Interactions of Insulin-Like Growth Factor-I, Insulin and Estradiol With Gnrh-Stimulated Luteinizing Hormone Release From Female Rat Gonadotrophs
Interactions of Insulin-Like Growth Factor-I, Insulin and Estradiol With Gnrh-Stimulated Luteinizing Hormone Release From Female Rat Gonadotrophs
Interactions of Insulin-Like Growth Factor-I, Insulin and Estradiol With Gnrh-Stimulated Luteinizing Hormone Release From Female Rat Gonadotrophs
EXPERIMENTAL STUDY
Abstract
Background: It is well established that ovarian steroids modulate gonadotropin secretion from anterior
pituitary cells. It has been speculated that insulin and IGF-I might influence gonadotropin secretion.
Objective: To investigate the effects of IGF-I and estradiol alone, or combinations of IGF-I with insulin
and estradiol on GnRH-stimulated LH release from female rat pituitary cells in serum-supplemented
and serum-free culture conditions.
Methods: Pituitary cells were incubated for 24 h or 48 h with a series of increasing concentrations of
IGF-I or estradiol and stimulated with 1 nmol/l GnRH for 3 h. To determine the interaction of IGF-I
and estradiol on GnRH-stimulated LH secretion, cells were exposed to increasing concentrations of
IGF-I and 100 pmol/l estradiol for 24 h. We also investigated the effects of combined treatment with
IGF-I and insulin on GnRH-stimulated LH secretion.
Results: Our findings indicate that long-term IGF-I treatment (24 h) alone has a significant
augmenting effect on GnRH-stimulated LH release in serum-free medium only, with a maximum at
low concentrations (10 and 100 pmol/l). Estradiol significantly increased GnRH-induced LH release
in a dose-dependent manner. The extent of GnRH-stimulated LH secretion by long-term estradiol
treatment (24 h) was significantly greater in serum-supplemented (+42%) medium than in serum-
free medium. Estradiol facilitated IGF-I-primed LH responses to GnRH in serum-free medium. In
contrast, in serum-supplemented medium, the facilitating potential of estradiol was lower. We also
found that, in GnRH-stimulated cells, LH release was augmented by insulin treatment, in contrast to
quiescent cells that had been pretreated with 100 pmol/l IGF-I alone and 1 nmol/l insulin alone.
Conclusions: IGF-I and to a lesser extent insulin stimulate GnRH-induced LH secretion from pituitary
gonadotrophs. This action is enhanced by estradiol treatment of the cells. However, the well known
stimulatory action of estradiol on LH secretion is dependent on the presence of growth factors.
q 2001 Society of the European Journal of Endocrinology Online version via http://www.eje.org
media of the rat in vitro. Ethanol suppresses hepatic penicillin and 10% horse serum pretreated with 2%
IGF-I mRNA levels in vivo, with a subsequent and charcoal (Norit A) and 0.2% Dextran T 70 (Pharmacia,
concomitant decrease in the circulating concentrations Uppsala, Sweden) (incubation medium). To allow cell
of IGF-I and LH (17, 18), indicating a possible linkage attachment, cells were kept for 36 h in humidified
between IGF-I and LH secretion. The findings of recent incubators at 37 8C in an atmosphere of 5% CO2 and
studies suggest a correlation between serum concen- 95% air.
trations of IGF-I and estradiol: estradiol treatment leads
to an increase in serum IGF-I concentration in mature
cows (19), gilts (20) and ewes (21), whereas it Treatment of cultured pituitary cells
suppresses serum IGF-I concentration in the rat (22).
Transdermal estradiol patches in postmenopausal After cell attachment, IGF-I, insulin (both dissolved in
women increase serum IGF-I concentrations; oral acidified PBS) and estradiol (dissolved in ethanol; all
administration, however, results in reduced serum Sigma, St Louis, MO, USA), were added to the culture
IGF-I concentrations (23), and serum IGF-I concentra- medium at appropriate concentrations for 24±48 h.
tions were significantly increased in the preovulatory The final concentration of ethanol in the culture
phase in normal women (24). Wilson (25) reported medium was 0.2%. Controls were exposed to phenol-
that, in female monkeys, the sensitivity to estradiol red-free medium 199 (Biochrom, Berlin, Germany),
feedback inhibition of serum LH was regulated by which was used in all cultures, containing the same
serum concentrations of IGF-I. Clapper et al. (21) quantity of ethanol, PBS, or both, without hormones
demonstrated that reduced concentrations of circulat- (vehicle). For certain experiments, cells were incubated
ing LH were accompanied by increased concentrations in serum-free media after the attachment period for up
of IGF-binding proteins (IGFBPs) in the pituitary of to 48 h. Their viability was not influenced as deter-
estradiol-treated ovariectomized ewes. There is accu- mined by the trypan blue exclusion method.
mulating evidence that estradiol indirectly regulates Before the cells were stimulated with GnRH, they
the synthesis and secretion of LH through alteration of were washed with serum-free medium (0.3% BSA
IGF-I action. These findings suggest that IGF-I might be instead of 10% horse serum) and incubated for 20 min.
a candidate target of estromedin (26, 27), and Then the medium was renewed and GnRH was directly
furthermore, it might contribute to the modulation of added to culture media. After incubation for 3 h, the
LH secretion by the anterior pituitary. medium was collected and stored at 220 8C. All
A limited amount of data indicate that IGF-I is able to experiments were performed in triplicate and repeated
increase LH release from male rat pituitary cells (4, 28). three or four times.
In the present study, we investigated the actions of IGF-I
and insulin on LH secretion from female rat pituitary
cells. In addition, we characterized the interactions of Effects of IGF-I on LH secretion from pituitary
IGF-I, insulin and estradiol on GnRH-stimulated LH cells cultured in serum-supplemented or
release.
serum-free medium
In the first series of experiments, we intended to
Materials and methods determine the effects of serum on IGF-I actions in
gonadotrophs. For this purpose, pituitary cells were
Pituitary cell preparation and culture
incubated for 24 h and 48 h with a series of increasing
conditions concentrations of IGF-I (10 and 100 pmol/l, 1 and
Pituitary glands were obtained from adult (200±300 g) 10 nmol/l) in serum-supplemented or serum-free
female Sprague±Dawley rats (Charles River, Kirch- medium and stimulated with 1 nmol/l GnRH during
borchen, Germany) at random stages of the estrous the last 3 h of incubation.
cycle. Single-cell suspensions were prepared by con-
trolled trypsinization as described previously (29).
Briefly, anterior pituitaries were minced into small Effects of estradiol on LH secretion from
pieces. After digestion with 0.5% trypsin, they were pituitary cells cultured in serum-
treated with DNAse and 0.1% trypsin inhibitor. After
gentle pipetting, dispersed cells were collected by
supplemented or serum-free medium
centrifugation. Cell viability was determined using To determine the influence of serum on the effects of
trypan blue exclusion method (usually more than estradiol on LH secretion, pituitary cell cultures were
95%). incubated with serum-supplemented or serum-free
Pituitary cells were cultured on multiwell dishes media and treated with increasing concentrations of
(200 000 cells/well) in medium 199 with Hanks' estradiol (1, 10 and 100 pmol/l, 1, 10 and 100 nmol/l)
salts and L-glutamine, supplemented with 1.4 g/l for 24 h, and finally stimulated with 1 nmol/l GnRH
sodium bicarbonate, 10 mg/ml streptomycin, 100 U/ml during the last 3 h of the incubation periods.
www.eje.org
www.eje.org
Additive effects of IGF-I and estradiol on LH Additive effects of IGF-I and insulin on LH
secretion from gonadotrophs in different secretion from gonadotrophs in serum-free
serum conditions medium
In serum-free medium, the combination of IGF-I and The combination of insulin and IGF-I led to a greater
estradiol led to a significantly greater basal and GnRH- GnRH-stimulated secretion of LH compared with that
stimulated LH secretion compared with either estradiol produced with IGF-I or insulin treatment alone. Although
or IGF-I treatment alone P , 0:05: We observed this the exposure of pituitary cells to 100 pmol/l IGF-I for
for all four different concentrations of IGF-I (10 pmol/l 48 h and 1 nmol/l insulin for 24 h in serum-free
to 10 nmol/l) combined with 100 pmol/l estradiol after medium led to a significant increase in GnRH-stimulated
www.eje.org
www.eje.org
and estradiol treatment suppressed serum IGF-I con- Our findings demonstrate that there are additive
centrations. Ovesen et al. (24) reported that serum IGF- effects of IGF-I and insulin on GnRH-stimulated LH
I concentrations were significantly increased in the release at the pituitary level. A recent study has also
preovulatory phase in normal women, which is in shown that treatment with either IGF-I or insulin alone
contrast to the findings of other recent studies that fails to increase the percentage of proliferation of
demonstrated no change in serum IGF-I concentrations pituitary cells, but combined treatment with IGF-I
throughout the menstrual cycle (6, 34). The mechan- plus insulin shows a significant increase (35). This
isms underlying this apparent divergence in IGF-I result suggests that low concentrations of insulin
responses after exogenous or endogenous increase in enhance the response of pituitary cells to IGF-I,
estradiol concentrations remain unclear. whereas at high concentrations, insulin binds to IGF-I
Our findings indicate that there are interactions receptors to trigger cell proliferation. Therefore, the
between IGF-I and estradiol at the pituitary level. The IGF-I receptor-mediated system and the insulin recep-
mechanism of such synergism on GnRH-stimulated LH tor-mediated system may work together in the regula-
secretion is still unclear, but several possibilities have tion of pituitary growth processes and promotion of the
been considered. Michels et al. (7) reported that expression of differentiated functions of the pituitary.
estradiol had prominent effects on components of the Thus the effects of insulin on the pituitary cells might
IGF system in the anterior pituitary, and that IGF-I be mediated through IGF-I receptors (35). Mason et al.
mRNA, IGF-I binding and IGFBPs in the rat pituitary (37) reported that insulin and subsequent IGF-I
gland were increased by estradiol treatment. The preincubation enhanced both basal and FSH-induced
greatest levels of pituitary IGF-I binding and IGFBP-1 release of estradiol from granulosa cells. The possible
expression could be demonstrated at proestrus in the mechanism of this synergistic effect may be via
rat ± a time when serum concentrations of estradiol are increased expression of IGF-I receptor at the cell surface
greatest. The findings of the present study, together by insulin, similar to the findings with IGF-II (37).
with these previous observations, suggest that estradiol In conclusion, the present study clearly demonstrates
alters the pituitary responsiveness to IGF-I. This additive actions between IGF-I and estradiol to enhance
supports the possibility that the amount of IGF-I GnRH-stimulated LH secretion from the female rat
receptor varies throughout the estrous cycle and anterior pituitary cells. Furthermore, insulin enhanced
pituitary IGF-I receptors are also regulated by estradiol the actions of IGF-I on secretory responses of gonado-
(20). Changes in pituitary IGF-I receptor numbers trophs. These interactions may be responsible for the
could partially explain the estradiol modulation of IGF- physiological regulation of gonadotropin secretion and
I-induced LH secretion described in this study, suggest- certain pathophysiological conditions that are asso-
ing a possible role for pituitary IGF-I as a mediator of ciated with hypersecretion of LH.
the effects of estradiol on the pituitary.
Recently a study by Sugino et al. (27) indicated that
this synergistic effect involves, at least in part, IGF-I-
stimulated estradiol receptor mRNA expression. There- References
fore, IGF-I may also sensitize pituitary cells to estradiol 1 Ortmann O, Emons G, Knuppen R & Catt KJ. Inhibitory actions of
keoxifene on luteinizing hormone secretion in pituitary gonado-
action by upregulating estradiol receptor expression. trophs. Endocrinology 1988 123 962±968.
This might explain in part the observation that IGF-I 2 Emons G, Hoffmann HG, Brack C, Ortmann O, Sturm R, Ball P.. et
and estradiol together facilitate the modulation of LH al. Modulation of gonadotropin-releasing hormone receptor
secretion. The results also indicate that there is a concentration in cultured female rat pituitary cells by estradiol
feedback mechanism between IGF-I and estradiol. The treatment. Journal of Steroid Biochemistry and Molecular Biology
1988 31 751±756.
steroid downregulates the concentration of the IGF-I 3 Stojilkovic SS & Catt KJ. Expression and signal transduction
receptor and its ligand (27). Therefore, synergistic pathways of gonadotrophin-releasing hormone receptors. Recent
actions of IGF-I and estradiol on GnRH-stimulated LH Progress in Hormone Research 1995 50 161±205.
secretion might occur predominantly at low concen- 4 Soldani R, Cagnacci A & Yen SS. Insulin, insulin-like growth
factor I (IGF-I) and IGF-II enhance basal and gonadotrophin-
trations of IGF-I, as observed in the present study. releasing hormone-stimulated luteinizing hormone release from
There are close interactions between IGF-I and rat anterior pituitary cells in vitro. European Journal of Endocrinol-
insulin (13). IGF-I shares 43% amino acid sequence ogy 1994 131 641±645.
homology with insulin, and biological activities of IGF-I 5 Parmer TG, Roberts CT Jr, LeRoith D, Adashi EY, Khan I,
are similar to those of insulin (35). Furthermore, Solan N.. et al. Expression, action, and steroidal regulation of
insulin-like growth factor-I (IGF-I) and IGF-I receptor in the rat
insulin can bind to IGF-I receptors, although with low corpus luteum: their differential role in the two cell populations
affinity, and IGF-I at high concentrations can also bind forming the corpus luteum. Endocrinology 1991 129 2924±
to insulin receptors (6). Moreover, IGF-I (4, 20, 28, 33) 2932.
and insulin (4, 31) are able to enhance basal and 6 Wang HS & Chard T. IGFs and IGF-binding proteins in the
regulation of human ovarian and endometrial function. Journal
GnRH-stimulated LH release in serum-free medium. of Endocrinology 1999 161 1±13.
Both, IGF-I receptors and insulin receptors are localized 7 Michels KM, Lee WH, Seltzer A, Saavedra JM & Bondy CA. Up-
in rat pituitary glands (8, 36). regulation of pituitary [125I]insulin-like growth factor (IGF-I)
www.eje.org
binding and IGF binding protein-2 and IGF-I gene expression by growth hormone (GH) secretion, insulin-like growth factor-I and
estrogen. Endocrinology 1993 132 23±29. GH-binding protein in post-menopausal women. Journal of
8 Bach MA & Bondy CA. Anatomy of the pituitary insulin-like Clinical Endocrinology and Metabolism 1991 72 374±381.
growth factor system. Endocrinology 1992 131 2588±2594. 24 Ovesen P, Vahl N, Fisker S, Veldhuis JD, Christiansen JS &
9 Cara JF, Fan J, Azzarello J & Rosenfield RL. Insulin-like growth Jorgensen JO. Increased pulsatile, but not basal, growth hormone
factor I enhances luteinizing hormone binding to rat ovarian secretion rates and plasma insulin-like growth factor I levels
theca-interstitial cells. Journal of Clinical Investigation 1990 86 during the periovulatory interval in normal women. Journal of
560±565. Clinical Endocrinology and Metabolism 1998 83 1662±1667.
10 Simone DA & Mahesh VB. An autoregulatory process for 25 Wilson ME. IGF-I administration advances the decrease in
androgen production in rat thecal-interstitial cells. Biology of hypersensitivity to estradiol negative feedback inhibition of
Reproduction 1993 48 46±56. serum LH in adolescent female rhesus monkeys. Journal of
11 Magoffin DA & Weitsman SR. Effect of insulin-like growth factor I Endocrinology 1995 145 121±130.
on cholesterol side-chain cleavage cytochrome P450 messenger 26 Adesanya OO, Zhou J & Bondy CA. Sex steroid regulation of
ribonucleic acid expression in ovarian theca-interstitial cells insulin-like growth factor system gene expression and prolifera-
stimulated to differentiate in vitro. Molecular and Cellular tion in primate myometrium. Journal of Clinical Endocrinology and
Endocrinology 1993 96 45±51. Metabolism 1996 81 1967±1974.
12 Magoffin DA & Weitsman SR. Insulin-like growth factor-I 27 Sugino N, Telleria CM, Tessier C & Gibori G. Regulation and role
regulation of luteinizing hormone (LH) receptor messenger of the insulin-like growth factor I system in rat luteal cells.
ribonucleic acid expression and LH-stimulated signal transduc- Journal of Reproduction and Fertility 1999 115 349±355.
tion in rat ovarian theca-interstitial cells. Biology of Reproduction 28 Soldani R, Cagnacci A, Paoletti AM, Yen SS & Melis GB.
1994 51 766±775. Modulation of anterior pituitary luteinizing hormone response to
13 Campbell BK, Baird DT & Webb R. Effects of dose of LH on gonadotropin-releasing hormone by insulin-like growth factor I
androgen production and luteinization of ovine theca cells in vitro. Fertility and Sterility 1995 64 634±637.
cultured in a serum-free system. Journal of Reproduction and 29 Ortmann O, Sturm R, Knuppen R & Emons G. Weak estrogenic
Fertility 1998 112 69±77. activity of phenol red in the pituitary gonadotroph: re-evaluation
14 Angervo M, Koistinen R, Suikkari AM & SeppaÈlaÈ M. Insulin-like of estrogen and antiestrogen effects. Journal of Steroid Biochem-
growth factor binding protein-I inhibits the DNA amplification istry and Molecular Biology 1990 35 17±22.
induced by insulin-like growth factor I in human granulosa- 30 Zapf A, Hsu D & Olefsky JM. Comparison of the intracellular
luteal cells. Human Reproduction 1991 6 770±773. itineraries of insulin-like growth factor-I and insulin and their
15 Kanzaki M, Hattori M & Kojima I. Growth of differentiation: receptors in rat-I fibroblasts. Endocrinology 1994 134 2445±
determination by FSH of the action of insulin-like growth factor-I 2452.
in cultured rat granulosa cells. Endocrine Journal 1996 43 15± 31 Adashi EY, Hsueh AJ & Yen SS. Insulin enhancement of
23. luteinizing hormone and follicle-stimulating hormone release
16 LaVoie HA, Garmey JC & Veldhuis JD. Mechanisms of insulin-like by cultured pituitary cells. Endocrinology 1981 108 1441±1449.
growth factor I augmentation of follicle-stimulating hormone- 32 Kanematsu T, Irahara M, Miyake T, Shitsukawa K & Aono T.
induced porcine steroidogenic acute regulatory protein gene Effect of insulin-like growth factor I on gonadotropin release from
promotor activity in granulosa cells. Endocrinology 1999 140 the hypothalamus-pituitary axis in vitro. Acta Endocrinologica
146±153. 1991 125 227±233.
17 Hiney JK, Srivastava V, Nyberg CL, Ojeda SR & Dees WL. Insulin- 33 Weil C, Carre F, Blaise O, Breton B & Le Bail PY. Differential effect
like growth factor I of peripheral origin acts centrally to of insulin-like growth factor I on in vitro gonadotropin (I and II)
accelerate the initiation of female puberty. Endocrinology 1996 and growth hormone secretions in rainbow trout (Oncorhynchus
137 3717±3728. mykiss) at different stages of the reproductive cycle. Endocrinology
18 Hiney JK, Srivastava V, Lara T & Dees WL. Ethanol blocks the 1999 140 2054±2062.
central action of IGF-I to induce luteinizing hormone secretion in 34 Thierry van Dessel HJ, Chandrasekher Y, Yap OW, Lee PD,
the prepubertal female rat. Life Sciences 1998 62 301±308. Hintz RL, Faessen Gh.. et al. Serum and follicular fluid levels of
19 Richards MW, Wettemann RP, Spicer LJ & Morgan GL. Nutri- insulin-like growth factor I (IGF-I), IGF-II, and IGF-binding
tional anestrus in beef cows: effects of body condition and protein-1 and -3 during the normal menstrual cycle. Journal of
ovariectomy on serum luteinizing hormone and insulin-like Clinical Endocrinology and Metabolism 1996 81 1224±1231.
growth factor-I. Biology of Reproduction 1991 44 961±966. 35 Oomizu S, Takeuchi S & Takahashi S. Stimulatory effect of
20 Whitley NC, Barb CR, Utley RV, Popwell JM, Kraeling RR & insulin-like growth factor I on proliferation of mouse pituitary
Rampacek GB. Influence of stage of the estrous cycle on insulin- cells in serum-free culture. Journal of Endocrinology 1998 157
like growth factor-I modulation of luteinizing hormone secretion 53±62.
in the gilt. Biology of Reproduction 1995 53 1359±1364. 36 Unger JW & Lange W. Insulin receptors in the pituitary gland:
21 Clapper JA, Snyder JL, Roberts AJ, Hanernik DL & Moss GE. morphological evidence for influence on opioid peptide-synthe-
Estradiol increases relative amounts of insulin-like growth factor sizing cells. Cell Tissue Research 1997 288 471±483.
binding protein (IGFBP)-3 in serum and expression of IGFBP-2 in 37 Mason HD, Willis DS, Holly JM & Franks S. Insulin preincubation
anterior pituitaries of ewes. Biology of Reproduction 1998 59 enhances insulin-like growth factor-II (IGF-II) action on ster-
124±130. oidogenesis in human granulosa cells. Journal of Clinical
22 Krattenmacher R, Knauthe R, Parczyk K, Walker A, Hilgenfeldt U Endocrinology and Metabolism 1994 78 1265±1267.
& Fritzemeier KH. Estrogen action on hepatic synthesis of
angiotensinogen and IGF-I: direct and indirect estrogen effects.
Journal of Steroid Biochemistry and Molecular Biology 1994 48
207±214.
23 Weissberger AJ, Ho KK & Lazarus L. Contrasting effects of oral Received 24 February 2000
and transdermal routes of estrogen replacement therapy on 24-h Accepted 7 September 2000
www.eje.org