Nicotinamide mononucleotide supplementation improves the quality of porcine oocytes under heat stress

Background:Elevated ambient temperature-caused heat stress is a major concern for livestock production due to its negative impact on animal feed intake,growth,reproduction,and health.Particularly,the germ cells are extremely sensitive to the heat stress.However,the effective approach and strategy regarding how to protect mammalian oocytes from heat stress-induced defects have not been determined.Methods:Germinal vesicle(GV)porcine oocytes were cultured at 41.5℃ for 24 h to induce heat stress,and then cultured at 38.5℃ to the specific developmental stage for subsequent analysis.Nicotinamide mononucleotide(NMN)was dissolved in water to 1 mol/L for a stock solution and further diluted with the maturation medium to the final concentrations of 10μmol/L,20μmol/L,50μmol/L or 100μmol/L,respectively,during heat stress.Immunostaining and fluorescence intensity quantification were applied to assess the effects of heat stress and NMN supplementation on the key processes during the oocyte meiotic maturation.Results:Here,we report that NMN supplementation improves the quality of porcine oocytes under heat stress.Specifically,we found that heat stress resulted in oocyte maturation failure by disturbing the dynamics of meiotic organelles,including the cytoskeleton assembly,cortical granule distribution and mitochondrial function.In addition,heat stress induced the production of excessive reactive oxygen species(ROS)and DNA damage,leading to the occurrence of apoptosis in oocytes and subsequent embryonic development arrest.More importantly,we validated that supplementation of NMN during heat stress restored the meiotic defects during porcine oocyte maturation.Conclusions:Taken together,our study documents that NMN supplementation is an effective approach to improve the quality of oocytes under heat stress by promoting both nuclear and cytoplasmic maturation.

Casein kinase 2 modulates the spindle assembly checkpoint to orchestrate porcine oocyte meiotic progression

Background:CK2(casein kinase 2)is a serine/threonine-selective protein kinase that has been involved in a variety of cellular processes such as DNA repair,cell cycle control and circadian rhythm regulation.However,its functional roles in oocyte meiosis have not been fully determined.Results:We report that CK2 is essential for porcine oocyte meiotic maturation by regulating spindle assembly checkpoint(SAC).Immunostaining and immunoblotting analysis showed that CK2 was constantly expressed and located on the chromosomes during the entire oocyte meiotic maturation.Inhibition of CK2 activity by its selective inhibitor CX-4945 impaired the first polar body extrusion and arrested oocytes at M I stage,accompanied by the presence of BubR1 at kinetochores,indicative of activated SAC.In addition,we found that spindle/chromosome structure was disrupted in CK2-inhibited oocytes due to the weakened microtubule stability,which is a major cause resulting in the activation of SAC.Last,we found that the level DNA damage as assessed byγH2A.X staining was considerably elevated when CK2 was inhibited,suggesting that DNA damage might be another critical factor leading to the SAC activation and meiotic failure of oocytes.Conclusions:Our findings demonstrate that CK2 promotes the porcine oocyte maturation by ensuring normal spindle assembly and DNA damage repair.

Proteome landscape and spatial map of mouse primordial germ cells

Primordial germ cells(PGCs)are precursors of both male and female gametes as fundamental materials for organism development.The transcriptome,methylome,and chromatin accessibility profiles of PGCs in both mice and humans have been recently reported.However,little is known about the characteristics of PGCs at the protein levels,which directly exert cellular functions.Here,we construct landscapes of both proteome and 3D spatial distribution of mouse PGCs at E11.5,E13.5 and E16.5 days,the three critical developmental windows for PGCs'sex differentiation,female meiosis initiation and male mitotic arrest.In each developmental stage of PGCs,nearly 2,000–3,000 proteins are identified,among which specific functional pathways such as oxidative phosphorylation,DNA damage repair,and meiotic cell cycle are involved for different events during PGCs development.Interestingly,by 3D modeling we find that PGCs spatially cluster into around 1,300 nests in genital ridge at E11.5 and the nest number is not increased by the exponential proliferation of PGCs.Comparative analysis of our proteomic data with published transcriptomic data does not show a close correlation,meaning that the practically executive factors are beyond the transcriptome.Thus,our work offers a valuable resource for the systematic investigations of PGC development at protein level and spatial map.

Supplementation of nicotinamide mononucleotide improves the quality of postovulatory aged porcine oocytes

Dear Editor,Postovulatory aging would occur to impair the oocyte quality in a timedependent manner if ovulated oocytes are not fertilized within the optimal timing in the oviduct or in the dishes(Trapphoff et al.,2016),which might result in the early pregnancy failure in vivo and adverse outcome of assisted reproductive technology(ART)in vitro(Nagy et al.,1993;Wilcox et al.,1998).However,the effective approaches to preventing the postovulatory aging-induced oocyte deterioration are still underexplored.We recently reported that supplementation of nicotinamide mononucleotide(NMN),a synthetic precursor of NAD+,which is an essential cofactorfor enzymes'functioning in almost all critical cellularmetabolic reactions(Imai and Guarente,2014;Canto et al.,2015),reverses the declining quality of maternally aged mouse oocytes(Miao et al.,2020).