Exploring the potential use of platelet rich plasma (PRP) from adult and umbilical cord blood in murine follicle culture.

Ovarian follicle culture is a powerful tool to study follicular physiology and has potential applications in clinical and commercial settings. Despite remarkable progress, recreating folliculogenesis in vitro remains challenging for many mammalian species. This study investigates the impact of platelet-rich plasma (PRP) derived from adult blood (human platelet lysate, hPL) and umbilical cord blood (Umbilical cord plasma, UCP) on murine pre-antral follicle culture and oocyte maturation. Pre-antral follicles were cultured individually for 10 days with fetal bovine serum (FBS) serving as the control and two PRP sources (hPL and UCP) and their activated forms (Ac-hPL and Ac-UCP). The results suggest that neither hPL nor UCP, regardless of activation status, improved follicle culture outcomes compared to FBS. Interestingly, activation did not significantly impact the main functional outcomes such as maturation rates, survival, and growth. Oestradiol secretion and oocyte diameter, often considered hallmarks of follicle quality, did not show significant differences between matured and non-matured oocytes across the treatment groups. However, gene expression analysis revealed a significant upregulation of Gdf-9 and Bmp-15 mRNA levels in oocytes from the Ac-UCP group, regardless of maturation stage, suggesting that the accumulation of the mRNA could be due to potential challenges in translation in the Ac-UCP group. In conclusion, this study challenges the hypothesis that PRP, as a serum source, could improve follicle culture outcomes compared to FBS, the gold standard in murine follicle culture. Further research is needed to understand the species-specific effects of PRP and explore other potential factors affecting follicle culture and oocyte quality.

Cellular senescence promotes progenitor cell expansion during axolotl limb regeneration.

Axolotl limb regeneration is accompanied by the transient induction of cellular senescence within the blastema, the structure that nucleates regeneration. The precise role of this blastemal senescent cell (bSC) population, however, remains unknown. Here, through a combination of gain- and loss-of-function assays, we elucidate the functions and molecular features of cellular senescence in vivo. We demonstrate that cellular senescence plays a positive role during axolotl regeneration by creating a pro-proliferative niche that supports progenitor cell expansion and blastema outgrowth. Senescent cells impact their microenvironment via Wnt pathway modulation. Further, we identify a link between Wnt signaling and senescence induction and propose that bSC-derived Wnt signals facilitate the proliferation of neighboring cells in part by preventing their induction into senescence. This work defines the roles of cellular senescence in the regeneration of complex structures.

Human platelet lysate improves the growth and survival of cultured human pre-antral follicles.

RESEARCH QUESTION: How do platelet-rich plasma products like human platelet lysate (HPL) and umbilical cord plasma (UCP) affect the growth and survival of isolated human pre-antral follicles in vitro? DESIGN: Human pre-antral follicles (n = 724; mean diameter: 75 µm; range: 46-237 µm) were isolated from ovarian medulla donated by 14 patients undergoing unilateral oophorectomy for ovarian tissue cryopreservation. Follicles were encapsulated in 0.5% alginate and cultured for 8 days in media supplemented with 5% fetal bovine serum (FBS) (n = 171), 2.5% human serum albumin (HSA) (n = 159), 5% HPL (n = 223) or 5% UCP (n = 171). RESULTS: The survival probability was significantly higher in the group supplemented with HPL (80%) compared with the other three groups: FBS (54%, P < 0.001); HSA (63%, P = 0.004) and UCP (29%, P < 0.001). Surviving follicles in the UCP group had less defined follicular membranes and decompacted granulosa cell layers. The median growth of surviving follicles was significantly (P < 0.001) larger in the HPL group (73 µm) compared with any of the other three groups: HSA (43 µm); FBS (40 µm) UCP (54 µm). A descriptive analysis of follicular secretion of anti-Müllerian hormone and oestradiol did not reveal any difference between the groups. The detectability of follicular genes was high for AR (100%), AMHR2 (100%) and FSHR (76%), whereas few follicles expressed LHR (20%). CONCLUSION: Human platelet lysate significantly improved survival and growth of cultured human pre-antral follicles compared with FBS, HSA and UCP. The use of HPL is a valuable improvement to culture human pre-antral follicles but further studies will have to prove whether the superiority of HPL translates into better quality oocytes.

Salamander-Eci: An optical clearing protocol for the three-dimensional exploration of regeneration.

BACKGROUND: Salamander limb regeneration is a complex biological process that entails the orchestration of multiple cellular and molecular mechanisms in a three-dimensional space. Hence, a comprehensive understanding of this process requires whole-structure level explorations. Recent advances in imaging and optical clearing methods have transformed the study of regenerative phenomena, allowing the three-dimensional visualization of structures and entire organisms. RESULTS: Here we introduce Salamander-Eci, a rapid and robust optical clearing protocol optimized for the widely used axolotl model, which allows simultaneous immunohistochemistry and Click-chemistry detection with minimal volume disruption. We provide examples of its application, from whole larva to adult limbs and organs, and complement it with an image analysis pipeline for volumetric cell quantification. Further, we offer a detailed 3D quantitation of cell proliferation throughout axolotl limb regeneration. CONCLUSIONS: Salamander-Eci enables the comprehensive volumetric analysis of regenerative phenomena at both local and systemic levels.