Interleukin 10 abolishes the growth inhibitory effects of all-trans retinoic acid on human myeloma cells

Recently, it was disclosed that all-trans retinoic acid (ATRA) inhibits myeloma cell growth by downregulating the interleukin 6 (IL-6)/IL-6 receptor (IL-6R) auto/paracrine loop, and upregulating p21/Cip1 cyclin-dependent kinase inhibitor (CDK-I), thereby inducing apoptosis with a decrease in Bcl-2 protein expression. To elucidate and generalize the effects of ATRA on the proliferation and cellular biology of myeloma cells, 12 human myeloma cell lines established in our laboratory were utilized. Two out of the 12 lines showed enhanced growth on supplementation of ATRA and were characterized by IL-10 production, downregulation of membrane Fas and reduced upregulation of p21/Cip1 CDK-I message. These characteristics may prove important for the clinical use of ATRA and should be considered before starting ATRA therapy for myeloma.     

The stimulatory and inhibitory effects of dopamine on prolactin secretion involve different G-proteins.

The reverse hemolytic plaque assay (RHPA) was used in this study to further characterize the mechanism whereby low concentrations of dopamine (DA) stimulate PRL secretion in vitro. Female Sprague-Dawley rats were used as a source of anterior pituitary cells for the RHPA. Pituitary cells were infused into Cunningham chambers along with a suspension of protein-A-coated ovine red blood cells. Excess cells were rinsed from the chambers leaving a monolayer of cells attached to the glass. The cells were then incubated with solutions containing PRL antiserum (1:40) and various concentrations of DA. After 4 h, a solution containing guinea pig complement (1:60) was infused into the chambers. Thirty minutes later, the cells were fixed and plaques (zones of hemolysis) surrounding PRL-producing cells (lactotrophs) were measured and used as an index of the amount of PRL secreted. Control cells that received no DA had a mean plaque area of 8,000 microns 2 and two distinct subpopulations of plaque sizes. This biphasic population of cells consisted of a small and a large plaque producing population. The mean plaque area surrounding lactotrophs was significantly (P less than 0.05) decreased if 1 microM or 10 microM DA was present (4,500 microns 2 and 3,500 microns 2, respectively). These cells which received inhibitory concentrations of DA demonstrated a monophasic distribution of plaque-forming cells. On the other hand, mean plaque area was significantly (P less than 0.05) increased if 0.1 nM or 1 nM DA was presented to the cells (15,000 microns 2 and 14,500 microns 2, respectively). These cells receiving stimulatory doses of DA exhibited a multiphasic distribution of plaque-forming cells. The possibility that the two physiological opposing actions of DA on PRL secretion might be mediated by different GTP binding proteins was also examined using cholera toxin (CTX) and pertussis toxin (PTX). Anterior pituitary cells were pretreated with either CTX (50 micrograms/ml) or PTX (5 micrograms/ml) for 1 h before initiation of the RHPA. In the RHPA, cells received no DA, a stimulatory dose of DA (0.1 nM), or a inhibitory dose of DA (10 microM). The effects of toxin pretreatment on mean plaque area of DA-treated cells was determined. PTX pretreatment significantly attenuated the inhibitory effects of DA while having no effect on the stimulatory effects of DA on PRL secretion. CTX significantly (P less than 0.05) potentiated the stimulatory effects of DA on PRL secretion and had no effect on inhibition.(ABSTRACT TRUNCATED AT 400 WORDS)     

Magnesium reverses inhibitory effects of calcium deprivation on coordinate response of 3T3 cells to serum

Deprivation of Ca(2+) in crowded cultures of 3T3 cells inhibits the onset of DNA synthesis. By raising [Mg(2+)] to 15 mM the inhibition produced by Ca(2+) deprivation can be fully overcome. Sparse cultures are not inhibited by a similar deprivation of Ca(2+), and therefore are not stimulated by supranormal [Mg(2+)]. The time course of stimulation of the onset of DNA synthesis by supranormal [Mg(2+)] in low [Ca(2+)] is the same as that produced by serum in physiological concentrations of Ca(2+) and Mg(2+). Concentrations of Mg(2+) > 20 mM in low [Ca(2+)] reverse the stimulation, and [Mg(2+)] >/= 30 mM kills many cells. In contrast to the stimulation by 15 mM Mg(2+), supranormal [Ca(2+)] has no effect on the onset of DNA synthesis in cultures inhibited by Mg(2+) deprivation, if the formation of insoluble Ca-P(i) complexes is prevented. Neither Na(+) nor K(+) reproduces the effects of Mg(2+). The uptake of uridine is another parameter of the coordinate response of 3T3 cells to serum stimulation that is inhibited by Ca(2+) deprivation, and supranormal [Mg(2+)] also reverses this inhibition. The results support the thesis that the coordinate response of growth and metabolism to external effectors is regulated by the availability of Mg(2+) within the cell and that the inhibitory effects of Ca(2+) deprivation are indirect and caused by a reduction in the availability of Mg(2+).     

Connecting liver and gut: murine liver sinusoidal endothelium induces gut tropism of CD4+ T cells via retinoic acid

Gut-activated T cells migrating into the liver can cause extraintestinal manifestations of inflammatory bowel disease. T cells acquire a gut-homing phenotype dependent on retinoic acid (RA) provided by intestinal dendritic cells (DC). We investigated whether liver antigen-presenting cells can induce gut tropism supporting an enterohepatic lymphocyte circulation. Priming of CD4(+) T cells by liver sinusoidal endothelial cells (LSEC) supported migration into gut and gut-associated lymphoid tissue. As observed for T cells primed by intestinal DCs, this gut tropism depended on α(4) β(7) integrin and CC chemokine receptor 9 (CCR9) expression by LSEC-primed CD4(+) T cells. The induction of gut-homing molecules was mediated by RA, a derivate of vitamin A that is stored in large amounts within the liver. LSECs expressed functional retinal dehydrogenases and could convert vitamin A to RA. Conversely, the lack of signaling via the RA receptor prevented the expression of α(4) β(7) integrin and CCR9 on LSEC-primed CD4(+) T cells, consequently reducing their in vivo migration to the intestine. Other liver antigen-presenting cells failed to support high expression of α(4) β(7) integrin on CD4(+) T cells, thus, the potential to induce gut homing is restricted to LSECs. CONCLUSION: The capacity to promote gut tropism via vitamin A use is not unique for intestinal DCs but is also a feature of LSECs. Our data support the assumption that CD4(+) T cells can migrate from the liver to the gut as one branch of a postulated enterohepatic lymphocyte circulation.     

The biphasic stimulatory and inhibitory effects of concanavalin A on thyroid activation induced by thyrotropin.

Concanavalin A (Con A) was tested for its ability to affect thyroid activation induced by TSH in mouse thyroid lobes. Pretreatment of thyroid lobes with Con A at concentrations from 1.55--400 microgram/ml was found to have biphasic stimulatory and inhibitory effects of the TSH-induced accumulation of cAMP and formation of colloid droplets. Low concentrations of Con A potentiated TSH activation of thyroidal formation of cAMP and endocytosis. In contrast, higher concentrations of Con A markedly inhibited these TSH effects. The inhibitory effects observed after preincubation with Con A were abolished by the addition of alpha-methyl-D-glucoside to the incubation medium. A high concentration of Con A also inhibited cAMP formation induced either by prostaglandin E2 or the long-acting thyroid stimulator. However, the basal and TSH-stimulated glucose oxidation in mouse thyroid lobes was not depressed by a high concentration of Con A. Uptake of 125 I-labeled Con A by thyroid tissues increased with time up to 1 h and was directly proportional to tissue weight. These findings suggest that the specific interaction between Con A and its receptors may lead to conformational changes in the structure of the membranes of the thyroid follicular cells which facilitate TSH-induced thyroid hormone secretion via the adenylate cyclase-cAMP system.     

GnRH agonist-induced inhibitory and stimulatory effects during ovarian follicular maturation

The in vivo regulation of ovarian gonadotropin and prolactin receptors and adenylate cyclase activity by FSH, and the potent GnRH agonist [D-Ala⁶]des-Gly¹⁰-GnRH N-ethylamide (GnRHa), was studied in immature hypophysectomized diethylstilbestrol-implanted rats. During FSH treatment over a 48 h period, FSH receptors increased 2-fold with the maximum response during the first 12 h, whereas LH and prolactin receptors increased by 10-fold and 6-fold with the maximum response from 12 to 48 h. Administration of GnRHa at any time during the 48 h period of FSH treatment inhibited the subsequent development of gonadotropin and PRL receptors. In contrast, administration of a single dose of 10 μg GnRHa after 48 h of FSH treatment stimulated follicular luteinization and caused increases in basal adenylate cyclase activity, ovarian weight and PRL receptor content, and concomitant decreases in gonadotropin receptors and adenylate cyclase responses. In the immature follicles of animals not primed with FSH, GnRHa caused progressive inhibition of FSH-sensitive adenylate cyclase activity, with a decrease in FSH receptors, but increased both basal and GMP-P(NH)P-stimulated adenylate cyclase activities. These results demonstrate that GnRHa causes marked inhibition of gonadotropin receptor expression in the basal and FSH-stimulated ovary. This decrease in gonadotropin receptors is an important component of the mechanism by which GnRH agonists inhibit ovarian gonadotropin-sensitive adenylate cyclase activity. In addition, these peptides exert stimulatory effects upon ovarian weight and basal adenylate cyclase activity, and cause an increase in PRL receptors and luteinization of mature ovarian follicles. The stimulatory actions of GnRH agonists upon ovarian function are more prominent with increasing maturation of the granulosa cell.     

Growth hormone inhibitory and stimulatory actions of L-dopa in patients with acromegaly

It is not clear whether dopamine (DA) has a central stimulating activity on GH secretion in patients with acromegaly, as it does in normal subjects. To clarify this, we compared the GH inhibitory potencies of DA, which does not cross the blood-brain barrier (BBB), and L-dopa or bromocriptine, which do cross the BBB, in 23 patients with acromegaly. Further, we examined the central effects of L-dopa after selectively blocking peripheral (median eminence and pituitary) DA receptors with domperidone (a DA D2 receptor blocker which does not cross the BBB). After the administration of DA (5 micrograms/kg X min, iv, for 90 min), L-dopa (500 mg, orally), or bromocriptine (2.5 mg, orally), the mean plasma GH decrease was greatest after DA [maximum decrement, 71.9 +/- 3.8% (+/- SEM); n = 21] compared to L-dopa (44.1 +/- 5.6%; n = 23; p less than 0.001) or bromocriptine (58.9 +/- 5.0%; n = 20; p less than 0.02). Eleven of these patients received a single infusion of domperidone (0.22 mg/min, iv, for 180 min) or a combination of domperidone and L-dopa. Mean plasma GH levels did not change during domperidone alone. However, plasma GH levels in these patients increased significantly when L-dopa was administered 30 min after the start of domperidone infusion (vs. control study: at 90 min, 137.3 +/- 10.8% vs. 100.2 +/- 3.9%, p less than 0.01; at 120 min, 138.8 +/- 19.7% vs. 106.5 +/- 3.1%, p less than 0.05). In contrast, one patient who had a distinct plasma GH increase in response to the domperidone-L-dopa test had no increase in plasma GH when given L-dopa 30 min after the start of a sulpiride infusion (DA D2 receptor blocker which crosses the BBB; 1.1 mg/min, iv, for 180 min). Unlike GH, plasma PRL responses to domperidone infusion were not modified by the additional administration of L-dopa. These results suggest that in acromegaly, DA has not only direct suppressive effects on the pituitary tumor somatotrophs, but also indirect stimulatory effects via the hypothalamus; therefore, the hypothalamic GH-releasing system is not entirely suppressed by excessive tumor GH secretion.     

Effects of retinoic acid on NIH3T3 cell transformation by the H-ras oncogene

Summary Exposure of NIH3T3 cells to retinoic acid resulted in a dose-dependent modulation of transformed focus formation after transfection with an activated H-ras oncogene. Inhibition induced by 10 M retinoic acid was maximal at 21.4% of control values. Maximal inhibition of transformation was found after exposure to 10 M retinoic acid between days 0 and 3 of the transfection period. This concentration was also inhibitory for colony formation upon transfection of the non-transforming geneaph, suggesting that retinoic acid acts primarily on the process of transfection to inhibit focus or colony formation. Exposure to retinoic acid during the late period of the transfection protocol (days 14–20) resulted in alterations in focus morphology. A transformed cell line containing H-ras underwent reversion of the transformed phenotype after 4 weeks of treatment with retinoic acid, as determined by alterations in cell morphology and anchorage-independent growth. Phenotypic reversion was not associated with changes in the expression of the exogenous H-ras or endogenous c-myc or c-fos oncogenes.Abbreviations DMEM Dulbecco's modified Eagle medium - SSC saline sodium citrate - TGF transforming growth factor This investigation was supported by grants CA 52925, CA 13343 and ES 00260     

Growth hormone and fibronectin expression in 3T3 preadipose cells.

In the present study we focused on the relationship between GH action and the extracellular matrix in 3T3-F442A preadipose cells. Results from Northern blotting indicated that in serum-free medium, the presence of 2 nM met-human GH down-regulated levels of fibronectin messenger RNA by approximately 40, 60, and 70% as compared with control levels on days 1, 2, and 4, respectively. GH-dependent reduction of levels of collagen alpha 1(I) mRNA expression occurred later and was less pronounced than effects on levels of fibronectin mRNA, suggesting a specificity in the matrix-altering function of GH. Western blot analyses and immunoprecipitation studies revealed that between 2 and 5 days of culture, matrix-associated fibronectin protein was reduced 70 to 90% by GH treatment. Down-regulation of fibronectin protein expression by met-human GH was dose-dependent between 2 and 0.02 nM. The presence of 2 nM insulin or insulin-like growth factor-1 promoted a 30-40% increase in fibronectin levels compared to control cells. The GH-promoted down-regulation of fibronectin expression was eliminated by concomitant addition of insulin. These data demonstrated that GH effects on matrix-associated fibronectin expression were independent of, and in opposition to, effects promoted by insulin and insulin-like growth factor-1. Treatment of culture dishes with fibronectin or collagen inhibited GH-stimulated adipogenesis 50 and 80%, respectively, compared with controls, as judged by levels of glycerol-3-phosphate dehydrogenase activity. Thus, composition of the extracellular matrix was a critical factor in GH-induced adipogenesis of 3T3-F442A fibroblasts. Our results demonstrate that GH action in 3T3 preadipose cells is intimately coupled to the biology of extracellular matrix.     

Inhibitory effects of serum and stimulatory effects of estrogen on prolactin mRNA levels in GH3 rat pituitary tumor cells.

We investigated the effects of serum and estrogen on prolactin (PRL) mRNA accumulation in GH3 cells under different cell culture conditions. Hybridization analysis of GH3 cellular RNA indicated that PRL mRNA levels decreased more than 20-fold in cells cultured for 1 week in medium containing dextran-charcoal-treated serum (stripped serum). No effects on actin mRNA levels were observed under these conditions. Furthermore, this inhibition of PRL mRNA accumulation depended on the concentration of stripped serum in the medium. Although incubation in stripped-serum medium inhibited cell growth, these culture conditions did not appear to irreversibly affect the GH3 cell population. These data indicate that a potent inhibitor of PRL mRNA accumulation is present in stripped serum. GH3 cells grown in stripped-serum medium were shown to be responsive to estrogen. Treatment of these cells with 10(-9) M estradiol resulted in a 6.6-fold stimulation of PRL gene expression. However, estrogen had no effect on cell growth under these conditions, suggesting that estrogen stimulates PRL gene expression and cell proliferation by independent mechanisms.     

Radiofrequency hyperthermia and topical retinoic acid therapy in murine melanoma

Malignant cells are known to be sensitive to increased temperature. The effects of hyperthermia (HT) on intradermally implanted S91 melanoma cells in syngeneic mice were investigated with a hand-held radiofrequency generator. The possible additive effects of topical retinoic acid (RA) in this system also were studied. Five millimeter diameter melanomas were treated with either HT alone, RA alone, or a combination of HT and RA and were then evaluated after 43 days and 59 days. Eighteen of 20 tumors treated with HT alone and all 20 melanomas treated with HT/RA were eradicated. RA alone caused complete regression in 11 of 19 treated tumors. It is concluded that radiofrequency HT is an effective treatment in intradermal murine melanoma and that the addition of RA does not significantly alter the outcome because of the extreme effectiveness of HT alone.     

The natural killer-related receptor for HLA-C expressed on T cells from CD3+ lymphoproliferative disease of granular lymphocytes displays either inhibitory or stimulatory function

Four patients with lymphoproliferative disease of granular lymphocytes (LDGL) coexpressing CD3 and the natural killer (NK)-related "p58" receptor for HLA-C alleles were studied. These CD3+p58+ LDGLs have been detected among a series of 44 CD3+ LDGLs analyzed. Two patients with LDGL (GI and BA) expressed only the p58 molecule defined by the GL-183 and CH-L monoclonal antibodies (MoAbs), while the cases of patients PU and MA also coexpressed the molecular form identified by EB6 anti-p58 MoAb. Three LDGL cases (GI, MA, and PU) displayed the CD8+4-CD16+ T- cell receptor (TCR)alpha/beta+ phenotype, while one patient (BA) was CD8+4+CD16+ TCRalpha/beta+. Freshly isolated granular lymphocytes (GL) from these cases displayed cytolytic activity in an anti-CD3 MoAb- triggered redirected killing assay against the Fcgamma-receptor+ (Fcgamma-R+) P815 target cell line. Lysis of P815 target cells, triggered by an anti-CD3 or by anti-CD16 MoAb, could be inhibited by the addition of anti-p58 MoAb in three fresh or interleukin (IL)-2- cultured GL tested (GI, MA, and PU). Triggering of cytotoxicity against the HLA-DR+ Fcgamma-R+ Daudi cell line induced by appropriate superantigens could also be inhibited by anti-p58 MoAb in patients PU and GI with LDGL. These data indicate that activation through the CD16, CD3, and TCR molecules can be modulated by p58 receptors in these LDGLs. On the contrary, IL-2-expanded cells of patient BA were induced to lyse P815 target cells by anti-p58 MoAb. In addition, anti-p58 MoAB enhanced anti-CD16 MoAb triggered lysis and did not inhibit activation via CD3. These data indicate that, in this particular patient with LDGL, p58 displays a stimulatory effect on cell triggering, rather than the typical inhibitory effect previously observed in p58+ T-cell clones derived from healthy donors. The anti-p58 MoAb did not induce CA++ mobilization in p58+ LDGLs and in a p58+CD3+ normal T-cell clone equipped with inhibitory p58 molecules, while Ca++ mobilization could be observed in cultured GL from patient BA, which could be activated by anti-58 MoAb. These findings suggest that stimulatory and inhibitory p58 molecules are equipped with different signal transducing properties, thus contributing to a better knowledge of the normal counterpart.     

Characterization of inhibitory and stimulatory forms of the murine natural killer cell receptor 2B4

The receptor 2B4 belongs to the Ig superfamily and is found on the surface of all murine natural killer (NK) cells as well as T cells displaying non-MHC-restricted cytotoxicity. Previous studies have suggested that 2B4 is an activating molecule because cross-linking of this receptor results in increased cytotoxicity and gamma-interferon secretion as well as granule exocytosis. However, it was recently shown that the gene for 2B4 encodes two different products that arise by alternative splicing. These gene products differ solely in their cytoplasmic domains. One form has a cytoplasmic tail of 150 amino acids (2B4L) and the other has a tail of 93 amino acids (2B4S). To determine the function of each receptor, cDNAs for 2B4S and 2B4L were transfected into the rat NK cell line RNK-16. Interestingly, the two forms of 2B4 had opposing functions. 2B4S was able to mediate redirected lysis of P815 tumor targets, suggesting that this form represents an activating receptor. However, 2B4L expression led to an inhibition of redirected lysis of P815 targets when the mAb 3.2.3 (specific for rat NKRP1) was used. In addition, 2B4L constitutively inhibits lysis of YAC-1 tumor targets. 2B4L is a tyrosine phosphoprotein, and removal of domains containing these residues abrogates its inhibitory function. Like other inhibitory receptors, 2B4L associates with the tyrosine phosphatase SHP-2. Thus, 2B4L is an inhibitory receptor belonging to the Ig superfamily.     

In vitro regulation of a SIN3-dependent DNA-binding activity by stimulatory and inhibitory factors.

The yeast SIN3 gene (also known as SDII, is a known negative regulator of the yeast HO gene. A DNA-binding activity, called SDP1, which binds to the HO promoter, is absent in extracts prepared from sin3 mutants and has been proposed to function as a repressor. We show that SIN3 does not encode SDP1 and that SDP1 DNA-binding activity is modulated in vitro by two factors, an inhibitory factor, I-SDP1, and a stimulatory factor, S-SDP1. I-SDP1 acts as an in vitro inhibitor of the SDP1 DNA-binding activity. Restoration of the DNA-binding activity is achieved by inclusion of a stimulatory factor, S-SDP1, which copurifies with the SIN3 protein. SDP1 DNA-binding activity was restored by treating a protein fraction containing SDP1 and I-SDP1 with the dissociating agent formamide.     

Growth factors from murine sarcoma virus-transformed cells.

Murine sarcoma virus-transformed mouse fibroblasts produce polypeptide growth factors and release them into serum-free medium. These factors stimulate cells to divide in monolayer cultures and also to form colonies that grow progressively soft agar. Three major peaks of activity are seen, with apparent molecular weights of 25,000, 12,000, and 7000. The sarcoma growth factors are heat-stable, trypsin-sensitive, and active in nanogram quantities when tested for growth stimulation of untransformed rat and mouse fibroblasts. All three molecular species are also capable of competing for membrane epidermal growth factor (EGF) receptors when tested with 125I-labeled EGF. They differ from mouse EGF, however, in their molecular weights, in their inability to react with anti-EGF antibodies, and in their ability to convert cells to anchorage independent (agar) growth. For the above reasons, we conclude that the sarcoma growth factors are a new class of polypeptide tropic factors that confer on fibroblasts in vitro properties associated with the transformed phenotype.     

Reticulocyte cytosol activator protein: effects on the stimulatory and inhibitory regulatory proteins of adenylate cyclase

Rat reticulocytes contain a cytosol activator protein (RCAP) that augments hormone-sensitive adenylate cyclase activity in the rat reticulocyte and other systems. In a previous publication, using a highly purified preparation of RCAP, we reported that the stimulatory guanine nucleotide-binding protein (Ns) was required for the actions of RCAP. We investigated this possibility by studying the actions of RCAP on cholera toxin-dependent ADP ribosylation of Ns. RCAP decreased cholera toxin-dependent ADP ribosylation of the 42,000-dalton subunit of Ns of reticulocyte [40.2 +/- 3.7 (+/-SEM) to 26.5 +/- 3.8 fmol/mg; n = 10; P less than 0.001], S49 wild-type (33.9 +/- 2.4 to 24.9 +/- 2.8 fmol/mg; n = 9; P less than 0.01), and UNC (25.3 +/- 3.5 vs. 17.6 +/- 3.1; n = 5; P less than 0.02) membranes. In contrast, pertussis toxin-dependent ADP-ribosylation of the inhibitory guanine nucleotide binding protein, Ni in reticulocyte, S49 wild-type lymphoma, and its UNC and cyc- variant membranes were all significantly augmented by RCAP. Moreover, when reticulocyte Ni was functionally ablated by exposure to pertussis toxin, RCAP no longer enhanced isoproterenol-responsive adenylate cyclase activity in reticulocyte membranes. These results suggest that RCAP stimulates adenylate cyclase activity by inhibiting Ni function, thus permitting enhanced Ns coupling to the adenylate cyclase enzyme (C).     

Dissociation between the direct stimulatory and inhibitory effects of a gonadotropin-releasing hormone analog on ovarian functions

The paradoxical effects of gonadotropin-releasing hormone (GnRH) on the ovary have hitherto been believed to result from different regimens of administration; an acute treatment was shown to stimulate the ovary while chronic administration of the hormone inhibited LH-induced responses. In the present report we demonstrate that a single injection of a GnRH analog (D-Ala6)des-Gly10-GnRH-N-ethylamide (GnRHa, 2 micrograms/rat) is sufficient to obtain a significant inhibition (75%) of hCG-induced ovulation in PMSG-primed, either intact or hypophysectomized, immature rats. Inhibition of ovarian development, in terms of growth and ovulation, by multiple injections with GnRHa (2 micrograms/rat, twice daily for 3 days) could be obtained only upon administration of the hormone at early stages of follicular development, i.e. concomitantly with the PMSG injection. When administered after PMSG, GnRHa could not inhibit the ovary but rather induced ovulation by itself in the absence of hCG. A 12-24 h delay in initiation of GnRHa treatment triggered 65% of the rats to ovulate while a delay of 48 h resulted in 100% ovulation. Under both regimes of GnRHa administration, either the inhibitory or the stimulatory, the oocytes of the treated rats were induced to resume meiotic maturation. Since under the inhibitory regime ovulation did not occur, maturation was followed by a massive degeneration of the oocytes trapped within their follicles. These findings demonstrate that the follicular stage of development rather than the dose and/or duration of GnRHa administration determines whether GnRHa inhibits ovarian growth and ovulation, while the competence of the oocytes to respond to the GnRHa stimulus and mature is independent of hormonal priming.