Biology subjects

Botigelli, R. C.

Publications and source records attributed to Botigelli, R. C..

4 recordsLinked to original sources

bioRxiv2026

Single-cell roadmap of bovine oogenesis and somatic niche interactions during fetal ovarian development

The major events of female germline establishment, from primordial germ cell (PGC) specification to assembly of primordial follicles, occur during embryonic/fetal development. This study presents a single-cell RNA-sequencing atlas of the bovine fetal ovary at four gestational timepoints: estimated day 50, and timed pregnancies at days 70, 90, and 120, capturing the progression of PGCs through commitment, meiotic entry, and early oocyte growth. Fourteen transcriptionally distinct cell populations were identified, including stromal, epithelial, endothelial, immune, somatic support cell, and germ cell lineages. Sub-clustering of the germ cell population resolved six developmental stages (PGCs, transitioning oogonia, proliferative oogonia, committed oogonia, meiotic prophase I oogonia, and oocytes), while that of the somatic support cell compartment revealed five granulosa cell subtypes (steroidogenic, pre-granulosa 1, pre-granulosa 2, pre-granulosa 3, and epithelial cells). Trajectory analysis reconstructed the developmental path of PGCs to oocytes, with sequential activation of meiotic and oocyte-specific gene programs. Representation of all six germ cell stages at day 120 pointed to asynchronous oogenesis in the fetal ovary, which was validated and shown to be region-specific by protein immunolocalization. Intercellular signaling networks between germ cells and the somatic niche were mapped, revealing strong interactions through BMP, KIT, IGF, IGFBP, WNT, and MDK pathways with temporal specificity across gestational ages. The bovine germ cell and pre-granulosa cell subtypes demonstrate significant transcriptional parallels with similarly-staged cells from human fetal ovaries, establishing the cow as a reliable model for human germ cell and ovarian development. Collectively, these data provide a developmental roadmap for bovine oogenesis at the single-cell resolution that advances fundamental understanding of gametogenesis and informs strategies for advanced assisted reproduction.

developmental biology↗

bioRxiv2025

Molecular profiling of bovine primordial germ cell specification and migration onset reveals a conserved program in bilaminar disc embryos

The goal of this study was to establish the timing and location of bovine primordial germ cell (PGC) specification, as well as the molecular characteristics that differentiate early PGCs from other cells in the embryo. A founder population of PGCs was identified via protein immunolocalization in the posterior epiblast (TBXT+) region at E16, by the unique co-expression of OCT4, SOX17, PRDM1, and TFAP2C with absent SOX2. At E20, PGCs were spread along the allantois and hindgut endoderm, indicative of migration onset. Single-cell RNA-sequencing at E20-22 revealed ten PGCs that were subset using the above markers. Further analysis of this population uncovered conserved and novel markers of the early germline and gave numerous insights into their developmental status, including markers associated with pluripotency maintenance, cell migration, repression of the somatic program, and initiation of epigenetic remodeling. This work establishes the region and transcription factor network of bovine PGC specification at day 16 of embryonic development and the first transcriptomic profile of migratory PGCs in cattle, demonstrating a conserved program with other species that form as a bilaminar embryonic disc leading to gastrulation.

developmental biology↗

bioRxiv2024

Primed bovine embryonic stem cell lines can be derived at diverse stages of blastocyst development with similar efficiency and molecular characteristics

In this study, we established bovine embryonic stem cell (bESC) lines from early (eBL) and full (BL) blastocysts to determine the efficiency of bESC derivation from an earlier embryonic stage and compare the characteristics of the resulting lines. Using established medium and protocols for derivation of primed bESCs from expanded blastocysts, we derived bESC lines from eBLs and BLs with the same efficiency (4/12 each, 33%). Regardless of original blastocyst stage, bESC lines had a similar phenotype, including differentiation capacity, stable karyotype, and pluripotency marker expression over feeder-free transition and long-term culture. Transcriptome and functional analyses indicated that eBL- and BL-derived lines were in primed pluripotency. We additionally compared RNA-sequencing data from our lines to bovine embryos and stem cells from other recent reports, finding that base medium was the predominant source of variation among cell lines. In conclusion, our results show that indistinguishable bESC lines can be readily derived from eBL and BL, widening the pool of embryos available for bESC establishment. Finally, our investigation points to sources of variation in cell phenotype among recently reported bESC conditions, opening the door to future studies investigating the impact of factors aside from signaling molecules on ESC derivation, maintenance, and performance. SUMMARY STATEMENTPrimed bovine embryonic stem cell lines can be readily established from early blastocysts and are indistinguishable from full blastocyst-derived lines, widening the pool of embryos available for stem cell derivation.

cell biology↗

bioRxiv2024

Three-dimensional culture in a bioengineered matrix and somatic cell complementation to improve growth and survival of bovine preantral follicles

STRUCTURED ABSTRACTO_ST_ABSPurposeC_ST_ABSHere we explored poly(ethylene glycol) (PEG) bioengineered hydrogels for bovine preantral follicle culture with or without ovarian cell co-culture and examined the potential for differentiation of bovine embryonic stem cells (bESCs) towards gonadal somatic cells to develop a system more similar to the ovarian microenvironment. MethodsBovine preantral follicles were first cultured in two-dimensional (2D) control or within PEG hydrogels (3D) and then co-cultured within PEG hydrogels with bovine ovarian cells (BOCs) to determine growth and viability. Finally, we tested conditions to drive differentiation of bESCs towards the intermediate mesoderm and bipotential gonad fate. ResultsPrimary follicles grew over the 10-day culture period in PEG hydrogels compared to 2D control. Early secondary follicles maintained a similar diameter within the PEG while control follicles decreased in size. Follicles lost viability after co-encapsulation with BOCs; BOCs lost stromal cell signature over the culture period within hydrogels. Induction of bESCs towards gonadal somatic fate under WNT signaling was sufficient to upregulate intermediate mesoderm (LHX1) and early coelomic epithelium/bipotential gonad markers (OSR1, GATA4, WT1). Higher BMP4 concentrations upregulated the lateral plate mesoderm marker FOXF1. PAX3 expression was not induced, indicating absence of the paraxial mesoderm lineage. ConclusionsCulture of primary stage preantral follicles in PEG hydrogels promoted growth compared to controls; BOCs did not maintain identity in the PEG hydrogels. Collectively, we demonstrate that PEG hydrogels can be a potential culture system for early preantral follicles pending refinements, which could include addition of ESC-derived ovarian somatic cells using the protocol described here. CAPSULE SUMMARYWe demonstrate that three-dimensional bioengineered hydrogels could aid in the survival and growth of small bovine preantral follicles. Moreover, bovine embryonic stem cells have the potential to differentiate towards precursors of somatic gonadal cell types, presenting an alternative cell source for preantral follicle co-culture.

developmental biology↗