The pBY520 containing the Hordeum vulgare HVA1 regulated by the rice actin promoter (Act1 5 ) or the JS101 containing the bacterial mannitol-1-phosphate dehydrogenase (mtlD) also regulated by rice Act1 5 and a combination of these two plasmids were transferred into the maize genome, and their stable expressions were confirmed through fourth generations. Plants transcribing a combination of the HVA1+mtlD showed higher leaf relative water content (RWC) and greater plant survival as compared with their single transgene transgenic plants and with their control plants under drought stress. When exposed to various salt concentrations, plants transcribing the HVA1+mtlD showed higher fresh and dry shoot and dry root matter as compared with single transgene transgenic plants and with their control plants. Furthermore, the leaves of plants expressing the mtlD accumulated higher levels of mannitol. Plants expressing the HVA1+mtlD improved plant survival rate under drought stress and enhanced shoot and root biomass under salt stress when compared with single transgene transgenic plants and with their wild-type control plants. The research presented here shows the effectiveness of coexpressing of two heterologous abiotic stress tolerance genes in the maize genome. Future field tests are needed to assure the application of this research.

This work was supported by grants from the National Institutes of Health Office of Research Infrastructure Programs Division of Comparative Medicine Grants R24 [OD012221 to K.E.L., OD011107/RR00169 (California National Primate Research Center), and OD010967/RR025880 to C.A.V.]; the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health under the award number T32HD087166; MSU AgBioResearch, Michigan State University. Authors have nothing to disclose.

Gene editing technologies offer new options for developing novel biomedical research models and for gene and stem cell based therapies. However, applications in many species demand high efficiencies, specificity, and a thorough understanding of likely editing outcomes. To date, overall efficiencies, rates of off-targeting and degree of genetic mosaicism have not been well-characterized for most species, limiting our ability to optimize methods. As a model gene for measuring these parameters of the CRISPR/Cas9 application in a primate species (rhesus monkey), we selected the β-hemoglobin gene (HBB), which also has high relevance to the potential application of gene editing and stem-cell technologies for treating human disease. Our data demonstrate an ability to achieve a high efficiency of gene editing in rhesus monkey zygotes, with no detected off-target effects at selected off-target loci. Considerable genetic mosaicism and variation in the fraction of embryonic cells bearing targeted alleles are observed, and the timing of editing events is revealed using a new model. The uses of Cas9-WT protein combined with optimized concentrations of sgRNAs are two likely areas for further refinement to enhance efficiency while limiting unfavorable outcomes that can be exceedingly costly for application of gene editing in primate species.

Trophoblast stem cells (TSCs) are crucial for embryo implantation and placentation. Environmental toxicants that compromise TSC function could impact fetal viability, pregnancy, and progeny health. Understanding the effects of low, chronic EDC exposures on TSCs and pregnancy is a priority in developmental toxicology. Differences in early implantation between primates and other mammals make a nonhuman primate model ideal. We examined effects of chronic low-level exposure to atrazine, tributyltin, bisphenol A, bis(2-ethylhexyl) phthalate, and perfluorooctanoic acid on rhesus monkey TSCs in vitro by RNA sequencing. Pathway analysis of affected genes revealed negative effects on cytokine signaling related to anti-viral response, most strongly for atrazine and tributyltin, but shared with the other three EDCs. Other affected processes included metabolism, DNA repair, and cell migration. Low-level chronic exposure of primate TSCs to EDCs may thus compromise trophoblast development in vivo, inhibit responses to infection, and negatively affect embryo implantation and pregnancy.

Synchronization of male and female locomotor activity plays a critical role in ensuring reproductive success, especially in semelparous species. The goal of this study was to elucidate the effects of individual chemical signals, or pheromones, on the locomotor activity in the sea lamprey (Petromyzon marinus). In their native habitat, adult preovulated females (POF) and ovulated females (OF) are exposed to sex pheromone compounds that are released from spermiated males and attract females to nests during their migration and spawning periods. In this study, locomotor activity of individual POF and OF was measured hourly in controlled laboratory conditions using an automated video-tracking system. Differences in the activity between a baseline day (no treatment exposure) and a treatment day (sex pheromone compound or control exposure) were examined for daytime and nighttime periods. Results showed that different pheromone compound treatments affected both POF and OF sea lamprey (p < 0.05) but in different ways. Spermiated male washings (SMW) and one of its main components, 7α,12α,24-trihydroxy-5α-cholan-3-one 24 sulfate (3kPZS), decreased activity of POF during the nighttime. SMW also reduced activity in POF during the daytime. In contrast, SMW increased activity of OF during the daytime, and an additional compound found in SMW, petromyzonol sulfate (PZS), decreased the activity during the nighttime. In addition, we examined factors that allowed us to infer the overall locomotor patterns. SMW increased the maximum hourly activity during the daytime, decreased the maximum hourly activity during the nighttime, and reduced the percentage of nocturnal activity in OF. Our findings suggest that adult females have evolved to respond to different male compounds in regards to their locomotor activity before and after final maturation. This is a rare example of how species-wide chemosensory stimuli can affect not only the amounts of activity but also the overall locomotor pattern in a vertebrate species.

Research Question: In the early stages of the COVID-19 pandemic, IVF clinics stopped the majority of patient treatment cycles to minimize the risk of disease transmission. The risk of SARS-CoV-2 viral exposure and potential cross contamination within the IVF lab remains largely unclear. To that end, the objective of this study was to examine follicular fluid (FF), culture media (M) and vitrification solution (VS) for SARS-CoV-2 in an IVF lab. Design : Prospective clinical study. All females undergoing transvaginal oocyte retrieval (TVOR) were required to have a negative SARS-CoV-2 RNA test 3-4 days prior to the procedure. Male partners were not tested. All cases utilized intracytoplasmic sperm injection (ICSI). The first tube of FF aspirated during oocyte retrieval, M drops following removal of embryos on day 5, and VS after blastocyst cryopreservation were analyzed for SARS-CoV-2 RNA. Results: In total, M from 61 patients, VS from 200 patients, and FF from 300 patients were analyzed. All samples were negative for SARS-CoV-2 viral RNA. Conclusion(s): With stringent safety protocols in place, including female patient testing and symptom-based screening of men, the presence of SARS-CoV-2 RNA was not detected in FF, M or VS. This work demonstrates the possibility of implementing a rapid laboratory screening assay for SARS-CoV-2 and has implications for safe laboratory operations, including cryostorage recommendations.

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Invitro culture (IVC) systems fail to completely recapitulate the invivo environment, resulting in metabolic stress during pre-implantation development and reduced blastocyst quality. We hypothesised that IVC-induced metabolic dysregulation in bovine embryos is mediated by changes in expression and/or activity of protein biomarkers associated with key metabolic pathways. Our objectives were to determine (1) expression of enzymes involved in glycolysis (HK-2, PKM2, LDHA, B and C isoforms), entry into the tricarboxylic acid (TCA) cycle (PDH), energy sensing/fatty acid oxidation (AMPK), and the metabolic signalling pathways (AKT, ERK, STAT3, 4EBP1) at the 1-cell (1C), 8- to 16-cell (8–16 C), and Day 7 blastocyst (d7BL) stage; and (2) evaluate the functional activity of these proteins both invivo (superovulated and flushed) and invitro (IVM/IVF/IVC) produced embryos using capillary Western blot (Protein-Simple, JESS; n=1 embryo/stage; n=3 replicates). For each protein, expression was normalized with total protein abundance in the same capillary and functional activity was determine based on the ratio of phosphorylated (p) to total (t) protein abundance in each sample. Data were analysed using a two-sample t-test. Results demonstrated significantly (P&lt;0.05) decreased LDHB expression at 1C, decreased functional activity of PDH at 8–16 C, and a trend (P&lt;0.09) for decreased activity of PDH and PKM2 enzymes in 1C embryos produced invitro. These results suggest a reduced ability of PKM2 to produce pyruvate in glycolysis, as well as reduced ability of LDHB to reversibly convert pyruvate into lactate and of PDH to convert pyruvate into acetyl-CoA for metabolism in the TCA cycle, indicating an overall slowing of aerobic metabolism. In contrast, expression of STAT3 and ERK1/2 in all stages examined, AKT in 8–16C and d7BL, and 4EBP1 in d7BL were significantly (P&lt;0.05) higher in IVP embryos. In addition to expression, decreased (P&lt;0.05) activity of ERK1/2, AKT, and 4EBP1 signalling at 1C, and a trend (P&lt;0.08) for decreased expression of 4EBP in 8–16C and d7BL produced invitro was observed. Activated AKT signalling enhances glucose uptake by stimulating hexokinase. Because the activity of glycolytic enzymes (PKM2, LDHB, PDH) is reduced at the 1-cell stage invitro, these embryos may be shifting metabolism to the pentose phosphate pathway, which might increase the ability of the embryo to protect against oxidative stress induced by the IVC environment. We observed a remarkable change in metabolic enzyme expression and activity invitro as early as the 1C stage, suggesting that bovine embryos are highly susceptible to metabolic stress even at this early stage of development. Collectively, these results point to specific abnormalities of metabolism in IVP embryos and suggest that differentially expressed proteins and their functional activity can be used as biomarkers in optimizing culture conditions to produce high-quality embryos invitro that more closely resemble their invivo counterparts.