GABAB receptors induce phasic release from medial habenula terminals through activity-dependent recruitment of release-ready vesicles.

GABAB receptor (GBR) activation inhibits neurotransmitter release in axon terminals in the brain, except in medial habenula (MHb) terminals, which show robust potentiation. However, mechanisms underlying this enigmatic potentiation remain elusive. Here, we report that GBR activation on MHb terminals induces an activity-dependent transition from a facilitating, tonic to a depressing, phasic neurotransmitter release mode. This transition is accompanied by a 4.1-fold increase in readily releasable vesicle pool (RRP) size and a 3.5-fold increase of docked synaptic vesicles (SVs) at the presynaptic active zone (AZ). Strikingly, the depressing phasic release exhibits looser coupling distance than the tonic release. Furthermore, the tonic and phasic release are selectively affected by deletion of synaptoporin (SPO) and Ca2+-dependent activator protein for secretion 2 (CAPS2), respectively. SPO modulates augmentation, the short-term plasticity associated with tonic release, and CAPS2 retains the increased RRP for initial responses in phasic response trains. The cytosolic protein CAPS2 showed a SV-associated distribution similar to the vesicular transmembrane protein SPO, and they were colocalized in the same terminals. We developed the "Flash and Freeze-fracture" method, and revealed the release of SPO-associated vesicles in both tonic and phasic modes and activity-dependent recruitment of CAPS2 to the AZ during phasic release, which lasted several minutes. Overall, these results indicate that GBR activation translocates CAPS2 to the AZ along with the fusion of CAPS2-associated SVs, contributing to persistency of the RRP increase. Thus, we identified structural and molecular mechanisms underlying tonic and phasic neurotransmitter release and their transition by GBR activation in MHb terminals.

A transchromosomic rat model with human chromosome 21 shows robust Down syndrome features.

Progress in earlier detection and clinical management has increased life expectancy and quality of life in people with Down syndrome (DS). However, no drug has been approved to help individuals with DS live independently and fully. Although rat models could support more robust physiological, behavioral, and toxicology analysis than mouse models during preclinical validation, no DS rat model is available as a result of technical challenges. We developed a transchromosomic rat model of DS, TcHSA21rat, which contains a freely segregating, EGFP-inserted, human chromosome 21 (HSA21) with >93% of its protein-coding genes. RNA-seq of neonatal forebrains demonstrates that TcHSA21rat expresses HSA21 genes and has an imbalance in global gene expression. Using EGFP as a marker for trisomic cells, flow cytometry analyses of peripheral blood cells from 361 adult TcHSA21rat animals show that 81% of animals retain HSA21 in >80% of cells, the criterion for a "Down syndrome karyotype" in people. TcHSA21rat exhibits learning and memory deficits and shows increased anxiety and hyperactivity. TcHSA21rat recapitulates well-characterized DS brain morphology, including smaller brain volume and reduced cerebellar size. In addition, the rat model shows reduced cerebellar foliation, which is not observed in DS mouse models. Moreover, TcHSA21rat exhibits anomalies in craniofacial morphology, heart development, husbandry, and stature. TcHSA21rat is a robust DS animal model that can facilitate DS basic research and provide a unique tool for preclinical validation to accelerate DS drug development.

Generation of rat pancreas in mouse by interspecific blastocyst injection of pluripotent stem cells.

The complexity of organogenesis hinders in vitro generation of organs derived from a patient's pluripotent stem cells (PSCs), an ultimate goal of regenerative medicine. Mouse wild-type PSCs injected into Pdx1(-/-) (pancreatogenesis-disabled) mouse blastocysts developmentally compensated vacancy of the pancreatic "developmental niche," generating almost entirely PSC-derived pancreas. To examine the potential for xenogenic approaches in blastocyst complementation, we injected mouse or rat PSCs into rat or mouse blastocysts, respectively, generating interspecific chimeras and thus confirming that PSCs can contribute to xenogenic development between mouse and rat. The development of these mouse/rat chimeras was primarily influenced by host blastocyst and/or foster mother, evident by body size and species-specific organogenesis. We further injected rat wild-type PSCs into Pdx1(-/-) mouse blastocysts, generating normally functioning rat pancreas in Pdx1(-/-) mice. These data constitute proof of principle for interspecific blastocyst complementation and for generation in vivo of organs derived from donor PSCs using a xenogenic environment.

Identification of TEKTIN1-expressing multiciliated cells during spontaneous differentiation of non-human primate embryonic stem cells.

Tektins are a group of microtubule-stabilizing proteins necessary for cilia and flagella assembly. TEKTIN1 (TEKT1) is used as a sperm marker for monitoring germ cell differentiation in embryonic stem (ES) and induced pluripotent stem (iPS) cells. Although upregulation of TEKT1 has been reported during spontaneous differentiation of ES and iPS cells, it is unclear which cells express TEKT1. To identify TEKT1-expressing cells, we established an ES cell line derived from cynomolgus monkeys (Macaca fascicularis), which expresses Venus controlled by the TEKT1 promoter. Venus expression was detected at 5 weeks of differentiation on the surface of the embryoid body (EB), and it gradually increased with the concomitant formation of a leash-like structure at the EB periphery. Motile cilia were observed on the surface of the Venus-positive leash-like structure after 8 weeks of differentiation. The expression of cilia markers as well as TEKT1-5 and 9 + 2 microtubule structures, which are characteristic of motile cilia, were detected in Venus-positive cells. These results demonstrated that TEKT1-expressing cells are multiciliated epithelial-like cells that form a leash-like structure during the spontaneous differentiation of ES and iPS cells. These findings will provide a new research strategy for studying cilia biology, including ciliogenesis and ciliopathies.

Lower developmental potential of rat zygotes produced by ooplasmic injection of testicular spermatozoa versus cauda epididymal spermatozoa.

Intracytoplasmic sperm injection (ICSI) is clinically used to treat obstructive/nonobstructive azoospermia. This study compared the efficacy of ICSI with cauda epididymal and testicular sperm in Wistar (WI) and Brown-Norway (BN) rats. The transfer of ICSI oocytes with cryopreserved epididymal and testicular WI sperm resulted in offspring production of 26.2% and 3.7%-4.7%, respectively (P < 0.05). Treatments for artificial oocyte activation (AOA) and acrosome removal improved pronuclear formation in BN-ICSI oocytes; however, only AOA treatment was effective in producing offspring (3.7%-6.5%). In the case of ICSI with testicular sperm (TESE-ICSI), one offspring (0.6%) was derived from the BN-TESE-ICSI oocytes. The application of AOA or a hypo-osmotic sperm suspension did not improve the production of TESE-ICSI offspring. Thus, outbred WI rat offspring can be produced by using ICSI and less efficiently by using TESE-ICSI. Challenges in producing offspring by using ICSI/TESE-ICSI in inbred BN strain require further investigation.

Erratum: Lower developmental potential of rat zygotes produced by ooplasmic injection of testicular spermatozoa versus cauda epididymal spermatozoa.

J Reprod Dev, Vol. 70, No. 4, p. 255 Table 2 have been corrected. For the bottom of Survived (%) row, the Survived value, which read "69", should be replaced with "86".

AVPV Kiss1 neuron-specific knockdown of purinergic P2X2 receptor suppresses LH surge and ovulation in Kiss1-Cre rats.

Ovulation disorders are a major cause of low pregnancy rates and infertility in humans and livestock. Kisspeptin neurons located in the anteroventral periventricular nucleus (AVPV) are responsible for the generation of the gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) surge and the consequent ovulation in female rodents. The present study aimed to examine whether purinergic neurons are direct upstream stimulators of AVPV kisspeptin neurons that trigger the GnRH/LH surge and consequent ovulation in Kiss1-Cre rats. We specifically knocked down the mRNA expression of the P2rx2 purinergic receptor in AVPV kisspeptin neurons by administering an adeno-associated virus (AAV) vector containing Cre-dependent P2rx2 short hairpin RNA (shRNA) into the AVPV region of ovariectomized (OVX) Kiss1-Cre rats treated with a proestrus level of estradiol-17ß (OVX + high E2) or ovary-intact Kiss1-Cre rats. The E2-induced afternoon LH surge was significantly suppressed by AVPV kisspeptin neuron-specific knockdown of P2rx2 in OVX + high E2 Kiss1-Cre rats compared with scrambled shRNA-treated control OVX + high E2 Kiss1-Cre rats. Furthermore, the specific knockdown of P2rx2 in AVPV kisspeptin neurons largely disrupted the estrous cycle, spontaneous LH surge, and ovulation in ovary-intact Kiss1-Cre rats. These findings suggest that purinergic neurons directly stimulate AVPV kisspeptin neurons via P2X2 receptors (P2RX2) to induce the GnRH/LH surge and consequent ovulation in female rats.

Direct evidence that KNDy neurons maintain gonadotropin pulses and folliculogenesis as the GnRH pulse generator.

The gonadotropin-releasing hormone (GnRH) pulse is fundamental for mammalian reproduction: GnRH pulse regimens are needed as therapies for infertile women as continuous GnRH treatment paradoxically inhibits gonadotropin release. Circumstantial evidence suggests that the hypothalamic arcuate KNDy neurons expressing kisspeptin (encoded by Kiss1), neurokinin B (encoded by Tac3), and dynorphin A serve as a GnRH pulse generator; however, no direct evidence is currently available. Here, we show that rescuing >20% KNDy neurons by transfecting Kiss1 inside arcuate Tac3 neurons, but not outside of these neurons, recovered folliculogenesis and luteinizing hormone (LH) pulses, an indicator of GnRH pulses, in female global Kiss1 knockout (KO) rats and that >90% conditional arcuate Kiss1 KO in newly generated Kiss1-floxed rats completely suppressed LH pulses. These results first provide direct evidence that KNDy neurons are the GnRH pulse generator, and at least 20% of KNDy neurons are sufficient to maintain folliculogenesis via generating GnRH/gonadotropin pulses.

LGI1-ADAM22-MAGUK configures transsynaptic nanoalignment for synaptic transmission and epilepsy prevention.

Physiological functioning and homeostasis of the brain rely on finely tuned synaptic transmission, which involves nanoscale alignment between presynaptic neurotransmitter-release machinery and postsynaptic receptors. However, the molecular identity and physiological significance of transsynaptic nanoalignment remain incompletely understood. Here, we report that epilepsy gene products, a secreted protein LGI1 and its receptor ADAM22, govern transsynaptic nanoalignment to prevent epilepsy. We found that LGI1-ADAM22 instructs PSD-95 family membrane-associated guanylate kinases (MAGUKs) to organize transsynaptic protein networks, including NMDA/AMPA receptors, Kv1 channels, and LRRTM4-Neurexin adhesion molecules. Adam22ΔC5/ΔC5 knock-in mice devoid of the ADAM22-MAGUK interaction display lethal epilepsy of hippocampal origin, representing the mouse model for ADAM22-related epileptic encephalopathy. This model shows less-condensed PSD-95 nanodomains, disordered transsynaptic nanoalignment, and decreased excitatory synaptic transmission in the hippocampus. Strikingly, without ADAM22 binding, PSD-95 cannot potentiate AMPA receptor-mediated synaptic transmission. Furthermore, forced coexpression of ADAM22 and PSD-95 reconstitutes nano-condensates in nonneuronal cells. Collectively, this study reveals LGI1-ADAM22-MAGUK as an essential component of transsynaptic nanoarchitecture for precise synaptic transmission and epilepsy prevention.

Germline development in rat revealed by visualization and deletion of Prdm14.

Primordial germ cells (PGCs), the founder cells of the germline, are specified in pre-gastrulating embryos in mammals, and subsequently migrate towards gonads to mature into functional gametes. Here, we investigated PGC development in rats, by genetically modifying Prdm14, a unique marker and an essential PGC transcriptional regulator. We trace PGC development in rats, for the first time, from specification until the sex determination stage in fetal gonads using Prdm14 H2BVenus knock-in rats. We uncover that the crucial role of Prdm14 in PGC specification is conserved between rat and mice, by analyzing Prdm14-deficient rat embryos. Notably, loss of Prdm14 completely abrogates the PGC program, as demonstrated by failure of the maintenance and/or activation of germ cell markers and pluripotency genes. Finally, we profile the transcriptome of the post-implantation epiblast and all PGC stages in rat to reveal enrichment of distinct gene sets at each transition point, thereby providing an accurate transcriptional timeline for rat PGC development. Thus, the novel genetically modified rats and data sets obtained in this study will advance our knowledge on conserved versus species-specific features for germline development in mammals.

Characterization of homozygous Foxn1 mutations induced in rat embryos by different delivery forms of Cas9 nuclease.

BACKGROUND: The Cas9 nuclease is delivered in the form of either Cas9 protein or mRNA along with CRISPR guide RNA (gRNA: dual-crRNA:tracrRNA or chimeric single-guide RNA) or in a plasmid package encoding both Cas9 and the CRISPR gRNA. METHODS AND RESULTS: We directly compared the efficiency of producing rat blastocysts with homozygous mutations of the Foxn1 locus by pronuclear injection of Cas9 in the form of protein, mRNA, or plasmid DNA. For highly efficient production of rat blastocysts with homozygous Foxn1 mutations, pronuclear injection of Cas9 protein at 60 ng/µl was likely optimal. While blastocyst harvest in the mRNA groups was higher than those in the protein and plasmid DNA groups, genotype analysis showed that 63.6%, 8.7-20.0%, and 25.0% of the analyzed blastocysts were homozygous mutants in the protein, mRNA, and plasmid DNA groups, respectively. The high efficiency of producing homozygous mutant blastocysts in the 60 ng/µl protein group may be associated with primary genome editing being initiated before the first cleavage. In most cases, homozygous mutations at the target Foxn1 locus are triggered by deletion and repair via nonhomologous end joining or microhomology-mediated end joining. Deletion downstream of the Cas9 break site was more likely than deletion in the upstream direction. CONCLUSIONS: The Cas9 nuclease in protein form, when coinjected with the CRISPR gRNA (ribonucleoprotein) into a rat zygote pronucleus, can access the target genome site and induce double-strand breaks promptly, resulting in the efficient production of homozygous mutants.

Interspecies organogenesis generates autologous functional islets.

Islet transplantation is an established therapy for diabetes. We have previously shown that rat pancreata can be created from rat pluripotent stem cells (PSCs) in mice through interspecies blastocyst complementation. Although they were functional and composed of rat-derived cells, the resulting pancreata were of mouse size, rendering them insufficient for isolating the numbers of islets required to treat diabetes in a rat model. Here, by performing the reverse experiment, injecting mouse PSCs into Pdx-1-deficient rat blastocysts, we generated rat-sized pancreata composed of mouse-PSC-derived cells. Islets subsequently prepared from these mouse-rat chimaeric pancreata were transplanted into mice with streptozotocin-induced diabetes. The transplanted islets successfully normalized and maintained host blood glucose levels for over 370 days in the absence of immunosuppression (excluding the first 5 days after transplant). These data provide proof-of-principle evidence for the therapeutic potential of PSC-derived islets generated by blastocyst complementation in a xenogeneic host.

Sex difference in developmental changes in visualized Kiss1 neurons in newly generated Kiss1-Cre rats.

Hypothalamic kisspeptin neurons are master regulators of mammalian reproduction via direct stimulation of gonadotropin-releasing hormone and consequent gonadotropin release. Here, we generated novel Kiss1 (kisspeptin gene)-Cre rats and investigated the developmental changes and sex differences in visualized Kiss1 neurons of Kiss1-Cre-activated tdTomato reporter rats. First, we validated Kiss1-Cre rats by generating Kiss1-expressing cell-specific Kiss1 knockout (Kiss1-KpKO) rats, which were obtained by crossing the current Kiss1-Cre rats with Kiss1-floxed rats. The resulting male Kiss1-KpKO rats lacked Kiss1 expression in the brain and exhibited hypogonadotropic hypogonadism, similar to the hypogonadal phenotype of global Kiss1 KO rats. Histological analysis of Kiss1 neurons in Kiss1-Cre-activated tdTomato reporter rats revealed that tdTomato signals in the anteroventral periventricular nucleus (AVPV) and arcuate nucleus (ARC) were not affected by estrogen, and that tdTomato signals in the ARC, AVPV, and medial amygdala (MeA) were sexually dimorphic. Notably, neonatal AVPV tdTomato signals were detected only in males, but a larger number of tdTomato-expressing cells were detected in the AVPV and ARC, and a smaller number of cells in the MeA was detected in females than in males at postpuberty. These findings suggest that Kiss1-visualized rats can be used to examine the effect of estrogen feedback mechanisms on Kiss1 expression in the AVPV and ARC. Moreover, the Kiss1-Cre and Kiss1-visualized rats could be valuable tools for further detailed analyses of sexual differentiation in the brain and the physiological role of kisspeptin neurons across the brain in rats.

Generation of Tfap2c-T2A-tdTomato knock-in reporter rats via adeno-associated virus-mediated efficient gene targeting.

Gene editing in mammalian zygotes enables us to generate genetically modified animals rapidly and efficiently. In this study, we compare multiple gene targeting strategies in rat zygotes by generating a novel knock-in reporter rat line to visualize the expression pattern of transcription factor AP-2 gamma (Tfap2c). The targeting vector is designed to replace the stop codon of Tfap2c with T2A-tdTomato sequence. We show that the combination of electroporation-mediated transduction of CRISPR/Cas9 components with adeno-associated virus-mediated transduction of the targeting vector is the most efficient in generating the targeted rat line. The Tfap2c-T2A-tdTomato fluorescence reflects the endogenous expression pattern of Tfap2c in preimplantation embryo, germline, placenta, and forebrain during rat embryo development. The reporter line generated here will be a reliable resource for identifying and purifying Tfap2c expressing cells in rats, and the gene targeting strategy we used can be widely applied for generating desired animals.

High survival of bovine mature oocytes after nylon mesh vitrification, as assessed by intracytoplasmic sperm injection.

Intracytoplasmic sperm injection (ICSI) is an alternative technique to in vitro fertilization (IVF) for producing transferable blastocysts, especially in combination with cryopreserved oocytes, when the IVF system does not work sufficiently. The present study was conducted to directly compare the efficacy of producing bovine blastocysts by ICSI and IVF from vitrified-warmed and fresh oocytes. Denuded oocytes with a detectable first polar body were vitrified-warmed using a nylon mesh device. In the non-vitrified control group, blastocyst yields 8 days after IVF and ICSI were 32.0 and 26.8%, respectively. Oocyte vitrification and subsequent IVF resulted in an impaired blastocyst yield (15.0%); however, such a loss of efficacy due to vitrification was not observed in the ICSI group (blastocyst yield, 25.2%). The alignment of cortical granules beneath the oolemma was comparable between the fresh control and vitrified-warmed oocytes. Here, we report the high survival of vitrified-warmed bovine oocytes, as assessed by ICSI.

Establishment of embryo transfer in the musk shrew (Suncus murinus).

The present study established techniques to induce pseudopregnancy, in vitro oocyte cultures from pronuclear to 2- to 4-cell stages, and embryo transfer in musk shrews, a reflex ovulator. Offspring were subsequently obtained by transferring in vivo-developed or in vitro-cultured embryos. Female musk shrews received human chronic gonadotropin (hCG), with or without mating stimuli, from vasectomized males to produce pseudopregnant recipients. Embryos at the 2- to 4-cell stage were collected 44-48 h after mating. Another set of embryos was collected 26-27 h after mating and then cultured for 20 h from the pronuclear to 2- to 4-cell stages. Subsequently, embryos were transferred into the oviducts of pseudopregnant recipients 24 or 48 h after the induction of pseudopregnancy. Offsprings were successfully obtained from recipients that received hCG 24 h before embryo transfer, regardless of mating stimuli. These techniques may be valuable for producing transgenic musk shrews.

Kiss1-dependent and independent release of luteinizing hormone and testosterone in perinatal male rats.

Prenatal and postnatal biphasic increases in plasma testosterone levels derived from perinatal testes are considered critical for defeminizing/masculinizing the brain mechanism that regulates sexual behavior in male rats. Hypothalamic kisspeptin neurons are indispensable for stimulating GnRH and downstream gonadotropin, as well as the consequent testicular testosterone production/release in adult male rats. However, it is unclear whether kisspeptin is responsible for the increase in plasma testosterone levels in perinatal male rats. The present study aimed to investigate the role of Kiss1/kisspeptin in generating perinatal plasma LH and the consequent testosterone increase in male rats by comparing the plasma testosterone and LH profiles of wild-type (Kiss1+/+) and Kiss1 knockout (Kiss1-/-) male rats. A biphasic pattern of plasma testosterone levels, with peaks in the prenatal and postnatal periods, was found in both Kiss1+/+ and Kiss1-/- male rats. Postnatal plasma testosterone and LH levels were significantly lower in Kiss1-/- male rats than in Kiss1+/+ male rats, whereas the levels in the prenatal embryonic period were comparable between the genotypes. Exogenous kisspeptin challenge significantly increased plasma testosterone and LH levels and the number of c-Fos-immunoreactive GnRH neurons in neonatal Kiss1-/- and Kiss1+/+ male rats. Kiss1 and Gpr54 (kisspeptin receptor gene) were found in the testes of neonatal rats, but kisspeptin treatment failed to stimulate testosterone release in the cultured testes of both genotypes. These findings suggest that postnatal, but not prenatal, testosterone increase in male rats is mainly induced by central kisspeptin-dependent stimulation of GnRH and consequent LH release.

Humanized UGT2 and CYP3A transchromosomic rats for improved prediction of human drug metabolism.

Although "genomically" humanized animals are invaluable tools for generating human disease models as well as for biomedical research, their development has been mainly restricted to mice via established transgenic-based and embryonic stem cell-based technologies. Since rats are widely used for studying human disease and for drug efficacy and toxicity testing, humanized rat models would be preferred over mice for several applications. However, the development of sophisticated humanized rat models has been hampered by the difficulty of complex genetic manipulations in rats. Additionally, several genes and gene clusters, which are megabase range in size, were difficult to introduce into rats with conventional technologies. As a proof of concept, we herein report the generation of genomically humanized rats expressing key human drug-metabolizing enzymes in the absence of their orthologous rat counterparts via the combination of chromosome transfer using mouse artificial chromosome (MAC) and genome editing technologies. About 1.5 Mb and 700 kb of the entire UDP glucuronosyltransferase family 2 and cytochrome P450 family 3 subfamily A genomic regions, respectively, were successfully introduced via the MACs into rats. The transchromosomic rats were combined with rats carrying deletions of the endogenous orthologous genes, achieved by genome editing. In the "transchromosomic humanized" rat strains, the gene expression, pharmacokinetics, and metabolism observed in humans were well reproduced. Thus, the combination of chromosome transfer and genome editing technologies can be used to generate fully humanized rats for improved prediction of the pharmacokinetics and drug-drug interactions in humans, and for basic research, drug discovery, and development.

Acellular matrix derived from rat liver improves the functionality of rat pancreatic islets before or after vitrification.

Cryopreservation of pancreatic islets can overcome the severe shortage of islet donors in clinical islet transplantation, but the impaired quality of post-warm islets need improvement. This present study was conducted to investigate whether the pre- or post-treatment of rat islets with liver decellularized matrix (LDM) for vitrification can improve the viability (FDA/PI double staining) and the functionality (glucose-stimulated insulin secretion [GSIS] assay). Rat LDM was prepared by high-hydrostatic pressure, lyophilization, and re-suspension in saline. Co-culturing of isolated islets with 0 (control), 30, 60, or 90 µg/ml LDM for 24 h resulted in the comparable viability among the 4 groups (98.7-99.6%) and the higher insulin secretion potential in 30 and 60 µg/ml LDM treatment groups than the control group (stimulation index [SI]: 12.1 and 12.7, respectively, vs. 6.5 in the control group, P < 0.05). When the islets co-cultured with 60 µg/ml LDM were vitrified-warmed on a nylon mesh cryodevice, the viability and the GSIS of the post-warm islets were not improved. Post-treatment of vitrified-warmed islets with 60 µg/ml LDM during the recovery culture for 12 h resulted in the comparable clearance of degenerating cell debris from the post-warm islets, while their insulin secretion potential was improved (SI: 5.0 vs. 3.5 in the control group, P < 0.05). These findings indicate that the components in LDM can enhance the insulin secretion potential of rat islets suffering damage by enzymatic stress during the islet isolation process or by cryoinjuries during the vitrification-warming process.

Rescue of vitrified-warmed bovine mature oocytes by short-term recovery culture with resveratrol.

Resveratrol, a well-known antioxidant, has been reported to protect mouse metaphase-II (M - II) stage oocytes from vitrification injuries when used as a treatment during a series of vitrification processes. The present study was conducted to investigate whether short-term treatment of post-warm bovine mature oocytes with resveratrol can increase blastocyst formation rate following in vitro fertilization and culture. Bovine denuded M - II oocytes were vitrified-warmed using Cryotop® or nylon mesh (pore size = 37 µm) as a cryodevice. The post-warm oocytes were treated for 2 h with 1 µM resveratrol in recovery culture medium. The resveratrol treatment had no harmful influence on morphological survival and cleavage rate of the oocytes vitrified-warmed with Cryotop® or nylon mesh. In the Cryotop® vitrification series, blastocyst formation rate of resveratrol-treated post-warm oocytes (39.0%) was not significantly different from that of non-treated post-warm oocytes (31.7%). However in the nylon mesh vitrification series, there was a significant increase in the blastocyst yield (42.4% vs. 31.3%, P < 0.05) when post-warm oocytes were treated with resveratrol. Blastocyst yield from fresh control oocytes was 49%. Levels of reactive oxygen species were comparable between post-warm and fresh control M - II oocytes, and decreased in oocytes after recovery culture with resveratrol. Mitochondrial activity of post-warm oocytes was restored to the pre-vitrification level during the recovery culture regardless of resveratrol supplementation. Thus, short-term recovery culture with resveratrol can rescue bovine M - II oocytes vitrified-warmed on a nylon mesh cryodevice.