Pluripotency and embryonic lineage genes expression in the presence of small molecule inhibitors of FGF, TGFß and GSK3 during pre-implantation development of goat embryos.

Generating stable livestock pluripotent stem cells (PSCs) can be used for complex genome editing, cellular agriculture, gamete generation, regenerative medicine and in vitro breeding schemes. Over the past decade, significant progress has been made in characterizing pluripotency markers for livestock species. In this study, we investigated embryo development and gene expression of the core pluripotency triad (OCT4, NANOG, SOX2) and cell lineage commitment markers (REX1, CDX2, GATA4) in the presence of three small molecules and their combination [PD0325901 (FGF inhibitor), SB431542 (TGFß inhibitor), and CHIR99021 (GSK3B inhibitor)] from day 2-7 post-insemination in goat. Significant reduction in rate of blastocyst formation was observed when SB was used along with PD or CHIR and their three combinations had more sever effect. SB and CHIR decreased the expression of SOX2 while increasing the GATA4 expression. PD decrease the relative expression of NANOG, OCT4 and GATA4, while increased the expression of REX1. Among the combination of two molecules, only SB + CHIR combination significantly decreased the expression of GATA4, while the combination of the three molecules significantly decreases the expression of NANOG, SOX2 and CDX2. According to these results, the inhibition of the FGF signaling pathway, by PD may lead to blocking the hypoblast formation as observed by reduction of GATA4. OCT4 and NANOG expressions did not show signs of maintenance pluripotency. GATA4, NANOG and OCT4 in the PD group were downregulated and REX1 as EPI-marker was upregulated thus REX1 may be considered as a marker of EPI/ICM in goat.

Effect of Mechanical Micro-Vibrations on The Efficiency of Leopard Inter-Species Somatic Cell Nuclear Transfer.

OBJECTIVE: Scarcity of oocytes for assisted reproduction in endangered species can be bypassed by interspecies somatic cell nuclear transfer (iSCNT). In Felids, domestic cat (Felis catus) oocytes can serve as recipients for the nucleus of the endangered Persian leopard (Panthera pardus saxicolor). However, in vitro oocyte maturation is still suboptimal in cats, whereas it has been reported to benefit from micro-vibration in non-felid species. Therefore, the present study is aimed to determine whether micro-vibration, applied during in vitro maturation (IVM), improves the embryogenic potential of cat oocytes transplanted with fibroblast nuclei of the Persian leopard. MATERIALS AND METHODS: In the experimental study, cat cumulus-oocyte complexes (COCs) were randomly assigned to the treatment group (micro-vibration) or control group (static culture). Resultant metaphase II (MII) oocytes were enucleated and reconstructed with nucleus transplants from leopard fibroblasts, followed by artificial oocyte activation and embryo culture under the same condition (static) for 7 days. RESULTS: While cumulus cell expansion and oocyte maturation profited from micro-vibration (P<0.05), the quantity and quality of blastocysts were significantly lower in micro-vibration than in the control group (P<0.05). The total number of blastocyst cells tended to be lower in the micro-vibration than in the control group (P=0.075). Nevertheless, the proportion of ICM and TE cells did not differ between the micro-vibration and control groups (P>0.05). CONCLUSION: The present study indicated that micro-vibration at a frequency of 44 Hz for 5 secs per hour enhanced nuclear maturation and cumulus cell expansion of cat oocytes. However, exposure to micro-vibration during IVM impaired the survival rate of reconstructed oocytes during the iSCNT process and their developmental competence toward the blastocyst stage.

Melatonin accelerates the developmental competence and telomere elongation in ovine SCNT embryos.

SCNT embryos suffer from poor developmental competence (both in vitro and in vivo) due to various defects such as oxidative stress, incomplete epigenetic reprogramming, and flaws in telomere rejuvenation. It is very promising to ameliorate all these defects in SCNT embryos by supplementing the culture medium with a single compound. It has been demonstrated that melatonin, as a multitasking molecule, can improve the development of SCNT embryos, but its function during ovine SCNT embryos is unclear. We observed that supplementation of embryonic culture medium with 10 nM melatonin for 7 days accelerated the rate of blastocyst formation in ovine SCNT embryos. In addition, the quality of blastocysts increased in the melatonin-treated group compared with the SCNT control groups in terms of ICM, TE, total cell number, and mRNA expression of NANOG. Mechanistic studies in this study revealed that the melatonin-treated group had significantly lower ROS level, apoptotic cell ratio, and mRNA expression of CASPASE-3 and BAX/BCL2 ratio. In addition, melatonin promotes mitochondrial membrane potential and autophagy status (higher number of LC3B dots). Our results indicate that melatonin decreased the global level of 5mC and increased the level of H3K9ac in the treated blastocyst group compared with the blastocysts in the control group. More importantly, we demonstrated for the first time that melatonin treatment promoted telomere elongation in ovine SCNT embryos. This result offers the possibility of better development of ovine SCNT embryos after implantation. We concluded that melatonin can accelerate the reprogramming of telomere length in sheep SCNT embryos, in addition to its various beneficial effects such as increasing antioxidant capacity, reducing DNA damage, and improving the quality of derived blastocysts, all of which led to a higher in vitro development rate.

Synergistic effects of myo-inositol and melatonin on cryopreservation of goat spermatozoa.

Overproduction of reactive oxygen species (ROS) during sperm cryopreservation has a detrimental effect on sperm parameters. Therefore, the use of antioxidants in the sperm freezing extender can reduce ROS destructive effects. In this study, we investigated whether co-supplementation of melatonin and myo-inositol into the semen extender can improve the post-cryopreservation quality of goat spermatozoa. After the freeze-thawing process, sperm motility, viability, plasma membrane and acrosome intact morphology were improved in the combined myo-inositol and melatonin group compared to both individual and the control groups (p < .05). In addition, the mean of sperm ROS, DNA damage and lipid peroxidation were reduced in co-supplementation of myo-inositol and melatonin compared to their individual counterparts (p < .05). Therefore, the synergistic effects of myo-inositol and melatonin on the cryopreserved spermatozoa are highly likely mediated through the reduction in important factors involved in the sperm lipid peroxidation. Finally, we used the cryopreserved spermatozoa for in vitro production of embryos. Results showed that combined group of myo-inositol and melatonin improved the cleavage rate compared to both individual and control groups, although blastocyst rate was improved using both individual and combined groups. In conclusion, co-supplementation of melatonin and myo-inositol is a promising approach for the improvement of goat sperm cryopreservation.

Ferulago angulata extract ameliorates epididymal sperm toxicity in mice induced by lead and diazinon.

BACKGROUND: The potential toxicity that results from environmental xenobiotics is not completely known. Increasing levels of heavy metals and the use of organophosphate pesticides (OPs) and their co-existence in the environment could be associated with an increasing incidence of male reproductive system disorders in humans and animals. Ferulago angulata is a dietary source of phenolic compounds with reported health benefits. OBJECTIVE: This study was conducted to investigate whether an extract of Ferulago angulata could protect adult male NMRI mice against reproductive toxicity induced by lead acetate (PbAc), diazinon (DZN), or PbAc + DZN. MATERIALS AND METHODS: Adult male NMRI mice were exposed to either 0.5% PbAc in drinking water, DZN (3 mg/kg/day, intraperitoneal [i.p.] injection), or PbAc + DZN in the presence or absence of 400 mg/kg/day Ferulago angulata hydroalcoholic extract (FAE) that was administered via gavage for 6 weeks. RESULTS: Chronic exposure to PbAc, DZN, and PbAc + DZN decreased sperm quality, sperm chromatin maturity and integrity, increased oxidative stress and lipid peroxidation, and could reduce male fertility indices. Co-administration of FAE could reduce these negative effects. CONCLUSION: The Ferulago angulata extract should be considered as a useful natural extract for the treatment of male infertility, especially in males exposed to conditions which induce reproductive toxicity.

Effect of DNA and Histone Methyl Transferase Inhibitors on Outcomes of Buffalo-Bovine Interspecies Somatic Cell Nuclear Transfer.

Somatic cell nuclear transfer (SCNT) derived embryos suffer from abnormal epigenetic reprogramming, which handicaps pre- and postimplantation development. It was hypothesized that epigenetic modifiers, including zebularine (DNA methyltransferase inhibitors) and BIX-01294 (histone methyltransferase inhibitors), could decrease the respective levels of 5-methylcytosine and H3K9me2 in reconstructed oocytes (RO). Accordingly, we investigated whether treating RO with zebularine and BIX-01294 for 16 hours after activation could improve developmental competence and quality of buffalo-bovine interspecies SCNT (iSCNT) embryos. Treatment of RO with zebularine but not BIX-01294 significantly increased two-cell formation at 16 hours postactivation. Conversely, early cleaved embryos had significantly lower rate of blastocyst formation in zebularine treated RO compared to their counterparts in control and BIX-01294 groups. Treatment of RO with zebularine and BIX-01294 did not improve blastocyst rate of buffalo-bovine iSCNT embryos compared to their control counterparts. However, these two epigenetic drugs might have some beneficial effects on buffalo-bovine iSCNT compared to bovine SCNT embryos. The quality of iSCNT blastocysts was improved due to significant expression of OCT4 and CDX2 in BIX-01294 and CDX2 in zebularine treated RO. Furthermore, treatment of RO with zebularine and BIX-01294 did not affect DNA fragmentation in derived blastocysts against control group. In conclusion, treatment with zebularine and BIX-01294 did not enhance developmental competence of iSCNT embryos, but may have some beneficial effects on epigenetic makeup and quality of derived blastocysts.