Robotic Label-Free Precise Oocyte Enucleation for Improving Developmental Competence of Cloned Embryos.

OBJECTIVE: The invisibility of domestic oocyte nucleus in bright field currently forces operators to blindly aspirate nucleus out in oocyte enucleation, usually causing large cytoplasm losses and poor developmental competences of cloned embryos. Although fluorescent labeling of nucleus allows for nucleus localization, the involved photobleaching problems and barriers to the execution of enucleation process limit its online-application in oocyte enucleation. This paper reports a novel label-free oocyte enucleation method for precise removal of the nucleus with less cytoplasm loss. METHODS: The relative positions between the injection pipette and nucleus for complete removal of nucleus with less cytoplasm loss were determined through a finite element modeling of nucleus aspiration. To position injection pipette to the above positions relative to nucleus, the appropriate oocyte orientation and trajectory of injection pipette inside oocyte were derived according to the offline-calibrated 3-D distribution of nucleus and the simulated dynamic drift of nucleus that occurs as injection pipette is maneuvered inside oocyte. Finally, a robotic label-free precise enucleation procedure was established. RESULTS: The experimental results on more than 1000 porcine oocytes proved that this system is capable of reducing cytoplasm loss by 60% at the same level of enucleation success rate and almost doubling the cleavage rate of clone embryos in comparison to blind aspiration method. CONCLUSIONS: The results prove that our method significantly improves the developmental competence of cloned embryos in comparison to manual enucleation method. SIGNIFICANCE: Our method is expected to improve the extremely low success rate of animal cloning in the future.

Protective Effects of Spermidine and Melatonin on Deltamethrin-Induced Cardiotoxicity and Neurotoxicity in Zebrafish.

Increased application of the pyrethroid insecticide deltamethrin has adverse effects on the cardiac system and neurobehavior on the non-target organisms, which has raised the public's attention. Because of spermidine and melatonin considered to have cardioprotective and neuroprotective characteristics, zebrafish were utilized as the model organism to explore the protective effects of spermidine and melatonin against deltamethrin-induced toxicity. We tested the neurobehavior of zebrafish larvae through a rest/wake behavior assay, and evaluated the levels of the expression of Scn5lab, gata4, nkx2.5, hcrt, hcrtr, and aanat2 by qRT-PCR. Besides that cmlc2 was evaluated by whole-mount in situ hybridization. Results have shown that compared with control group, 0.025 mg/L deltamethrin could significantly disturb the cardiac development, downregulating the expression of Scn5lab and transcriptional factors gata4 and nkx2.5, disturbing cardiac looping, resulting in defects in cardiac morphology and function. Moreover, deltamethrin could alter the expression levels of rest/wake genes and cause hyperactivity in zebrafish larvae. Besides, compared with deltamethrin group, the exogenous 0.01 mg/L spermidine and 0.232 mg/L melatonin could significantly rescue the adverse effects of deltamethrin on the cardiac system and neurobehavior in zebrafish. This indicated that spermidine and melatonin have neuroprotective and cardioprotective effects against deltamethrin-induced adverse effects in zebrafish.

Melatonin attenuates 17ß-trenbolone induced insomnia-like phenotype and movement deficiency in zebrafish.

17ß-trenbolone (17ß-TBOH) is one of the dominant metabolites of trenbolone acetate, which is widely applied in beef cattle operations around the globe. The effects of environmental concentrations of 17ß-trenbolone on the early development of zebrafish embryos have received very little attention. Melatonin could regulate sleep-wake cycle and plays a protective role in various adverse conditions. Here, environmentally realistic concentrations of 17ß-trenbolone (1 ng/L, 10 ng/L, 50 ng/L) has been exposure to zebrafish embryos at 2 h postfertilization (hpf). The results showed that 10 ng/L and 50 ng/L 17ß-trenbolone disturbed the distribution of caudal primary motoneurons and downregulated expression of motoneuron development related genes along with locomotion decreasing. While melatonin could recover the detrimental effects caused by 17ß-trenbolone. Interestingly, 17ß-trenbolone exposure increased waking activity and decreased rest even in a low dose (1 ng/L). Moreover, it upregulated hypocretin/orexin (Hcrt) signaling which promotes wakefulness. Melatonin restored the insomnia-like alternation induced by 17ß-trenbolone exposure. Collectively, we conclude that 17ß-trenbolone disturbed motoneuron development and altered sleep/wake behavior, while melatonin could alleviate the deleterious influence on motoneuron development and recover the circadian rhythm.

Combined treatment of melatonin and sodium tanshinone IIA sulfonate reduced the neurological and cardiovascular toxicity induced by deltamethrin in zebrafish.

The pyrethroid insecticide deltamethrin has been reported to have an effect on vertebrate development and cardiovascular disease. Sodium tanshinone IIA sulfonate (STS) is considered to have cardioprotective effects and melatonin is known to regulate sleep-waking cycles. In this experiment, we used transgenic zebrafish Tg (kdrl:mCherry) and Tg (myl7:GFP) to investigate whether STS and melatonin could reverse the cardiovascular toxicity and neurotoxicity induced by deltamethrin. Zebrafish embryos were exposed to 25 µg/L deltamethrin at 10 hpf and treated with 100 mmol/L STS and 1 µmol/L melatonin showed that deltamethrin treatment affected normal cardiovascular development. In situ hybridization and qRT-PCR results showed that deltamethrin could interfere with the normal expression of cardiovascular development-related genes vegfr2, shh, gata4, nkx2.5, causing functional defects in the cardiovascular system. In addition, deltamethrin could affect the sleep-waking behavior of larvae, increasing the activity of larvae, decreasing the rest behavior and the expression of hcrt, hcrtr, aanat2 were down-regulated. The addition of melatonin and STS can significantly alleviate cardiovascular toxicity and sleep-waking induced by deltamethrin, while restoring the expression of related genes to normal levels. Our study demonstrates the role of STS and melatonin in protecting cardiovascular and sleep-waking behavior caused by deltamethrin.

Fluorene-9-bisphenol exposure induces cytotoxicity in mouse oocytes and causes ovarian damage.

Fluorene-9-bisphenol (BHPF), a substitute for bisphenol A, is a chemical component of plastics for industrial production. There is evidence that BHPF exerts an antioestrogenic effect on mice, induces endometrial atrophy and leads to adverse pregnancy outcomes. However, the effects of BHPF on oocyte maturation and ovary development as well as its possible mechanisms remain unclear. The objective of this study was to investigate the toxicity and mechanism of BHPF exposure in mouse oocytes in vitro and in vivo. Our results showed that BHPF could inhibit the maturation of oocytes in vitro by reducing the protein level of p-MAPK and destroying the meiotic spindle. We found that in vitro, BHPF-treated oocytes showed increased ROS levels, DNA damage, mitochondrial dysfunction, and expression of apoptosis- and autophagy-related genes, such as Bax, cleaved-caspase 3, LC 3 and Atg 12. In addition, in vivo experiments showed that BHPF exposure could induce the expression of oxidative stress genes (Cat, Gpx 3 and Sod 2) and apoptosis genes (Bax, Bcl-2 and Cleaved-caspase 3) and increase the number of atresia follicles in the ovaries. Our data showed that BHPF exposure affected the first polar body extrusion of oocytes, increased oxidative stress, destroyed spindle assembly, caused DNA damage, altered mitochondrial membrane potentials, induced apoptosis and autophagy, and affected ovarian development.

Resveratrol reverses the adverse effects of a diet-induced obese murine model on oocyte quality and zona pellucida softening.

Reproductive dysfunction associated with obesity is increasing among women of reproductive age, including infertility and increasing risk of miscarriage. In females, reproductive disorders are linked to declining quality of oocytes. Using a model of diet-induced obesity, we have investigated the possible effects of obesity on oocyte quality, including metabolism, lipid accumulation, ROS levels, meiosis and changes in spindle structure in Metaphase II. Our study showed that obesity induced by a high fat diet can impair oocyte meiosis, destroy spindle assembly, and promote oxidative stress and abnormal mitochondrial distribution. With the addition of resveratrol, the negative impact of diet-induced obesity on the quality of oocytes was alleviated to some extent. In addition, we found that obesity causes mouse oocytes to soften, and resveratrol can restore the zona pellucida of oocytes to the same state as the control group. In conclusion, resveratrol can reverse the adverse effects of obesity on oocytes, which is beneficial for subsequent embryonic development.