups were obtained from three recipients. The present work demonstrates that vitrified rat oocytes surrounded by cumulus cells can be fertilized in vitro with cryopreserved sperm, and that 2PN embryos derived from cryopreserved gametes can develop to term. To our knowledge, this is the first report of successful generation of rat offspring derived from vitrified oocytes that were fertilized in vitro with cryopreserved sperm.

Historically, the conservation or preservation of mammalian genetic resources, especially farm animals, has been conducted under in situ conditions by maintaining living individuals as "livestock." However, systems for laboratory in vitro embryo production using gametes such as spermatozoa and oocytes are now available, in addition to ex situ preservation methods for mammalian genetic resources. One of these methods is the cryopreservation of gametes, embryos, and gonadal tissues. In pigs, freezing of sperm is the most reliable and well-established method for this purpose. On the other hand, cryopreservation of female gametes (oocytes) and gonadal tissues-usually by vitrification-has been associated with very low efficacies. Recently, in our laboratory, some research themes related to this issue have been pursued. We have been focusing on advances in porcine in vitro embryo production systems, and here, we introduce recent data on the vitrification of porcine immature oocytes and gonadal tissues followed by their xenografting into host mice to produce gametes.

In pigs, the efficiency of embryo production after intracytoplasmic sperm injection (ICSI) is still low because of frequent failure of normal fertilization, which involves formation of two polar bodies and two pronuclei. To clarify the reasons for this, we hypothesized that ICSI does not properly trigger sperm-induced fertilization events, especially intracellular Ca2+ signaling, also known as Ca2+ oscillation. We also suspected that the use of in vitro-matured oocytes might negatively affect fertilization events and embryonic development of sperm-injected oocytes. Therefore, we compared the patterns of Ca2+ oscillation, the efficiency of oocyte activation and normal fertilization, and embryo development to the blastocyst stage among in vivo- or in vitro-matured oocytes after ICSI or in vitro fertilization (IVF). Unexpectedly, we found that the pattern of Ca2+ oscillation, such as the frequency and amplitude of Ca2+ rises, in oocytes after ICSI was similar to that in oocytes after IVF, irrespective of the oocyte source. However, half of the oocytes failed to become activated after ICSI and showed no Ca2+ oscillation. Moreover, the embryonic development of normal fertilized oocytes was reduced when in vitro-matured oocytes were used, irrespective of the fertilization method employed. These findings suggest that low embryo production efficiency after ICSI is attributable mainly to poor developmental ability of in vitro-matured oocytes and a lack of Ca2+ oscillation, rather than the pattern of oscillation.

In pigs, the damaged sperm membrane leads to leakage of phospholipase C-ζ (PLCζ), which has been identified as a sperm factor, and a reduction of oocyte-activating ability. In this study, we investigated whether sperm selected by Percoll gradient centrifugation (Percoll) have sufficient PLCζ, and whether the efficiency of fertilization and blastocyst formation after intracytoplasmic sperm injection (ICSI) using Percoll-selected sperm can be improved. Percoll-selected sperm (Percoll group) or sperm without Percoll selection (Control group) were used. A proportion of the oocytes injected with control sperm were subjected to electrical stimulation at 1 h after ICSI (Cont + ES group). It was found that the Percoll group showed a large amount of PLCζ in comparison with the Control group. Furthermore, application of Percoll-selected sperm for ICSI increased the efficiency of fertilization and embryo development. Thus, these results indicate the Percoll-selected sperm have sufficient PLCζ and high oocyte-activating ability after ICSI in pigs.

In mammals, spermatozoa activate oocytes by triggering a series of intracellular Ca2+ oscillations with phospholipase C zeta (PLCζ), a sperm-borne oocyte-activating factor. Because the introduction of PLCζ alone can induce oocyte activation, it might be a promising reagent for assisted reproductive technologies. To test this possibility, we injected human PLCζ (hPLCζ) mRNA into mouse oocytes at different concentrations. We observed the oocyte activation and subsequent embryonic development. Efficient oocyte activation and embryonic development to the blastocyst stage was achieved only with a limited range of mRNA concentrations (0.1 ng/μl). Higher concentrations of mRNA caused developmental arrest of most embryos, suggesting that excessive PLCζ protein might be harmful at this stage. In a second series of experiments, we aimed to regulate the PLCζ protein concentration in oocytes by applying auxin-inducible degron (AID) technology that allows rapid degradation of the target protein tagged with AID induced by auxin. Injection of the hPLCζ protein tagged with AID and enhanced green fluorescent protein (hPLCζ-AID-EGFP) demonstrated that high EGFP expression levels at the late 1-cell stage were efficiently reduced by auxin treatment, suggesting efficient hPLCζ degradation by this system. Furthermore, the defective development observed with higher concentrations of hPLCζ-AID-EGFP mRNA was rescued following auxin treatment. Full-term offspring were obtained by round spermatid injection with optimized hPLCζ-AID activation. Our results indicate that this AID technology can be applied to regulate the protein levels in mouse oocytes and that our optimized PLCζ system could be used for assisted fertilization in mammals.

In mammals, pregnancy is an irreversible and complicated event. The mammalian uterus requires many physiological and morphological changes for pregnancy-associated events including implantation, decidualization, placentation and parturition. The failure to complete any events results in implantation failure, spontaneous miscarriage or abnormal parturition, including preterm birth. These events are primarily regulated by ovarian estrogen and progesterone (P4). P4 and estrogen are produced in the ovary throughout pregnancy in mice, but in humans, hormonal support switches from the ovary to the placenta. The first direct interaction between embryo and uterus is implantation. In humans, about 75% of unsuccessful pregnancies are believed to result from defective implantation. Therefore, a better understanding of the molecular mechanisms associated with implantation would be helpful for the further improvement of clinical treatments. Recent studies using genetically modified mice have given us conside...

Our aim was to optimize the cryoprotectant treatment for the preservation of immature porcine cumulus-oocyte complexes (COCs) by solid surface vitrification. In each experiment, the vitrification solution consisted of 50 mg/ml polyvinyl pyrrolidone, 0.3 M of the actual sugar and in total 35% (v/v) of the actual permeating cryoprotectant (pCPA) combination. After warming, the COCs were subjected to in vitro maturation, fertilization and embryo culture. In Experiment 1, trehalose and sucrose were equally effective during vitrification and warming in terms of facilitating oocyte survival and subsequent embryo development. In Experiment 2, when equilibration was performed at 38.5 C in a total of 4% (v/v) pCPA for 15 min, the combination of ethylene glycol and propylene glycol (EG + PG = 1:1) was superior to EG and dimethyl sulfoxide (EG + DMSO = 1:1) in terms of oocyte survival after vitrification and the quality of resultant blastocysts. In Experiment 3, equilibration in 4% (v/v) pCPA for 15 min before vitrification was superior to that in 15% (v/v) CPA for 5 min for achievement of high survival rates irrespective of the pCPA combination used. In Experiment 4, when equilibration was performed in 4% EG + PG for 5 min, 15 min or 25 min, there was no difference in oocyte survival and subsequent embryo development after vitrification and warming; however, the developmental competence of cleaved embryos was tendentiously reduced when equilibration was performed for 25 min. In conclusion, trehalose and sucrose were equally effective in facilitating vitrification, and the optimum pCPA treatment was 5-15 min equilibration in 4% (v/v) of EG + PG followed by vitrification in 35% (v/v) EG + PG.

Vitrification is a powerful tool for the efficient production of offspring derived from cryopreserved oocytes or embryos in mammalian species including domestic animals. Genome editing technologies such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated (Cas)9 are now available even for domestic species, suggesting that the vitrification of embryos at the pronuclear stage (PN) will be more important because they could provide genomic host cells to be targeted by TALENs or CRISPR/Cas9. Although we reported the successful production of piglets derived from vitrified PN embryos by a solid-surface vitrification method with glutathione supplementation, further improvements are required. The cryoprotective agent (CPA) carboxylated ε-poly-L-lysine (COOH-PLL) was introduced in 2009. COOH-PLL reduces the physical and physiological damage caused by cryopreservation in mammalian stem cells and the vitrification of mouse oocytes and embryos. Those results suggested that vitrification of COOH-PLL may help improve the developmental ability of pig embryos vitrified at the PN stage. However, it remains unclear whether COOH-PLL is available as a CPA for the vitrification of embryos in domestic species. In this study, we evaluated COOH-PLL as a CPA with ethylene glycol (EG) and Cryotop as a device for the vitrification of PN pig embryos. Exposure to vitrification solution supplemented with COOH-PLL up to 30% did not decrease developmental ability to the 2-cell stage and the blastocyst stage. After warming, most of the vitrified embryos survived regardless of the concentration of COOH-PLL (76.0 ± 11.8% to 91.8 ± 4.6%). However, the vitrified embryos without COOH-PLL showed a lower development rate up to the blastocyst stage (1.3 ± 1.0%) compared to the fresh embryos (28.4 ± 5.0%) (p

Targeted genome editing is a widely applicable approach for efficiently modifying any sequence of interest in animals. It is very difficult to generate knock-out and knock-in animals except for mice up to now. Very recently, a method of genome editing using zinc-finger nucleases (ZFNs) has been developed to produce knockout rats. Since only injection of ZFNs into the pronuclear (PN) embryo is required, it seems to be useful for generating gene-targeted animals, including domestic species. However, no one has reported the successful production of knockout pigs by direct injection of ZFNs into PN embryos. We examined whether ZFN works on editing the genome of porcine growth hormone receptor in two kinds of cell lines (ST and PT-K75) derived from the pig as a preliminary study. Our data showed that pZFN1/2 vectors were efficiently transfected into both ST and PT-K75 cells. In both cell lines, results from Cel-I assay showed that modification of the targeted gene was confirmed. We injected ZFN1/2 mRNAs into the nucleus of PN stage embryos and then they were transferred to the recipients. However, pups were not delivered. Taken together, ZFN can be an available technology of genome editing even in the pig but further improvement will be required for generating genome-modified pigs.

At fertilization, inositol 1,4,5-trisphosphate receptor type 1 (IP3 R1) has a crucial role in Ca(2+) release in mammals. Expression levels, localization and phosphorylation of IP3 R1 are important for its function, but it still remains unclear which molecule(s) regulates IP3 R1 behavior in pig oocytes. We examined whether there was a difference in localization of IP3 R1 after in vitro or in vivo maturation of pig oocytes. In mouse oocytes, large clusters of IP3 R1 were formed in the cortex of the oocyte except in a ring-shaped band of cortex adjacent to the spindle. However, no such clusters of IP3 R1 were observed in pig oocytes and there was no difference in its localization between in vitro and in vivo matured oocytes. We next tried to clarify which factor(s) regulates IP3 R1 localization, phosphorylation and expression using M-phase stage-dependent kinase inhibitors. Our results show that treatments with roscovitine (p34(cdc2) kinase inhibitor) or U0126 (mitogen-activated protein kinase inhibitor) did not affect IP3 R1 expression or localization in pig oocytes, although the latter strongly inhibited phosphorylation. However, treatment with BI-2536, an inhibitor of polo-like kinase 1 (Plk1), dramatically decreased the expression level of IP3 R1 in pig oocytes in a dose-dependent manner. From these results, it is suggested that Plk1 is involved in the regulation of IP3 R1 expression in pig oocytes.

Transgenic animals are generally produced by microinjection of exogeneous DNA into embryos at the pronuclear (PN) stage. PN embryos also can be used for knockout animals because artificial nucleases such as zinc-finger nuclease or transcription activator-like effector nuclease are now available for modification of the targeted gene. If the embryos can be vitrified with multiple rounds, the remaining embryos without microinjection can be reused. In this study, we examined the developmental competence of repetitively vitrified mouse embryos at the PN stage using Cryotop. It was also examined whether a new cryoprotective agent (CPA), carboxylated ε-poly-l-lysine (COOH-PLL), is available for vitrification of mouse embryos. PN embryos were vitrified with dimethyl sulfoxide (DMSO) and ethylene glycol (EG) as CPAs. After warming, some embryos were re-vitrified up to three times. The re-vitrification did not affect survival and in vitro developmental ability. PN embryos were also vitrified with COOH-PLL instead of DMSO up to three times. The embryos re-vitrified with COOH-PLL and EG also maintained high survival and developmental ability. However embryos vitrified with COOH-PLL and EG at three times significantly showed higher developmental ability (61.2±3.1%) than those vitrified with DMSO and EG at three times (44.2±2.7%) which was equivalent to that of fresh embryos (70.0±3.6%). Taken together, our results show that re-vitrification of mouse PN embryos did not have a detrimental effect on the in vitro and in vivo development of the embryos. In addition, COOH-PLL is available as a CPA for vitrification of mouse PN embryos.

In pigs, the embryonic developmental ability after intracytoplasmic sperm injection (ICSI) is inferior to that resulting from in vitro fertilization (IVF). We evaluated the timing of cell division up to blastocyst formation on embryonic development after ICSI using either whole sperm (w-ICSI) or the sperm head alone (h-ICSI) and IVF as a control. At 10 h after ICSI or IVF, we selected only zygotes, and each of the zygotes/embryos was evaluated for cleavage every 24 h until 168 h. We then observed a delay in the 1st and 2nd cleavages of h-ICSI embryos and also in blastocoele formation by w-ICSI embryos in comparison with IVF embryos. The rate of blastocyst formation and the quality of blastocysts in both ICSI groups were inferior to those in the IVF group. In conclusion, the delay in cleavage of porcine ICSI embryos shows poorer embryonic development.

Our aim was to optimize a cryoprotectant treatment for vitrification of immature porcine cumulus-oocyte complexes (COCs). Immature COCs were vitrified either in 35% ethylene glycol (EG), 35% propylene glycol (PG) or a combination of 17.5% EG and 17.5% PG. After warming, the COCs were in vitro matured (IVM), and surviving oocytes were in vitro fertilized (IVF) and cultured. The mean survival rate of vitrified oocytes in 35% PG (73.9%) was higher (P

A sperm-specific phospholipase C (PLC), PLCzeta (PLCζ), is thought to underlie the initiation of calcium ([Ca(2+) ]i ) oscillations that induce egg activation in mammals. In large domestic species, only bovine, porcine and recently equine PLCζ have been cloned, and the physiological functions of these molecules have not been fully characterized. Here, we evaluated the physiological functions of equine PLCζ (ePLCζ) in mouse oocytes. ePLCζ was cloned from testis using RT-PCR. The expression of ePLCζ messenger RNA was confirmed in testis but not in other tissues. Microinjection of ePLCζ complementary RNA (cRNA) into mouse oocytes induced long-lasting [Ca(2+) ]i oscillations, and most of the injected oocytes formed pronuclei (PN). The injection of cRNAs encoding horse, mouse, human and cow PLCζ into mouse oocytes showed that ePLCζ had the highest [Ca(2+) ]i oscillation-inducing activity among the species tested. Mutation of D202R, which renders the protein inactive, abrogated the activity of ePLCζ. The nuclear translocation ability of ePLCζ was defective when expressed in mouse oocytes. Taken together, our findings show for the first time that ePLCζ has highest activity of the mammalian species studied to date. Our findings will be useful for the improvement of reproductive technologies in the horse.

At fertilization, the sperm triggers resumption from the arrest, extrusion of the second polar body and pronuclear formation, the events of which are collectively acknowledged as 'oocyte activation'. In all species up to date, oocyte activation requires a fertilization-associated increase in the intracellular concentration of calcium. Especially in mammals, the signal of intracellular calcium rise at fertilization consists of periodical rises, which are also referred to as calcium oscillations. Our recent results suggest that these calcium oscillations have an important role in not only oocyte activation but also development of mammals. Pigs are animals of great agricultural value and ones in which assisted reproductive techniques, including somatic cell nuclear transfer, to produce gene-modified pigs. Although reconstructed embryos require artificial activation stimuli which mimic fertilization-associated increase of intracellular calcium in the oocytes, it has been known that the developmental ability of the oocytes after artificial activation is low and the regimen seems to be required for improvement. Recently we focused on two molecules, phospholipase C zeta and inositol 1,4,5-triphosphate receptor which have important roles in regulation of calcium oscillations during fertilization in mammals, including pigs. In this review, we will discuss the present status and future perspective of molecular mechanisms during fertilization in pigs.

In vitro production of embryos, including in vitro maturation, fertilization of oocytes and their subsequent culture to the embryo stage, has become the most popular method of studying gametogenesis and embryogenesis in pigs. As well as their utility for basic studies, these procedures now enable us to generate viable embryos and offspring as a means of conserving genetic resources and rare animal breeds. Recently, more advanced technologies such as xenografting of gonadal (testicular and ovarian) tissues into immunodeficient experimental animals have been developed. In combination with in vitro embryo production techniques, this approach may provide many benefits. We have been carrying out studies to acquire basic information about the application of this method to porcine species, and to improve the existing techniques. Recently, we obtained oocytes from ovarian tissue xenografted and grown in nude mice that had the capacity to be fertilized and the ability to develop into early-stage embryos. We also obtained spermatozoa from the xenografted testicular tissues and injected them intracytoplasmically into in vitro-matured oocytes to produce piglets. Here we discuss the further possibilities of conservation and utilization of porcine gonadal tissue by xenografting into immunodeficient mice.

Unfertilized oocytes are one of the most desired germ cell stages for cryopreservation because these cryopreserved oocytes can be used for assisted reproductive technologies, including in vitro fertilization (IVF) and intracytoplasmic sperm injection. However, in general, the fertility and developmental ability of cryopreserved oocytes are still low. The aim of the present study was to improve vitrification of mouse oocytes. First, the effects of calcium and cryoprotectants, dimethyl sulfoxide and ethylene glycol (EG), in vitrification medium on survival and developmental ability of vitrified oocytes were evaluated. Oocytes were vitrified by a minimal volume cooling procedure using different cryoprotectants. Most of the vitrified oocytes were morphologically normal after warming, but their fertility and development were low independently of calcium and cryoprotectants. Second, the effect of cumulus cells on ability of oocytes to be fertilized and develop in vitro was examined. The fertility and developmental ability of denuded oocytes (DOs) after IVF were reduced compared with cumulus-oocyte complexes (COCs) both in fresh and cryopreserved groups. Vitrified COCs showed significantly (P

It has been reported that phosphatidylinositol 3-kinase (PI3K)-protein kinase B (PKB) pathway plays a crucial role in the meiotic resumption and progression to the metaphase II (MII) stage of oocytes. However, the role of this pathway in meiotic arrest at the MII stage (cytostatic activity) is not well understood. In this study the effect of a PI3K inhibitor, LY294002, on the MAPK and p34(cdc2) kinase activities of matured porcine oocytes was examined. After maturation culture, both the MAPK and p34(cdc2) kinase activities in the oocytes were gradually decreased in a time-dependent manner. Although 25 micromol/L LY294002 did not affect either the MAPK or p34(cdc2) kinase activities, 50 micromol/L LY294002 suppressed the PKB phosphorylation and slightly decreased MAPK activity, but not the p34(cdc2) kinase activity. Therefore the effect of 10 micromol/L Ca(2+) ionophore which was reported as inducing a transient decrease of p34(cdc2) kinase but not MAPK activities, was also examined in LY294002-treated oocytes. By additional treatment with LY294002 after Ca(2+) ionophore, both the MAPK and p34(cdc2) kinase activities were decreased in a time-dependent manner, concomitantly with improvement of pronuclear formation. Therefore, we concluded that PI3K is involved in the maintenance of MAPK activity in matured porcine oocytes.

We investigated survival, meiotic competence, cytoplasmic maturation, in vitro fertilization, and development of immature porcine (Sus scrofa) oocytes cryopreserved by a modified solid surface vitrification protocol. Cumulus-oocyte complexes (COCs) collected from follicles 3 to 6mm in diameter in abattoir-derived ovaries of prepubertal gilts were either vitrified (Vitrified group), subjected to cryoprotectant treatment (CPA group), or used without any treatment (Control group). Oocyte viability was assayed by staining with fluorescein diacetate. Live oocytes were matured in vitro and their meiotic progression investigated by nuclear staining. In a series of experiments, the glutathione (GSH) content of in vitro-matured oocytes and viability of cumulus cells were assayed simultaneously. The in vitro-matured oocytes were also fertilized and cultured in vitro to assess their ability to be fertilized and to develop to the blastocyst stage, respectively. The proportion of viable oocytes in the Vitrified group was significantly lower than that in the CPA and Control groups (27.7%, 90.4%, and 100%, respectively). Among the three groups, there were no differences in meiotic competence, cumulus viability, and GSH levels at the end of in vitro maturation. Fertilization parameters (i.e., rates of male pronucleus formation, monospermy, and second polar body extrusion) were also similar among groups. However, comparison of the developmental abilities of oocytes in the Vitrified, CPA, and Control groups revealed that the Vitrified group had a significantly reduced ability to undergo first cleavage (34.4%, 63.3%, and 69.0%) and to develop to the blastocyst stage (5.1%, 25.5%, and 34.6%). The mean total cell numbers in blastocysts after 6 d of culture were not significantly different among the Vitrified, CPA, and Control groups (40.3, 42.8, and 43.4). In conclusion, despite low survival rates and impaired development in the Vitrified group, meiotic competence, cytoplasmic maturation, and subsequent fertilization characteristics of surviving germinal vesicle oocytes were unaffected by vitrification, and high-quality blastocysts were produced from vitrified immature oocytes.

It is anticipated that the utilization of spermatogonia through testicular xenografting will open new avenues for the conservation of male gametes. With the aim of establishing this new technique for genetic preservation of pigs, we used it in combination with intracytoplasmic sperm injection (ICSI). Testicular tissues derived from neonatal piglets, which contained seminiferous cords consisting of only gonocytes/spermatogonia, were transplanted under the back skin of castrated nude mice. Between 125 and 192 d after xenografting, sperm (morphologically similar to epididymal sperm) were recovered from 41 of the 65 host mice (63.1%). Testicular spermatozoa from adult boars were used as a positive control. A single spermatozoon was injected into an in vitro matured porcine oocyte, and the oocytes were electro-stimulated and cultured (graft-ICSI and testis-ICSI, respectively). Blastocyst rates in both ICSI groups (24.9% and 37.4%, respectively) were higher (P < 0.05) than those without the injection procedure (parthenogenetic; 12.7%) and after injection of a small amount of injection buffer (sham; 13.0%). Rates of diploid blastocysts in both graft-ICSI and testis-ICSI groups (48.9% and 60.6%) were higher (P < 0.05) than those in the parthenogenetic and sham groups (13.5% and 28.0%). Therefore, we demonstrated that porcine oocytes injected with xenogeneic sperm have in vitro developmental ability to the blastocyst stage.

To preserve genetic resources efficiently in rats, sperm cryopreservation is essential. This study aimed to confirm the ability of cryopreserved and transported rat spermatozoa to fertilize through intrauterine insemination. The Komeda miniature rat Ishikawa is a mutant caused by the autosomal recessive mutation mri. The epididymal sperm was frozen with egg yolk medium and banked at the National BioResource Project (NBRP), Kyoto University. The sperm was transported to Azabu University, then thawed at 37℃. The thawed semen was inseminated into the uterine horns of recipients; its motility was around 10%. Seven of 15 inseminated female rats became pregnant and 13 live pups were born. The results indicate that rat spermatozoa cryopreserved at NBRP are capable of restoring genetic resources through intrauterine insemination. We also confirmed the usefulness of assisted reproductive technologies for the rat including sperm cryopreservation and intrauterine insemination.

In rats, the success of in vitro fertilization (IVF) was reported 40 years ago. Although it has been demonstrated in papers that these IVF oocytes using sperm freshly collected from cauda epididymides can be developed to term via embryo transfer, successful IVF with cryopreserved rat sperm has never been reported to date. Here, we report establishment of a successful IVF system using frozen/thawed rat spermatozoa. Our data showed that intracellular cAMP and free cholesterol levels in frozen/thawed rat sperm were maintained low, suppressing capacitation-associated tyrosine phosphorylation. The treatment of methyl-beta-cyclodextrin improved removal of free cholesterol from the membrane in frozen/thawed sperm but not induction of capacitation-associated tyrosine phosphorylation in the sperm. Treatment with a phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xanthin (IBMX), dramatically increased cAMP and tyrosine phosphorylation levels in frozen/thawed rat sperm. When the IBMX-treated frozen/thawed sperm were used for IVF, the proportions of pronuclear formation and blastocyst formation were significantly higher than those of frozen/thawed sperm treated without IBMX (P < 0.05). The embryos were developed to term at a high success rate equivalent to the rate obtained with IVF using fresh sperm. Thus, we established for the first time a successful IVF system in rats using cryopreserved spermatozoa.