Melatonin Alleviates Hypoxia-Induced Apoptosis of Granulosa Cells by Reducing ROS and Activating MTNR1B-PKA-Caspase8/9 Pathway.

In mammalian ovaries, the avascular environment within follicular cavity is supposed to cause hypoxic status in granulosa cells (GCs), leading to apoptotic cell death accompanied by cumulative reactive oxygen species (ROS) production. Melatonin (N-acetyl-5-methoxytryptamine, MT), a broad-spectrum antioxidant that exists in porcine follicle fluid, was suggested to maintain GCs survival under stress conditions. In this study, using the established hypoxic model (1% O2) of cultured porcine GCs, we explored the effect of MT on GCs apoptosis. The results showed that MT restored cell viability and reduced the apoptosis of GCs during hypoxia exposure. In addition, GCs treated with MT exhibited decreased ROS levels and increased expression of antioxidant enzymes including heme oxygenase-1 (HO-1), glutathione S-transferase (GST), superoxide dismutase 1 (SOD1), and catalase (CAT) upon hypoxia incubation. Moreover, the hypoxia-induced expression of cleaved caspase 3, 8, and 9 was significantly inhibited after MT treatment. In contrast, blocking melatonin receptor 2 (MTNR1B) with a competitive antagonist 4-phenyl-2-propionamidotetralin (4P-PDOT) diminished the inhibitory effects of MT on caspase 3 activation. By detecting levels of protein kinase (PKA), a downstream kinase of MTNR1B, we further confirmed the involvement of MT-MTNR1B signaling in mediating GCs protection during hypoxia stress. Together, the present data provide mechanistic evidence suggesting the role of MT in defending GCs from hypoxia-induced apoptosis.

Growth performance, reproductive traits and offspring survivability of genetically modified rams overexpressing toll-like receptor 4.

Genetic modification provides a means to enhancing disease resistance in animals. Toll-like receptor 4 (TLR4), a member of the TLR family, is critical for the recognition of lipopolysaccharide (LPS)/endotoxin from Gram-negative bacteria by host immune cells, which initiates cell activation and subsequently triggers a proinflammatory response to the invading pathogens. In this study, the first generation of genetically modified (GM) sheep overexpressing TLR4 was produced by microinjection for better disease resistance. Compared with wild-type (WT) rams, the GM rams have similar growth performance, basic semen quality and spermatozoon ultrastructure. The offspring birth rates after cervical artificial insemination were also similar between GM (90.32%) and WT (92.38%) rams. Overall, the presence and expression of the TLR4 transgene in the genome did not appear to interfere with normal semen production, reproductive traits and the ability of transgene transmission to offspring. The expression levels of TLR4, tumor necrosis factor and interferon gamma genes in monocyte/macrophages from GM sheep were significantly higher than that from WT sheep at early stages after LPS stimulation. The GM offspring born from the founder transgenic ram inseminated ewes had similar survival rate with WT offspring (88.89% vs 84.86%) at weaning. The TLR4 transgene showed no deleterious effects on growth performance, reproductive traits and offspring survivability of GM rams. Therefore, the GM sheep overexpressing TLR4 provide a powerful experimental model for analyzing function of TLR4 in vivo during infection and inflammation.

Melatonin promotes development of haploid germ cells from early developing spermatogenic cells of Suffolk sheep under in vitro condition.

Promotion of spermatogonial stem cell (SSC) differentiation into functional sperms under in vitro conditions is a great challenge for reproductive physiologists. In this study, we observed that melatonin (10(-7) M) supplementation significantly enhanced the cultured SSCs differentiation into haploid germ cells. This was confirmed by the expression of sperm special protein, acrosin. The rate of SSCs differentiation into sperm with melatonin supplementation was 11.85 ± 0.93% which was twofold higher than that in the control. The level of testosterone, the transcriptions of luteinizing hormone receptor (LHR), and the steroidogenic acute regulatory protein (StAR) were upregulated with melatonin treatment. At the early stage of SSCs culture, melatonin suppressed the level of cAMP, while at the later stage, it promoted cAMP production. The similar pattern was observed in testosterone content. Expressions for marker genes of meiosis anaphase, Dnmt3a, and Bcl-2 were upregulated by melatonin. In contrast, Bax expression was downregulated. Importantly, the in vitro-generated sperms were functional and they were capable to fertilize oocytes. These fertilized oocytes have successfully developed to the blastula stage.

Effects of seasonal changes on the ovulation rate and embryo quality in superovulated Black Suffolk ewes.

OBJECTIVE: The objective of this study was to investigate the effects of seasonal changes on the superovulation in Black Suffolk ewes, particularly the ovulation rate and embryo quality. DESIGN: Black Suffolk ewes were superovulated either in May (n=22) or in September (n=21), 2013. After estrus synchronization with CIDR, the donor ewes were superovulated with PMSG and seven decreasing doses of FSH (twice daily at 07:00 and 19:00 for four consecutive days. Then, they were subjected to laparoscopic intrauterine artificial insemination. The viable morula and blastocysts were recovered and immediately transferred to recipients. RESULTS: Ewes that were superovulated in May had a much higher ovulation rate than those were superovulated in September (16.8 ± 3.23vs. 10.2 ± 2.94, p<0.01); however, the viability rate of the embryo was lower than that of September (56.0 ± 1.92% vs. 92.5 ± 3.26%, p<0.01). There was no significant difference in the survival rate of the transferred viable embryos (33.9 ± 1.00% vs. 36.7 ± 1.64%, p>0.05) and the number of offspring per donor ewe (3.1 ± 0.54 vs. 2.9 ± 0.72, p>0.05) between May and September. In contrast, the offspring/ova ratio of the donor ewes superovulated in May was lower than that of September (18.5 ± 1.64% vs. 32.8 ± 2.14%, p<0.01). CONCLUSIONS: The superovulation of Black Suffolk ewes may be affected by the seasonal changes. Generallly, The ewe's ovulation rate was higher in May, whereas the viability rate of embryo was higher in September.

Effects of melatonin on the proliferation and apoptosis of sheep granulosa cells under thermal stress.

The cross-talk between oocyte and somatic cells plays a crucial role in the regulation of follicular development and oocyte maturation. As a result, granulosa cell apoptosis causes follicular atresia. In this study, sheep granulosa cells were cultured under thermal stress to induce apoptosis, and melatonin (MT) was examined to evaluate its potential effects on heat-induced granulosa cell injury. The results demonstrated that the Colony Forming Efficiency (CFE) of granulosa cells was significantly decreased (heat 19.70% ± 1.29% vs. control 26.96% ± 1.81%, p < 0.05) and the apoptosis rate was significantly increased (heat 56.16% ± 13.95% vs. control 22.80% ± 12.16%, p < 0.05) in granulosa cells with thermal stress compared with the control group. Melatonin (10⁻7 M) remarkably reduced the negative effects caused by thermal stress in the granulosa cells. This reduction was indicated by the improved CFE and decreased apoptotic rate of these cells. The beneficial effects of melatonin on thermal stressed granulosa cells were not inhibited by its membrane receptor antagonist luzindole. A mechanistic exploration indicated that melatonin (10⁻7 M) down-regulated p53 and up-regulated Bcl-2 and LHR gene expression of granulosa cells under thermal stress. This study provides evidence for the molecular mechanisms of the protective effects of melatonin on granulosa cells during thermal stress.

Melatonin promotes superovulation in sika deer (Cervus nippon).

In this study, the effects of melatonin (MT) on superovulation and reproductive hormones (melatonin, follicle-stimulating hormone (FSH), luteinizing hormone (LH) and PRL) were investigated in female sika deer. Different doses (40 or 80 mg/animal) of melatonin were subcutaneously implanted into deer before the breeding season. Exogenous melatonin administration significantly elevated the serum FSH levels at the time of insemination compared with levels in control animals. During superovulation, the serum LH levels in donor sika deer reached their highest values (7.1±2.04 ng/mL) at the point of insemination, compared with the baseline levels (4.98±0.07 ng/mL) in control animals. This high level of LH was sustained until the day of embryo recovery. In contrast, the serum levels of PRL in the 80 mg of melatonin-treated group were significantly lower than those of control deer. The average number of corpora lutea in melatonin-treated deer was significantly higher than that of the control (p<0.05). The average number of embryos in the deer treated with 40 mg of melatonin was higher than that of the control; however, this increase did not reach significant difference (p>0.05), which may be related to the relatively small sample size. In addition, embryonic development in melatonin-treated groups was delayed.

Melatonin promotes embryonic development and reduces reactive oxygen species in vitrified mouse 2-cell embryos.

Two-cell embryos of mouse were vitrified by the open-pulled straw (OPS) method. The vitrified embryos were warmed and introduced into M16 medium for culture that contains melatonin at different concentrations (10(-3), 10(-5), 10(-7), 10(-9), 10(-11) m). This process caused reactive oxygen species (ROS) formation and jeopardized the development of the embryos. Melatonin, at different concentrations, significantly suppresses ROS production and promotes embryonic development in vitrified embryos compared with untreated ones. The mechanistic studies indicated that the beneficial effects of melatonin on vitrified 2-cell embryos of mouse were melatonin receptor (MT1 and MT2) independent. The direct free radical scavenging activity, the enhancement of endogenous glutathione levels, and the anti-apoptotic capacity of melatonin may account for its protective effects on vitrified embryonic development.

Effects of melatonin on in vitro development of mouse two-cell embryos cultured in HTF medium.

Melatonin is capable of improving the developmental capacity of ovine, porcine and bovine embryos in vitro. However, whether melatonin possesses similar benefits to the in vitro mouse embryonic development has yet to be determined. In this study, we assessed the effects of various concentrations of melatonin (10-13 to 10-3 M) on the in-vitro development of mouse embryos cultured in HTF medium for 96 hr; embryos cultured without melatonin were used as control. The in vitro development of mouse two-cell embryos significantly benefited from treatment with melatonin in a concentration-dependent manner. The effects of melatonin on the rates of blastocyst formation, hatching/hatched blastocysts and cell number per blastocyst were bi-phasic; all significantly increased by melatonin at 10-13 to 10-5 M and decreased by melatonin at 10-3 M. Maximal benefit of melatonin on in vitro mouse 2-cell embryo development was achieved at a concentration of 10-9 M. In comparison to control, 10-9 M melatonin increased blastocyst formation rate from 48.08 +/- 5.25% to 82.08 +/- 2.34% (p < 0.05), hatched blastocyst rate from 25.65 +/- 11.79% to 66.47 +/- 4.94% (p < 0.05), and cell number per blastocyst 62.71 +/- 5.97 to 77.91 +/- 10.63 (p < 0.05). Thus, our datas demonstrated firstly that melatonin has beneficial effects on the in vitro development of 2-cell mouse embryos cultured in HTF medium.

Melatonin exists in porcine follicular fluid and improves in vitro maturation and parthenogenetic development of porcine oocytes.

This study focused on the effect of melatonin on in vitro maturation of porcine oocytes and their parthenogenetic embryonic development. Melatonin was measured in porcine follicular fluid of follicles of different sizes in the same ovary. Melatonin exists in follicular fluid, and the concentration is approximately 10(-11) m. Its concentration decreased as the diameter of follicle increased, which suggests an effect of melatonin on oocyte maturation. Therefore, immature oocytes were cultured in vitro in maturation medium supplemented with melatonin (10(-11), 10(-9), 10(-7), 10(-5) and 10(-3) m) or without melatonin. The oocytes at maturation stage were collected and activated. The parthenogenetic embryos were cultured and observed in medium supplemented with or without melatonin. Fresh immature oocytes without melatonin treatment were used as control. When only maturation medium was supplemented with 10(-9) m melatonin, the cleavage rate, blastocyst rate and the cell number of blastocyst (70 +/- 4.5%, 28 +/- 2.4% and 50 +/- 6.5%) were significantly higher (P < 0.05) than that of controls; when only culture medium was supplemented with melatonin, the highest cleavage rate, blastocyst rate and the cell number of blastocyst was observed at 10(-7) m melatonin, which were significantly higher than that of controls (P < 0.05). The best results (cleavage rates 79 +/- 8.4%, blastocyst rates 35 +/- 6.7%) were obtained when both the maturation and culture medium were supplemented with 10(-9) m melatonin respectively (P < 0.05). In conclusion, exogenous melatonin at the proper concentration may improve the in vitro maturation of porcine oocytes and their parthenogenetic embryonic development. Further research is needed to identify the effect of melatonin on in vitro and in vivo oocyte maturation and embryo development in porcine.

Optimal concentration of calcium and electric field levels improve tetraploid embryo production by electrofusion in mice.

Polyploid embryo production is an important technique in generating mice directly from embryonic stem (ES) cells. The present study was designed to assess the effect of different calcium concentrations and electric field intensities on the production of tetraploid embryos with higher developmental potential by electrofusion. Two-cell mouse embryos were electrofused in fusion solution containing different concentrations of calcium ion (0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 and 1.4 mM). The rates of blastomere fusion, and subsequent cleavage and development of tetraploids to the blastocyst stage were highest when two-cell embryos were electrically stimulated in a fusion medium containing 1.0 mM calcium. Therefore, we tested electric field intensities (0.6, 0.8, 1.0, 1.2 and 1.4 kV/cm) for electrofusion of two-cell embryos and subsequent development to the blastocyst stage in 1.0 mM calcium. The highest rates of fusion and blastocyst formation were observed when the electric field strength was 0.8 kV/cm. The present results showed that mouse two-cell embryos stimulated with 0.8 kV/cm in a fusion medium containing 1.0 mM calcium had the highest rates of fusion and development to the blastocyst stage.

Vitrification of farmed blue fox oocytes in ethylene glycol and DMSO-based solutions using open-pulled straw (OPS).

Farmed blue fox was used as a model to develop cryopreservation protocol for nondomestic canine species. We report here the developmental potential of farmed blue fox oocytes after vitrification with a two-step OPS method. Oocytes were collected and pre-cultured for 0, 24, 48, 72 hours respectively before cryopreservation. Vitrification of oocytes was achieved by a 30 sec treatment in 10% ethylene glycol (EG) or 10% EG + 10% dimethyl sulfoxide (DMSO) at 25 degree C followed by a 25 sec equilibration in EFS30 (30% (v/v) EG +21% (w/v) Ficoll +0.35M sucrose) or EDFS30 (15% (v/v) EG +15% (v/v) DMSO +21% (w/v) Ficoll +0.35M sucrose), before plunging into liquid nitrogen. The survival of oocytes after vitrification was assessed morphologically immediately after warming, and cultured for in vitro maturation. For comparison, control oocytes were cultured for in vitro maturation for 96 hours. The best result was obtained when oocytes were pre-cultured for 72 hours, first exposed to 10 percent EG + 10% DMSO and vitrified in EDFS30. The survival percentage of oocytes under these conditions was not significantly different (P > 0.05) from that of the control.

Tetraspanin CD9 in bovine oocytes and its role in fertilization.

This study was conducted in bovine to investigate whether CD9 (a member of the tetraspanin superfamily of proteins) is present on oocytes and whether it functions in sperm-oocyte binding and fusion. First, the presence of CD9 in bovine matured oocytes was examined by immunofluorescence with the anti-CD9 monoclonal antibody (mAb) and fluorescein isothiocyanate-conjugated goat anti-mouse antibody, and the results showed that CD9 was expressed on the plasma membrane of matured oocytes. Sperm binding and fusion with oocytes was then examined by in vitro fertilization. When the zona pellucida-free matured oocytes were fertilized, both sperm binding to ooplasma and sperm penetrating into oocytes were significantly (P<0.01) reduced in anti-CD9 antibody-treated oocytes (6.3 +/- 0.7 per oocyte and 41.6%, respectively) compared with untreated control oocytes (19.0 +/- 0.7 per oocyte and 81.3%, respectively), indicating that the anti-CD9 mAb potentially inhibits sperm-oocyte binding and fusion. These results demonstrated that the CD9 present on bovine matured oocytes is involved in sperm-oocyte interaction during fertilization.

Surgical embryo transfer resulted in birth of live offspring in farmed blue fox.

Finland blue fox (Alopex lagopus) has great reputation in pelt industry around the world for its large size and top-ranking fur quality; however, both the herd size and the average survival rate of purebred offspring are rather low in production systems in China. Surgical transfer of blue fox embryos was investigated as a means to increase the population fox and also as a possible means to conserve endangered canine species. The animals were chosen on the basis of synchrony in natural oestrus. During the reproductive season of blue fox, 59 embryos were flushed from 6 farmed donors 9-11 days after the first insemination, and 53 embryos were transferred surgically into the uteri of the 6 paired recipients with natural synchronized oestrous. Two of the recipients littered 46-49 days after embryo transfer; one gave birth to 7 pups and the other 1 pup. This report describes the first successful embryo transfer in the farmed blue fox in China.

Effects of oocyte activation and sperm preparation on the development of porcine embryos derived from in vitro-matured oocytes and intracytoplasmic sperm injection.

The objective was to determine the effects of various methods of oocyte activation and sperm pretreatment on development of porcine embryos derived from in vitro-matured oocytes and intracytoplasmic sperm injection (ICSI). The second polar body was extruded in the majority (>78.4%) of in vitro-matured (IVM) oocytes 4h after electrical pulse activation. In embryos generated by ICSI and sham-ICSI, a combination of an electrical pulse, with various chemical activators 4 h later, improved (P < 0.05) blastocyst formation rate compared to activation only with a pulse. Treatment with 6-dimethylaminopurine (DMAP) after electrical activation significantly increased the oocyte activation rate. The effects of exposure of sperm to repeated freeze-thaw cycles (without cryoprotectant) on oocyte activation and the effects of sperm pre-incubated with dithiothreitol (DTT) or Triton X-100 on early embryo development were also examined. Blastocyst formation rates after ICSI did not differ between motile sperm and those rendered immotile by one-time freezing and thawing without cryoprotectant. However, sperm rendered immotile by three cycles of freezing/thawing without cryoprotectant had a significantly lower blastocyst formation rate. Although oocytes injected with sperm pre-incubated with Triton X-100 had a higher normal fertilization rate than those pre-incubated with DTT or one-time frozen/thawed sperm, rates of blastocyst formation and cell numbers were similar among the three groups. In conclusion, various methods of oocyte activation and sperm preparation significantly affected the developmental capacity of early porcine embryos derived from IVM and ICSI.

Experimental study on mechanism and protection of stress ulcer produced by explosive noise.

AIM:To establish an experimental model of stress ulcer produced by explosive noise, and to probe into its mechanism and protection.METHODS:The country standard Wistar white rats were randomly divided into control group(n =8), which were neither stimulated nor protected, and stimulating group (divided into subgroups A, B and C, including 8 rats each which were decapitated to draw blood for test immediately, 12 hours and 24 hours after stimulation) and prevention group (divided into subgroups A, B and C, having 8 rats each, subgroup A was given cimetidine, B anisodamine and C both drugs). Firing noises of submachine guns were used as inflicting factor. The rats were fasted for 24 hours and stimulated by firing noise for 12 hours. The change of ulcer index, gastric mucosal and related serum hormones were observed.RESULTS:Stress ulcer was significant in the stimulating group, and its ulcer index (8.6 ± 0.6) was remarkably higher than that in both the control group and prevention group (0.3 ± 0.1, P < 0.01).Its serum gastrin (Gas ng/L, 294 ± 163 vs 63 ± 40,P< 0.01) and endothelin (ET ng/L, 181 ± 57 vs 135 ± 42, P < 0.1) were apparently higher than those in the control group, and its serum nitric oxide (NO) level was conspicuously lower than that in the control group (ng/L, 0.3 ± 0.1 vs 0.8 ± 0.5 P <0.5), while the serum gastrin level (ng/L, 556 ± 225) in prevention group was distinctly higher than that in both the control (P<0.1) and stimulating group (P < 0.5). There were no significant differences in the changes of ET and NO between the control and the stimulating groups.CONCLUSION:Stress ulcer model of rats can be successfully established by the stimulation of explosive noise. Gas, ET and NO are related to the formation of stress ulcer, and play an important role in its mechanism. Hepatic function affected by noise is observed in this experiment.