Structures of the ADGRG2-Gs complex in apo and ligand-bound forms.

Adhesion G protein-coupled receptors are elusive in terms of their structural information and ligands. Here, we solved the cryogenic-electron microscopy (cryo-EM) structure of apo-ADGRG2, an essential membrane receptor for maintaining male fertility, in complex with a Gs trimer. Whereas the formations of two kinks were determinants of the active state, identification of a potential ligand-binding pocket in ADGRG2 facilitated the screening and identification of dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate and deoxycorticosterone as potential ligands of ADGRG2. The cryo-EM structures of DHEA-ADGRG2-Gs provided interaction details for DHEA within the seven transmembrane domains of ADGRG2. Collectively, our data provide a structural basis for the activation and signaling of ADGRG2, as well as characterization of steroid hormones as ADGRG2 ligands, which might be used as useful tools for further functional studies of the orphan ADGRG2.

Control of spontaneous activation of rat oocytes by regulating plasma membrane Na+/Ca2+ exchanger activities.

Inhibiting oocyte spontaneous activation (SA) is essential for successful rat cloning by nuclear transfer (NT). This study tested the hypothesis that activities of the Na(+)/Ca(2+) exchanger (NCX) would decrease with oocyte aging and that SA of rat oocytes could be inhibited if the intraoocyte Ca(2+) rises were prevented by activating the NCX through increasing Na(+) concentrations in the culture medium. Elevating Na(+) levels in culture medium by supplementing NaCl inhibited SA of rat oocytes, while maintaining a constant level of maturation-promoting factor and mitogen-activated protein kinase activities. Experiments using the NCX inhibitor bepridil, the Na(+)/K(+)-ATPase inhibitor ouabain, and an assay for intraoocyte Ca(2+) concentrations showed that extracellular Na(+) inhibited rat oocyte SA by enhancing NCX activity and preventing intracellular Ca(2+) rises. Immunohistochemical quantification indicated that the density of NCX1 decreased significantly in aged oocytes that were prone to SA compared with that in freshly ovulated oocytes whose SA rates were low during in vitro culture. Cumulus cell NT showed that sham enucleation caused marked SA in freshly ovulated rat oocytes and that Na(+) supplementation prevented the manipulation-induced SA and improved the in vitro and in vivo development of rat somatic cell NT embryos. Taken together, the results have confirmed our hypothesis that the NCX is active in rat oocytes and its activity decreases with oocyte aging and that activating the NCX by increasing extracellular Na(+) inhibits SA of rat oocytes and improves the development of rat somatic cell NT embryos. These data are also important for understanding the mechanisms of oocyte aging.

Environmentally relevant concentrations of Mn2+ disrupts the endocrine regulation of growth in juvenile Yunlong groupers (Epinephelus moara♀×Epinephelus lanceolatus♂).

Even though manganese is a bioelement essential for metabolism, excessive manganese levels in water can be detrimental to fish development and growth. Therefore, the aim of this study was to evaluate the effects of Mn2+ (0, 0.5,1, 2, and 4 mg·L-1) exposure for 30 d on the growth performance, growth hormone/insulin-like growth factor (GH/IGF) axis, hypothalamic-pituitary-thyroid (HPT) axis, and monoaminergic neurotransmitters of Epinephelus moara♀×Epinephelus lanceolatus♂(Yunlong grouper). Compared with the control and low Mn2+concentration groups of (0.5 and 1 mg·L-1), the high concentration of Mn2+ (4 mg·L-1) significantly reduced body weight (BW), body length (BL), weight gain rate (WGR), and specific growth rate (SGR), increased the feed coefficient rate (FCR) and mortality of Yunlong groupers (P < 0.05). Further, the levels of GH and IGF, along with the expression of ghra and ghrb were significantly reduced after exposure to 2 and 4 mg·L-1 Mn2+for 30 d, whereas the expression of sst5 was significantly up-regulated after exposure to 2 and 4 mg·L-1 Mn2+for 20 and 30 days. Moreover, Mn2+exposure increased thyroid hormone (T3) and thyroid stimulating hormone (TSH) contents, accompanied by increased mRNA levels of dio1 and dio2, however, the T4 level was decreased. Finally, dopamine (DA) and serotonin (5-HT) levels significantly decreased after long-term exposure to higher concentrations of Mn2+, and the levels their metabolites changed as well, suggesting that the synthesis and metabolism of DA and 5-HT were affected. Accordingly, changes in the GH/IGF and HPT axes-related parameters may be the cause of growth inhibition in juvenile groupers under Mn2+ exposure, indicating that the relationship between endocrine disorder and growth inhibition should not be ignored.

[Impacts of Spartina alterniflora invasion on the benthic food web in the Yellow River Delta during autumn]. / äº’èŠ±ç±³è‰å ¥ä¾µå¯¹é»„æ²³ä¸‰è§’æ´²ç§‹å­£åº•æ –é£Ÿç‰©ç½‘çš„å½±å“.

Spartina alterniflora was introduced into the Yellow River Delta (YRD) in 1990 with the purpose of shore protection and siltation accretion. However, it spread rapidly and became a severe threat to the local coastal wetland ecosystem. To assess the impacts of S. alterniflora invasion on the benthic food web, we sampled the potential food sources of macrobenthos in November 2020, analyzed the trophic level and the benthic food web structure based on stable isotope technique. Results showed that the average δ13C values of macrobenthic food sources followed an order: sediment organic matter (SOM) > S. alterniflora > benthic microalgae > particulate organic matter (POM) > Suaeda salsa. The average δ15N values significantly differed among food sources, ranging from 1.24‰ to 9.03‰. The trophic levels of different macrobenthos ranged from 1.73 to 4.19, of which the bivalve species was the lowest one. S. alterniflora and the decayed debris were the most important food sources for macrobenthos, but without any impact on the trophic level structure of macro-benthos. In conclusion, Spartina alterniflora invasion distinctly changed the composition of food sources of macrobenthos through a "bottom-up" effect, which would probably impact the local food web structure in the YRD wetland.

RNA-Binding Protein IGF2BP2/IMP2 is a Critical Maternal Activator in Early Zygotic Genome Activation.

A number of genes involved in zygotic genome activation (ZGA) have been identified, but the RNA-binding maternal factors that are directly related to ZGA in mice remain unclear. The present study shows that maternal deletion of Igf  2bp2 (also commonly known as Imp2) in mouse embryos causes early embryonic developmental arrest in vitro at the 2-cell-stage. Transcriptomics and proteomics analyses of 2-cell-stage embryos in mice reveal that deletion of IMP2 downregulates the expression of Ccar1 and Rps14, both of which are required for early embryonic developmental competence. IGF2, a target of IMP2, when added in culture media, increases the proportion of wild-type embryos that develop successfully to the blastocyst stage: from 29% in untreated controls to 65% (50 × 10-9 m IGF2). Furthermore, in an experiment related to embryo transfer, foster mothers receiving IGF2-treated embryos deliver more pups per female than females who receive untreated control embryos. In clinically derived human oocytes, the addition of IGF2 to the culture media significantly enhances the proportion of embryos that develop successfully. Collectively, the findings demonstrate that IMP2 is essential for the regulation and activation of genes known to be involved in ZGA and reveal the potential embryonic development-related utility of IGF2 for animal biotechnology and for assisted reproduction in humans.

The histone modification reader ZCWPW1 is required for meiosis prophase I in male but not in female mice.

Meiosis is a specialized type of cell division that creates haploid germ cells and ensures their genetic diversity through homologous recombination. We show that the H3K4me3 reader ZCWPW1 is specifically required for meiosis prophase I progression in male but not in female germ cells in mice. Loss of Zcwpw1 in male mice caused a complete failure of synapsis, resulting in meiotic arrest at the zygotene to pachytene stage, accompanied by incomplete DNA double-strand break repair and lack of crossover formation, leading to male infertility. In oocytes, deletion of Zcwpw1 only somewhat slowed down meiosis prophase I progression; Zcwpw1-/- oocytes were able to complete meiosis, and Zcwpw1-/- female mice had normal fertility until mid-adulthood. We conclude that the H3K4me3 reader ZCWPW1 is indispensable for meiosis synapsis in males but is dispensable for females. Our results suggest that ZCWPW1 may represent a previously unknown, sex-dependent epigenetic regulator of germ cell meiosis in mammals.

Meiotic arrest with roscovitine and follicular fluid improves cytoplasmic maturation of porcine oocytes by promoting chromatin de-condensation and gene transcription.

The developmental capacity of in vitro matured oocytes is inferior to that of the in vivo matured ones due to insufficient cytoplasmic maturation. Although great efforts were made to accomplish better cytoplasmic maturation by meiotic arrest maintenance (MAM) before in vitro maturation (IVM), limited progress has been achieved in various species. This study showed that MAM of porcine oocytes was better achieved with roscovitine than with dibutyryl cyclic adenosine monophosphate (db-cAMP) or 3-isobutyl-1-methylxanthine. Oocyte developmental competence after IVM was significantly improved following MAM in 199 + FF medium (TCM-199 containing 10% porcine follicular fluid and 25 µM roscovitine) to a level even higher than that in control oocytes matured without pre-MAM. Observations on other markers further confirmed the positive effects of MAM in 199 + FF on oocyte cytoplasmic maturation. During MAM culture in 199 + FF, re-decondensation (RDC) of condensed chromatin occurred, and transcription of genes beneficial to cytoplasmic maturation was evident in some of the oocytes with surrounded nucleoli (SN). However, MAM with db-cAMP neither induced RDC nor improved oocyte developmental potential. Together, the results suggest that MAM in the presence of FF and roscovitine improved the developmental competence of porcine oocytes by promoting a pre-GVBD chromatin de-condensation and expression of beneficial genes.

Cumulus cells accelerate oocyte aging by releasing soluble Fas ligand in mice.

Although previous studies have suggested that cumulus cells (CCs) accelerate oocyte aging by secreting soluble and heat-sensitive paracrine factors, the factors involved are not well characterized. Because Fas-mediated apoptosis represents a major pathway in induction of apoptosis in various cells, we proposed that CCs facilitate oocyte aging by releasing soluble Fas ligand (sFasL). In this study, we reported that when the aging of freshly ovulated mouse oocytes were studied in vitro, both the apoptotic rates of CCs and the amount of CCs produced sFasL increased significantly with the culture time. We found that oocytes expressed stable levels of Fas receptors up to 24 h of in vitro aging. Moreover, culture of cumulus-denuded oocytes in CCs-conditioned CZB medium (CM), in CZB supplemented with recombinant sFasL, or in CM containing sFasL neutralizing antibodies all showed that sFasL impaired the developmental potential of the oocytes whereas facilitating activation and fragmentation of aging oocytes. Furthermore, CCs from the FasL-defective gld mice did not accelerate oocyte aging due to the lack of functional FasL. In conclusion, we propose that CCs surrounding aging oocytes released sFasL in an apoptosis-related manner, and the released sFasL accelerated oocyte aging by binding to Fas receptors.

Role of Na+/Ca2+ exchanger (NCX) in modulating postovulatory aging of mouse and rat oocytes.

We studied the role of the Na+/Ca2+ exchanger (NCX) in modulating oocyte postovulatory aging by observing changes in NCX contents and activities in aging mouse and rat oocytes. Whereas the NCX activity was measured by observing oocyte activation following culture with NCX inhibitor or activator, the NCX contents were determined by immunohistochemical quantification. Although NCX was active in freshly-ovulated rat oocytes recovered 13 h post hCG injection and in aged oocytes recovered 19 h post hCG in both species, it was not active in freshly-ovulated mouse oocytes. However, NCX became active when the freshly-ovulated mouse oocytes were activated with ethanol before culture. Measurement of cytoplasmic Ca2+ revealed Ca2+ increases always before NCX activation. Whereas levels of the reactive oxygen species (ROS) and the activation susceptibility increased, the density of NCX member 1 (NCX1) decreased significantly with oocyte aging in both species. While culture with H2O2 decreased the density of NCX1 significantly, culture with NaCl supplementation sustained the NCX1 density in mouse oocytes. It was concluded that (a) the NCX activity was involved in the modulation of oocyte aging and spontaneous activation; (b) ROS and Na+ regulated the NCX activity in aging oocytes by altering its density as well as functioning; and (c) cytoplasmic Ca2+ elevation was essential for NCX activation in the oocyte.

Carrying-over effects of GVBD blocking on post-blocking meiotic progression of oocytes: species difference and the signaling pathway leading to MPF activation.

Efforts to improve the quality of in vitro matured oocytes by blocking germinal vesicle breakdown (GVBD) and allowing more time for ooplasmic maturation have achieved little due to a lack of knowledge on the molecular events during GVBD blocking. Such knowledge is also important for studies aimed at regulating gene expression in maturing oocytes prior to GVBD. We studied species difference and signaling pathways leading to the carrying-over effect of GVBD blocking on post-blocking meiotic progression (PBMP). Overall, GVBD-blocking with roscovitine decelerated PBMP of mouse oocytes but accelerated that of pig oocytes. During blocking culture, whereas cyclin B of pig oocytes increased continuously, that of mouse oocytes declined first and then increased slowly. In both species, (a) whereas active CDC2A showed a dynamics similar to cyclin B, inactive CDC2A decreased continuously; (b) when oocytes were blocked in blocking medium containing cycloheximide, PBMP was decelerated significantly while cyclin B and active CDC2A decreasing to the lowest level; (c) whereas sodium vanadate in blocking medium reduced PBMP, epidermal growth factor (EGF) in blocking medium accelerated PBMP significantly with no effect on cyclin B levels. In conclusion, the EGF signaling cascade accelerated PBMP by promoting the pre-MPF (M-phase-promoting factor) to MPF conversion during GVBD blocking with roscovitine. The significant difference in PBMP observed between mouse and pig oocytes was caused by species difference in cyclin B dynamics during blocking culture as no species difference was observed in either pre-MPF to MPF conversion or the EGF signaling activity.

Roles of MAPK and spindle assembly checkpoint in spontaneous activation and MIII arrest of rat oocytes.

Rat oocytes are well known to undergo spontaneous activation (SA) after leaving the oviduct, but the SA is abortive with oocytes being arrested in metaphase III (MIII) instead of forming pronuclei. This study was designed to investigate the mechanism causing SA and MIII arrest. Whereas few oocytes collected from SD rats at 13 h after hCG injection that showed 100% of mitogen-activated protein kinase (MAPK) activities activated spontaneously, all oocytes recovered 19 h post hCG with MAPK decreased to below 75% underwent SA during in vitro culture. During SA, MAPK first declined to below 45% and then increased again to 80%; the maturation-promoting factor (MPF) activity fluctuated similarly but always began to change ahead of the MAPK activity. In SA oocytes with 75% of MAPK activities, microtubules were disturbed with irregularly pulled chromosomes dispersed over the spindle and the spindle assembly checkpoint (SAC) was activated. When MAPK decreased to 45%, the spindle disintegrated and chromosomes surrounded by microtubules were scattered in the ooplasm. SA oocytes entered MIII and formed several spindle-like structures by 6 h of culture when the MAPK activity re-increased to above 80%. While SA oocytes showed one Ca(2+) rise, Sr(2+)-activated oocytes showed several. Together, the results suggested that SA stimuli triggered SA in rat oocytes by inducing a premature MAPK inactivation, which led to disturbance of spindle microtubules. The microtubule disturbance impaired pulling of chromosomes to the spindle poles, caused spindle disintegration and activated SAC. The increased SAC activity reactivated MPF and thus MAPK, leading to MIII arrest.

Synthesis and analgesic activity of secondary amine analogues of pyridylmethylamine and positional isomeric analogues of ABT-594.

A series of highly sterically hindered secondary amine analogues of pyridylmethylamine (7a-f, 8a-c) and positional isomeric analogues of ABT-594 (9a-c) were synthesized and evaluated for their in vivo analgesic activity. The compounds 7a and 7d show potent analgesic activity and lower toxicity. Some interesting structure-activity relationships have been revealed.

[Expression of IRP2 mRNA, TfR mRNA and Fn mRNA in HL-60 cells].

To explore the mechanism of iron metabolism and its regulation as well as the roles of IRP(2) in ion metabolism of HL-60 cells, HL-60 cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, which was treated with ferric chloride (FeCl(3)) or deferoxamine (DFO). The cells were harvested at 12, 24 and 48 hours of proliferation, and total RNA was isolated; cDNA was synthesized by reverse transcription (RT), and relative expression levels of IRP(2) mRNA, Fn mRNA and TfR mRNA were determined by RT-PCR. The results showed at follows: (1) the level of IRP(2) mRNA remained constant in all cells, whether or not treated with DFO or FeCl(3). However, the expression of IRP(2) mRNA decreased when the time of cell culture was prolonged. There was no significant difference between groups (F(B-S) = 1.199, P > 0.05), but there was significant difference among the different time culture (F(W-S) = 43.418, P < 0.01). (2) Cells which treated neither with DFO nor ferri chloride showed significant difference from the control (F(W-S) = 7.184, F(B-S) = 113.926; P < 0.01). The level of TfR mRNA increased in the cells treated with DFO. Surprisingly, when cells treated with FeCl(3), there was not decline of TfR mRNA expression, but it increased lightly at 12 hours and peaked at 24 hours and declined drastically at 48 hours. (3) The level of Fn mRNA in the cells treated with FeCl(3) was approximately 2-fold as the control cells. In contrast with the control cells, there was significant difference (P < 0.05). The level of Fn mRNA of the cells treated with DFO had little change. As compared with the control cells, no significant difference was seen (P > 0.05). (4) There was not any significant correlation between IRP(2) mRNA and TfR mRNA or Fn mRNA in HL-60 cells (r = -0.005; r = 0.074; P > 0.05). It is concluded that (1) IRP(2) may regulate the iron metabolism in HL-60 cells by altering amounts of the IRP(2) 3.7- or 6.4-kb mRNA at the transcriptional level, or by IRP(2) degradation at the post transcriptional level. (2) Both of Fn mRNA and TfR mRNA participated, more or less, in the iron metabolism in HL-60 cells.