Homocysteine-Thiolactone links metabolic stress to ovarian function by regulating steroid biosynthesis and receptor expression

Introduction: Elevated homocysteine leads to increased levels of homocysteine thiolactone (HCTL), a highly active thioester that alters protein structure and function by N-homocysteinylation. The most common causes for elevated homocysteine/HCTL are malnutrition and MTHFR mutations. Having reported that the 677 C >T MTHFR mutation is associated with poor ovarian response in patients undergoing controlled ovarian stimulation, we investigated the effect of HCTL on granulosa cell biology.

Methods: Pooled cultures (collected from 5 – 10 patients) of human primary granulosa cells (GCs) were used in the current study. GCs were allowed to adapt to serum-free/folate-free conditions and then treated with HCTL (10^-1, 1, 10, 10², 10³ µM). We quantified intracellular ROS synthesis and apoptosis by visualizing DHF formation and Caspase activation, respectively. MTHFR, LHR and ERα were assessed by immunocytochemistry. Estradiol and progesterone production was quantified in GC supernatants by EIA.

Result: Intracellular ROS content was increased in GCs treated with HCTL (up to 1.5-fold, p < 0.05) without inducing apoptosis. At the same time MTHFR (10µM: 1.8-fold), LHR (10µM: 3.4-fold) and ERα (10µM: 1.8-fold) were also found to be significantly (p < 0.05) up-regulated by HCTL. Though HCTL increased estradiol production (10µM: 1.4-fold, p < 0.05), it down-regulated progesterone synthesis (10µM: 0.7-fold, p < 0.05) in GCs.

Conclusion: HCTL-triggered oxidative stress alters basic elements of GC steroid synthesis and function (E2/P4 synthesis, MTHFR, LHR and ERα expression). We hypothesize that HCTL could be a crucial mediator for ovarian dysfunction, subfertility as well as recurrent miscarriage in patients with MTHFR mutations.