INVESTIGATING MALE INFERTILITY: EDITING GENES IMPORTANT FOR SPERMATOGENESIS IN A CELL CULTURE SYSTEM

Abstract The four levels of aging include the specific disease, the systemic complications that lead to the disease, the loss of cellular integrity that affects the system, and macromolecule malfunction that alters cellular integrity. Similarly, this project investigates infertility and testicular cancer, misregulation of the endocrine system, problems with germ and Sertoli cell maintenance, and ultimately gene expression changes that alter cellular proliferation and death. Infertility affects 4.5–6% of North American males and up to 15% of couples worldwide. Infertility may be caused by unknown genetic factors, as up to 2,300 genes are pertinent to male fertility. This research project aims to create the molecular toolbox needed to evaluate gene function in cultured male germ cells. We will use mouse primary spermatogonial stem cells and a human testicular cancer cell line to knock out the functions of genes Phosphoprotein 1 (Spp1) and Inhibitor of DNA Binding 4 (Id4) in the testis to determine the effect of the knockouts on male germ cell proliferation and cell death. We designed single guide RNAs (gRNAs) using online bioinformatic tools and amplified the genes from human and mouse genomic DNA to demonstrate the effectiveness of our gRNAs in vitro. These gRNAs and Cas9 compose the beginnings of the molecular toolbox used to electroporate cultured cells. This research will contribute to the greater scientific community by providing insight into the function of Spp1 and Id4 relating to male fertility and providing a methodology for future research in aging-related fertility diseases and testicular cancer.

a clinical questionnaire (Canine Frailty Index;CFI;Banzato et al., 2019) to assess frailty and an owner assessment tool (VetMetrica HRQL) to evaluate HRQL in 451 adult companion dogs. Results support evidence of validity for the tools by confirming expectations that frailty and HRQL deteriorate with age. CFI scores were significantly higher (higher frailty) and HRQL scores significantly lower (worse HRQL) in old dogs (≥ 7 years of age) compared to young dogs (≥ 2 and < 6 years of age). Body size (small < 25lbs or large > 50lbs) was not associated with CFI or total HRQL score. However, older, larger dogs showed faster age-related decline in HRQL scores specific to owner-reported activity and comfort. Findings suggest that the clinician-assessed CFI and owner-reported VetMetrica HRQL may be useful tools to evaluate two determinants of healthspan in dogs: the accumulation of frailty and the progressive decline in quality of life. Establishing validated tools that operationalize the assessment of canine healthspan is critical for the linking pathophysiological mechanisms to aging phenotypes in the companion dog and for accelerating the development of gerotherapeutics that benefit both human and veterinary medicine.

SEX DIFFERENCES IN LIFESPAN RESPONDING TO EARLY AND LATE ONSET DIETARY RESTRICTION
Nisi Jiang, Vivian Diaz, Randy Strong, and James Nelson,

UT Health San Antonio, San Antonio, Texas, United States
Dietary restriction (DR) extends lifespan in many animal models. In mice, a range of factors affects the magnitude of the pro-longevity effect of DR, including the strain, restriction level, sex, and age of treatment onset. Early onset DR dramatically prolongs lifespan, whereas DR started later in life has diminished or no significant effect. The lifespan responses of males and females to DR also differ. Whether there is a sex difference in the lifespan response to the age of onset of DR has not been examined. Probing the basis for the variable response to DR can help elucidate the underlying mechanisms that mediate lifespan extension. We diet restricted UM-HET3 mice of both sexes from either 5 months (early-onset) or 20 months (lateonset) of age and monitored their survival. Early onset DR significantly prolonged lifespan in both males and females, which increased median lifespans by 54% and 18.5%, respectively. By contrast, although late onset DR prolonged lifespans in both sexes, the magnitude of increase in males (1.4%) was significantly lower than in females (15.8%, p=0.0474). Moreover, the decrease in the magnitude of the response to late-onset DR compared to that of earlyonset DR is more dramatic in males than females. Further analysis found a significant interaction (p=0.0001) between early and late-onset DR in males but not in females (p=0.3818). Together, these results indicate that the lifespan response to delayed onset of DR diminishes more in males than in females, providing another model to probe the underlying mechanisms of DR.

FOOD AND SUPPLEMENT COMBINATIONS TO PREVENT OR POSTPONE AGING, MCI, AND ALZHEIMER'S DISEASE.
Alec Pruchnicki, 1 Barrie Kavasch, 2 Barrie Sachs, 3 Roseanne Schnoll, 4 and Rolf Martin 3 , 1. Vista on 5th, New York,New York,United States,2. Blueberry Study MMT Corporation,Sherman,Connecticut,United States,3. MMT Corporation,Sherman,Connecticut,United States,4. Brooklyn College,CUNY,Sherman,Connecticut,United States Evidence of many interacting causes of aging has renewed interest in food and nutrient combinations that may blunt the main causes[PMC9184560, PMC8889622] and postpone aging, mild cognitive impairment and Alzheimer's ("A-MCI-Alz") in contrast to prior failures [PMC9232186]. We present an updated list of anti-aging/pro-longevity strategies to address limitations of previous food/nutrient combinations. Our list has two unusual features: (i) all interventions should be administered concurrently so no fundamental causes of A-MCI-Alz are without remediation and can inflame and leave smoldering important causal factors, (ii) all interventions should be provided daily or weekly to help avoid re-ignition of key factors driving A-MCI-Alz. this project investigates infertility and testicular cancer, misregulation of the endocrine system, problems with germ and Sertoli cell maintenance, and ultimately gene expression changes that alter cellular proliferation and death. Infertility affects 4.5-6% of North American males and up to 15% of couples worldwide. Infertility may be caused by unknown genetic factors, as up to 2,300 genes are pertinent to male fertility. This research project aims to create the molecular toolbox needed to evaluate gene function in cultured male germ cells. We will use mouse primary spermatogonial stem cells and a human testicular cancer cell line to knock out the functions of genes Phosphoprotein 1 (Spp1) and Inhibitor of DNA Binding 4 (Id4) in the testis to determine the effect of the knockouts on male germ cell proliferation and cell death. We designed single guide RNAs (gRNAs) using online bioinformatic tools and amplified the genes from human and mouse genomic DNA to demonstrate the effectiveness of our gRNAs in vitro. These gRNAs and Cas9 compose the beginnings of the molecular toolbox used to electroporate cultured cells. This research will contribute to the greater scientific community by providing insight into the function of Spp1 and Id4 relating to male fertility and providing a methodology for future research in aging-related fertility diseases and testicular cancer.

P16-EXPRESSING SENESCENT CELLS ARE A DOUBLE-EDGED SWORD IN SHAPING IMMUNE RESPONSES WITH AGE
Blake Torrance, Hunter Panier, Andreia Cadar, Dominique Martin, Erica Lorenzo, Jenna Bartley, and Laura Haynes, UConn Health, Farmington, Connecticut, United States Aging results in the accumulation of senescent cells which can cause dysfunction in many contexts but the effects on immune responses remain unclear. Here, we aimed to probe the effects of clearing senescent cells in aged mice on the immune response to influenza infection. We utilized a powerful p16 trimodality reporter mouse model (p16-3MR): under the control of the p16 promoter, these mice express cassettes encoding luciferase, RFP, and herpesvirus thymidine kinase (HSV-TK). p16 is commonly upregulated in senescent cells so this model allows us to selectively delete those cells by treating with ganciclovir (GCV), which will induce apoptosis in cells expressing HSV-TK. We hypothesized that while p16-expressing senescent cells may exacerbate dysfunctional responses to a primary infection, they may play a protective role in resolving inflammation and fostering memory cell generation. We found that deletion of p16-expressing cells enhanced viral clearance and decreased infiltration of pro-inflammatory flu-specific CD8 T cells during the primary response to infection. Conversely, at 30 days post infection, there were fewer flu-specific CD8 memory T cells and lower amounts of anti-viral antibodies in the lungs of GCV treated mice. We also observed perturbations in memory T cell trafficking in GCV treated mice. Furthermore, GCV treated mice were unable to mount an effective memory response and were unable to control viral load following a heterosubtypic challenge. This suggests that targeting senescent cells may potentiate primary responses while limiting the ability to form durable and protective immune memory with age.

ARE WE GOING TO FEEL COMFORTABLE THERE?: EMERGENT FINDINGS FROM THE DIVERSITY IN LIFE PLAN COMMUNITIES STUDY
Mushira Khan, 1 Pankaja Desai, 2 and Catherine O'Brien 3 , 1. Mather Institute,Evanston,Illinois,United States,2. Rush University Medical Center,Chicago,Illinois,United States,3. Mather,Evanston,Illinois,United States Life Plan Communities (LPCs), formerly known as Continuing Care Retirement Communities, offer an array of amenities as well as a continuum of care where residents may live independently or access assisted living or skilled care if needed. Racial/ethnic minority older adults are under-represented among LPC residents. One likely contributor to this lack of diversity is that racial/ethnic minorities make up a relatively small proportion of higher income groups. However, there may be additional reasons for their relative absence in most LPCs. The purpose of the 'Diversity in Life Plan Communities' study is to (1) identify barriers that impact the decision to move into an LPC for racial/ ethnic minorities, and (2) identify strategies to increase resident diversity in LPCs. In this presentation we share preliminary findings from in-depth, semi-structured, qualitative interviews with community-dwelling Black, Latinx, and South Asian adults (n=10) living in Chicagoland and case study interviews with current LPC 'resident champions' (n=3) who are actively involved in efforts to increase resident diversity at their respective LPCs. Thematic analysis of the data showed that lack of awareness about LPCs, concerns about racism, desire to age-in-place among loved ones, and perceived non-availability of culturally-congruent activities were key barriers to moving into an LPC. These findings suggest that in order to increase resident diversity within their communities, LPC operators, staff, and current residents may consider targeted outreach efforts in racial/ethnic communities, take steps to create a welcoming environment and engage in more culturally congruent activities, and involve adult children/grandchildren in the decision-making process. The COVID-19 pandemic has exposed older adults to complex healthcare situations, via personal experience or media stories about serious illness. Hearing about lengthy intubation, sedation, rapid decline, and distress at the end of life has the potential to prompt people to reevaluate their perspective on their own end-of-life care. This study explored advance care planning (ACP) among older adults and whether COVID-19 experiences altered their healthcare preferences and planning. One hundred and fifty-one respondents (M age = 71.2 yrs, range = 55-93) completed an online survey about ACP completion, ACP conversations, and life-prolonging interventions. Respondents were mainly Innovation in Aging, 2022, Vol. 6, No. S1