NMN rescued porcine oocyte mitochondrial dysfunction caused by aged.

Images (A) of mitochondrial content level (B) at 0 h, AGED-24 h, AGED+NMN-24 h, AGED-48 h and AGED+NMN-48 h oocytes. Scale bars represented 100 μm. Images (C) of mitochondrial membrane potential level (D) at 0 h, AGED-24 h, AGED+NMN-24 h, AGED-48 h and AGED+NMN-48 h oocytes. *p < 0.05 indicated significant differences. Scale bars represented 100 μm.</p

NMN rescued aged-induced fragmentation of porcine oocytes.

(A) Oocyte morphologies and fragmentation rate (B) in aged 0 h, 24 h, and 48 h groups; (C) Oocyte morphologies at CON-48 h and 100 μm NMN groups. The red circles represent aging oocytes; (D) The fragmentation rate of aged 48 h oocytes treated with different concentrations of NMN (0, 1, 10 or 100 μm). The red dotted circle indicated fragmented oocytes. Scale bars represent 100 μm. *p < 0.05, **p < 0.01.</p

NMN improved the effects of aging on embryonic development.

The D7 embryo morphologies (A) and blastocyst rate (B) at 0 h, AGED-24 h, AGED+NMN-24 h groups. Scale bars represented 100 μm. Expression of pluripotent genes NANOG (C) OCT4 (D) and SOX2 (E) at 0 h, AGED-24 h, AGED+NMN-24 h groups. *p < 0.05, **p < 0.01 indicated significant differences. (F) and (G) Statistics of the number of blastocysts at 0 h, AGED-24 h, AGED+NMN-24 h groups. Scale bars represented 50 μm.</p

NMN rescued the cytoskeletal damage of aged porcine oocytes.

Representative confocal images (A) and fluorescence intensity (B) of actin at 0 h, AGED-24 h, AGED+NMN-24 h, AGED-48 h and AGED+NMN-48 h oocytes. *p < 0.05 indicated significant differences. Scale bars represented 50 μm.</p

NMN reduced the level of ROS in aged porcine oocytes.

Images (A) and ROS level (B) at 0 h, AGED-24 h, AGED+NMN-24 h, AGED-48 h and AGED+NMN-48 h oocytes. Scale bars represented 100 μm. The expression of anti‐oxidative stress genes SOD1 (C) and Cat (D) in fresh and aged oocytes. *p < 0.05, **p < 0.01 indicated significant differences.</p

NMN maintained the chromosome morphology of aged porcine oocytes.

(A) Representative confocal images of normal and abnormal spindles and DNA. (B, C) The ratio of abnormal spindle and misaligned chromosome at 0 h, AGED-24 h, AGED+NMN-24 h, AGED-48 h and AGED+NMN-48 h oocytes. *p < 0.05, **p < 0.01 indicated significant differences. Scale bars represented 2 μm.</p

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Oocyte senescence alters the shape and function, thereby weakening the fertilization potential. Nicotinamide mononucleotide (NMN) reverses age-related dysfunctions in various organs. Studies had shown long-term administration of NMN reduced the physiological decline associated in aged mice and reversed the aging of the ovaries. However, the protective effect of NMN on aged porcine oocytes is still unclear. In this study, we investigated the effects of NMN on aging porcine oocytes and subsequent embryonic development. We established a model of senescence of porcine oocytes after ovulation by extending the culture time in vitro. NMN supplementation significantly reduced reactive oxygen species (ROS) levels in senescence oocytes and increased the mRNA levels of antioxidant genes SOD1 and Cat. The mitochondrial membrane potential of aged oocytes treated with NMN was increased compared with that of untreated oocytes. In addition, the mRNA level of apoptosis-related gene Bax was significantly decreased in senescence oocytes treated with NMN, while the mRNA level of anti-apoptosis-related gene BCL-2 was significantly increased. Furthermore, NMN supplementation enhanced the subsequent development ability of senescent oocytes during in vitro aging. Compared with untreated senescent oocytes, the blastocyst formation rate and pluripotent genes of senescent oocytes treated with NMN were significantly increased. Taken together, these results suggest that NMN is beneficial for delaying the aging process in porcine oocytes.</div

NMN decreased the mRNA level of apoptosis-related genes of aged porcine oocytes.

(A) The expression of anti‐apoptosis genes (Bcl2) at 0 h, AGED-24 h, AGED+NMN-24 h, AGED-48 h and AGED+NMN-48 h oocytes. (B) The expression of pro‐apoptosis genes (Bax) at 0 h, AGED-24 h, AGED+NMN-24 h, AGED-48 h and AGED+NMN-48 h oocytes. (C) Effect of NMN on Bcl-2/ Bax ratio in aged oocytes. *p < 0.05, **p < 0.01 indicated significant differences.</p

DataSheet_1_Commercial scale genetic transformation of mature seed embryo explants in maize.docx

A novel, efficient maize genetic transformation system was developed using Agrobacterium-mediated transformation of embryo explants from mature seeds. Seeds from field grown plants were sterilized and crushed to isolate embryo explants consisting of the coleoptile, leaf primordia, and shoot apical meristem which were then purified from the ground seed bulk preparation. The infection of relevant tissues of seed embryo explants (SEEs) by Agrobacterium was improved by the centrifugation of the explants. Transgenic plants were obtained by multiple bud induction on high cytokinin media, followed by plant regeneration on hormone-free medium. Three different selectable markers (cp4 epsps, aadA, and nptII) were successfully used for producing transgenic plants. Stable integration of transgenes in the maize genome was demonstrated by molecular analyses and germline transmission of the inserted transgenes to the next generation was confirmed by pollen segregation and progeny analysis. Phenotypic evidence for chimeric transgenic tissue was frequently observed in initial experiments but was significantly reduced by including a second bud induction step with optimized cytokinin concentration. Additional improvements, including culturing explants at an elevated temperature during bud induction led to the development of a revolutionary system for efficient transgenic plant production and genome editing. To our knowledge, this is the first report of successful transgenic plant regeneration through Agrobacterium-mediated transformation of maize mature SEEs. This system starts with mature seed that can be produced in large volumes and the SEEs explants are storable. It has significant advantages in terms of scalability and flexibility over methods that rely on immature explants.</p