The neonatal southern white rhinoceros ovary contains oogonia in germ cell nests

The study addresses the urgent need for assisted reproductive strategies to conserve the functionally extinct northern white rhinoceros (Ceratotherium simum ssp. cottoni), for which only two females remain. A critical prerequisite for developing advanced assisted reproductive technologies (aART), including in vitro gametogenesis and follicle culture, is species-specific knowledge of ovarian physiology and folliculogenesis. While ultrasound and post-mortem histology have been used to assess reproductive pathologies in rhinoceros species, detailed histological descriptions of healthy rhinoceros ovaries are lacking, and a prior report did not include ovarian histology. This work aims to provide a comprehensive structural and molecular characterization of neonatal and adult southern white rhinoceros (Ceratotherium simum simum) ovaries, the closest relative to the northern white rhinoceros, to inform the development of in vitro techniques to culture follicles and generate oocytes for conservation.

Assisted reproductive technologies have been explored in rhinoceros species to overcome infertility and enhance genetic diversity, with ex situ populations often showing irregular cycles and long non-reproductive periods. Previous attempts at in vitro maturation and fertilization in Sumatran and black rhinoceroses had limited success but indicate potential for gamete rescue. Some species, such as the naked mole rat, display postnatal neo-oogenesis with persistent germ cell nests into adulthood, offering a model for in vitro gametogenesis. Studies have investigated using fetal and adult tissues, as well as embryonic and induced pluripotent stem cells, for generating gametes. However, foundational histological data on rhinoceros ovaries is sparse; a 2001 report on rhinoceros reproductive tract anatomy omitted ovarian histology. Comparative observations in related species (e.g., tapirs, horses) and in other mammals include germ cell nests postnatally in some species, variations in ovarian architecture, and roles for extracellular matrix components such as hyaluronan (HA) during follicle development.

Study design and samples: Ovarian tissue was collected from one stillborn neonatal female (rhino 1) and three adult southern white rhinoceroses (rhinos 2–4; ages 39.5, 30, and 38 years) euthanized for health reasons. The neonate had no infectious or congenital causes of death identified; perinatal hypoxia was suspected. Adult reproductive histories varied; one had prior GnRF-based contraception and ceased cycling. Collection and processing: Post-mortem ovaries were dissected, transported on ice, and fixed. The neonatal ovaries were dissected and fixed the day after birth. Cross-sections or whole ovaries were fixed. Fixatives and embedding: Tissue was fixed in Bouin's, 4% paraformaldehyde, 10% neutral buffered formalin (NBF), or Form-Acetic (NBF with 5% acetic acid). Fixed samples were paraffin-embedded and sectioned at 5 µm. Histological staining and imaging: Sections were stained with hematoxylin and eosin (H&E), Periodic acid-Schiff (PAS), and Masson's trichrome. Hyaluronan was detected using hyaluronic acid-binding protein (HABP) with DAB detection. Bright-field microscopy (Leica DM2500, Lumenera Infinity 5 camera) and whole-slide scanning (ZEISS Axioscan 7, 20x) were used. Immunohistochemistry: Antigen retrieval was performed (low pH sodium citrate for collagen I, Ki-67, MCM2, SOX2, DDX4, CD20, NaKATPase; high pH Vector solution for Oct4, AMH, CB1). Endogenous peroxidase was blocked with 3% H2O2; non-specific binding blocked with 5% normal goat serum. Primary antibodies (collagen I, Ki-67, MCM2, SOX2, Oct4/POU5F1, DDX4/VASA, AMH, CB1, CD20, NaKATPase) were applied for 2 h at room temperature, followed by biotinylated secondaries, ABC Elite detection, and DAB; slides were counterstained with Harris hematoxylin. TUNEL assay (FragEL) detected DNA fragmentation. Follicle classification and measurements: Follicles were classified as primordial, transitional, primary, secondary, pre-antral, or antral using established criteria. Only follicles with a visible oocyte nucleus were measured. Parameters included granulosa cell number, mean oocyte diameter, mean follicle diameter, oocyte and follicle areas, and the oocyte-to-follicle size ratio, quantified in ImageJ. When shrinkage artifacts were present, measurements included the shrinkage space. Descriptive statistics were reported when n ≥ 3. Statistics and reproducibility: Given low follicle numbers and limited samples (1 neonatal, 3 adults), no inferential statistics were performed; findings are descriptive and all observed data included.

- Macroscopic ovarian architecture: Adult southern white rhinoceros ovaries were unusually flat with a pronounced two-sided organization: a smooth cortical side and a more vascular medullary side, rather than the typical outer cortex and inner medulla arrangement. Neonatal ovaries showed asymmetry but lacked a clear cortex–medulla demarcation, instead containing stroma-separated cell areas. - Follicle presence and stages: Follicles were present in all four rhinoceroses, including the aged adults. All developmental stages (primordial to antral) were observed in both neonatal and adult ovaries. Follicle density was highest in the neonate and lower in older adults. - Quantitative follicle metrics: The number of granulosa cells ranged from 6 in an adult primordial follicle to 1,955 in an adult antral follicle with a diameter of 786.14 µm. Mean oocyte diameter increased with follicle stage, from 28.70 ± 4.03 µm in adult secondary follicles to approximately 93.08 µm in an adult antral follicle. The ratio of oocyte to follicle size decreased with follicle maturation. - Follicle morphology and distribution: Neonatal ovaries exhibited some ellipsoidal follicles with variable granulosa layering within the same follicle and frequent follicle clustering; adults also showed local clustering but less conspicuously due to low density. - Hyaluronan distribution: In neonates, HA was detected in follicular fluid and extracellular matrix around granulosa cells of smaller antral follicles, and strongly delineated basal laminae. In adults, blood vessels were HA-rich; basal laminae of larger antral follicles were weak or negative for HA, and HA distribution was more uniform across cortex and medulla. - Neonatal cell nests indicative of germ cells: Neonatal ovaries contained stroma-bounded cell areas consistent with ovigerous cords/germ cell nests. These cells showed proliferation (Ki-67, MCM2 positive), pluripotency (SOX2, Oct4 positive), and germ cell identity (DDX4 positive). DDX4-positive oocytes within small follicles were also observed. - Additional molecular features: TUNEL assay indicated low levels of apoptosis within nests; AMH was present in granulosa cells and heterogeneously within cell areas; CB1 showed strong expression in blood vessels and moderate expression in nests, suggesting potential steroidogenic activity; NaKATPase demarcated cell membranes in follicles and undefined cell populations.

This study provides the first structural and molecular characterization of rhinoceros ovaries, revealing that neonatal southern white rhinoceros ovaries contain mitotically active, pluripotent germ cell-like cells within nests, strongly suggesting the presence of oogonia at birth. This is unusual compared to most mammals where germ cell nests break down into primordial follicles prenatally or perinatally, and aligns more with species exhibiting prolonged germ cell nest presence. The presence of DDX4, SOX2, Oct4, and proliferation markers in neonatal nests supports their identity as germ cells undergoing early development rather than luteinized stromal cells proposed in related species. Low apoptosis (TUNEL) suggests nests had not yet undergone extensive germ cell attrition typical of meiotic prophase I. The confirmation of follicles at all stages in both neonatal and aged adult ovaries, despite low overall follicle density and abundant stroma, is significant for conservation strategies. The adult ovaries’ atypical two-sided cortex–medulla orientation is distinct from horses (closest relatives) and may reflect age or non-reproductive status. HA localization patterns differed between neonatal and adult tissues, suggesting developmental regulation of extracellular matrix components during folliculogenesis. Follicle clustering and ellipsoidal morphology observed in neonates are consistent with patterns in several mammals and may indicate follicle–follicle interactions that influence development. These findings directly address the study’s goal of informing aART development for rhinoceros conservation. The availability of neonatal oogonia and adult follicles provides potential starting material for in vitro gametogenesis and follicle culture, respectively, with implications for rescuing genetic diversity in critically endangered rhinoceros populations.

Neonatal southern white rhinoceros ovaries contain mitotically active, pluripotent, germ cell marker-positive cells in nests, consistent with oogonia persisting at birth, while adult ovaries, despite advanced age, still contain follicles across stages. Adult ovaries exhibit an unusual flattened, two-sided cortex–medulla organization. These insights provide a foundation for developing in vitro gametogenesis and follicle culture approaches to support conservation, highlighting stillborn neonatal ovaries as valuable sources of germ cells and underscoring the need to examine neonatal ovaries of other endangered species. Future work should expand sample sizes across ages and reproductive states, standardize tissue handling to minimize artifacts, and functionally test in vitro culture systems for rhinoceros germ cells and follicles.

The study is descriptive with a small sample size (one neonatal and three adult ovaries) and low follicle numbers, limiting statistical inference and generalizability. Samples were from aged or non-reproductive individuals, potentially influencing ovarian morphology and follicle density. Variable fixation and processing (including international transport) introduced shrinkage artifacts and precluded reliable follicle health assessments. Findings should be validated in additional individuals and standardized protocols.