Developmental competence and neonatal outcomes of nonpronuclear zygotes following single vitrified-warmed blastocyst transfers using propensity score matching analysis.

PURPOSE: To investigate developmental competence and neonatal outcomes of nonpronuclear (0PN) zygotes following single vitrified-warmed blastocyst transfers (VBT). METHODS: The clinical, laboratorial and neonatal data of 996 patients with ≤ 38 years who underwent blastocyst culture and single VBT were retrospectively analyzed. The pregnancy and neonatal outcomes of VBT were compared between 0PN and 2PN blastocysts using propensity score matching (PSM). Moreover, Day 3 (D3) embryo development and blastocyst formation were compared between 0PN and 2PN zygotes. RESULTS: There were no significant differences in clinical pregnancy rate (CPR), live birth rate (LBR) and neonatal outcomes of VBT between the 0PN and 2PN blastocysts irrespectively of whether PSM was used. However, early abortion rate (EAR) was higher in blastocysts from 0PN D3 embryos > 10 cells (p < 0.05) before PSM. Moreover, the early developmental competence of 0PN zygotes was different from that of 2PN zygotes presenting higher percentages of D3 embryos ≤ 6 cells (p < 0.01) and > 10 cells (p < 0.01), lower available blastocyst formation rate (ABFR) (p < 0.01) and good-quality blastocyst formation rate (GBFR) (p < 0.01) in D3 embryos with 4-6 cells. ABFR and GBFR increased with cell number when compared among embryos with 4-6 cells, 7-10 cells and > 10 cells, irrespectively of 0PN or 2PN embryos. CONCLUSION: The early developmental competence of 0PN zygotes was different from that of 2PN zygotes, but did not influence pregnancy and neonatal outcomes following VBT. ABFR and GBFR increased with cell number, irrespectively of 0PN or 2PN embryos.

Association of serum progesterone levels on the transfer day with pregnancy outcomes in hormone replacement frozen-thawed cycles with oral dydrogesterone for strengthened luteal phase support.

OBJECTIVE: To investigate the relationship between serum progesterone (P) levels on the day of blastocyst transfer and pregnancy outcomes in frozen-thawed embryo transfer (FET) cycles using hormone replacement therapy (HRT) with oral dydrogesterone for strengthened luteal phase support (LPS). MATERIALS AND METHODS: This was a retrospective study including 1176 FET cycles. All patients received 40 mg of intramuscular (IM) P daily for endometrium transformation plus oral dydrogesterone 10 mg BID from transfer day for strengthened LPS. Pregnancy outcomes were compared between serum P levels on the transfer day ≥10 ng/ml and <10 ng/ml. Furthermore, cycles were divided into 10 groups by deciles of P and ongoing pregnancy rate (OPR) was calculated in each group. Analyses using deciles of serum P were completed to see if these could create further prognostic power. RESULTS: No differences were observed in clinical pregnancy rates (CPRs), OPRs and live birth rates (LBRs) between serum P levels ≥10 ng/ml and <10 ng/ml. Patients with serum P levels <5.65 ng/ml (10th percentile) had a significantly lower OPR (48.31% vs. 58.98%, p = 0.03) and LBR (43.22% vs. 57.75%, p = 0.003) than the rest of the patients. Multivariate logistic regression analysis showed serum P levels on the transfer day were not associated with pregnancy outcomes. CONCLUSION: Measuring serum P levels on the day of HRT-FET is of clinical importance. Lower serum P levels impact the success of HRT-FET cycles, suggesting that there may be a threshold below which it is difficult to improve pregnancy outcomes via oral dydrogesterone to strengthen LPS.

A Comprehensive Sequencing Analysis of Testis-Born miRNAs in Immature and Mature Indigenous Wandong Cattle (Bos taurus).

Micro RNAs (miRNAs) have been recognized as important regulators that are indispensable for testicular development and spermatogenesis. miRNAs are endogenous transcriptomic elements and mainly regulate the gene expression at post-transcriptional levels; however, the key role of miRNA in bovine testicular growth is not clearly understood. Thus, supposing to unveil the transcriptomics expression changes in the developmental processes of bovine testes, we selected three immature calves and three sexually mature bulls of the local Wandong breed for testicular-tissue sample collection. The cDNA libraries of experimental animals were established for RNA-sequencing analysis. We detected the miRNA expression in testes by using high-throughput sequencing technology, and bioinformatics analysis followed. The differentially expressed (DE) data showed that 151 miRNAs linked genes were significantly DE between immature and mature bull testes. Further, in detail, 64 were significantly up-regulated and 87 were down-regulated in the immature vs. mature testes (p-value < 0.05). Pathway analyses for miRNA-linked genes were performed and identified JAG2, BCL6, CFAP157, PHC2, TYRO3, SEPTIN6, and BSP3; these genes were involved in biological pathways such as TNF signaling, T cell receptor, PI3KAkt signaling, and functions affecting testes development and spermatogenesis. The DE miRNAs including MIR425, MIR98, MIR34C, MIR184, MIR18A, MIR136, MIR15A, MIR1388 and MIR210 were associated with cattle-bull sexual maturation and sperm production. RT-qPCR validation analysis showed a consistent correlation to the sequencing data findings. The current study provides a good framework for understanding the mechanism of miRNAs in the development of testes and spermatogenesis.

Chromatin remodeler INO80 mediates trophectoderm permeability barrier to modulate morula-to-blastocyst transition.

Inositol requiring mutant 80 (INO80) is a chromatin remodeler that regulates pluripotency maintenance of embryonic stem cells and reprogramming of somatic cells into pluripotent stem cells. However, the roles and mechanisms of INO80 in porcine pre-implantation embryonic development remain largely unknown. Here, we show that INO80 modulates trophectoderm epithelium permeability to promote porcine blastocyst development. The INO80 protein is highly expressed in the nuclei during morula-to-blastocyst transition. Functional studies revealed that RNA interference (RNAi)-mediated knockdown of INO80 severely blocks blastocyst formation and disrupts lineage allocation between the inner cell mass and trophectoderm. Mechanistically, single-embryo RNA sequencing revealed that INO80 regulates multiple genes, which are important for lineage specification, tight junction assembly, and fluid accumulation. Consistent with the altered expression of key genes required for tight junction assembly, a permeability assay showed that paracellular sealing is defective in the trophectoderm epithelium of INO80 knockdown blastocysts. Importantly, aggregation of 8-cell embryos from the control and INO80 knockdown groups restores blastocyst development and lineage allocation via direct complementation of the defective trophectoderm epithelium. Taken together, these results demonstrate that INO80 promotes blastocyst development by regulating the expression of key genes required for lineage specification, tight junction assembly, and fluid accumulation.

Integrated Analysis of Long Non-Coding RNA and mRNA Expression Profiles in Testes of Calves and Sexually Mature Wandong Bulls (Bos taurus).

The mRNAs and long non-coding RNAs axes are playing a vital role in the regulating of post-transcriptional gene expression. Thereby, elucidating the expression pattern of mRNAs and long non-coding RNAs underlying testis development is crucial. In this study, mRNA and long non-coding RNAs expression profiles were investigated in 3-month-old calves and 3-year-old mature bulls' testes by total RNA sequencing. Additionally, during the gene level analysis, 21,250 mRNAs and 20,533 long non-coding RNAs were identified. As a result, 7908 long non-coding RNAs (p-adjust < 0.05) and 5122 mRNAs (p-adjust < 0.05) were significantly differentially expressed between the distinct age groups. In addition, gene ontology and biological pathway analyses revealed that the predicted target genes are enriched in the lysine degradation, cell cycle, propanoate metabolism, adherens junction and cell adhesion molecules pathways. Correspondingly, the RT-qPCR validation results showed a strong consistency with the sequencing data. The source genes for the mRNAs (CCDC83, DMRTC2, HSPA2, IQCG, PACRG, SPO11, EHHADH, SPP1, NSD2 and ACTN4) and the long non-coding RNAs (COX7A2, COX6B2, TRIM37, PRM2, INHBA, ERBB4, SDHA, ATP6VOA2, FGF9 and TCF21) were found to be actively associated with bull sexual maturity and spermatogenesis. This study provided a comprehensive catalog of long non-coding RNAs in the bovine testes and also offered useful resources for understanding the differences in sexual development caused by the changes in the mRNA and long non-coding RNA interaction expressions between the immature and mature stages.

Dynamic changes of miRNAs in skeletal muscle development at New Zealand rabbits.

BACKGROUND: miRNA is one of the crucial roles in the complex and dynamic network that regulates the development of skeletal muscle. The landscape of skeletal muscle miRNAs from fetus to adult in New Zealand rabbits has not been revealed yet. RESULTS: In this study, nine RNA-seq libraries of fetus, child and adult rabbits' leg muscles were constructed. A total of 278 differentially expressed miRNAs (DEmiRNAs) were identified. In the fetus vs. child group, the main functional enrichments were involved in membrane and transport. Pathway enriched terms of up-regulated DEmiRNAs were connected with the differentiation and hypertrophy of skeletal muscle, and down-regulated ones were related to muscle structure and metabolic capacity. In the child vs. adult group, functions were associated to positioning and transportation, and pathways were relevant to ECM, muscle structure and hypertrophy. Finally, ocu-miR-185-3p and ocu-miR-370-3p, which had the most target genes, were identified as hub-miRNAs in these two groups. CONCLUSIONS: In short, we summarized the highly expressed and uniquely expressed DEmiRNAs of fetus, child and adult rabbits' leg muscles. Besides, the potential functional changes of miRNAs in two consecutive stages have been explored. Among them, the ocu-miR-185-3p and ocu-miR-370-3p with the most target genes were selected as hub-miRNAs. These data improved the understanding of the regulatory molecules of meat rabbit development, and provided a novel perspective for molecular breeding of meat rabbits.

Histone Arginine Methyltransferase CARM1-Mediated H3R26me2 Is Essential for Morula-to-Blastocyst Transition in Pigs.

Coactivator-associated arginine methyltransferase 1 (CARM1) is involved in both establishment of first pluripotent lineage and pluripotency maintenance of embryonic stem cells (ESCs) in mice. However, the histone substrates and role of CARM1 in early embryonic development remain largely unknown. Here, we show that CARM1 specifically catalyzes H3R26me2 to promote porcine blastocyst formation. The putative histone substrates of CARM1, including H3R2me2, H3R17me2, and H3R26me2, are present in pig early embryos. The changes of CARM1 mRNA during early embryogenesis parallel that of H3R26me2. Functional studies using a combinational approach of chemical inhibition and RNA interference (RNAi) showed that catalytic activity inhibition of CARM1 protein or knockdown (KD) of CARM1 mRNA did not alter the levels of both H3R2me2 and H3R17me2, but significantly reduced H3R26me2 levels in porcine embryos. Furthermore, CARM1 inhibition or KD did not affect embryo development to the 2-cell, 4-cell, 8-cell, and morula stages, but severely compromised blastocyst development. CARM1 knocked down embryos that developed to the blastocyst stage had fewer total cells, inner cell mass (ICM), and trophectoderm (TE) cells. Mechanistically, single embryo RNA-sequencing analysis revealed that CARM1 KD altered the transcriptome characterized by downregulation of key genes associated with Hippo and PI3K-AKT signaling pathways. Taken together, these results demonstrate that CARM1 specifically catalyzes H3R26me2 in porcine embryos and participates in blastocyst development.

The protective effects of human umbilical cord mesenchymal stem cell-derived extracellular vesicles on cisplatin-damaged granulosa cells.

OBJECTIVE: Long term exposure to gonadotoxic chemotherapy is becoming a major cause of premature ovarian failure/insufficiency (POF/POI) with the increasing cancer incidence among young women. The present study was designed to investigate the protective effects of human cord mesenchymal stem cells (HUCMSCs)-derived extracellular vesicles (EVs) on cisplatin (CDDP)-damaged granulosa cells (GCs) in vitro. MATERIALS AND METHODS: EVs were obtained from supernatant of cultured HUCMSCs by ultracentrifugation method, purified by Sucrose density gradient centrifugation, and then were co-cultured with cisplatin-damaged GCs of 3-weeks female Sprague-Dawley (SD) rats. PKH26 labeled EVs could be observed in normal and CDDP-damaged GCs after 6 h co-culture. RESULTS: The surviving GCs were significantly higher and apoptotic GCs were significantly lower in EVs + CDDP group compared with CDDP group. Meanwhile, the levels of E2 and StAR (the key gene related to synthesis of steroid hormone) were significantly higher in EVs + CDDP group compared with CDDP group. Furthermore, the mRNA expression of Caspase 3 was down-regulated significantly and the ratio of Bcl-2/Bax was up-regulated significantly in EVs + CDDP group. Moreover, the protective effect of EVs on CDDP-damaged GCs showed a dose-dependent effect. CONCLUSION: HUCMSCs-derived EVs could become incorporated to CDDP-damaged GCs, and increase the number of living cells, therefore playing important roles in promoting resistance to cisplatin-induced GCs apoptosis and restoring synthesis and secretion of steroid hormone in GCs. This study might provide a theoretical and experimental basis for use of mesenchymal stem cells (MSCs) derived EVs instead of MSCs as a cell-free therapeutic strategy for the patients with POI induced by chemotherapeutic agents.

Extracellular Vesicles Derived from Mesenchymal Stem Cells Recover Fertility of Premature Ovarian Insufficiency Mice and the Effects on their Offspring.

It has been reported that extracellular vesicles (EVs) derived from human umbilical cord mesenchymal stem cells (HUCMSCs) can promote the proliferative and secretive functions of granulosa cells. In vivo study further demonstrated that EVs derived from HUCMSCs can not only promote the angiogenesis of ovarian tissue but also restore the function of an ovary of chemically induced premature ovarian insufficiency (POI) mice. However, no study investigates the effects of HUCMSCs derived EVs on fertility recovery of POI mice and evaluating their offspring. This study investigates the effects of HUCMSCs derived EVs on fertility recovery and the cognitive function of their offspring. A POI model was established by intraperitoneal injection of cyclophosphamide (CTX) and busulfan (BUS), and randomly divided into EVs-transplantation group (a single injection of 150 µg EVs proteins which suspended in 0.1 ml phosphate buffer saline [PBS] via tail vein), POI group (a single injection of 0.1 ml PBS via tail vein), and normal control group (a single injection of 0.1 ml PBS via tail vein without intraperitoneal injection of CTX and BUS). After EVs treatment, not only the ovarian function of POI mice recovered but also the fertility increased with less time to get pregnant, evaluating by in vitro fertilization and mating test. Cognitive behaviors of the offspring were similar among the three groups through the Y-maze test and novel object recognition task. An anti-apoptotic effect was identified through immunohistochemistry, real-time polymerase chain reaction and western blot. These findings indicate that HUCMSCs derived EVs can improve the fertility of POI mice without adverse effects on the cognitive behavior of their offspring, highlighting the potential value of EVs to be a cell-free therapy for patients suffering from POI.

Cumulative live birth rate of advanced-age women more than 40 with or without poor ovarian response.

OBJECTIVE: The aim of this study was to investigate cumulative live birth rate (CLBR) per oocyte retrieval cycle and per patient in women over 40 years old undergoing IVF/ICSI treatments, stratified for age, ovarian response and oocyte retrieval cycle number. MATERIALS AND METHODS: 244 patients with poor ovarian response (POR) and 372 patients with normal ovarian response (NOR) were retrospectively investigated. RESULTS: Of the patients aged 40 to 43 years, CLBR per oocyte retrieval cycle and per patient (4.3%; 8.8%) in POR group were both lower than those in NOR group (15.8%; 24.8%) (P < 0.01). No significant differences in live birth rate (LBR) per oocyte retrieval cycle or CLBR per patient were observed in the group of POR patients irrespective of oocyte retrieval cycles they underwent. Similarly, CLBR per patient in NOR group did not increase significantly with the oocyte retrieval cycle number. However, LBR per oocyte retrieval cycle in the first cycle (Cycle 1, 20.3%) was significantly higher than that in the second cycle (Cycle 2, 9.2%) and the third cycle (Cycle 3, 4.4%) (P < 0.01). And 94.8% (73/77) of live births were achieved during the first two cycles. Of the patients aged 44 to 45 years and over 45 years old, there were no significant differences in CLBR per oocyte retrieval cycle or per patient between POR and NOR groups. CONCLUSION: Relatively higher cumulative live birth rate was only found in the patients aged 40 to 43 years without poor ovarian response. These findings may provide some information that further sub-classification of advance-age women according to ovarian response may help both clinicians and patients to balance decision-making about their infertility treatment.

Double ovarian stimulation during the follicular and luteal phase in women ≥38 years: a retrospective case-control study.

Previous studies have shown that double ovarian stimulation could obtain more oocytes in women with poor ovarian response. This retrospective case-control study aimed to investigate the efficacy of double ovarian stimulation in older women. One hundred and sixteen women aged ≥38 years who received double ovarian stimulation were assigned to the study, with 103 divided into four groups according to follicular-phase ovarian stimulation protocols, including gonadotrophin-releasing hormone agonist short protocol (n = 27), gonadotrophin-releasing hormone antagonist protocol (n = 32), mild stimulation protocol (n = 21) and medrocyprogesterone acetate (MPA) pituitary down-regulation protocol (n = 23). Numbers of oocytes retrieved and available embryos after double ovarian stimulation were more than double those obtained after follicular-phase ovarian stimulation alone. In total 81.90% of patients had available embryos, and the cancellation rate decreased from 37.07% to 18.10%. Forty-eight cases underwent 50 cryopreserved embryo transfer cycles, with a 22.00% clinical pregnancy rate. The implantation rate (10.53% versus 10.67%) was similar between the embryos derived from first and second stimulations. The results suggest that double ovarian stimulation could increase the chances of achieving pregnancy by accumulating more oocytes/embryos in a short time, which might serve as a useful strategy for older women.

The effects of blastocyst morphological score and blastocoele re-expansion speed after warming on pregnancy outcomes.

OBJECTIVE: The aim of this study was to investigate associations between the morphology score of blastocysts and blastocoele re-expansion speed after warming with clinical outcomes, which could assist in making correct and cost-effective decisions regarding the appropriate time to vitrify blastocysts and to transfer vitrified-warmed blastocysts. METHODS: A total of 327 vitrified-warmed two-blastocyst transfer cycles in women 38 years old and younger were included in this retrospective study. RESULTS: The clinical pregnancy rate (CPR) and implantation rate (IR) of transfers of two good-morphology grade 4 blastocysts vitrified on day 5 (64.1% and 46.8%, respectively) were significantly higher than the CPR and IR associated with the transfers of two good-morphology grade 3 blastocysts vitrified on day 5 (46.7% and 32.2%, respectively). No significant differences were found in the CPR and IR among the transfers of two good-morphology grade 4 blastocysts regardless of the day of cryopreservation. Logistic regression analysis showed that blastocoele re-expansion speed after warming was associated with the CPR. CONCLUSION: The selection of a good-morphology grade 4 blastocyst to be vitrified could be superior to the choice of a grade 3 blastocyst. Extending the culture of grade 3 blastocysts and freezing grade 4 or higher blastocysts on day 6 could lead to a greater likelihood of pregnancy. Since re-expansion was shown to be a morphological marker of superior blastocyst viability, blastocysts that quickly re-expand after warming should be prioritized for transfer.

Vitrification of in vitro matured oocytes collected from surplus ovarian medulla tissue resulting from fertility preservation of ovarian cortex tissue.

PURPOSE: The aim of the study was to investigate the maturation rate of immature oocytes collected from ovarian medulla tissue normally discarded during preparation of ovarian cortical tissue for fertility preservation. Further we evaluated survival of derived MII oocytes following vitrification and warming. METHODS: 36 patients aged from 8 to 41 years who had one ovary excised for fertility preservation were included. Oocytes were collected from the medulla tissue and matured in vitro 44-48 h followed by vitrification. Number of oocytes collected, the rates of maturation and post-warming survival were assessed. RESULTS: On average, 11 immature oocytes were collected per patient. The overall maturation rate was 29 % irrespective of whether the ovary was transported 4-5 h on ice or obtained immediately after oophorectomy. The maturation rate in patients below 20 years of age (55 %) was significantly higher than that of patients aged 20-30 years (29 %) and above 30 years (26 %). The post-warming survival rate was 64 %. No significant relationship was observed between the number of collected oocytes and the age of patients. CONCLUSIONS: Approximately three MII oocytes were obtained per patient following in vitro maturation (IVM) of immature oocytes collected from medulla tissue, of which two survived vitrification and warming. This approach represents an add-on method to potentially augment the fertility opportunity for cancer patients, especially in young women with cancer where transplantation of cortical tissue may pose a risk of relapse, but the IVM approach is currently too inefficient to be the only method used for fertility preservation.

[Application Prospect of Stem Cell-derived Microvesicles in Regeneration of Injured Tissues].

More and more evidence indicates that microvesicles (MVs) play a key role in cell-to-cell communication. The MVs are circular fragments of membrane released from the endosomal compartment as exosomes or shed from the cell surface membranes of most types. Components of donor cells are incorporated into MVs that contain bioactive lipids, proteins, genetic cargoes. MVs derived from stem cells may reprogram cells that survived in injury tissue and favor tissue regeneration by delivering their bioactive cargoes to influence the behaviors of recipient cells. Compared with mesenchymal stem cells (MSCs), MVs derived from MSCs were found to mimic the beneficial effects of these cells. These proregenerative effects mediated by MVs can be explained by the fact that MVs are enriched in bioactive lipids, anti-apoptotic and pro-stimulatory growth factors or cytokines, and deliver mRNAs, regulatory miRNAs and proteins that improve overall cell function. Therefore, it opens novel perspectives in exploiting these MVs in tissue regeneration and repair. In addition, the use of MVs instead of stem cells could represent a safe and potentially more advantageous alternative to cell-therapy approaches.

The effects of fertilization mode, embryo morphology at day 3, and female age on blastocyst formation and the clinical outcomes.

The aim of this study was to evaluate the influence of in vitro fertilization (IVF) versus intracytoplasmic sperm injection (ICSI), fertilization mode embryonic morphology at day 3, and female age on blastocyst development, on the clinical outcomes of pregnancy after blastocyst transfer. A total of 471 cycles were retrospectively investigated. The rates of blastocyst formation and of good blastocyst morphology were higher in IVF than in ICSI cycles but there were no significant differences in the clinical pregnancies or in the miscarriage rates. The rates of formation of blastocyst and of blastocysts with good morphology were significantly higher from good-morphology embryos than from poor-morphology embryos. Nevertheless, 16.9% of the poor-morphology embryos reached the blastocyst stage. The total rates of blastocyst formation, and rates of clinical pregnancy and implantation were statistically similar in the age <35, 35-39, and >39 year groups, although tending to decrease with increasing age. When equal numbers of embryos were transferred on day 3, the rates of clinical pregnancy and implantation after blastocyst transfer were significantly higher in the <35 year age group than in the 35-39 and >39 year age groups, which were not significantly different. The miscarriage rates after embryo or blastocyst transfers were not statistically different in groups of similar age. Therefore, extended embryo culture up to the blastocyst stage could be implemented for women aged younger than 35 years to increase the pregnancy rate. For older women, transfer and vitrification of available embryos at day 3 and extended culture of morphologically poor embryos to the blastocyst stage for cryopreservation may improve the clinical outcome.

[Expression of Myc-R9-EGFP fusion protein and validation of its transduction activity].

To construct, express, purify and identify the Myc-R9-EGFP fusion protein and validate its transduction activity in the cultured porcine embryo fibroblasts. cDNA of pig c-Myc gene was amplified by RT-PCR with specific primers of 9 arginine (R9) from the primordial genital ridges and inserted into prokaryotic expression vector pET-28a-EGFP. After DNA sequencing confirmation, the recombinant plasmid was then transformed into BL21 (Escherichia coli) strain. After IPTG induction, the target fusion protein was efficiently induced to express, successfully purified by Novagen His-Bind kit, identified by SDS-PAGE and Western blotting. Finally, its high transduction activity in the porcine embryo fibroblasts was validated. The purified Myc-R9-EGFP fusion protein and the validation of its transduction activity in fibroblasts have provided an experimental foundation for further studies on the biological characterization of Myc protein, and soundly facilitated the further study of establishing pig induced pluripotent stem cells by recombinant protein.

Characterization of bovine induced pluripotent stem cells by lentiviral transduction of reprogramming factor fusion proteins.

Pluripotent stem cells from domesticated animals have potential applications in transgenic breeding. Here, we describe induced pluripotent stem (iPS) cells derived from bovine fetal fibroblasts by lentiviral transduction of Oct4, Sox2, Klf4 and c-Myc defined-factor fusion proteins. Bovine iPS cells showed typical colony morphology, normal karyotypes, stained positively for alkaline phosphatase (AP) and expressed Oct4, Nanog and SSEA1. The CpG in the promoter regions of Oct4 and Nanog were highly unmethylated in bovine iPS cells compared to the fibroblasts. The cells were able to differentiate into cell types of all three germ layers in vitro and in vivo. In addition, these cells were induced into female germ cells under defined culture conditions and expressed early and late female germ cell-specific genes Vasa, Dazl, Gdf9, Nobox, Zp2, and Zp3. Our data suggest that bovine iPS cells were generated from bovine fetal fibroblasts with defined-factor fusion proteins mediated by lentivirus and have potential applications in bovine transgenic breeding and gene-modified animals.