12674 Spatiotemporal Analysis Of Steroidogenesis Pathway Genes Expression In Mouse Fetal Leydig Cells

Abstract Disclosure: K. Jiang: Other; Self; NIH ORIP P51OD011106&S10OD028626. A. Kothandapani: None. Z. Fu: None. T.W. Kearse: None. J. Jorgensen: Grant Recipient; Self; NIH R01HD090660. Male mouse embryos experience a masculinization programming window (MPW, embryonic day, E13-16), during which a critical threshold of androgens is produced to ensure male-pattern development. Insufficient fetal androgens during the MPW leads to variations of masculinization, such as cryptorchidism and hypospadias. In rodents, fetal Leydig cells (FLC) are the primary source of androgens; however, our knowledge about their maturation and steroid synthesis regulation is limited. Hence, the objective for this research is to reveal spatiotemporal patterns of steroidogenic pathway genes in mouse fetal testes using high-resolution technologies. To understand the temporal aspect of steroidogenesis, we quantified transcript numbers of steroidogenic pathway genes via copy number qPCR from E11 to postnatal day 6 (P6). Results showed a gradual increase in transcripts as FLCs differentiate until E14, which then surge during the MPW until E16, followed by a rapid drop in expression to pre-FLC differentiation levels by the time of birth (P0). The profound changes persisted after data were normalized to FLC numbers, suggesting active stimulation and downregulation phases of transcription regulation. Next, to assess androgen output in relation to gene transcript levels, we collected testes at 24 hour intervals from E12 to P3 to measure progesterone, androstenedione, and testosterone via LC/MS/MS; results are pending. Guided by the temporal expression profile, we examined the spatial patterns of Star (steroidogenic acute regulatory protein) at the onset (E13), peak (E16), and end (P0) of steroidogenesis using single-molecule fluorescent in situ hybridization (smFISH). We were surprised to observe three different stages for Star transcription based on the subcellular localization of it: Stage I-only at gene loci in the nucleus, newly differentiated; Stage II-in both the nucleus and cytoplasm, peak activity; and Stage III-in cytoplasm only, repressed transcription. The proportion of Stage I FLC decreased over time (15% at E13, 2% at E16, and 0.4% at P0). Most (55%) FLCs at E16 were at Stage II, when testes exhibit maximum steroidogenic gene transcript numbers. Stage III FLCs dominated in P0 testes with 68% of the total FLC number, reflecting diminishing steroidogenesis. Of note, we also discovered that the global testis pattern for Star transcripts exhibited a unique pattern. At the onset of differentiation (E13), FLCs accumulated in the middle of the dorsal-ventral axis and at either pole of the anterior-posterior axis. By E16 and at P0, FLCs were evenly distributed throughout the testis. In conclusion, we observed that FLC steroidogenic pathway gene transcription is not synchronized but individual FLCs contribute to global testis androgen output as they differentiate in a coordinated pattern towards maximal output to coordinate masculinization. Presentation: 6/1/2024