Progress 07/01/24 to 06/30/25

Outputs
Target Audience:The primary target audiences for this project are 1. Other researchers and sceintists in the fields of animal science and 2. Farmers and producers whose systems may ultimately be impacted by the findings. In this early stage of the project, other researchers scientists were reached through presentations of this work at the USDA PD meeting and my Hatch Multistate meeting. These presentations provided valuable opportunities to share the work and recieve feedback. In the next reporting period, we plan to publish this work in an academic journal. Changes/Problems:All experiments in this project are based onability to collect fetalovarian samples from a local slaughterhouse, Nicholas Meats. The collection of these samples was slower than anticipated, thus delaying several of the major experiments proposed, most notably the single cell sequencing experiments, and delaying our ability to submit publications. At this time, we now have all necessary samples (from all fetal age groups) for all IHC and RNA experiments banked in our lab, resulting from many trips to the slaughterhouse over the last year. Moreover, we haveregular trips to the slaughterhouse scheduled every two weeks, for the remainder of the project period. These will provide adequate samples for the in vitro and single cell experiments proposed. What opportunities for training and professional development has the project provided?This project has provided significant opportunities for professional development. 1. An undergraduate honors student was on this project and involved in sample collection, processing, and storage. This project provided opportunities for her to learn skills including tissue processing, sectioning, immunohistochemistry (IHC), RNA isolation, and qPCR. She also completed an honors thesis in my laboratory, providing an opportunityfor her to develop writing and communications skills. 2. Two undergraduate students just startedworking on this project and are learning skills including tissue culture, processing, and embedding. 3. A graduate student working on this project has developed significant numbers of new skills including laboratory skills (flow cytometry, IHC, tissue dissociation, tissue culture), experimental design and troubleshooting skills, presentation skills (from biweekly lab meetings) and bioinformatics skills. She has also submitted an abstract to a conference based on this project, and in the next reporting period, this conference presentation will be reported. 4. A technician working on this project has developed laboratory skills (dissection, tissue processing), organizational skills, and communication skills working with the local slaughterhouse we have used for tissue collection. 5. Given that this is a new investigator seed grant, the PD has had the opportunity to develop significant skills directing and leading a project and has given the presentations described below. How have the results been disseminated to communities of interest?1. Preliminary results were presented by the PD at the PI's Hatch Multistate meeting in Ohio in May 2025. This was to a group of peer reproductive biologists and provided an excellent opportunity to receive feedback on experiments in progress. 2. Preliminary results were presented by thePD at the USDA PD meeting in June. This also was to a group of peer reproductive biologists and also provided an excellent opportunity to receive feedback on experiments in progress.? What do you plan to do during the next reporting period to accomplish the goals?1. The single cell sequencing experiments will be performed. This will provide detailed information on both phenotypes and abundance of immune cell subtypes in the developing ovary and their interactions with other immune cell types. 2. In vitro ovarian cortical culture experiments will be completed to provide mechanistic information on immune cell effects on ovarian function, including oocyte death, primordial follicle assembly, and primordial follicle activation. 3. A manuscript detailing the first set of experiments is currently in preparation. It will be submitted during the next reporting period. The results will also be presented at the Society of the Study of Reproduction conference.

Impacts
What was accomplished under these goals? 1. We determined that PTPRC, the transcript encoding CD45 (a total immune cell marker) declines temporally in the developing ovary, at the same time as oocyte attritionand primordial follicle assembly. Indeed, the decline in PTPRC between fetal day 90 and fetal day 150is correlated with a decline in dead cells per section between fetal day 90 and fetal day 150. This suggests that the greater immune cell populations on day 90 are associated with, and perhaps driving,the greater oocyte death on day 90. We have also used immunohistochemistry and identified CD45 cells in the developing ovary, colocalizing with DDX4cells (oocytes). Quantification of CD45 cells by both flow cytometry and by counting CD45+ cells per section in IHC is currently underway. 2. We determined that markers of total T cells (CD3D) and T helper cells (CD4) do notchange during ovarian development, either at the time of primordial follicle assembly or at the time of follicular activation. In contrast, markers of CD8 cytotoxic cells do change.CD8 alpha beta T cells areclassical cytotoxic proinflammatory CD8 T cells, whereasCD8 alpha alphaT cells arehomeostatic pro-resolving CD8 T cells. Interestingly, CD8A increased with the process of ovarian development and was at its greatest abundance during follicular activation. In contrast, CD8B was most abundant during oocyte attrition and then declined by the time of follicular activation. This suggests that theremay be a switch from inflammatoryCD8AB to pro-resolving CD8AA cells as follicular assembly is completed. Experiments are underway to validate this by flow cytometry. 3. We determined that CD68, a macrophage marker, is most abundant in the ovary on day 120,during follicular assembly. Abundance of M1 proinflammatory and M2 pro-resolving macrophages was unchanged. We hypothesize that macrophages may be involved in phagocytosis or tissue remodeling in the ovary during this time. 4. We identified threecytokines that tended to decline between the time of follicular assembly (day 90-110) and the time of follicular activation (Day 175-200). These included the IL36 inhibitor IL36RA, the proinflammatory cytokine IL1A, and the immune cell chemotactic cytokine CCL2. In addition, CCL3, another immune cell chemotactic cytokine, tended to declinebetween day 120-135 and day 175-200. Overall, this suggests a more proinflammatory environment in the ovary during follicular assembly and a less inflammatory environment during early activation.

Publications