Effects of Insulin and Insulin-Like Growth Factors on Proliferation of Rat Ovarian Theca-Interstitial Cells

Abstract

Hyperplasia of the theca-interstitial (T-I) compartment, such as observed in polycystic ovary syndrome, is associated with ovarian dysfunction. Yet the mechanisms regulating proliferation of T-I cells are virtually unknown. This study was an investigation of the effects of insulin and insulin-like growth factors (IGF-I and IGF-II) on proliferation of rat T-I cells. Purified T-I cells were incubated in chemically defined media. Insulin (1-100 nM) and both IGFs (0.3-30 nM) dose-dependently stimulated DNA synthesis as determined by radiolabeled thymidine incorporation assay. IGF-I was most potent with EC50 = 1.4 +/- 0.4 nM, while IGF-II had EC50 = 4.3 +/- 0.18 nM and insulin had EC50 = 8.4 +/- 3.9 nM. The maximal effects of all three treatments were comparable. A combination of IGF-I at 10 nM (a concentration producing a near-maximal effect) with insulin or IGF-II resulted in DNA synthesis comparable to that achieved by IGF-I alone. IGF-I mutants with decreased affinity to IGF-binding proteins (IGFBPs)-long R3-IGF-I and des(1-3)IGF-I-produced greater effects on DNA synthesis than did IGF-I. The effects of insulin and IGFs on cell proliferation were confirmed by counting the steroidogenically active cells (stained positive for 3 beta-hydroxysteroid dehydrogenase [3 beta-HSD]) and steroidogenically inactive cells (3 beta-HSD negative). The number of steroidogenically active T-I cells was increased by insulin (by 3.7-fold, p < 0.001), IGF-I (by 3.2-fold, p < 0.001), and IGF-II (by 2.1-fold, p < 0.001). The number of steroidogenically inactive cells was not significantly altered. These findings indicate that 1) insulin- and IGF-dependent synthesis of DNA by T-I cells is stimulated via a common pathway, probably via type I IGF receptors; 2) endogenous IGFBPs may modify the effects of IGF-I; and 3) the increased DNA synthesis is reflected by an increase in the number of steroidogenically active cells. Insulin and the IGFs may play a role in the regulation of proliferation and differentiation of T-I cells under physiological and pathological conditions. In particular, the present observations may explain thecal and stromal hyperplasia accompanying hyperinsulinemic conditions such as polycystic ovary syndrome or hyperthecosis.

Insulin, leptin, IGF-I and insulin-dependent protein concentrations after insulin-sensitizing therapy in obese women with polycystic ovary syndrome

“…One could speculate that, despite similar total IGF-I levels, the IGFBP-1 concentration is decreased in PCOS women, which in turn leads to an increase in the free fraction of IGF-I (24). Moreover, Duleba reported that insulin diminishes not only hepatic IGFBP-1 synthesis but also the intraovarian pool (25). It could promote higher local IGF-I activity.…”

Regulation of adiponectin and its receptors in rat ovary by human chorionic gonadotrophin treatment and potential involvement of adiponectin in granulosa cell steroidogenesis

“…The remaining ovarian tissues were washed thrice with medium 199 containing 25 mmol/l Hepes (pH 7.4), L-glutamine (2 mmol/l), BSA (0.1%), penicillin (100 U/ml) and streptomycin (100 mg/l) to release remaining granulosa cells. To obtain T-I cells, the ovarian tissue was minced actively and repeatedly and incubated for 60 min at 37 8C in the same medium 199, supplemented with collagenase type 1 (5.1 mg/ml) plus 10 mg/ml DNase, as described by Duleba et al (1997).The T-I cells released by this digestion were centrifuged at 250 g for 5 min and washed in collagenase-free medium twice to eliminate remaining collagenase. The dispersed cells were then resuspended in the same McCoy's 5A medium used for the culture of the granulosa cells.…”

Follicular survival, activation of primordial follicles and DNA fragmentation after storage of goat ovaries at 35ºC in supplemented Minimal Essential Medium

“…Finally, insulin acts synergistically with FSH to promote granulosa cell differentiation and proliferation. It also facilitates FSH-dependent steroid production and LH receptor induction in cultured granulosa cells (DULEBA et al, 1997;MAY et al, 1980). This combination of hormones also stimulated follicular development and maintained follicular survival, as well as increased estradiol secretion and promoted oocyte meiosis resumption in goat preantral follicles (CHAVES et al, 2012).…”