Deciphering Sperm-Carried IGF2 Transcript Variants: Cloning, qPCR Detection, and Variant Analysis.

Abstract

Context: The insulin-like growth factor 2 (IGF2) gene, a paternally imprinted gene inactive in oocytes, plays a vital role in early embryo development. While 5 IGF2 variants have been described, the specific variants expressed in human spermatozoa compared to granulosa cells (GCs) remain unclear.

Objective: To characterize the quantity and variants of IGF2 transcripts expressed in human spermatozoa.

Methods: Post-gradient sperm samples were collected from 2 healthy, fertile men with normal semen parameters, while GCs were isolated following an oocyte retrieval procedure of a woman undergoing in vitro fertilization due to male factor infertility. RNA extraction, cDNA synthesis, PCR amplification, and cloning were performed. PCR products were ligated into PCR4-TOPO vectors and transformed into Escherichia coli DH-10α. A total of 96 positive clones (32 per sample) were characterized via Sanger sequencing to identify variants. Quantitative PCR (qPCR) with gene-specific primers analyzed transcript quantities, single nucleotide polymorphisms (SNPs), product sizes, and melting temperatures.

Results: Of the 96 true-positive IGF2 cDNA clones, 14 distinct variants were identified, including deletions, insertions, and SNPs, resulting in amino acid sequence changes. Two common variants were present in both sperm and GCs, while 2 were GC-specific, and the remaining were exclusive to spermatozoa. Some clustered with known NCBI variants, while others formed 2 novel phylogenetic clusters.

Conclusion: This study expands the repertoire of IGF2 variants and highlights differences between spermatozoa and GC transcripts. It is the first to analyze IGF2 variants in sperm from fertile men, paving the way for future research into their role in embryogenesis.