PRSSLY-Based Molecular Sex Determination of Syrian Hamster (Mesocricetus auratus) Pups Using Placental Tissues

Molecular sex determination in Syrian hamsters (<i>Mesocricetus auratus</i>) has been limited by the incomplete annotation of Y-linked loci in currently available genome assemblies. Here, we evaluate the Y-linked gene <i>PRSSLY,</i> which encodes a testis-specific serine protease-like protein, as a molecular marker for genetic sexing of Syrian hamster embryonic and placental tissues. Primers flanking a conserved <i>PRSSLY</i> coding region produced a male-specific amplicon showing 100% concordance with results from the established <i>KDM5C/KDM5D</i> PCR assay in E15.5 tail biopsies. SYBR Green-based qPCR enables the accurate detection of <i>PRSSLY</i>, characterized by a unique melt-curve profile, exclusively in male samples, allowing for efficient and sensitive mid-throughput analysis. Application of the PRSSLY assay to 417 placental samples from 39 dams demonstrated its suitability for large-scale sex genotyping, enabling sex assignment in the majority of samples despite the intrinsic complexity of placental tissue containing both maternal and embryonic genetic material. This assay provides a robust and reproducible approach for accurate sex genotyping in developmental and reproductive studies using Syrian hamsters.