Zinc transporter ZnT3/Slc30a3 has a potential role in zinc ion influx in mouse oocytes.

Zinc is an essential trace element for various physiological functions, including reproduction. The influx/efflux of zinc ions is regulated by zinc transporters (Zip1-14 and ZnT1-8, 10). However, the precise roles of zinc transporters and zinc dynamics in reproductive functions are unknown. In this study, ZnT3/Slc30a3 gene knockout (KO) mice were used to analyze the role of ZnT3. In ZnT3 KO mice, intracellular zinc ions in oocytes/zygotes were significantly reduced compared to those in controls, and free zinc ions did not accumulate in the oocyte cytoplasm. However, fertilization of these oocytes and the average litter size were comparable to those of control mice. Our results suggest that ZnT3 plays an important role in the accumulation of zinc ions in oocytes but not in the developmental ability of mice. ZnT3 KO mice will be useful for examining zinc dynamics in oocytes and other tissues.

A combined treatment with progesterone, anti-inhibin serum, and equine chorionic gonadotropin improves number of ovulated oocytes in young C57BL/6J mice.

Superovulation procedures are routinely and widely used in mouse reproductive technology. Previous studies have shown that a large number of oocytes can be obtained from adult mice (> 10 weeks old) using a combined treatment with progesterone (P4) and anti-inhibin serum (AIS). However, these effects have not been fully investigated in young (4 weeks) C57BL/6J mice. Here, we found that a modified superovulation protocol (combined treatment with P4, AIS, eCG (equine chorionic gonadotropin), and hCG (human chorionic gonadotropin); P4D2-Ae-h) improved the number of oocytes compared to the control (eCG and hCG) (39.7 vs. 21.3 oocytes/mouse). After in vitro fertilization, pronuclear formation rates were 69.3% (P4D2-Ae-h group) and 66.2% (control group). After embryo transfer, 46.4% (116/250) of the embryos in the P4D2-Ae-h group successfully developed to term, which was comparable to the control group (42.9%; 123/287 embryos). In conclusion, our protocol (P4D2-Ae-h) was effective for superovulation in young C57BL/6J mice.

Successful production of offspring derived from mouse zygotes vitrified with carboxylated ε-poly-L-lysine and polyvinyl alcohol without serum.

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.

Chromosomal analyses of human giant diploid oocytes by next-generation sequencing.

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.

Carboxylated ε-poly-L-lysine, a cryoprotective agent, is an effective partner of ethylene glycol for the vitrification of embryos at various preimplantation stages.

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.

Aromatase inhibitor use during ovarian stimulation suppresses growth of uterine endometrial cancer in xenograft mouse model.

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.

Application of auxin-inducible degron technology to mouse oocyte activation with PLCζ.

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.

Molecular mechanisms of embryonic implantation in mammals: Lessons from the gene manipulation of mice.

BACKGROUND: Human infertility has become a serious and social issue all over the world, especially in developed countries. Numerous types of assisted reproductive technology have been developed and are widely used to treat infertility. However, pregnancy outcomes require further improvement. It is essential to understand the cross-talk between the uterus (mother) and the embryo (fetus) in pregnancy, which is a very complicated event. METHODS: The mammalian uterus requires many physiological and morphological changes for pregnancy-associated events, including implantation, decidualization, placentation, and parturition, to occur. Here is discussed recent advances in the knowledge of the molecular mechanisms underlying these reproductive events - in particular, embryonic implantation and decidualization - based on original and review articles. MAIN FINDINGS RESULTS: In mice, embryonic implantation and decidualization are regulated by two steroid hormones: estrogen and progesterone. Along with these hormones, cytokines, cell-cycle regulators, growth factors, and transcription factors have essential roles in implantation and decidualization in mice. CONCLUSION: Recent studies using the gene manipulation of mice have given considerable insight into the molecular mechanisms underlying embryonic implantation and decidualization. However, as most of the findings are based on mice, comparative research using different mammalian species will be useful for a better understanding of the species-dependent differences that are associated with reproductive events, including embryonic implantation.

Efficient pig ICSI using Percoll-selected spermatozoa; evidence for the essential role of phospholipase C-ζ in ICSI success.

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.

Lack of calcium oscillation causes failure of oocyte activation after intracytoplasmic sperm injection in pigs.

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.

Optimization of cryoprotectant treatment for the vitrification of immature cumulus-enclosed porcine oocytes: comparison of sugars, combinations of permeating cryoprotectants and equilibration regimens.

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.

The effect of a novel cryoprotective agent, carboxylated ε-poly-L-lysine, on the developmental ability of re-vitrified mouse embryos at the pronuclear stage.

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.

Delay in cleavage of porcine embryos after intracytoplasmic sperm injection (ICSI) shows poorer embryonic development.

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.

Recent advances of oocyte/embryo vitrification in mammals from rodents and large animals.

Vitrification is a valuable technology that enables semipermanent preservation and long-distance or international transportation of genetically modified and native animals. In laboratory mice, vitrification maintains and transports embryos, and many institutions and companies sell vitrified embryos. In contrast, despite numerous papers reporting on vitrification in livestock over the past decade, practical implementation has yet to be achieved. However, with advances in genome editing technology, it is anticipated that the number of genetically modified domestic animals will increase, leading to a rise in demand for vitrification of oocytes and embryos. Here, we provide an objective overview of recent advancements in vitrification technology for livestock, drawing a comparison with the current developments in laboratory animals. Additionally, we explore the future prospects for vitrification in livestock, focusing on its potential benefits and drawbacks.

Comparison of ethylene glycol and propylene glycol for the vitrification of immature porcine oocytes.

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<0.05) than that in 35% EG (27.8%). Oocyte maturation rates did not differ among vitrified and non-vitrified control groups. Blastocyst formation in the vitrified EG group (10.8%) was higher (P<0.05) than that in the vitrified PG group (2.0%) but was lower than that in the control group (25.0%). Treatment of oocytes with 35% of each cryoprotectant without vitrification revealed a higher toxicity of PG on subsequent blastocyst development compared with EG. The combination of EG and PG resulted in 42.6% survival after vitrification. The maturation and fertilization rates of the surviving oocytes were similar in the vitrified, control and toxicity control (TC; treated with EG+PG combination without cooling) groups. Blastocyst development in the vitrified group was lower (P<0.05) than that in the control and TC groups, which in turn had similar development rates (10.7%, 18.1% and 23.3%, respectively). In conclusion, 35% PG enabled a higher oocyte survival rate after vitrification compared with 35% EG. However, PG was greatly toxic to oocytes. The combination of 17.5% EG and 17.5% PG yielded reasonable survival rates without toxic effects on embryo development.

Uterine epithelial Gp130 orchestrates hormone response and epithelial remodeling for successful embryo attachment in mice.

Leukemia inhibitory factor (LIF) receptor, an interleukin 6 cytokine family signal transducer (Il6st, also known as Gp130) that is expressed in the uterine epithelium and stroma, has been recognized to play an essential role in embryo implantation. However, the molecular mechanism underlying Gp130-mediated LIF signaling in the uterine epithelium during embryo implantation has not been elucidated. In this study, we generated mice with uterine epithelium specific deletion of Gp130 (Gp130 ecKO). Gp130 ecKO females were infertile due to the failure of embryo attachment and decidualization. Histomorphological observation revealed that the endometrial shape and embryo position from Gp130 ecKO were comparable to those of the control, and uterine epithelial cell proliferation, whose attenuation is essential for embryo implantation, was controlled in Gp130 ecKO. Comprehensive gene expression analysis using RNA-seq indicates that epithelial Gp130 regulates the expression of estrogen- and progesterone-responsive genes in conjunction with immune response during embryo implantation. We also found that an epithelial remodeling factor, snail family transcriptional repressor 1 (Snai1), was markedly reduced in the pre-implantation uterus from Gp130 ecKO. These results suggest that not only the suppression of uterine epithelial cell proliferation, but also Gp130-mediated epithelial remodeling is required for successful implantation in mice.

Successful Production of Offspring Derived from Phospholipase C Zeta-Deficient Sperm by Additional Artificial Activation.

During mammalian fertilization, repetitive rises of intracellular calcium called calcium oscillations are required for full activation of oocytes. Therefore, oocytes such as round spermatid injected or somatic cell nuclear transferred require additional artificial activation which mimics the calcium oscillations. It is well recognized that sperm specific phospholipase C (PLCζ) is a strong candidate as the sperm factor which can induce calcium oscillations and, at least in mammals, the genetic mutation of PLCζ in human causes male infertility due to the lack of calcium oscillations in the oocytes. Recent studies showed that the sperm lacking PLCζ (Plcz1-/-) still could induce rise(s) of intracellular calcium in the oocytes after IVF but not intracytoplasmic sperm injection (ICSI). In the ICSI oocytes, no pronuclear formation or development to the two-cell stage was observed. However, it is still unclear whether additional activation treatment can rescue the low developmental ability of Plcz1-/--sperm-derived oocytes after ICSI. In this study, we examined whether oocytes injected with a Plcz1-/- sperm can develop to term by additional artificial activation. In oocytes injected a Plcz1-/- sperm and Plcz1-/- and eCS (another candidate of the sperm factor) double knockout sperm (Plcz1-/-eCS-/-), the rates of pronuclear formation were very low (2.0 ± 2.3% and 6.1 ± 3.7%, respectively) compared to control (92.1 ± 2.6%). However, these rates were dramatically improved by additional procedures of PLCζ-mRNA injection or SrCl2 treatment (Plcz1-/- sperm + PLCζ mRNA, Plcz1-/- sperm + SrCl2 and Plcz1-/-eCS-/- sperm + PLCζ mRNA; 64.2 ± 10.8%, 89.2 ± 2.4% and 72.6 ± 5.4%, respectively). Most of the oocytes were developed to the two-cell stage. After embryo transfer, healthy pups were obtained in all these groups (Plcz1-/- sperm + PLCζ mRNA:10.0 ± 2.8%, Plcz1-/- sperm + SrCl2:4.0 ± 4.3% and Plcz1-/-eCS-/- sperm + PLCζ mRNA: 10.0 ± 5.7%). The rate in Plcz1-/- sperm + SrCl2 group was significantly lower than that in control (26.0 ± 2.4%). Taken together, our present results show that additional activation treatment such as SrCl2 and PLCζ mRNA can fully support to develop to term even in oocyte injected Plcz1-/- sperm. In addition, PLCζ-induced oocyte activation is more suitable for successful development to term compared to that such as phenomenon induced by SrCl2. These findings will contribute to improvement for male-dependent human infertility and reproductive technologies in other mammalian species.

Successful production of offspring using cryopreserved sperm via nonsurgical artificial insemination in rats.

In rats, artificial insemination (AI) is surgically performed as a general tool to obtain offspring using cryopreserved spermatozoa. Nonsurgical AI is a more desirable technology because it does not require any surgical procedures. However, there has never been a successful nonsurgical AI since frozen-thawed rat spermatozoa show low motility. We show here for the first time successful nonsurgical AI in rats using oxytocin treatment. Intraperitoneal injection of oxytocin (1/800 IU) immediately before nonsurgical AI significantly increased the number of sperm collected from the oviducts compared with that without oxytocin treatment. Therefore, to obtain pups, oxytocin was intraperitoneally injected into females mated with vasectomized males, and the rats were then used for nonsurgical AI. Seven of the 12 oxytocin-treated rats became pregnant after nonsurgical AI, and 37 pups were obtained. Only one rat (1/13) without oxytocin treatment was pregnant after nonsurgical AI, and only 1 pup was delivered. These results show success for the first time in obtaining offspring using frozen-thawed rat spermatozoa via nonsurgical AI. Our results also suggest the possibility that oxytocin treatment is effective for improvement of nonsurgical AI even in other species.

Dynamic changes of intracellular zinc ion level during maturation, fertilization, activation, and development in mouse oocytes.

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, SrCl2 , and TPEN also reduced the amounts of zinc ions in the PN stage. On the other hand, PN embryos derived from sperm injection still showed high level of zinc ions. However, the amounts of zinc ions rapidly increased at the blastocysts regardless of activation method. We showed here that the amounts of zinc ions dramatically changed during maturation, fertilization, activation, and development in mouse oocytes.

Pre-treatment of sperm reduces success of ICSI in the pig.

In pigs, although ICSI is a feasible fertilization technique, its efficiency is low. In general, injected pig sperm are insufficient to induce oocyte activation and embryonic development. Pretreatments for disrupting sperm membranes have been applied to improve the fertility of ICSI oocytes; however, we hypothesize that such pretreatment(s) may reduce the ability of the sperm to induce oocyte activation. We first evaluated the effects of sperm pretreatments (sonication (SO) to isolate the sperm heads from the tails, Triton X-100 (TX), and three cycles of repeated freezing/thawing (3×-FT) for disrupting sperm membranes) on the rate of pronucleus (PN) formation after ICSI. We found that oocytes injected with control (whole) sperm had higher rates of PN formation than those obtained after subjecting the sperm to SO, TX, and 3×-FT. The amounts of phospholipase Cζ (PLCζ), which is thought to be the oocyte-activating factor in mammalian sperm, in sperm treated by each method was significantly lower than that in whole untreated sperm. Furthermore, using immunofluorescence, it was found that in pig sperm, PLCζ was localized to both the post-acrosomal region and the tail area. Thus we demonstrated for the first time that sperm pretreatment leads to a reduction of oocyte-activating capacity. Our data also show that in addition to its expected localization to the sperm head, PLCζ is also localized in the tail of pig sperm, thus raising the possibility that injection of whole sperm may be required to attain successful activation in pigs.