(258) Two of a Kind: How the Equine Fetal Gonad Balances DHCR7 to Yield Estrone and Equilin

Two of a Kind: How the Equine Fetal Gonad Balances DHCR7 to Yield Estrone and Equilin

Katarzyna Malin¹; Margo Verstraete^1,2; William Holl³; Machteld van Heule^1,2; Jamie Norris¹; Kirsten Scoggin⁴; Barry Ball⁴; Alan J. Conley¹; Mariano Carossino³; Pouya Dini¹

1. Department of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, CA, USA

2. Department of Morphology, Imaging, Orthopedics, Rehabilitation and Nutrition, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium

3. Department of Pathobiological Sciences and Louisiana Animal Disease Diagnostic Laboratory, School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana, USA

4. Department of Veterinary Science, Gluck Equine Research Center, University of Kentucky, Lexington, Kentucky, USA


Abstract Text:

 The eleven-months-long equine pregnancy is marked by the existence of the only known non-primate feto-placental unit, in which a cooperation between the fetal gonad and the placenta paints a peculiar sex hormone landscape. The equine fetal gonad is a unique organ: utilizing the same steroidogenic enzymes (CYP11A1 and CYP17A1), the interstitium synthesizes two different estrogen precursors. The cholesterol-derived dehydroepiandrosterone (DHEA) is a substrate for the placental estrone, and 7-dehydroDHEA(7-dhDHEA), derived from cholesterol’s precursor, 7-dehydrocholesterol (7-DHC), is a substrate for equilin. Furthermore, the gonad grows until circa 8^th gestational month and then undergoes involution until birth, which is likely due to a dramatic loss of the interstitial cell volume shown in previous morphometric studies. Estrone and equilin peak at different gestational periods; estrone between 120 and 180, and equilin between 210 and 240, when the fetal gonad size is the largest. Both decline until the parturition. However, it is not known what factors regulate the changes in estrone and equilin levels throughout the pregnancy. To identify the potential limiting steps at the gonad level, equine fetal gonads (gestational age: 4MO, 6MO, 9MO, 10MO, 11MO) were subjected to RNA seq, and consequently, to RNA scope (DHCR7), and immunofluorescence imaging (DHCR7, CYP11A1, CYP17A1). Twenty-eight genes from the cholesterol and steroid synthesis pathway and regulatory were evaluated for absolute and differential expression in RNA seq. The highest expression was seen in CYP17A1, DHCR24 and MSMO1, and the lowest expression was seen in LBR. However, only DHCR7, whose loss-of-function mutations are associated with pathological accumulation of 7-DHC in the Smith-Lemli-Opitz Syndrome (SLOS), was differentially expressed (Log₂FC=-4.52 (p_adj< 0.001)) from 4^th to 11^th gestational month; no differential expression between 9^th, 10^th and 11^thgestational months). This pattern mirrors estrone levels in the pregnant mare plasma reported in the literature. In the immunofluorescent staining, CYP11A1 and CYP17A1 were used as markers of the interstitial cells participating in DHEA and 7-dhDHEA synthesis. DHCR7 compartmentalization was the highest in the 6^th gestational month testes interstitium. RNA scope DHCR7 evaluation showed the highest compartmentalization of DHCR7 mRNA in the gonads of both sexes in the 4^th gestational month, declining with the progression of the pregnancy. The lack of differential expression upstream and downstream of DHCR7 and the compartmentalization of its mRNA and protein expression suggests that: (1) the synthesis of 7-dhDHEA and DHEA may be separated spatially, (2) two populations of the equine fetal gonad interstitium may be maintained for this purpose, (3) the decrease in the interstitial cell volume may be a candidate for the limiting factor of equilin synthesis. These findings also suggest that the potentially cytotoxic 7-DHC is successfully transported within the interstitial cell of the equine gonad for the synthesis of 7dh-DHEA. Typically, DHCR7 co-localizes with enzymes that synthesize 7-DHC to yield cholesterol (primarily in the endoplasmic reticulum), which only then is transported to the mitochondrial CYP11A1. However, in the conditions of downregulated DHCR7 that allow for equilin synthesis, 7-DHC would require to be transported to CYP11A1 in the place of cholesterol. As this unique profile is not found in any other physiological setting, its investigation could be beneficial to the SLOS research. This work was supported by the National Institutes of Health (NIH) under grant number T32 OD 011147 and the UC Davis Center for Equine Health.