7397 Characterization of Sperm-Carried IGF2 Transcript Variants

Abstract Disclosure: R. Cannarella: None. M. Suryavanshi: None. A. Miller: None. S.D. Lundy: None. A.E. Calogero: None. Background: We have previously identified the expression of the highly-conserved paternally-imprinted insulin-like growth factor 2 (IGF2) gene in human spermatozoa and suggested its role in early embryo development. Although there are 5 different variants of the IGF2, there is no evidence of the variants expressed in human spermatozoa. Objective: The present basic research study was undertaken to characterize the IGF2 transcript variant(s) expressed in human spermatozoa and to develop a sensitive method that can distinguish variants based on quantitative polymerase chain reaction (qPCR). Methods: Two post-gradient sperm samples from healthy, fertile men with proven paternity were used. Granulosa cells (GCs) obtained after the oocyte retrieval procedure and subsequently isolated and purified from follicular fluid were used as controls. Each sample underwent RNA extraction, cDNA synthesis, and PCR. The PCR product was ligated with the pCR4-TOPO vector and used to transform TOPO10 one-shot electro-competent cells of E. coli DH-10α. For each sample, 32 white colonies (true positive clones selected by blue-white screening method) were grown per sample (total in 96). All 96 clones were fully characterized by Sanger sequencing and single nucleotide polymorphism (SNP) were detected. Variations in products from single IGF2 gene primers pair were recorded on Sybr green-based qPCR, such as variations in melting temperatures (Tm), product size, and number of copies. Results: We successfully isolated and identified 96 true positive colonies, which were confirmed as IGF2 cDNA clones. This comprehensive analysis revealed 11 distinct genetic variants within the cDNA clones. Specifically, these variants comprised 6 SNPs and 5 deletions, highlighting the diversity in the sequences. These findings were particularly significant in relation to the IGF2 primer employed in the study, as they indicated the presence of 11 different sequence variants. By Sybr green qPCR, clones derived from GCs and sperm samples predominantly showed an IGF2 transcript variant with Tms of 89.01°C, 88.86°C, and 88.71°C. In a surprising observation, the sperm samples exhibited three sperm-specific variants with a range of a Tm ranging from 89.01°C to 88.26°C. Furthermore, the deletions were characterized by a significantly lower Tm, ranging between 78.86°C and 71.11°C. Conclusion: The IGF2 transcript is intrinsically present in sperm samples, albeit with notable variations. The detected variants likely arise from SNPs within the transcripts and the deletions could be attributed to splicing activity during maturation or post-transcriptional modifications. To our knowledge, this is the first study targeting the IGF2 transcript variants in spermatozoa from fertile men. Basic research is needed to understand the implications of different sperm-specific IGF2 transcript variants on early embryo development. Presentation: 6/2/2024