Induction of hyaluronan synthase 2 by human chorionic gonadotropin in mural granulosa cells of equine preovulatory follicles.

This research examined how the hyaluronan synthase 2 (HAS2) gene is activated in the granulosa cells of horses during ovulation, triggered by the human chorionic gonadotropin (hCG) hormone. The study cloned and characterized the equine HAS2, analyzed its regulation during hCG-induced ovulation, and identified it as the key factor in the visible expansion of the granulosa cell layer in horses, compared to other species.

Investigation of equine HAS2

• The researchers cloned and characterized equine HAS2, a gene critically involved in the production of hyaluronan (HA), which plays a crucial role in tissue hydration, space filling and maintaining the architecture in tissues.
• The equine HAS2 cDNA was made up of a 5′-untranslated region of 436 base pairs, an open reading frame of 1659 base pairs, and a 3′-untranslated region of 707 base pairs.
• The open reading frame encoded a 552-amino acid protein which was highly conserved (98-99% identity) when compared to other known mammalian homologues.

Regulation of HAS2 during hCG-induced ovulation

• The researchers observed the regulation of HAS2 mRNA in equine follicles during estrus (the period when the mare’s reproductive system is receptive to breeding) from 0 and 39 hours after an ovulatory dose of hCG, and in corpora lutea (the remnant of the follicle after ovulation) on the 8th day of the estrous cycle.
• The amount of HAS2 transcript, determined through semiquantitative RT-PCR/Southern blotting analyses, went through a transient induction during the ovulatory process. It was undetectable before hCG treatment, increased significantly after the hormone treatment (P < 0.05), and returned to undetectable levels in corpora lutea.

Anatomical location and impact of HAS2 induction

• The granulosa cell layer was identified as the key site of HAS2 expression based on analyses performed on isolated preparations of theca interna and granulosa cells.
• The induction of HAS2 transcript was accompanied by a dramatic increase in HA production following hCG treatment, as demonstrated via immunohistochemical approach.

Genomic Sequencing of HAS2

• A 1.8-kilobase fragment of genomic sequence located just ahead of equine HAS2 was isolated and characterized.
• Comparisons with equivalent sequences from the human and mouse genomes revealed several conserved putative cis-acting elements, suggesting similar control structures across these species.

The research contributed valuable insights into the regulation of HAS2 in the granulosa cells during ovulation in horses and presented a useful model for studying the molecular control of HAS2 gene expression.