Gamma-glutamylcysteine synthetase, also known as glutamate-cysteine ligase (EC 6.3.2.2 ), is the first rate-limiting enzyme in glutathione biosynthesis.
Human liver gamma-glutamylcysteine synthetase consists of 2 subunits: a heavy catalytic subunit (GCLC; OMIM 606857) and a light regulatory subunit.
NCBI Summary:
Glutamate-cysteine ligase, also known as gamma-glutamylcysteine synthetase is the first rate-limiting enzyme of glutathione synthesis. The enzyme consists of two subunits, a heavy catalytic subunit and a light regulatory subunit. This locus encodes the catalytic subunit, while the regulatory subunit is derived from a different gene located on chromosome 1p22-p21. Mutations at this locus have been associated with hemolytic anemia due to deficiency of gamma-glutamylcysteine synthetase and susceptibility to myocardial infarction.[provided by RefSeq, Oct 2010]
Developmental potential of prepubertal mouse oocytes is compromised due mainly to their impaired synthesis of glutathione. Jiao GZ et al. Although oocytes from prepubertal animals are found less competent than oocytes from adults, the underlying mechanisms are poorly understood. Using the mouse oocyte model, this paper has tested the hypothesis that the developmental potential of prepubertal oocytes is compromised due mainly to their impaired potential for glutathione synthesis. Oocytes from prepubertal and adult mice, primed with or without eCG, were matured in vitro and assessed for glutathione synthesis potential, oxidative stress, Ca reserves, fertilization and in vitro development potential. In unprimed mice, abilities for glutathione synthesis, activation, male pronuclear formation, blastocyst formation, cortical granule migration and polyspermic block were all compromised significantly in prepubertal compared to adult oocytes. Cysteamine and cystine supplementation to maturation medium significantly promoted oocyte glutathione synthesis and blastocyst development but difference due to maternal age remained. Whereas reactive oxygen species (ROS) levels increased, Ca storage decreased significantly in prepubertal oocytes. Levels of both catalytic and modifier subunits of the ?-glutamylcysteine ligase were significantly lower in prepubertal than in adult oocytes. Maternal eCG priming improved all the parameters and eliminated the age difference. Together, the results have confirmed our hypothesis by showing that prepubertal oocytes have a decreased ability to synthesize glutathione leading to an impaired potential to reduce ROS and to form male pronuclei and blastocysts. The resulting oxidative stress decreases the intracellular Ca store resulting in impaired activation at fertilization, and damages the microfilament network, which affects cortical granule redistribution leading to polyspermy.
Espey and Richards 2002 reviewed the literature on ovulation-related genes.
A moderate amount of -Glutamylcysteine Synthetase (GCS) mRNA was distributed randomly (i.e., nonuniformly) in the granulosa layer of mature follicles even before the ovulatory process was induced by hCG. In addition, there was limited, but distinct, expression of GCS mRNA in the theca interna surrounding the granulosa cells. After stimulation of the ovaries with hCG, the onset of ovarian GCS mRNA expression in mural granulosa cells was slightly later than that of ALAS and Egr-1. There was a transient increase during the first several hours of the ovulatory process in the granulosa of most, but not all, of the follicles that appeared to be large enough to progress to rupture. However, expression was minimal in most follicles at 12 h after hCG, when the follicles began to rupture. GCS expression was substantially elevated between 4 and 8 h after hCG specifically in the thecal tissue as well as in some localized areas of the ovarian stroma.
Expression regulated by
FSH, LH, Steroids
Comment
Gonadotropin Regulation of Glutamate Cysteine Ligase Catalytic and Modifier Subunit Expression in the Rat Ovary is Subunit and Follicle Stage-Specific. Tsai-Turton M, et al have observed that levels of the antioxidant glutathione (GSH) and protein levels of the catalytic and modifier subunits of the rate-limiting enzyme in GSH synthesis, GCLc and GCLm, increase in immature rat ovaries after treatment with gonadotropin. The goals of the present studies were to delineate the time course and intraovarian localization of changes in GSH and GCL after PMSG and after an ovulatory gonadotropin stimulus. 24h after PMSG there was a shift from predominantly granulosa cell expression of gclm mRNA, and to a lesser extent gclc, to predominantly theca cell expression. GCLc immunostaining increased in granulosa and theca cells and in interstitial cells. Next, prepubertal female rats were primed with PMSG, followed 48h later by 10 IU hCG. GCLm protein and mRNA levels increased dramatically from 0h to 4h after hCG, then declined rapidly. There was minimal change in GCLc. The increase in gclm mRNA expression was localized mainly to granulosa and theca cells of preovulatory follicles. To verify that GCL responds similarly to an endogenous preovulatory gonadotropin surge, we quantified ovarian GCL mRNA levels during the periovulatory period in adult rats. Gclm mRNA levels increased after the gonadotropin surge on proestrus and then declined rapidly. Finally, we assessed the effects of gonadotropin on ovarian GCL enzymatic activity. GCL enzymatic activity increased significantly at 48h after PMSG injection and did not increase further after hCG. These results demonstrate that gonadotropins regulate follicular GCL expression in a follicle stage-dependent manner and in a GCL subunit-dependent manner.
Gene expression decreased. Luteinization of porcine preovulatory follicles leads to systematic changes in follicular gene expression. Agca C et al. The LH surge initiates the luteinization of preovulatory follicles and causes hormonal and structural changes that ultimately lead to ovulation and the formation of corpora lutea. The objective of the study was to examine gene expression in ovarian follicles (n = 11) collected from pigs (Sus scrofa domestica) approaching estrus (estrogenic preovulatory follicle; n = 6 follicles from two sows) and in ovarian follicles collected from pigs on the second day of estrus (preovulatory follicles that were luteinized but had not ovulated; n = 5 follicles from two sows). The follicular status within each follicle was confirmed by follicular fluid analyses of estradiol and progesterone ratios. Microarrays were made from expressed sequence tags that were isolated from cDNA libraries of porcine ovary. Gene expression was measured by hybridization of fluorescently labeled cDNA (preovulatory estrogenic or -luteinized) to the microarray. Microarray analyses detected 107 and 43 genes whose expression was decreased or increased (respectively) during the transition from preovulatory estrogenic to -luteinized (P<0.01). Cells within preovulatory estrogenic follicles had a gene-expression profile of proliferative and metabolically active cells that were responding to oxidative stress. Cells within preovulatory luteinized follicles had a gene-expression profile of nonproliferative and migratory cells with angiogenic properties. Approximately, 40% of the discovered genes had unknown function.
Ovarian localization
Oocyte, Granulosa, Luteal cells, Stromal cells
Comment
Follicle-Stimulating Hormone and Estradiol Interact to Stimulate Glutathione Synthesis in Rat Ovarian Follicles and Granulosa Cells. Hoang YD et al. Glutathione (GSH), the most abundant intracellular non-protein thiol, is critical for many cellular functions. The rate-limiting step in GSH synthesis is catalyzed by glutamate cysteine ligase (GCL), a heterodimer composed of a catalytic (GCLC) and a modifier (GCLM) subunit. The tissue-specific regulation of GSH synthesis is poorly understood. We previously showed that gonadotropin hormones regulate ovarian GSH synthesis. Herein we sought to clarify the ovarian cell type-specific effects of follicle stimulating hormone (FSH) and estradiol on GSH synthesis. Immature female rats were treated with estradiol to stimulate development of small antral follicles. Granulosa cells (GCs) from these follicles or whole follicles were cultured in serum-free media, with or without FSH and 17beta-estradiol. GSH and GCLC protein and mRNA levels increased in GCs treated with FSH alone. The effects of FSH on GCLC and GCLM protein and mRNA levels, GCL enzymatic activity, and GSH concentrations in GCs were significantly enhanced by the addition of estradiol. Estradiol alone had no effects on GSH. Dibromo cAMP mimicked and protein kinase A (PKA) inhibitors prevented FSH stimulation of GCL subunit protein levels. In cultured small antral follicles, FSH stimulated estradiol synthesis and robustly increased GCL subunit mRNA and protein levels and GSH concentrations. GCL subunit mRNA expression increased in both the granulosa cells and theca cells of follicles with FSH stimulation. These data demonstrate that maximal stimulation of GSH synthesis by FSH in granulosa cells and follicles requires estradiol. Without estradiol, FSH causes lesser increases in GCL subunit expression via a PKA dependent pathway.
Luderer U, et al reported he localization of glutamate cysteine ligase subunit mRNA within the rat ovary and relationship to follicular apoptosis.
Ovarian levels of the antioxidant tripeptide glutathione (GSH) increase following gonadotropin administration, suggesting that GSH synthesis in the ovary may be associated with follicular growth. In situ hybridization with (35)S-labeled riboprobes was used to localize ovarian mRNA expression of the catalytic and modulatory subunits of glutamate cysteine ligase (Gclc and Gclm), the rate-limiting enzyme in GSH synthesis, during each stage of the rat estrous cycle. Gclm was highly expressed in the granulosa cells and oocytes of healthy, growing follicles, not in atretic follicles. Gclc was also highly expressed in follicles; however, unlike Gclm, Gclc was also expressed in corpora lutea and interstitial cells. In a subsequent experiment, the hypothesis that GSH synthesis occurs in healthy, but not in apoptotic, follicles was tested. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) was used to detect apoptotic cells in the ovaries, and in situ hybridization for Gclm and Gclc was performed in adjacent sections of the same ovaries. TUNEL staining was found to be significantly associated with absence of Gclm hybridization in granulosa cells and oocytes and with lack of strong Gclc hybridization in granulosa cells. These results suggest that follicular apoptosis may be associated with down-regulation of Gclm and Gclc transcription in granulosa cells and oocytes.