NLRP7 is expressed in the ovine ovary and associated with in vitro pre-implantation embryo development.

NLRP (NACHT, LRR and PYD domain-containing proteins) family plays pivotal roles in mammalian reproduction. Mutation of NLRP7 is often associated with human recurrent hydatidiform moles. Few studies regarding the functions of NLRP7 have been performed in other mammalian species rather than humans. In the current study, for the first time, the function of NLRP7 has been explored in ovine ovary. NLRP7 protein was mainly located in ovarian follicles and in in vitro pre-implantation embryos. To identify its origin, 763 bp partial CDS of NLRP7 deriving from sheep cumulus oocyte complexes (COCs) was cloned, it showed a great homology with Homo sapiens. The high levels of mRNA and protein of NLRP7 were steadily expressed in oocytes, parthenogenetic embryos or IVF embryos. NLRP7 knockdown by the combination of siRNA and shRNA jeopardized both the parthenogenetic and IVF embryo development. These results strongly suggest that NLRP7 plays an important role in ovine reproduction. The potential mechanisms of NLRP7 will be fully investigated in the future.

Melatonin regulates the activities of ovary and delays the fertility decline in female animals via MT1/AMPK pathway.

Female fertility irreversibly declines with aging, and this is primarily associated with the decreased quality and quantity of oocytes. To evaluate whether a long-term of melatonin treatment would improve the fertility of aged mice, different concentrations of melatonin (10-3 , 10-5 , 10-7  mol/L) were supplemented into drinking water. Melatonin treatments improved the litter sizes of mice at the age of 24 weeks. Mice treated with 10-5  mol/L melatonin had the largest litter size among other concentrations. At this optimal concentration, melatonin not only significantly increased the total number of oocytes but also their quality, having more oocytes with normal morphology that could generate more blastocyst after in vitro fertilization in melatonin (10-5  mol/L)-treated group than that in the controls. When these blastocysts were transferred to recipients, the litter size was also significantly larger in melatonin treated mice than that in controls. The increases in TAOC and SOD level and decreases in MDA were detected in ovaries and uterus from melatonin-treated mice compared to the controls. Melatonin reduced ROS level and maintained mitochondrial membrane potential in the oocytes cultured in vitro. Mechanistically studies revealed that the beneficial effects of melatonin on oocytes were mediated by MT1 receptor and AMPK pathway. Thereafter, MT1 knocking out (MT1-KO) were generated and shown significantly reduced number of oocytes and litter size. The expression of SIRT1, C-myc, and CHOP were downregulated in the ovary of MT1-KO mice, but SIRT1 and p-NF-kB protein level were elevated in response to disturbed redox balance. The results have convincingly proven that melatonin administration delays ovary aging and improves fertility in mice via MT1/AMPK pathway.

Effects of melatonin administration on embryo implantation and offspring growth in mice under different schedules of photoperiodic exposure.

BACKGROUND: Embryo implantation is crucial for animal reproduction. Unsuccessful embryo implantation leads to pregnancy failure, especially in human-assisted conception. Environmental factors have a profound impact on embryo implantation. Because people are being exposed to more light at night, the influence of long-term light exposure on embryo implantation should be explored. METHODS: The effects of long photoperiodic exposure and melatonin on embryo implantation and offspring growth were examined. Long photoperiodic exposure (18:6 h light:dark) was selected to resemble light pollution. Melatonin (10-2, 10-3, 10-4, 10-5 M) was added to the drinking water of mice starting at Day 1 (vaginal plugs) until delivery. RESULTS: Melatonin treatment (10-4,10-5 M) significantly increased litter sizes compared to untreated controls (12.9 ± 0.40 and 12.2 ± 1.01 vs. 11.5 ± 0.43; P < 0.05). The most effective concentration of melatonin (10-4 M) was selected for further investigation. No remarkable differences were found between melatonin-treated mice and controls in terms of the pups' birth weights, weaning survival rates, and weaning weights. Long photoperiodic exposure significantly reduced the number of implantation sites in treated mice compared to controls (light/dark, 12/12 h), and melatonin rescued this negative effect. Mechanistic studies revealed that melatonin enhanced the serum 17ß-estradiol (E2) levels in the pregnant mice and upregulated the expression of the receptors MT1 and MT2 and p53 in uterine tissue. All of these factors may contribute to the beneficial effects of melatonin on embryo implantation in mice. CONCLUSION: Melatonin treatment was associated with beneficial effects in pregnant mice, especially those subjected to long photoperiodic exposure. This was achieved by enhanced embryo implantation. At the molecular level, melatonin administration probably increases the E2 level during pregnancy and upregulates p53 expression by activating MT1/2 in the uterus. All of the changes may improve the microenvironment of the uterus and, thus, the outcomes of pregnancy.

An AANAT/ASMT transgenic animal model constructed with CRISPR/Cas9 system serving as the mammary gland bioreactor to produce melatonin-enriched milk in sheep.

Melatonin as a potent antioxidant exhibits important nutritional and medicinal values. To produce melatonin-enriched milk will benefit the consumers. In this study, a sheep bioreactor which generates melatonin-enriched milk has been successfully developed by the technology that combined CRISPR/Cas9 system and microinjection. The AANAT and ASMT were cloned from pineal gland of Dorper sheep (Ovis aries). The in vitro studies found that AANAT and ASMT were successfully transferred to the mammary epithelial cell lines and significantly increased melatonin production in the culture medium compared to the nontransgenic cell lines. In addition, the Cas9 mRNA, sgRNA, and the linearized vectors pBC1-AANAT and pBC1-ASMT were co-injected into the cytoplasm of pronuclear embryos which were implanted into ewes by oviducts transferring. Thirty-four transgenic sheep were generated with the transgenic positive rate being roughly 35% which were identified by Southern blot and sequencing. Seven carried transgenic AANAT, two carried ASMT, and 25 carried both of AANAT and ASMT genes. RT-PCR and Western blot demonstrated that the lambs expressed these genes in their mammary epithelial cells and these animals produced melatonin-enriched milk. This is the first report to show a functional AANAT and ASMT transgenic animal model which produce significantly high levels of melatonin milk compared to their wild-type counterparts. The advanced technologies used in the study laid a foundation for generating large transgenic livestock, for example, the cows, which can produce high level of melatonin milk.

Effects of Melatonin on Early Pregnancy in Mouse: Involving the Regulation of StAR, Cyp11a1, and Ihh Expression.

To test whether melatonin plays an important role in the process of early pregnancy, melatonin was given in drinking water to pregnant mice at different gestation stages. These included mice who were given melatonin 14 days prior to their successful mating (confirmed by vaginal plug) (Group A), after successful mating (Group B), and 14 days prior to and until after successful mating (Group C). Melatonin administration significantly enhanced serum as well as ovarian melatonin levels in the mice. It was observed that melatonin did not affect the natural estrous of mice. On day 0.5 of gestation (D0.5), melatonin not only elevated progesterone (P) secretion, but also upregulated expressions of StAR and Cyp11a1, the two marker genes of corpus luteum in ovaries (p < 0.05). Group A had a significantly lower estradiol (E2) secretion and a higher number of implantation sites as well as litter size than controls (p < 0.05) and also had an increased Ihh expression in endometrium of D7.5 gestation. Melatonin treatment after successful mating improved the progesterone (P) secretion at D7.5 of gestation (p < 0.05) and significantly induced leukaemia inhibitory factor (LIF) expression (p < 0.05). Our study indicates that melatonin treatment up-regulated the genes involved in pregnenolone synthesis in ovary and Ihh expression in uterine endometrium. The mechanisms of melatonin to improve embryo implantation related to their actions on promoting the development of corpus luteum before gestation and helping to specify uterine receptivity in early pregnant mice.

Melatonin Promotes the In Vitro Development of Microinjected Pronuclear Mouse Embryos via Its Anti-Oxidative and Anti-Apoptotic Effects.

CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats) combined with pronuclear microinjection has become the most effective method for producing transgenic animals. However, the relatively low embryo developmental rate limits its application. In the current study, it was observed that 10-7 M melatonin is considered an optimum concentration and significantly promoted the in vitro development of murine microinjected pronuclear embryos, as indicated by the increased blastocyst rate, hatching blastocyst rate and blastocyst cell number. When these blastocysts were implanted into recipient mice, the pregnancy rate and birth rate were significantly higher than those of the microinjected control, respectively. Mechanistic studies revealed that melatonin treatment reduced reactive oxygen species (ROS) production and cellular apoptosis during in vitro embryo development and improved the quality of the blastocysts. The implantation of quality-improved blastocysts led to elevated pregnancy and birth rates. In conclusion, the results revealed that the anti-oxidative and anti-apoptotic activities of melatonin improved the quality of microinjected pronuclear embryos and subsequently increased both the efficiency of embryo implantation and the birth rate of the pups. Therefore, the melatonin supplementation may provide a novel alternative method for generating large numbers of transgenic mice and this method can probably be used in human-assisted reproduction and genome editing.

Beneficial Effects of Melatonin on the In Vitro Maturation of Sheep Oocytes and Its Relation to Melatonin Receptors.

(1) Background: The binding sites of melatonin, as a multifunctional molecule, have been identified in human, porcine, and bovine samples. However, the binding sites and mechanisms of melatonin have not been reported in sheep; (2) Methods: Cumulus-oocyte complexes (COCs) were cultured in TCM-199 supplemented with melatonin at concentrations of 0, 10-3, 10-5, 10-7, 10-9, and 10-11 M. Melatonin receptors (MT1 and MT2) were evaluated via immunofluorescence and Western blot. The effects of melatonin on cumulus cell expansion, nuclear maturation, embryo development, and related gene (GDF9, DNMT1, PTX3, HAS2, and EGFR) expression were investigated. The level of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) were evaluated in oocytes and cumulus, respectively; (3) Results: Both MT1 and MT2 were expressed in oocytes, cumulus cells, and granulosa cells. Melatonin with a concentration of 10-7 M significantly enhanced the rates of nuclear maturation, cumulus cells expansion, cleavage, and blastocyst. Melatonin enhanced the expression of BMP15 in oocytes and of PTX3, HAS2, and EGFR in cumulus cells. Melatonin decreased the cAMP level of oocytes but enhanced the cGMP level in oocytes and cumulus cells; (4) Conclusion: The higher presence of MT1 in GV cumulus cells and the beneficial effects of melatonin indicated that its roles in regulating sheep oocyte maturation may be mediated mainly by the MT1 receptor.

Mitochondria Synthesize Melatonin to Ameliorate Its Function and Improve Mice Oocyte's Quality under in Vitro Conditions.

The physiology of oocyte in vitro maturation remains elusive. Generally, the oocytes have a very low maturation rate under in vitro conditions. In the current study, we found that melatonin promotes the maturation of oocytes in which mitochondria play a pivotal role. It was identified that; (1) mitochondria are the major sites for melatonin synthesis in oocytes and they synthesize large amounts of melatonin during their maturation; (2) melatonin improves mitochondrial function by increased mtDNA copy, mitochondrial membrane potential (ΔΨm) and mitochondrial distribution and ATP production in oocytes; (3) the meiotic spindle assembly is enhanced; (4) melatonin reduces ROS production and inhibits 8-oxodG formation, thereby protecting potential DNA mutation from oxidative damage. As a result, melatonin improves the quality of oocytes, significantly accelerates the developmental ability of IVF embryo. The results provide novel knowledge on the physiology of oocyte's maturation, especially under in vitro conditions.

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.

Beneficial effects of melatonin on bovine oocytes maturation: a mechanistic approach.

This study was performed to investigate the effect of melatonin on bovine oocyte maturation and subsequent embryonic development in vitro. The endogenous melatonin concentration in bovine follicular fluid is approximately 10(-11) M. To examine the potential beneficial effects of melatonin on bovine oocyte maturation in vitro, germinal vesicle (GV) oocytes were incubated with different concentrations of melatonin (10(-11), 10(-9), 10(-7), 10(-5), 10(-3) M). Melatonin supplementation at suitable concentrations significantly promoted oocyte maturation. The development of embryos and the mean cell number/blastocyst produced after in vitro fertilization were remarkably improved. The most effective melatonin concentrations obtained from the studies ranged from 10(-9) to 10(-7) M. The expression of melatonin receptor MT1 and MT2 genes was identified in cumulus cells, granulosa cells, and oocytes using reverse transcription PCR, immunofluorescence, and Western blot. The mechanistic studies show that the beneficial effects of melatonin on bovine oocyte maturation are mediated via melatonin membrane receptors as the melatonin receptor agonist (IIK7) promotes this effect while the melatonin receptor antagonist (luzindole) blocks this action. Mechanistic explorations revealed that melatonin supplementation during bovine oocyte maturation significantly up-regulated the expressions of oocyte maturation-associated genes (GDF9, MARF1, and DNMT1a) and cumulus cells expansion-related gene (PTX3, HAS1/2) and that LHR1/2, EGFR are involved in signal transduction and epigenetic reprogramming. The results obtained from the studies provide new information regarding the mechanisms by which melatonin promotes bovine oocyte maturation in vitro and provide an important reference for in vitro embryo production of bovine and the human-assisted reproductive technology.

Beneficial effects of melatonin on in vitro bovine embryonic development are mediated by melatonin receptor 1.

In the current study, a fundamental question, that is, the mechanisms related to the beneficial effects of melatonin on mammalian embryonic development, was addressed. To examine the potential beneficial effects of melatonin on bovine embryonic development, different concentrations of melatonin (10(-11), 10(-9), 10(-7), 10(-5), 10(-3) M) were incubated with fertilized embryos. Melatonin in the range of 10(-11) to 10(-5) M significantly promoted embryonic development both in early culture medium (CR1aa +3 mg/mL BSA) and in later culture medium (CR1aa + 6%FBS). The most effective concentrations applied in the current studies were 10(-9) and 10(-7) M. Using quantitative real-time PCR with immunofluorescence and Western blot assays, the expression of melatonin receptor MT1 and MT2 genes was identified in bovine embryos. Further studies indicate that the beneficial effects of melatonin on bovine embryo development were mediated by the MT1 receptor. This is based on the facts that luzindole, a nonselective MT1 and MT2 antagonist, blocked the effect on melatonin-induced embryo development, while 4-P-PDOT, a selective MT2 antagonist, had little effect. Mechanistic explorations uncovered that melatonin application during bovine embryonic development significantly up-regulated the expression of antioxidative (Gpx4, SOD1, bcl-2) and developmentally important genes (SLC2A1, DNMT1A, and DSC2) while down-regulating expression of pro-apoptotic genes (P53, BAX, and Caspase-3). The results obtained from the current studies provide new information regarding the mechanisms by which melatonin promotes bovine embryonic development under both in vitro and in vivo conditions.

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 the in vitro development of pronuclear embryos and increases the efficiency of blastocyst implantation in murine.

When a defect occurs in the in vitro development of a pronuclear embryo, the interruption of the subsequent implantation limits the success of assisted conception. This common problem remains to be solved. In this study, we observed that melatonin at its physiological concentration (10(-7)  m) significantly promoted the in vitro development of murine pronuclear embryos. This was indicated by the increased blastocyst rate, hatching blastocyst rate, and blastocyst cell number with melatonin treatment. In addition, when these blastocysts were implanted into female recipient mice, the pregnancy rates (95.0% versus control 67.8%), litter sizes (4.1 pups/litter versus control 2.7 pups/litter), and postnatal survival rates of offspring (96.84% versus control 81.24%) were significantly improved compared with their non-melatonin-treated counterparts. Mechanistic studies revealed that melatonin treatment upregulates gene expression of the antioxidant enzyme, superoxide dismutase (SOD), and the anti-apoptotic factor bcl-2 while downregulating the expression of pro-apoptotic genes p53 and caspase-3. Due to these changes, melatonin treatment reduces ROS production and cellular apoptosis during in vitro embryo development and improves the quality of blastocysts. The implantation of blastocysts with higher quality leads to more healthy offspring and increased pup survival.

Effects of melatonin on superovulation and transgenic embryo transplantation in small-tailed han sheep (Ovis aries).

OBJECTIVE: In this study, the effects of melatonin on superovulation and the transfer of transgenic embryos were investigated in Small-Tailed Han sheep. DESIGN: Different doses of melatonin (0, 40 or 80 mg/animal) were subcutaneously implanted into both multiparous (4-5 years old) donors and recipients before superovulation and estrus synchronization. The one-year-old young ewes without melatonin treatment served to evaluate the reproductive efficiency of the adult multiparous ewes. Ewes with superovulation were used as embryo donors. The estrus were induced in embryo recipients after embryo transpimplanted. RESULTS: The results showed that the number of corpora lutea of the ewes received subcutaneous 40 or 80 mg melatonin implant (13.4±1.05/ewe, 15.1±1.62/ewe) were significantly higher than that of in control group (8.8±0.37/ewe) (p<0.05). Similarily the number of recovered embryos from the ewes received subcutaneous 40 or 80 mg melatonin implant (10.3±0.84/ewe, 10.9±1.21/ewe) was significantly higher than the control group (6.2±0.60/ewe) (p<0.05). The transimplantd embryos from 40 or 80 mg melatonin treated donors dramatically improved the pregnancy and birth rates compared to control ewes. In addition, both 40 mg and 80 mg melatonin implatation lead to more lambs born per embryo. CONCLUSIONS: These observations provide valuable information for the application of melatonin in increasing superovulation and transgenic embryo transplantation efficiency in sheep.

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.

Removal of both anionic and cationic dyes from wastewater using pH-responsive adsorbents of L-lysine molecular-grafted cellulose porous foams.

High adsorption efficiency, active to both anionic and cationic dyes and simple desorption are three main challenges of the existed adsorbents for decolorization of the dye-contained wastewaters. Porous foams based on L-lysine (Lys) molecular-grafted cellulose were firstly designed and fabricated to overcome those challenges. Cellulose were grafted with Lys in 1-butyl-3-methylimidazolium chloride (BMIMCl) via a chemical connection resulted from glycidyl methacrylate (GMA). The synthesized cellulose derivative (Cell-g-PGMA-Lys) was regenerated in the morphology of foam by non-solvent induced phase inversion from the BMIMCl-based solutions. The presence of Lys moieties and porous structure of Cell-g-PGMA-Lys were confirmed with a series of instrumental analysis. Both anionic reactive brilliant red X-3B (RBR X-3B) and cationic methylene blue (MB) were effectively adsorbed on and desorbed from Cell-g-PGMA-Lys by adjusting the solution pH value. Cell-g-PGMA-Lys had higher adsorption capacities than most of the reported adsorbents and was easy to separate from the decolorized water. It could be reused many times with little reduction of the adsorption capacity, which remained 86.9% and 92.5% for RBR X-3B and MB respectively after six adsorption-desorption cycles. The isothermal and kinetic adsorption proved that dyes were adsorbed single-layered on Cell-g-PGMA-Lys depending upon the electrostatic interaction between adsorbent and adsorbate.

Highly ordered asymmetric cellulose-based honeycomb membrane for moisture-electricity generation and humidity sensing.

Moisture-trigged electricity generator (MEG) that can convert ubiquitous moisture into electricity are highly desirable for developing renewable energy supply and ameliorating the crisis in energy. Constructing an asymmetric ordered, namely gradient ordered porous membrane has great potential in MEG. Herein, a series of cellulose acetate (CA)-based membranes with ordered asymmetric honeycomb membranes were fabricated by Breath Figure method, along with silver nanowires (AgNWs) coating. The asymmetric gradient honeycomb pores were achieved by graft modification of lauroyl chloride and adjustment of relative humidity, which not only endowed the MEG with sensitive sensing signals transport under tension and humidity fluctuations but also enhanced voltages generation speed under flowing moisture. The current study provides a facile and scalable strategy for constructing asymmetric gradient ordered porous materials and creates more possibilities for MEG self-powered flexible wearable electronic devices.

α-ketoglutarate delays age-related fertility decline in mammals.

The fecundity reduction with aging is referred as the reproductive aging which comes earlier than that of chronological aging. Since humans have postponed their childbearing age, to prolong the reproductive age becomes urgent agenda for reproductive biologists. In the current study, we examined the potential associations of α-ketoglutarate (α-KG) and reproductive aging in mammals including mice, swine, and humans. There is a clear tendency of reduced α-KG level with aging in the follicle fluids of human. To explore the mechanisms, mice were selected as the convenient animal model. It is observed that a long term of α-KG administration preserves the ovarian function, the quality and quantity of oocytes as well as the telomere maintaining system in mice. α-KG suppresses ATP synthase and alterations of the energy metabolism trigger the nutritional sensors to down-regulate mTOR pathway. These events not only benefit the general aging process but also maintain ovarian function and delay the reproductive decline. Considering the safety of the α-KG as a naturally occurring molecule in energy metabolism, its utility in reproduction of large mammals including humans deserves further investigation.

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.