Follicle-like and other novel structures found in ovaries of aged white rhinoceroses and their potential impact on oocyte recovery rate

Abstract

Female rhinoceroses have an ovarian reserve that consists of immature oocytes in primordial follicles. Establishing methods to grow and culture these oocytes from those follicles in the laboratory might fuel efforts towards in vitro embryo production in this species without the need for repeated in vivo oocyte collection. In depth understanding of the biology behind and improvement of assisted reproductive techniques (ARTs) is the only option for preventing the disappearance of functionally extinct species such as the northern white rhinoceros. In vitro follicle development can only be obtained by in depth knowledge on rhinoceros ovarian histology. In addition to known ovarian features, we identified numerous undescribed structural and molecular ovarian characteristics from adult southern white rhinoceros (n=3). Ovarian sections were stained with H&E, PAS or Masson Trichrome and molecular analyses (hyaluronic acid detection, immunohistochemistry and TUNEL assay) were performed to identify proliferation, extracellular matrix, pluripotency markers, hormones, enzymes, markers for inflammation or endocrine glands, blood vessel presence, oocyte markers and apoptosis. Besides degenerating, deformed, or luteinized follicles, analysis revealed several ‘follicle-like structures’ (FLS) that deviated from the expected follicle appearance. Most importantly, the majority of FLS did not contain any oocyte and were comprised of a collection of cells organised around fluid islands with or without an antral cavity. The discovery and description of FLS in aged southern white rhinoceroses may play a role in poor oocyte recovery rates in ovum pick-up (OPU) in older rhinoceroses. It remains to be investigated whether FLS might be present on ovaries from other rhinoceros species and whether they might serve as a general indicator for oocyte recovery success in aged females.