Enhancing the Spermidine Synthase-Based Polyamine Biosynthetic Pathway to Boost Rapid Growth in Marine Diatom Phaeodactylum tricornutum.

Diatoms, efficient carbon capture organisms, contribute to 20% of global carbon fixation and 40% of ocean primary productivity, garnering significant attention to their growth. Despite their significance, the synthesis mechanism of polyamines (PAs), especially spermidine (Spd), which are crucial for growth in various organisms, remains unexplored in diatoms. This study reveals the vital role of Spd, synthesized through the spermidine synthase (SDS)-based pathway, in the growth of the diatom Phaeodactylum tricornutum. PtSDS1 and PtSDS2 in the P. tricornutum genome were confirmed as SDS enzymes through enzyme-substrate selectivity assays. Their distinct activities are governed primarily by the Y79 active site. Overexpression of a singular gene revealed that PtSDS1, PtSDS2, and PtSAMDC from the SDS-based synthesis pathway are all situated in the cytoplasm, with no significant impact on PA content or diatom growth. Co-overexpression of PtSDS1 and PtSAMDC proved essential for elevating Spd levels, indicating multifactorial regulation. Elevated Spd content promotes diatom growth, providing a foundation for exploring PA functions and regulation in diatoms.

Role of kisspeptin on cell proliferation and steroidogenesis in luteal cells in vitro and in vivo.

BACKGROUND: Kisspeptin (KISS1) and kisspeptin receptor (KISS1R) are essential gatekeepers of the reproductive system. The functions of KISS1 and KISS1R in corpus luteal cells remain ambiguous. The objective was to observe normal physiologic functions of corpus luteal cells in vivo and clarify the functions of KISS1 in vitro. METHODS: We conducted an in vivo observation of cellular patterns as well as the levels of steroidogenic enzymes and KISS1/KISS1R in corpus luteal cells obtained from female crossbred Taiwan native goats in the estrous cycle; the observation was performed using hematoxylin and eosin and immunohistochemistry staining. Subsequently, we used kisspeptin-10 (Kp-10) to stimulate temperature sensitive-caprine luteal cell line (ts-CLC-D) cells to investigate the progesterone (P4) levels, steroidogenic messenger RNA (mRNA)/protein levels, cell survival rate, intracellular Ca2+ concentration, and cell proliferation-related mRNA/protein levels in the mitogen-activated protein kinase pathway in vitro by applying immunofluorescence staining, Western blotting, 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide assay, and real-time polymerase chain reaction. RESULTS: We observed the presence of proteins and mRNAs for STAR, CYP11A1, HSD3B, KISS1, and KISS1R in the corpus luteal cells from goats in vivo. In vitro, the addition of Kp-10 reduced the P4 levels (p < 0.01) and increased cell proliferation (p < 0.05) of the ts-CLC-D cells. Furthermore, we found that the levels of proteins and mRNA for STAR, CYP11A1, and HSD3B decreased significantly when Kp-10 was added (p < 0.05). However, adding Kp-10 did not affect the mRNA levels for PLCG2, DAG1, PRKCA, KRAS, RAF1, MAP2K1, MAP2K2, MAPK3, MAPK1, and MAPK14. CONCLUSION: We determined that KISS1 could affect the P4 levels, steroidogenesis, and cell proliferation in luteal cells. However, further research is required to clarify how KISS1 regulates proliferation and steroid production in luteal cells.