The FSH-mTOR-CNP signaling axis initiates follicular antrum formation by regulating tight junction, ion pumps, and aquaporins.

The initial formation of the follicular antrum (iFFA) serves as a dividing line between gonadotropin-independent and gonadotropin-dependent folliculogenesis, enabling the follicle to sensitively respond to gonadotropins for its further development. However, the mechanism underlying iFFA remains elusive. Herein, we reported that iFFA is characterized by enhanced fluid absorption, energy consumption, secretion, and proliferation and shares a regulatory mechanism with blastula cavity formation. By use of bioinformatics analysis, follicular culture, RNA interference, and other techniques, we further demonstrated that the tight junction, ion pumps, and aquaporins are essential for follicular fluid accumulation during iFFA, as a deficiency of any one of these negatively impacts fluid accumulation and antrum formation. The intraovarian mammalian target of rapamycin-C-type natriuretic peptide pathway, activated by follicle-stimulating hormone, initiated iFFA by activating tight junction, ion pumps, and aquaporins. Building on this, we promoted iFFA by transiently activating mammalian target of rapamycin in cultured follicles and significantly increased oocyte yield. These findings represent a significant advancement in iFFA research, further enhancing our understanding of folliculogenesis in mammals.

An ordinal radiomic model to predict the differentiation grade of invasive non-mucinous pulmonary adenocarcinoma based on low-dose computed tomography in lung cancer screening.

OBJECTIVES: To construct a radiomic model of low-dose CT (LDCT) to predict the differentiation grade of invasive non-mucinous pulmonary adenocarcinoma (IPA) and compare its diagnostic performance with quantitative-semantic model and radiologists. METHODS: A total of 682 pulmonary nodules were divided into the primary cohort (181 grade 1; 254 grade 2; 64 grade 3) and validation cohort (69 grade 1; 99 grade 2; 15 grade 3) according to scanners. The radiomic and quantitative-semantic models were built using ordinal logistic regression. The diagnostic performance of the models and radiologists was assessed by the area under the curve (AUC) of the receiver operating characteristic curve and accuracy. RESULTS: The radiomic model demonstrated excellent diagnostic performance in the validation cohort (AUC, 0.900 (95%CI: 0.847-0.939) for Grade 1 vs. Grade 2/Grade 3; AUC, 0.929 (95%CI: 0.882-0.962) for Grade 1/Grade 2 vs. Grade 3; accuracy, 0.803 (95%CI: 0.737-0.857)). No significant difference in diagnostic performance was found between the radiomic model and radiological expert (AUC, 0.840 (95%CI: 0.779-0.890) for Grade 1 vs. Grade 2/Grade 3, p = 0.130; AUC, 0.852 (95%CI: 0.793-0.900) for Grade 1/Grade 2 vs. Grade 3, p = 0.170; accuracy, 0.743 (95%CI: 0.673-0.804), p = 0.079), but the radiomic model outperformed the quantitative-semantic model and inexperienced radiologists (all p < 0.05). CONCLUSIONS: The radiomic model of LDCT can be used to predict the differentiation grade of IPA in lung cancer screening, and its diagnostic performance is comparable to that of radiological expert. KEY POINTS: • Early identifying the novel differentiation grade of invasive non-mucinous pulmonary adenocarcinoma may provide guidance for further surveillance, surgical strategy, or more adjuvant treatment. • The diagnostic performance of the radiomic model is comparable to that of a radiological expert and superior to that of the quantitative-semantic model and inexperienced radiologists. • The radiomic model of low-dose CT can be used to predict the differentiation grade of invasive non-mucinous pulmonary adenocarcinoma in lung cancer screening.

Radiomic and quantitative-semantic models of low-dose computed tomography for predicting the poorly differentiated invasive non-mucinous pulmonary adenocarcinoma.

PURPOSE: Poorly differentiated invasive non-mucinous pulmonary adenocarcinoma (IPA), based on the novel grading system, was related to poor prognosis, with a high risk of lymph node metastasis and local recurrence. This study aimed to build the radiomic and quantitative-semantic models of low-dose computed tomography (LDCT) to preoperatively predict the poorly differentiated IPA in nodules with solid component, and compare their diagnostic performance with radiologists. MATERIALS AND METHODS: A total of 396 nodules from 388 eligible patients, who underwent LDCT scan within 2 weeks before surgery and were pathologically diagnosed with IPA, were retrospectively enrolled between July 2018 and December 2021. Nodules were divided into two independent cohorts according to scanners: primary cohort (195 well/moderate differentiated and 64 poorly differentiated) and validation cohort (104 well/moderate differentiated and 33 poorly differentiated). The radiomic and quantitative-semantic models were built using multivariable logistic regression. The diagnostic performance of the models and radiologists was assessed by area under curve (AUC) of receiver operating characteristic (ROC) curve, accuracy, sensitivity, and specificity. RESULTS: No significant differences of AUCs were found between the radiomic and quantitative-semantic model in primary and validation cohorts (0.921 vs. 0.923, P = 0.846 and 0.938 vs. 0.911, P = 0.161). Both the models outperformed three radiologists in the validation cohort (all P < 0.05). CONCLUSIONS: The radiomic and quantitative-semantic models of LDCT, which could identify the poorly differentiated IPA with excellent diagnostic performance, might provide guidance for therapeutic decision making, such as choosing appropriate surgical method or adjuvant chemotherapy.

[Association Between Medication Compliance and Various Risky Behaviors in Patients With Schizophrenia]. / ç²¾ç¥žåˆ†è£‚ç—‡æ‚£è€ æœè¯ä¾ä»Žæ€§ä¸Žä¸åŒç±»åž‹å±é™©è¡Œä¸ºçš„å ³è”ç ”ç©¶.

Objective: To investigate the status of medication adherence and various types of risky behaviors of schizophrenia patients in a certain area of western China and to explore accordingly the correlation between the two. Methods: A total of 292 667 patients with schizophrenia were enrolled in a follow-up survey between 2006 and 2018. In addition, based on the outcome-wide analysis strategy, a multivariate Cox proportional risk regression model was used to estimate and compare the impact of medication adherence on different types of risky behaviors in schizophrenia patients. Results: In this 13-year prospective cohort, 65 175 patients (31.4%) showed good medication adherence, while 142 394 patients (68.6%) showed poor medication adherence. The incidence rates of various risky behaviors during the follow-up period were as follows, minor nuisances, 12.25%, violation of the Law of the People's Republic of China on Penalties for Administration of Public Security (APS law), 3.82%, violation of criminal law, 0.94%, suicide completed, 0.28%, self-harm, 1.42%, and attempted suicide, 0.82%. Schizophrenia patients who had poor medication adherence had higher risks of committing violence against others and self-inflicted injury compared to patients with good medication adherence did, with the associated effects being minor nuisances (hazard ratio [HR]=1.31, 95% confidence interval [CI]: 1.27-1.35), violation of APS law (HR=1.47, 95% CI: 1.38-1.56), violation of criminal law (HR=1.17, 95% CI: 1.05-1.31), and self-harm (HR=1.43, 95% CI: 1.32-1.56), respectively, while the risk of suicide completed is lower in schizophrenia patients with poor medication adherence than that in patients with good medication adherence (HR=0.56, 95% CI: 0.47-0.66). There was no statistically significant association between attempted suicide and medication adherence. Conclusion: There are variations in the direction and strength of the association between medication adherence and different types of risky behaviors and further research is needed to elucidate the mechanisms of the association.

A Prepubertal Mice Model to Study the Growth Pattern of Early Ovarian Follicles.

Early folliculogenesis begins with the activation of the follicle and ends with the formation of the follicular antrum, which takes up most of the time of folliculogenesis. In this long process, follicles complete a series of developmental events, including but not limited to granulosa cell (GC) proliferation, theca folliculi formation, and antrum formation. However, the logical or temporal sequence of these events is not entirely clear. This study demonstrated in a mouse model that completion of early folliculogenesis required a minimum of two weeks. The oocyte reached its largest size in the Type 4-5 stage, which was therefore considered as the optimum period for studying oogenesis. Postnatal days (PD) 10-12 were regarded as the crucial stage of theca folliculi formation, as Lhcgr sharply increased during this stage. PD13-15 was the rapid growth period of early follicles, which was characterized by rapid cell proliferation, the sudden emergence of the antrum, and increased Fshr expression. The ovarian morphology remained stable during PD15-21, but antrum follicles accumulated gradually. Atresia occurred at all stages, with the lowest rate in Type 3 follicles and no differences among early Type 4-6 follicles. The earliest vaginal opening was observed at PD24, almost immediately after the first growing follicular wave. Therefore, the period of PD22-23 could be considered as a suitable period for studying puberty initiation. This study objectively revealed the pattern of early folliculogenesis and provided time windows for the study of biological events in this process.

Melatonin Administration Accelerates Puberty Onset in Mice by Promoting FSH Synthesis.

Although melatonin has been extensively studied in animal reproduction, the mechanism of melatonin in puberty remains elusive. This study was designed to explore the effect of intraperitoneal administration of melatonin on puberty onset in female mice. The injection of melatonin into postnatal days 10 mice at a dose of 15 mg/kg accelerated the puberty onset in mice. Mechanistically, there was no difference in physical growth and serum Leptin levels after melatonin administration. Meanwhile, the serum levels of reproductive hormones involved in hypothalamic-pituitary-ovarian axis, such as FSH and estrogen level in serum were increased. The mRNA levels of GnRH and GnRHr were not affected by melatonin, while the expressions of FSHß in pituitary and Cyp19a1 in ovary were significantly up-regulated. In addition, melatonin still promoted FSH synthesis after ovariectomy. Furthermore, the enhanced activity of ERK1/2 signaling verified that the expression of FSHß increased in pituitary. We confirmed that melatonin promoted the FSH synthesis in pituitary, thereby increased serum estrogen levels and ultimately accelerated puberty onset. However, these effects of melatonin may be pharmacological due to the high dose. This study would help us to understand the functions of melatonin in pubertal regulation comprehensively.

A mouse model reveals the events and underlying regulatory signals during the gonadotrophin-dependent phase of follicle development.

During folliculogenesis, the gonadotrophin (GTH)-dependent phase begins at the small antral follicle stage and ends with Graafian follicles. In this study, pregnant mare's serum GTH was used to induce GTH-dependent folliculogenesis in mice, following which the developmental events that follicles undergo, as well as the underlying regulatory signals, were investigated at both the morphological and transcriptomic level. GTH-dependent folliculogenesis consisted of three phases: preparation, rapid growth and decelerated growth. In the preparation phase, comprising the first 12 h, granulosa cells completed the preparations for proliferation and differentiation, shifted energy metabolism to glycolysis, and reduced protein synthesis and processing. The rapid growth phase lasted from 12 to 24 h; in this phase, granulosa cells completed their proliferation, and follicles acquired the capacity for estradiol secretion and ovulation. Meanwhile, the decelerating growth phase occurred between 24 and 48 h of GTH-dependent folliculogenesis. In this phase, the proliferation and expansion of the follicular antrum were reduced, energy metabolism was shifted to oxidative phosphorylation, and cell migration and lipid metabolism were enhanced in preparation for luteinization. We also revealed the key signaling pathways that regulate GTH-dependent folliculogenesis and elucidated the activation sequence of these pathways. A comparison of our RNA-sequencing data with that reported for humans suggested that the mechanisms involved in mouse and human folliculogenesis are evolutionarily conserved. In this study, we draw a detailed atlas of GTH-dependent folliculogenesis, thereby laying the foundation for further investigation of the regulatory mechanisms underlying this process.

Accuracy of Pulmonary Nodule Volumetry Using Noise-Optimized Virtual Monoenergetic Image and Nonlinear Blending Image Algorithms in Dual-Energy Computed Tomography: A Phantom Study.

OBJECTIVE: The aim of the study was to assess accuracy of pulmonary nodule volumetry using noise-optimized virtual monoenergetic image (VMI+) and nonlinear blending image (NBI) algorithms in dual-energy computed tomography (DECT). METHODS: An anthropomorphic chest phantom with 10 simulated nodules (5 solid nodules and 5 ground-glass opacities) was scanned using DECT80/Sn140kV, DECT100/Sn140kV, and single-energy CT (SECT120kV/200mAs), respectively. The dual-energy images were reconstructed using VMI+ (70 keV) and NBI algorithms. The contrast-to-noise ratio and absolute percentage error (APE) of nodule volume were measured to assess image quality and accuracy of nodule volumetry. The radiation dose was also estimated. RESULTS: The contrast-to-noise ratio of SECT120kV/200mAs was significantly higher than that of NBI80/Sn140kV and VMI+80/Sn140kV (both corrected P < 0.05), whereas there were no significant differences between NBI100/sn140kV and SECT120kV/200mAs and between VMI+100/sn140kV and SECT120kV/200mAs (both corrected P > 0.05). The APE of SECT120kV/200mAs was significantly lower than that of NBI80/Sn140kV and VMI+80/Sn140kV in both types of nodules (all corrected P < 0.05), whereas there were no significant differences between VMI+100/sn140kV and SECT120kV/200mAs in solid nodules and between NBI100/Sn140kV and SECT120kV/200mAs in ground-glass opacities (both corrected P > 0.05). The radiation dose of DECT100/Sn140kV and DECT80/Sn140kV were significantly lower than that of SECT120kV/200mAs (both corrected P < 0.05). CONCLUSIONS: The DECT100/sn140kV can ensure image quality and nodule volumetry accuracy with lower radiation dose compared with SECT120kV/200mAs. Specifically, the VMI+ algorithm could be used in solid nodules and NBI algorithm in ground-glass opacities.

Evaluation and mining the applicable methods of roughage digestibility determination for buffalo (Bubalus bubalis).

Buffalo is an amazing ruminant with high tolerance to low-quality roughage. Due to the special breeding environment, there are few reports on the digestibility of roughage in buffalo because it is difficult to quantify ingestion and egestion. To find more applicable method to determine the digestibility of low-quality roughage in buffalo, this study was conducted to compare total feces collection (TFC) method with the following three indirect techniques: Cr2O3 (chromic oxide), AIA (acid-insoluble ash), and ADL (acid detergent lignin), to determine rice straw digestibility in buffalo. Six non-pregnant, non-lactating female buffaloes were used in this experiment and the nutritional compositions of the rice straw and feces were measured. Using Cr2O3 and AIA methods, the digestibility of dry matter (DM), ash, organic matter (OM), nitrogen free extract (NFE), crude protein (CP), ether extract (EE), crude fiber (CF), neutral detergent fiber (NDF), acid detergent fiber (ADF), cellulose (CEL), hemicellulose (HC), and ADL did not have statistically significant differences compared with TFC (P > 0.05). However, the digestibility of DM, ash, OM, NFE, EE, CF, NDF, ADF, and ADL determined using ADL method were significantly lower than those using the TFC (P < 0.05). The feces recovery of Cr2O3 and AIA was 95.89% and 97.14%, which were higher than that of ADL (88.90%). In summary, compare with ADL method, TFC, Cr2O3, and AIA methods are more accurate to determine the roughage digestibility of buffalo. Furthermore, Cr2O3 and AIA methods are applicable and convenient to evaluate the roughage digestibility of buffalo under extensive feeding system.

Alpha-ketoglutarate affects murine embryo development through metabolic and epigenetic modulations.

α-Ketoglutarate (α-KG) is an intermediary metabolite in the tricarboxylic acid (TCA) cycle and functions to inhibit ATPase and maintain the pluripotency of embryonic stem cells (ESCs); however, little is known regarding the effects of α-KG on the development of preimplantation embryos. Herein, we report that α-KG (150 µM) treatment significantly promoted the blastocyst rate, the number of inner cell mass (ICM) cells and foetal growth after embryo transfer. Mechanistic studies revealed two important pathways involved in the α-KG effects on embryo development. First, α-KG modulates mitochondria function by inducing relatively low ATP production without modification of mitochondrial copy number. The relatively low energy metabolism preserves the pluripotency and competence of the ICM. Second, α-KG modifies epigenetics in embryos cultured in vitro by affecting the activity of the DNA demethylation enzyme TET and the DNA methylation gene Dnmt3a to increase the ratio of 5hmC/5mC ratio. Elevation of the 5hmC/5mC ratio not only promotes the pluripotency of the ICM but also leads to a methylation level in an in vitro embryo close to that in an in vivo embryo. All these functions of α-KG collectively contribute to an increase in the number of ICM cells, leading to greater adaptation of cultured embryos to in vitro conditions and promoting foetal growth after embryo transfer. Our findings provide basic knowledge regarding the mechanisms by which α-KG affects embryo development and cell differentiation.

Accuracy of Pulmonary Nodule Volumetry at Different Exposure Parameters in Low-Dose Computed Tomography: A Phantom Study.

OBJECTIVE: To explore the exposure parameters with minimized radiation dose for accurate pulmonary nodule volumetry using low-dose computed tomography (LDCT). METHODS: An anthropomorphic chest phantom with 11 pulmonary nodules (6 solid nodules and 5 ground-glass opacities) was scanned using 256-slice multidetector computed tomography scanner at various tube voltage and current (combinations of 80, 100 and 120 kV with 10 to 30 mAs). Raw data sets were reconstructed using the hybrid iterative reconstruction method and nodule volume was calculated by a semiautomatic software. The absolute percentage error (APE) of nodule volume relating to the reference acquisition and contrast-to-noise ratio was measured. RESULTS: Nodule characteristic and tube voltage (P < 0.0001) as well as the interaction between nodule characteristic and tube voltage (P = 0.0026) contributed significantly to the mean difference of APE, while tube current did not (P = 0.21). Post hoc analysis revealed no significant difference was found between the APE at 100 kV and 120 kV in both solid nodules (2.3 ± 0.4% vs 1.8 ± 0.6%, P = 0.14) and ground-glass opacities (6.0 ± 0.5% vs 4.9 ± 0.6%, P = 0.11). Exploratory analyses further showed that the APE at 100 kV with 10 mAs did not differ from that at 120 kV with 30 mAs in both solid nodules (2.5 ± 0.5% vs 1.7 ± 0.3%, P = 0.025, corrected P = 0.20) and ground-glass opacities (6.4 ± 0.4% vs 4.8 ± 1.0%, P = 0.0084, corrected P = 0.068). CONCLUSIONS: In our study, the exposure parameters with minimized radiation dose for accurate pulmonary nodule volumetry were found at 100 kV with 10 mAs, and the estimated effect radiation dose was as low as 0.2 mSv, suggesting the feasibility of further reducing radiation dose by decreasing tube voltage and current in LDCT lung screening.

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.

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.

The Regulatory Mechanism of MLT/MT1 Signaling on the Growth of Antler Mesenchymal Cells.

Melatonin (MLT) plays an important role in regulating the physiological cycle of seasonal breeding animals. Melatonin receptor I (MT1) is effectively expressed in the cambium layer of deer antler. However, the function and metabolic mechanism of MLT/MT1 signaling in the mesenchymal cells of sika deer remain to be further elucidated. In this work, we detected the effects of MLT/MT1 signaling on mesenchymal cells proliferation and the interaction between MLT/MT1 and IGF1/IGF1-R signaling. The results show that (1) deer antler mesenchymal cells actually express MT1; (2) exogenous melatonin significantly promotes mesenchymal cells proliferation, while MT1 knock-down significantly impairs the positive effects of melatonin; and (3) melatonin significantly enhanced IGF1/IGF1-R signaling, as both the expression of IGF1 and IGF-1R increased, while MT1 knock-down significantly decreased IGF1-R expression and IGF1 synthesis. In summary, these data verified that MLT/MT1 signaling plays a crucial role in antler mesenchymal proliferation, which may be mediated by IGF1/IGF1-R.

Melatonin Improves the Quality of Inferior Bovine Oocytes and Promoted Their Subsequent IVF Embryo Development: Mechanisms and Results.

The inferior oocytes (IOs), which are not suitable for embryo development, occupy roughly one-third or more of the collected immature bovine oocytes. The IOs are usually discarded from the in vitro bovine embryo production process. Improving the quality of the inferior oocytes (IOs) and make them available in in vitro embryo production would have important biological, as well as commercial, value. This study was designed to investigate whether melatonin could improve the quality of IOs and make them usable in the in vitro maturation (IVM) and subsequent (in vitro fertilization) IVF embryo development. The results indicated that: the maturation rate of IOs and their subsequent IVF embryo developments were impaired compared to cumulus-oocyte complexes and melatonin treatment significantly improved the quality of IOs, as well as their IVF and embryo developments. The potential mechanisms are that: (1) melatonin reduced reactive oxygen species (ROS) and enhanced glutathione (GSH) levels in the IOs, thereby protecting them from oxidative stress; (2) melatonin improved mitochondrial normal distribution and function to increase ATP level in IOs; and (3) melatonin upregulated the expression of ATPase 6, BMP-15, GDF-9, SOD-1, Gpx-4, and Bcl-2, which are critical genes for oocyte maturation and embryo development and downregulated apoptotic gene expression of caspase-3.

Melatonin and its receptor MT1 are involved in the downstream reaction to luteinizing hormone and participate in the regulation of luteinization in different species.

The functions of melatonin in preovulatory fluid remain elusive. In the current study, we observed that the extremely high level of expression of MT1 in mice granulosa cells was rapidly induced by hCG (equivalent LH) within 2 hours and this was referred as MT1 surge. In cumulus cells, serotonin N-acetyltransferase (SNAT) was also upregulated by hCG and led to elevated melatonin levels in ovarian follicle fluid. Melatonin application before MT1 surge significantly promoted embryo implantation, and this was probably attributed to a rise in progesterone levels in the serum. The mechanistic studies indicated that melatonin/MT1 (MLT/MT1) signaling remarkably improved the expression of corpus luteum marker genes, that is, Akr1c18 and Cyp11a1. High-throughput sequencing results suggested that extracellular matrix (ECM) receptor interaction, focal adhesion, and activation of PI3K/Akt pathway which are involved in granulosa cell luteinization might mediate the actions of MLT/MT1 signal. In addition, this effect on luteinization was compared in different species. It was verified that high melatonin levels exist in serum at estrum of cows and help to improve the first estrus fecundation rate. These results suggested that both melatonin and MT1 are involved in the downstream reaction of hCG (LH) and they play important roles in luteinization. These findings provide the novel information on the physiology of melatonin in animal reproduction.

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.

Melatonin protects porcine oocyte in vitro maturation from heat stress.

Melatonin is a pleiotropic molecule which plays an important role in animal reproductive activities. Because of the increased global warming, the impact of heat stress (HS) on stockbreeding has become an inevitable issue to be solved. To investigate the potential effects of melatonin on the in vitro maturation of porcine oocyte under the HS, a HS model for porcine oocyte maturation has been used in this study and the different concentrations of melatonin (10(-6) -10(-9)  m) were also tested for their protective effects on oocytes. The polar body rate, the index of the nuclear maturation of the oocytes, and the cleavage rate as well as the blastocyst rate were measured to evaluate the developmental competence of the oocytes after parthenogenetic activation (PA). The results showed that HS [in vitro maturation (IVM) 20-24 hr, 42°C] significantly reduced the polar body rate of oocytes and the blastocyte rate of porcine PA embryos, while melatonin (10(-7)  m) application not only improved polar body rate and blastocyte rate, but also preserved the normal levels of steroid hormone which is disrupted by HS. The presence of melatonin (10(-7)  m) during the oocyte maturation under the HS reduced reactive oxygen species (ROS) formation, enhanced glutathione (GSH) production, inhibited cell apoptosis, and increased the gene expressions of SIRT1, AKT2, and Polg2. Importantly, the endogenously occurring melatonin of cumulus-oocyte complexes was significantly induced by HS. The results indicated that melatonin application effectively protected the oocytes from HS. These observations warranted the further studies in vivo regarding to improve the reproductive activities of animals under the global warming environment.

Melatonin-related genes expressed in the mouse uterus during early gestation promote embryo implantation.

Melatonin, a superior antioxidant, is an important molecule which regulates female reproduction due to its receptor-mediated and receptor-independent antioxidant actions. In this study, we investigated the effect of melatonin on early gestation in a mouse model. During early gestation, the expression of the melatonin's rate-limiting enzyme, AANAT, gradually increased - in the uterus while the MT2 melatonin receptor was only expressed at day 2 of gestation and no MT1 was detected. Based on these findings, we conducted a melatonin injection experiment which demonstrated that 15 mg/kg melatonin significantly improved the number of implantation sites and the litter size. Also, the blastocyst and uterus were collected to identify the local action of melatonin. In the melatonin-treated mice, the endometrium was thicker than in the control mice; melatonin also caused an increase in density of uterine glands, and the uterine gland index (UGI) was significantly elevated over that of the control. Serum steroid hormone measurements revealed that at day 6 of gestation (postimplantation), melatonin significantly downregulated the E2 level, with no obvious effects on progesterone. Gene expression assay revealed that melatonin significantly upregulated expression of HB-EGF, a crucial gene involved in implantation as well as its receptor ErbB1 in the blastocyst. In addition, PRA, an important gene which influences the decidual response and luminal cell differentiation, p53, which regulates uterine through leukaemia inhibitory factor (LIF), were both increased after melatonin treatment. These data suggest that melatonin and its MT2 receptor influence early gestation. Exogenous melatonin treatment can improve mouse embryo implantation and litter size, which may have important applications in human reproductive health and animal husbandry.

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.