Intrafollicular injection of nanomolecules for advancing knowledge on folliculogenesis in livestock

Authors: FEUGANG, JEAN - Mississippi State University; ISHAK, GHASSAN - Southern Illinois University; EGGERT, MATTHEW - Auburn University; ARNOLD, ROBERT - Auburn University; RIVERS, ORION - Mississippi State University; WILLARD, SCOTT - Mississippi State University; RYAN, PETER - Mississippi State University; GASTAL, EDUARDO - Southern Illinois University

Submitted to: Theriogenology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/22/2022
Publication Date: 8/27/2022
Citation: Feugang, J.M., Ishak, G.M., Eggert, M.W., Arnold, R.D., Rivers, O.S., Willard, S.T., Ryan, P.L., Gastal, E.L. 2022. Intrafollicular injection of nanomolecules for advancing knowledge on folliculogenesis in livestock. Theriogenology. 192:132-140. https://doi.org/10.1016/j.theriogenology.2022.08.032.
DOI: https://doi.org/10.1016/j.theriogenology.2022.08.032

Interpretive Summary: The oocyte quality is a major bottleneck in the success of assisted reproduction in mammals. Numerous studies have indicated that the immediate environment of the oocyte within the follicle, the follicular fluid (FF), significantly influences the oocyte developmental competence. Therefore, developing strategies for intrafollicular assessment of the follicle health status for possible in situ therapeutic interventions to improve oocyte quality before ovulation may offer a unique avenue to revolutionize assisted reproduction in veterinary and human infertility clinics. Here we used a syringe for intrafollicular injection followed by the biopsy of fragments of follicle wall in live mares. Both the biopsy and FF were subjected to further microscopy evaluations. Results indicated that bioactive molecules could be injected within the follicle with minimal invasion to inspect their impacts on the follicle characteristics for enhancing knowledge of the intrafollicular environment during folliculogenesis. The proposed technological approach may better harness oocyte maturation for improved assisted reproductive outcomes.

Technical Abstract: Despite the progress in assisted reproductive techniques, there is still a lack of rapid and minimally invasive in situ approaches for further enhancements of female fertility. Therefore, we synthesized clinically relevant liposome nanoparticles for ovarian intrafollicular injection to allow in vivo cellular imaging for future drug delivery, using the mare as an animal model. Ovarian follicles of living mares were injected in vivo with fluorescently labeled liposomes. Samples of the follicular wall (mural granulosa, theca interna, and theca externa), granulosa cells, and follicular fluid were harvested 24 h post-injection through the follicle wall biopsy (FWB), flushing, and aspiration techniques, respectively, using a transvaginal ultrasound-guided approach. In parallel, post-mortem dissected, and cultured porcine antral follicles were microinjected with doxorubicin-encapsulated liposomes to assess intracellular delivery potential. All injected mare and pig follicles were macroscopically healthy, and fluorescence imaging revealed successful intrafollicular binding to mural granulosa cells and progressive migration of liposomes to other follicle cell layers (theca interna, and theca externa), regardless of the follicle size. Intracellular delivery of doxorubicin was confirmed in all porcine follicle wall cell types. We conclude that the intrafollicular injection of nanomolecules is a promising approach for real-time monitoring of intrafollicular processes and potential utilization of in vivo cellular drug delivery to assist in follicle disease treatments and fertility improvement.