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Calcium signaling is a critical regulator of sperm activation and function during the processes of capacitation and fertilization. Here, we describe a combined method for calcium imaging of single, live human sperm in response to stimuli administered with a precisely targeted delivery technique. This protocol is an adaptation of techniques developed for studies of murine sperm [1, 2], and enables real-time monitoring of human sperm calcium dynamics with high spatiotemporal resolution and concurrent detection of acrosome exocytosis (AE), a functional endpoint of sperm capacitation and requirement for physiological fertilization.The described imaging technique provides a valuable tool for exploration of calcium regulation in human sperm, which is essential to answer important questions and knowledge gaps regarding the link between calcium dynamics, AE, and fertilization. The versatility of this technique can be amplified through use of various indicator dyes or integration with pharmacological strategies such as pre-treating sperm with inhibitors or activators targeting specific receptors, channels, or intracellular signaling pathways of interest. Beyond fundamental inquiries into sperm physiology, this method can also be applied to assess the impact of potential contraceptive compounds on calcium signaling, AE, and membrane integrity., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Sperm maturation depends on exposure to microenvironments within the different segments of the epididymis, but mechanisms underlying how these microenvironments are produced or maintained are not well understood. We hypothesized that epididymal extracellular vesicles could play a role in the process of maintaining microenvironments in different regions of the epididymis. Specifically, we tested whether the extracellular vesicles from different regions of the epididymis can ensure paracrine communication between cells in different segments. Domestic cat tissues were used to develop a reproducible in vitro culture system for corpus epididymis explants that were then exposed to extracellular vesicles collected from upstream (i.e., caput) segments. Impacts of different culture or exposure conditions were compared by analyzing the morphology, apoptosis, transcriptional activity, and gene expression in the explants. Here, we report the development of the first in vitro culture system for epididymal tissue explants in the domestic cat model. Using this system, we found that extracellular vesicles from the caput segment have a significant effect on the transcriptional profile of tissue from the corpus segment (1233 differentially expressed genes due to extracellular vesicle supplementation). Of note, expressions of genes associated with regulation of epithelial cell differentiation and cytokine signaling in the epididymis were influenced by the presence of extracellular vesicles. Together, our findings comprise the first report in any species of paracrine control of segmental gene regulation by epididymal extracellular vesicles. These results contribute to a better understanding of epididymis biology and could lead to strategies to enhance or suppress male fertility., (Published by Oxford University Press on behalf of Society for the Study of Reproduction 2024.)

Resource-poor areas with moisture deficit lands following aerobic and direct seeded rice (DSR) methods of cultivation face severe problems of iron deficiency. In this study, Bengal and Assam Aus rice panel was phenotyped at the seedling stage using an iron-deprived hydroponic medium for various shoot and root traits. A novel iron deficiency scoring scale was used to classify the tolerance reaction and could range anywhere between 0 and 9, indicating the most tolerant and susceptible, respectively. The GWAS results identified four putative candidate genes; OsFLA for number of leaves and shoot length, OsBIDK1 for root traits; average diameter, volume, biomass, projected area, and surface area, OsHPL3 for chlorophyll index of the third leaf and AKR2B (XBOS252) was for Fe score, (which was earlier reported in relation to Xa21). The nsSNP (nsSNPs) variations in these gene sequences were used to group the panel and identify superior haplotypes and donors. BR16 was identified as a superior donor, with higher chlorophyll index and shoot length than RA23, also higher values for root traits like root average diameter, root volume, root projected area and root surface area followed by Shete Bhado. The impact of identified nsSNPs on protein structure and stability was investigated. The conserved domains detected in the mutated proteins of the superior haplotypes are very informative, highlighting that natural selection favors abiotic stress tolerant variants in resource poor areas. Thus, justifying our choice of Aus landraces for association mapping of Fe deficiency tolerant genes in rice., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Context: The COVID-19 pandemic spurred significant government investments for hiring public health workers. There are clear opportunities to help build capacities among both current and incoming public health workers, closing well-elucidated skill gaps., Objective: To report on the development process, methods used, and outcomes seen from a point-in-time public health workforce capacity-building initiative, Public Health Essentials (PHE) ., Design: Capacity-building outcomes evaluation using pre/postintervention measures., Setting: The United States., Participants: A total of 512 learners working in roles (government or adjacent to) that support public health., Intervention: PHE, a cohort-based facilitated asynchronous online course comprising 5 units, 18 modules, 54 learning outcomes, and 266 teaching and applied assessment elements designed to build public health strategic skills., Main Outcome Measures: Two outputs and 3 outcomes were used to assess and improve progress in achieving our goal of building generalizable and transferrable public health ability and confidence among diverse public health workers: Use of PHE , PHE completion rate , Learner competence , Change in self-assessed ability , and Benefits of PHE., Results: From September 2021 to December 2022, 4 agencies used PHE for fellowship training or employee capacity building. Some 79% (n = 512) of learners completed the training, demonstrating competence in all 54 areas assessed by expert course facilitators. Of those, 79% (n = 321) completed both optional pre- and post-PHE surveys, reporting statistically significant gains in all strategic skill domains assessed (n = 9), regardless of demographics and public health experience. Learners gained new skills and knowledge (92%), developed a better understanding of public health (86%), and broadened their public health skill base (84%). A majority can apply the knowledge and skills gained directly to their work (94%), which benefits their team (92%), and have increased their confidence as public health practitioners (49%)., Conclusions: PHE can significantly improve learners' ability across 9 strategic skill areas in as few as 15 weeks, regardless of their demographics, training, or experience., Competing Interests: Conflicts of Interest: N/A., (Copyright © 2023 The Authors. Published by Wolters Kluwer Health, Inc.)

The murine epididymis has 10 distinct segments that provide the opportunity to identify compartmentalized cell physiological mechanisms underlying sperm maturation. However, despite the essential role of the epididymis in reproduction, remarkably little is known about segment-specific functions of this organ. Here, we investigate the dramatic segmental localization of the ganglioside GM1, a glycosphingolipid already known to play key roles in sperm capacitation and acrosome exocytosis. Frozen tissue sections of epididymides from adult mice were treated with the binding subunit of cholera toxin conjugated to AlexaFluor 488 to label GM1. We report that GM1-enriched vesicles were found exclusively in principal and clear cells of segment 2. These vesicles were also restricted to the lumen of segment 2 and did not appear to flow with the sperm into segment 3, within the limits of detection by confocal microscopy. Interestingly, this segment-specific presence was altered in several azoospermic mouse models and in wild-type mice after efferent duct ligation. These findings indicate that a lumicrine factor, itself dependent on spermatogenesis, controls this segmental differentiation. The RNA sequencing results confirmed global de-differentiation of the proximal epididymal segments in response to efferent duct ligation. Additionally, GM1 localization on the surface of the sperm head increased as sperm transit through segment 2 and have contact with the GM1-enriched vesicles. This is the first report of segment-specific vesicles and their role in enriching sperm with GM1, a glycosphingolipid known to be critical for sperm function, providing key insights into the segment-specific physiology and function of the epididymis., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Acrosome exocytosis (AE), in which the sperm's single exocytotic vesicle fuses with the plasma membrane, is a complex, calcium-dependent process essential for fertilization. However, our understanding of how calcium signaling regulates AE is still incomplete. In particular, the interplay between intra-acrosomal calcium dynamics and the intermediate steps leading to AE is not well-defined. Here, we describe a method that provides spatial and temporal insights into acrosomal calcium dynamics and their relationship to membrane fusion and subsequent exocytosis of the acrosome vesicle. The method utilizes a novel transgenic mouse expressing an Acrosome-targeted Sensor for Exocytosis (AcroSensE). The sensor combines a genetically encoded calcium indicator (GCaMP) fused with mCherry. This fusion protein was specifically designed to enable the concurrent observation of acrosomal calcium dynamics and membrane fusion events. Real-time monitoring of acrosomal calcium dynamics and AE in live AcroSensE sperm is achieved using a combination of high frame-rate imaging and a stimulant delivery system that can target single sperm. This protocol also provides several examples of basic methods to quantify and analyze the raw data. Because the AcroSensE model is genetically encoded, its scientific significance can be augmented by using readily available genetic tools, such as crossbreeding with other mouse genetic models or gene-editing (CRISPR) based methods. With this strategy, the roles of additional signaling pathways in sperm capacitation and fertilization can be resolved. In summary, the method described here provides a convenient and effective tool to study calcium dynamics in a specific subcellular compartment-the sperm acrosome-and how those dynamics regulate the intermediate steps leading to membrane fusion and acrosome exocytosis.

Research Question: How do capacitation ability, measured by Cap-Score™, and traditional semen analysis measures (volume, concentration, motility) change with age in men questioning their fertility (MQF)?, Design: Cap-Score and semen analysis measures were obtained from MQF (n = 2652; multicentric design: 35 reproductive endocrinologist prescribers, n = 16 clinics). Morphology was not included due to differences among clinics. A Mann-Whitney test was used to compare Cap-Scores between MQF and men with known recent paternity (n = 76). The following age groups were constructed for MQF: 20-24, 25-29, 30-34, 35-39, 40-44, 45-49 and 50+. Associations between semen analysis, Cap-Score and age groups were evaluated using mixed-model analysis of variance to identify possible influence of Cap-Score collection kit type (n = 763 collected at home; n = 1889 collected at clinics)., Results: MQF had reduced capacitation ability (mean ± SE; 29.25 ± 0.15 versus 35.34 ± 0.88; P < 0.001). No change in Cap-Score (P = 0.916) or concentration (P = 0.926) was detected with age group. In contrast, both volume (P = 0.008) and % motility (P < 0.001) declined with age., Conclusions: Men presenting because of difficulties in generating pregnancy showed equivalent reductions in capacitation ability regardless of age. In contrast, motility and volume declined with age. These data suggest that capacitation ability is a more sensitive indicator of male fertility across age groups than traditional semen analysis and should not be reserved for older men. Importantly, these data do not address whether sperm fertilizing ability declines in the general population as men age. Instead, they indicate that if men are having difficulty conceiving, no matter what their age, then defects in sperm fertilizing ability are equally likely to be the cause., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Curbing the coronavirus disease 2019 (COVID-19) pandemic requires a thorough understanding of risk factors for transmission of SARS-CoV-2, the etiologic agent. Institutions of higher education present unique challenges for controlling disease spread because of features inherent to these settings. Our objective was to determine risk factors for SARS-CoV-2 infection among a university student population in the northeastern USA during the spring and fall 2021 semesters, using the case-control study design. Cases were defined as students with a newly diagnosed SARS-CoV-2 infection detected either through the robust PCR-based surveillance testing program on campus or through healthcare testing if symptoms compatible with COVID-19 were present. Controls were defined as students with negative SARS-CoV-2 status, based on consistently negative PCR results at the time of selection. A comprehensive questionnaire was administered to each student enrolled in the study, covering a broad range of campus life activities. A total of 446 cases and 1,185 controls were included in this study. Multivariable logistic regression analysis showed that recent party attendance (adjusted OR = 2.3, p < .0001), recently visiting a bar (aOR = 1.6, p = .007), living in a campus residence hall (aOR = 1.6, p = .001), fraternity/sorority membership (aOR = 1.8, p = .002), and recent travel (aOR = 1.3, p = .04) were associated with being a COVID-19 case. Having an on-campus job was negatively associated with being a COVID-19 case (aOR = 0.6, p = .0003). Among cases, the most commonly reported symptoms were cough (43.9%), fatigue (38.1%) and sore throat (30.3%). These findings can be used to inform the development of COVID-19 mitigation strategies and public health outreach efforts in university settings, thus reducing SARS-CoV-2 transmission among students and helping to preserve the vital education and research missions of these institutions., (© 2022 Wiley-VCH GmbH.)

Water and land resources have been aggressively exploited in the recent decades to meet the growing demands for food. The changing climate has prompted rice scientists and farmers of the tropics and subtropics to adopt the direct seeded rice (DSR) system. DSR system of rice cultivation significantly reduces freshwater consumption and labor requirements, while increasing system productivity, resource use efficiency, and reducing greenhouse gas emissions. Early root vigor is an essential trait required in an ideal DSR system of rice cultivation to ensure a good crop stand, adequate uptake of water, nutrients and compete with weeds. The aus subpopulation which is adapted for DSR was evaluated to understand the biology of early root growth under limited nitrogen conditions over two seasons under two-time points (14 and 28 days). The correlation study identified a positive association between shoot dry weight and root dry weight. The genome-wide association study was conducted on root traits of 14 and 28 days with 2 million single-nucleotide polymorphisms (SNPs) using an efficient mixed model. QTLs over a significant threshold of p < 0.0001 and a 10% false discovery rate were selected to identify genes involved in root growth related to root architecture and nutrient acquisition from 97 QTLs. Candidate genes under these QTLs were explored. On chromosome 4, around 30 Mbp are two important peptide transporters ( PTR5 and PTR6 ) involved in mobilizing nitrogen in the root during the early vegetative stage. In addition, several P transporters and expansin genes with superior haplotypes are discussed. A novel QTL from 21.12 to 21.46 Mb on chromosome 7 with two linkage disequilibrium (LD) blocks governing root length at 14 days were identified. The QTLs/candidate genes with superior haplotype for early root vigor reported here could be explored further to develop genotypes for DSR conditions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Anandan, Panda, Sabarinathan, Travis, Norton and Price.)

To minimize the impacts of COVID-19 and to keep campus open, Cornell University's Ithaca, NY, campus implemented a comprehensive process to monitor COVID-19 spread, support prevention practices, and assess early warning indicators linked to knowledge, behaviors, and attitudes of campus community members. The integrated surveillance approach informed leadership and allowed for prompt adjustments to university policies and practices through evidence-based decisions. This approach enhanced healthy behaviors and promoted the well-being and safety of all community members. ( Am J Public Health . 2022;112(7):980-984. https://doi.org/10.2105/AJPH.2022.306838).

Secretion of the acrosome, a single vesicle located rostrally in the head of a mammalian sperm, through a process known as "acrosome exocytosis" (AE), is essential for fertilization. However, the mechanisms leading to and regulating this complex process are controversial. In particular, poor understanding of Ca2+ dynamics between sperm subcellular compartments and regulation of membrane fusion mechanisms have led to competing models of AE. Here, we developed a transgenic mouse expressing an Acrosome-targeted Sensor for Exocytosis (AcroSensE) to investigate the spatial and temporal Ca2+ dynamics in AE in live sperm. AcroSensE combines a genetically encoded Ca2+ indicator (GCaMP) fused with an mCherry indicator to spatiotemporally resolve acrosomal Ca2+ rise (ACR) and membrane fusion events, enabling real-time study of AE. We found that ACR is dependent on extracellular Ca2+ and that ACR precedes AE. In addition, we show that there are intermediate steps in ACR and that AE correlates better with the ACR rate rather than absolute Ca2+ amount. Finally, we demonstrate that ACR and membrane fusion progression kinetics and spatial patterns differ with different stimuli and that sites of initiation of ACR and sites of membrane fusion do not always correspond. These findings support a model involving functionally redundant pathways that enable a highly regulated, multistep AE in heterogeneous sperm populations, unlike the previously proposed "acrosome reaction" model., Competing Interests: Conflict of interest A. J. T. discloses that he is a founder and an officer of Androvia LifeSciences, LLC, a biotechnology company investigating solutions for male infertility, in which he holds a minor equity stake. There are no conflicts with the current work. All other coauthors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Iron (Fe) deficiency is one of the common causes of anaemia in humans. Improving grain Fe in rice, therefore, could have a positive impact for humans worldwide, especially for those people who consume rice as a staple food. In this study, 225-269 accessions of the Bengal and Assam Aus Panel (BAAP) were investigated for their accumulation of grain Fe in two consecutive years in a field experiment under alternative wetting and drying (AWD) and continuous flooded (CF) irrigation. AWD reduced straw Fe by 40% and grain Fe by 5.5-13%. Genotype differences accounted for 35% of the variation in grain Fe, while genotype by irrigation interaction accounted for 12% of the variation in straw and grain Fe in year 1, with no significant interactions detected in year 2. Twelve rice accessions were identified as having high grain Fe for both years regardless of irrigation treatment, half of which were from BAAP aus subgroup 3 which prominently comes from Bangladesh. On average, subgroup 3 had higher grain Fe than the other four subgroups of aus . Genome-wide association mapping identified 6 genomic loci controlling natural variation of grain Fe concentration in plants grown under AWD. For one QTL, nicotianamine synthase OsNAS3 is proposed as candidate for controlling natural variation of grain Fe in rice. The BAAP contains three haplotypes of OsNAS3 where one haplotype (detected in 31% of the individuals) increased grain Fe up to 11%. Haplotype analysis of this gene in rice suggests that the ability to detect the QTL is enhanced in the BAAP because the high Fe allele is balanced in aus , unlike indica and japonica subgroups., (© 2021 The Authors. Food and Energy Security published by John Wiley & Sons Ltd.)