Influence of Ovarian Ornithine Decarboxylase in Folliculogenesis and Luteinization
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内分泌学杂志 2005年第2期
Departments of Biochemistry and Molecular Biology (A.J.L.-C., C.L.-G., F.T., R.P.), Pharmacology (C.M.B., A.C.), and Cell Biology (M.T.C.), Faculty of Medicine, University of Murcia, 30100 Murcia, Spain
Address all correspondence and requests for reprints to: Dr. Rafael Pe?afiel, Department of Biochemistry and Molecular Biology, Faculty of Medicine, Campus de Espinardo, 30100 Murcia, Spain. E-mail: rapegar@um.es.
Abstract
LH plays a relevant role in folliculogenesis, ovulation, and luteinization. Although ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis, is a target of LH in the ovary, the functional significance of ODC induction has remained elusive. Our study reveals that the blockade of the induction of ovarian ODC by means of the specific inhibitor -difluoromethylornithine (DFMO) affects folliculogenesis and luteinization. In immature female mice, DFMO was found to inhibit ovarian growth, the formation of Graafian follicles, and the secretion of progesterone and estradiol. In adult cycling females, the administration of DFMO on the evening/night of proestrus markedly decreased plasma progesterone levels at diestrus, which was associated to the decrease in the expression of steroidogenic factor 1, cytochrome cholesterol side chain cleavage enzyme, and steroidogenic acute regulatory protein in the ovary and to a reduced vascularization of the corpora lutea. These effects were not reverted by the administration of gonadotropins or prolactin. ODC immunoreactivity was also stimulated by LH in theca and granulosa cells of antral follicles but not in preantral follicles. Overall, these experiments demonstrate that elevated ODC values found in the ovary of immature and adult mice play a relevant function in ovarian physiology and that ODC/polyamines must be considered as important mediators of some of the effects of LH on follicular development and luteinization.
Introduction
FOLLICLE DEVELOPMENT IS a complex and dynamic process requiring the coordinate interactions of multiple intragonadal and extragonadal factors (1, 2). Pituitary hormones as well as growth factors and steroids derived from the gonads play key functions in regulating specific aspects of folliculogenesis. Current evidence suggests that, although preantral follicles are dependent on local regulators with gonadotropins having a facilitatory role, antral follicles require gonadotropins as essential factors for their development and locally produced hormones and growth factors play a facilitatory role (3, 4, 5, 6, 7, 8, 9). In rodents, ovulation and luteinization are initiated by the pituitary surge of LH that takes place at the evening of proestrus (1, 10, 11). The biochemical mechanisms that participate in the cytological, histological, and functional changes that gonadotropins produce in the ovary are complex and only partially understood. It is widely accepted that gonadotropins either directly or indirectly regulate the expression of many proteins in the ovary, including growth factors, enzymes, and transcription factors that may impact multiple signaling cascades (12, 13, 14).
Polyamines are ubiquitous polycationic substances that play an important role in cell growth, differentiation, and gene expression (15, 16, 17, 18). Early studies demonstrated that ornithine decarboxylase (ODC), a key enzyme in the biosynthetic pathway of polyamines, was induced in the rat ovary in response to the administration of human chorionic gonadotropin (hCG) (19, 20, 21) and that, in cycling adult females, there was a marked increase of ovarian ODC activity that followed the preovulatory surge of LH (19). The functional significance of ODC induction in the ovary has remained elusive. In the last decade, several transgenic mouse models for studying the role of polyamines in normal, hypertrophic, and neoplastic growth have been developed (22). The high frequency of infertile animals observed in these transgenic mice suggested that polyamine regulation may be an important factor for the correct development of reproductive tissues (23, 24, 25). Moreover, we recently found that the blockade of ovarian ODC induction by treating adult cycling female mice with -difluoromethylornithine (DFMO), a potent and specific inhibitor of ODC (26), produced a marked fall in the levels of plasma progesterone at diestrus, suggesting that the preovulatory rise of ovarian ODC may be critical for the process of luteinization (27). On the basis of these considerations, the present study was undertaken to explore further the role of ODC in ovarian physiology. In this work, we have studied the influence of gonadotropins on the induction of ODC by determining enzyme activity and immunolocalization of the ODC protein in the mouse ovary at different stages of follicular development as well as the effect of ODC blockade in folliculogenesis and steroid hormone secretion in both normal cycling adult female mice and immature animals primed with pregnant mare serum gonadotropin (PMSG) and hCG. Our results indicate that ODC is required for both the development of antral follicles and the luteinization of granulosa cells in Graafian follicles, suggesting the implication of this enzyme in the signaling pathway promoted by LH in the ovary.
Materials and Methods
Animals and treatments
Adult and immature Swiss CD1 female mice were used in these experiments. Animals were fed standard chow (UAR A03; Panlab, Barcelona, Spain) and water ad libitum. Animals were maintained at 22 C ambient temperature and 50% relative humidity under a controlled 12-h light, 12-h dark cycle (lights on at 0800 h). Estrous cycles were monitored by daily vaginal smears, and only mice exhibiting at least two consecutive 4-d estrous cycles were included in the study. Blood samples were collected under light ether anesthesia by cardiac puncture at 0900–1000 h of proestrus, estrus, and diestrus (one puncture per animal). Plasma was obtained by centrifugation at 4 C and was kept frozen at –70 C until analysis. Animals were killed by cervical dislocation under ether anesthesia, and the ovaries were quickly removed, weighed, and processed. Procedures involving animals and their care were conducted in conformity with the institutional guidelines that are in compliance with national (RD 223/1988) and international laws and policies (European Economic Community Council Directive 86/009; National Institutes of Health Guide for the Care and Use of Laboratory Animals, 7th ed.).
For collecting ovulated eggs, oviducts were removed and placed in the well of a dish containing phosphate buffer (pH 7.4). To collect oocyte-cumulus complexes, the ampullary region of the oviduct was identified and torn open with fine steel tweezers under a dissecting microscope. The oocyte-cumulus complexes were isolated, and the cumulus cells were removed by digestion for 5 min with sheep testicular hyaluronidase (1 mg/ml). The oocytes were identified and counted.
The ODC inhibitor DFMO (Illex Products Inc., San Antonio, TX) was given to adult mice under different schedules. For chronic studies, DFMO was injected (500 mg/kg, sc) at the day of proestrus, estrus, or diestrus I during the afternoon/evening (see Results), and mice were killed at diestrus II (0900–1000 h). In acute experiments, DFMO (500 mg/kg, sc) was given 30 min before hormone administration. In immature animals, a group of 24-d-old female mice was treated with PMSG (10 IU, ip). Fifty-two hours after PMSG, the mice were injected with hCG (5 IU, im) and killed 48 h later. Another group received the same treatment, but DFMO (500 mg/kg, sc) was given 30 min before PMSG injection and was also in the drinking water (2% solution) during the whole period. Control animals were injected with vehicle. To study the influence of gonadotropins on ovarian ODC induction, adult female mice were injected with 5 IU of LH, FSH, or hCG and killed 5 h after the injection. To study the influence of pituitary hormones on the effect of DFMO on progesterone secretion, mice were treated with LH (6 IU, im), FSH (6 IU, im), or prolactin (20 IU, sc) at 2000 h of proestrus. PMSG, LH, and prolactin were obtained from Sigma Chemical Co. (St. Louis, MO); hCG and FSH were supplied by Serono (Madrid, Spain).
Enzymatic measurements
For enzyme determination, ovaries were homogenized with the aid of a Polytron homogenizer (Kinematica GmbH, Kriens/Luzern, Switzerland) in buffer containing 25 mM Tris (pH 7.2), 2 mM dithiothreitol, 0.1 mM pyridoxal phosphate, 0.1 mM EDTA, and 0.25 M sucrose. The extract was centrifuged at 20,000 x g for 20 min, and ODC activity was determined in the supernatant. ODC activity was determined basically as described by Russell and Snyder (28) by measuring 14CO2 release from L-[1-14C] ornithine. The incubation mixture contained 20 mM Tris (pH 7.2), 0.1 mM pyridoxal phosphate, 0.1 mM EDTA, 2 mM dithiothreitol, and 0.4 mM L-[1-14C] ornithine (NEN Life Science Products, Boston, MA; specific activity, 2.6 Ci/mol) in a total volume of 62.5 μl. The samples were incubated at 37 C for 30 min, and the reaction was stopped by adding 0.5 ml of 2 M citric acid. Activity was expressed as nanomoles of CO2 produced per hour and per gram of wet weight.
Polyamine analysis
For polyamine analysis, the dansylation method of Seiler (29) was used. Dansylated polyamines were separated by HPLC using a Lichrosorb 10-RP-18 column (4.6 x 250 mm; Merck, Darmstadt, Germany) and acetonitrile-water mixtures (running from 70:30 to 96:4 ratio during 25 min of analysis) as mobile phase. 1,6-Hexanediamine was used as internal standard. Detection of the derivatives was achieved using a fluorescence detector, with a 340-nm excitation filter and a 435-nm emission filter.
RT-PCR
Total RNA was extracted from tissues with the RNAqueous-Midi Kit (Ambion Inc., Austin, TX) and purified following the manufacturer’s specifications. Total RNA was reverse transcribed using oligo(dT)18 as primer and MMLV reverse transcriptase (Ambion Inc). Products were amplified by means of Taqpolymerase (Sigma) using specific primer pairs within the linear range for each gene product. Amplified products were resolved by electrophoresis in 2% agarose gel containing 40 mM Tris/acetate and 1 mM EDTA (pH 8.0) in a horizontal slab gel apparatus using 1x Tris/acetate/EDTA buffer. The gel was stained with ethidium bromide (0.2 μg/ml for 15 min), followed by destaining in water, and photographed by UV transillumination using a Gel Doc system camera (Bio-Rad, Hercules, CA); the bands were quantified using the Multi-Analyst PC software for Bio-Rad. Primers (Sigma-Genosys, Suffolk, UK) were as follows: mouse ?-actin (accession no. NM_007393), 606-1142: forward, 5'-tgcgtctggacctggctg; reverse, 5'-ctgctggaaggtggacag; mouse aromatase (accession no. D00658), 1022–1614: forward, 5'-cctgaaggagatccacactg; reverse, 5'-tggtttccatgtaattacgg; mouse 3?-hydroxysteroid dehydrogenase 1 (3? HSD; accession no. M58567), 361-1050: forward, 5'-tgttgtcatccacactgctg; reverse, 5'-ggacgtagcaggaagctcac; mouse cytochrome cholesterol side chain cleavage enzyme (P450scc; accession no. NM_019779), 129-1250: forward, 5'-gtacctctactagcagtcct; reverse, 5'-aagtcttggctggaatcttg; mouse steroidogenic factor 1 (SF-1; accession no. AF511594), 441-1070: forward, 5'-actacatgttaccccctagc; reverse, 5'-tgtgcagcagggagccagcc; and mouse steroidogenic acute regulatory protein (StAR; accession no. GI:1236242), 251–905: forward, 5'-ctcaactggaagcaacactc; reverse, 5'-gggctggcttccaggcgctt.
Analytical methods
Progesterone and estradiol were determined by ELISA using the Enzymun-Test kit supplied by Boehringer Mannheim Immunodiagnostics (Mannheim, Germany). Concentrations of progesterone and estradiol in plasma were measured in duplicate. Ovarian progesterone was determined after homogenization of ovaries using a Polytron (Brinkmann Instruments, Westbury, NY) in ice-cold ethanol (1:20 wt/vol). The extract was centrifuged at 10,000 x g for 20 min, the supernatant was diluted in 50% ethanol containing 0.9% NaCl, and progesterone was measured in duplicate. The intraassay variation and sensitivity were approximately 10% and 5 pg/tube, respectively.
Histology and cytochemistry
Ovaries from the different experimental groups were removed and fixed in 10% formalin in PBS (0.01 M PBS; pH 7.4) for 10 h. After washing in PBS, samples were processed routinely and cut in 5-μm paraffin sections. Sections were stained with hematoxylin and eosin, lectin histochemistry, and immunocytochemistry.
For lectin histochemistry, tissue sections from paraffin blocks were deparaffinized in xylene and hydrated in a graded ethanol series. Endogenous peroxidase activity was destroyed by treatment with 0.3% hydrogen peroxide in PBS for 30 min. Sections were washed in three 5-min changes of PBS and then incubated in a moist chamber for 2 h at room temperature in horseradish peroxidase-conjugated lectin from Dolichus biflorus (12 μg/ml), a specific marker for endothelial cells. After washing in PBS, the peroxidase activity was visualized with PBS containing 0.05% 3,3-diaminobenzidine tetrahydrochloride and 0.015% hydrogen peroxide. Sections were counterstained with hematoxylin, dehydrated, cleared, and mounted in DPX medium (BDH Chemicals, Poole, UK).
For immunocytochemistry, a two-step method was applied. After blocking endogenous peroxidase activity in the same way that was described for lectin histochemistry, sections were incubated with normal goat serum (1:30) for 1 h and then with rabbit polyclonal antibody to ODC (Euro-Diagnostica, Malm?, Sweden; 1:100) overnight at 4 C. After washing in PBS, sections were incubated with peroxidase-conjugated goat antirabbit Ig (Chemicon International Inc., Temecula, CA; 1:100) for 1 h and developed with 3,3-diaminobenzidine tetrahydrochloride and 0.015% hydrogen peroxide. After immunostaining, sections were counterstained with hematoxylin. In the control, anti-ODC antibody was substituted by PBS. Similar results were obtained using a rabbit polyclonal antibody to ODC obtained from Affiniti (Exeter, UK).
Image analysis
Density of blood vessels in corpora lutea was performed with a computer-assisted image analysis system, using MIP-4.5 software (Consulting Image Digital, Barcelona, Spain). A square field (134-μm side) was superimposed upon the captured image to be used as reference area. The area of blood vessels included in this square was used for estimating the percentage of blood vessels. Thirty random fields were evaluated in each experimental group, and the mean and SEM were calculated.
Statistics
Results are given as the mean ± SD. The significance of differences observed was assessed by ANOVA, followed by the post hoc Newman-Keuls multiple range test. P < 0.05 was considered statistically significant.
Results
Effects of the inhibition of ODC activity on plasma steroid levels in adult female mice
Figure 1 shows the values of ODC activity in the ovary of immature mice and adult females at different stages of the estrous cycle. In adult cycling females, there was a marked increase in ovarian ODC activity in the evening of proestrus that was associated with the rise in putrescine and spermidine concentrations (from 85 ± 11 to 158 ± 39 nmol/g for putrescine and 716 ± 104 to 929 ± 42 nmol/g for spermidine; P < 0.05). When the rise in ODC activity at the evening of proestrus was blocked by administration of DFMO, a specific inhibitor of ODC (26), no significant changes either in the estrous cycle or in the number of ova found in the oviducts the next morning were observed (data not shown). The influence of the inhibition of ODC by DFMO, given in the evening of proestrus, on plasma concentration of progesterone and estradiol at the different days of the estrous cycle is shown in Fig. 2. In agreement with results in rats reported by others, there was a transient increase in plasma progesterone concentration in the evening of proestrus and a sustained rise of progesterone values during diestrus in control animals. Our results showed that the treatment with DFMO did not affect plasma progesterone values at proestrus or estrus but produced a marked decrease of this hormone during diestrus (25% of control values). This effect of the ODC inhibitor on plasma progesterone at diestrus was not observed when DFMO was given in the evening of estrus or metaestrus (data not shown). DFMO treatment also produced a moderate fall in plasma estradiol at diestrus. Because the analysis of progesterone concentration in the ovaries at diestrus in both control and DFMO-treated animals showed a clear decrease of 27% (data not shown), which was considerably smaller than the fall observed in plasma, we tested the possible influence of DFMO treatment in ovarian angiogenesis by using a histochemical analysis of blood vessel density by means of lectins that react and stain vascular endothelial cells. This analysis revealed a significant decrease (35%) of the density of blood vessels in the corpora lutea from ovaries of DFMO-treated mice relative to controls (Fig. 3).
FIG. 1. Changes in ovarian ODC activity as a function of age. Immature and peripubertal mice were killed in the morning of diestrus when possible. Adult animals were killed in the morning of estrus (E), diestrus (DE), and proestrus (PE) and between 1800 and 2300 h of proestrus (PE*). Ovaries were collected at the time indicated, and homogenized and ODC activity was measured by a radiometric method. Results are the mean ± SD of four to eight animals per point.
FIG. 2. Changes in progesterone and estradiol levels during the estrous cycle produced by treatment of adult female mice with DFMO at proestrus. DFMO (500 mg/kg, sc) was injected into adult female at 1200, 1600, and 2000 h of proestrus, and progesterone was measured the next estrus, diestrus, and proestrus (0900–1000 h). Blood was collected by cardiac puncture, and steroid hormones were determined by ELISA. Results are the mean ± SD of six to eight animals per group. a, P < 0.001 vs. control group.
FIG. 3. Blood vessel staining of corpus luteum in adult female mice at diestrus. Lectin from Dolichus biflorus was used to specifically label blood vessels in corpus luteum. A, Control mice. B, Mice treated with DFMO at proestrus (500 mg/kg at 1200, 1600, and 2000 h, sc) and killed on the diestrus morning. Note the decrease in the density of vessels in DFMO-treated samples. The percentage of stained blood vessels were 8.0 ± 0.5% in controls vs. 5.2 ± 0.3% in DFMO-treated mice (P < 0.01).
Regulation of ovarian ODC by gonadotropins and their influence on the effect of DFMO on plasma steroid levels in adult female mice
To explore the effects of gonadotropins on ovarian ODC activity, female mice were treated with LH, FSH, or both, and enzyme activity was determined 5 h after hormone administration. Figure 4 shows that FSH produced a small increase in ODC activity, whereas LH and hCG increased the activity of the decarboxylase by about 10-fold. The simultaneous administration of LH and FSH produced additive effects. To determine whether the effect produced by DFMO treatment on plasma progesterone levels could be directly related to the blockade of ovarian ODC activity in the evening of proestrus or be the consequence of the alteration of the periovulatory surge of gonadotropins or prolactin, female mice treated with DFMO on the evening of proestrus received a simultaneous administration of pituitary hormones, and plasma progesterone was determined the next diestrus. Table 1 shows that neither LH plus FSH nor prolactin administration were able to prevent the decrease in plasma progesterone produced by DFMO during diestrus. Figure 5 also shows the effect of DFMO on the acute changes produced by hCG administration in the morning of proestrus on both ovarian ODC activity and plasma steroid concentrations, measured 5 h after hormone administration. Although DFMO completely abolished the remarkable rise of ovarian ODC activity, this compound did not affect the changes in plasma progesterone induced by the gonadotropin. Interestingly, hCG produced a marked decrease in plasma estradiol levels that was totally prevented by DFMO administration.
FIG. 4. Influence of gonadotropins on ovarian ODC activity. LH, FSH, and hCG (6 IU each, im) were given to adult female mice in the morning of diestrus, and animals were killed 5 h later. Ovaries were collected and homogenized, and ODC activity was measured by a radiometric method. Results are the mean ± SD of five to six animals per group. a, P < 0.001 vs. control; b, P < 0.05 vs. control; c, P < 0.05 vs. LH.
TABLE 1. Effect of DFMO on diestrus plasma progesterone levels and influence of gonadotropins or prolactin administration
FIG. 5. Influence of DFMO on ovarian ODC activity and plasma steroid levels after acute hCG treatment. Adult female mice were treated in the morning of proestrus with 10 IU of hCG, and animals were killed 5 h after hormone administration. DFMO (500 mg/kg, sc) was given 30 min before hCG administration. Control animals were injected with vehicle. Blood was obtained by cardiac puncture, and steroid hormones were determined by ELISA. Ovaries were collected at the time indicated and homogenized, and ODC activity was measured by a radiometric method. P, Progesterone; E2, estradiol. Results are the mean ± SD of five to six animals per group. a, P < 0.001 vs. control and hCG+DFMO; b, P< 0.05 vs. control; c, P< 0.05 vs. control and hCG+DFMO.
Influence of ODC in ovarian development
Figure 1 shows the variation of ovarian ODC activity along the postnatal development. The activity, which was low before weaning, increased during the peripubertal period and fell again in the mature animals except in the evening of proestrus. To study the influence of ovarian ODC activity in ovarian development, two groups of 24-d-old female mice were primed with PMSG and hCG, and the influence of DFMO, which was given to one group, was determined by comparison with control mice and with mice receiving the hormone treatment alone. Ovarian ODC activity raised from 7.2 ± 1.4 nmol/h/g in controls to 134.5 ± 5.2 nmol/h/g in the hormone-primed mice. In the DFMO-treated group, ODC activity was lower than 1 nmol/h/g. Figure 6A shows three representative sections of ovaries from control and treated mice. DFMO slightly diminished (28%) the increase of the ovarian size elicited by the hormone treatment but almost abolished the formation of antral follicles. The analysis of progesterone and estradiol in the plasma of mice under the different experimental conditions revealed that DFMO inhibited the marked increase in ovarian progesterone promoted by the hormones and also diminished the modest increase in ovarian estradiol (Fig. 6B).
FIG. 6. A, Influence of DFMO in the growth of the ovary of immature mice. The 24-d-old female mice were injected with 10 IU of PMSG to stimulate follicular growth, followed 52 h later with 5 IU of hCG to stimulate ovulation and luteinization; the mice were killed 48 h after hCG administration. a, Control animals. Mainly preantral small follicles were observed; note the small size of the ovary. b, Hormone-stimulated animals. An increase of antral follicles (Af) was shown. c, Hormone-stimulated animals that received an injection of DFMO (500 mg/kg, sc) 30 min before PMSG administration and DFMO (2% in the drinking water) until killing. A regression of the maturation process was detected. Scale bar, 500 μm. B, Influence of DFMO on plasma steroid hormones of immature mice. Plasma progesterone and estradiol were determined by ELISA. Results are the mean ± SD of four to six animals per group. a, P < 0.001 vs. other groups.
Immunocytochemical analysis of ODC in the mouse ovary
Cellular localization of ODC protein in the ovary was studied by immunocytochemical techniques using antibodies directed against mouse ODC. Figure 7A shows a negative control of the technique obtained when the ovarian section was incubated with the second antibody alone. When the two antibodies were used, a light and diffuse immunoreactivity was found in the granulosa cells of antral follicles and in the luteal cells of control ovaries (Fig. 7, B and C). The treatment with hCG produced a marked increase in ODC immunoreactivity in the oocyte and in the cytosol of theca and granulosa cells of antral and ovulatory follicles (Fig. 7, D and F). Granulosa cells from preantral follicles failed to express ODC in response to hCG (Fig. 7G). In the oocyte, immunoreactivity was localized in the cytoplasm and was absent in the nucleus and in the zona pellucida (Fig. 7, D and F).
FIG. 7. Localization of ODC in the ovary of adult mice. A, Negative control of the technique. Section was incubated only with secondary antibody; no reaction was observed. B, Control ovary. A slight labeling of ODC was evident in the cytoplasm of oocyte and granulosa cells of antral follicles. C, Corpus luteum in control ovary. Luteal cells were slightly immunoreactive. D–F, Antral follicles of hCG-treated animals. hCG (10 IU) was given in the morning, and ovaries were obtained 5 h after hCG administration. An increased immunoreactivity was observed in the theca and granulosa cells and in the cytoplasm of oocyte of antral follicles. No reaction was detected in zona pellucida and nucleus of oocyte. G, Preantral follicle of hCG-treated mice at proestrus. ODC reaction in primary follicles was only restricted to cytoplasm of oocyte. No labeling was observed in granulosa cells. O, Oocyte; G, granulosa; T, theca; arrowhead, zona pellucida. Scale bar, 22 μm.
Effect of DFMO on the levels of mRNAs of steroidogenic proteins in the ovary
The levels of mRNAs of SF-1, StAR, P450scc, 3? HSD, aromatase, and ?-actin were analyzed by means of semiquantitative RT-PCR in the ovaries of adult female mice primed with PMSG and hCG that had received either saline or DFMO. Figure 8 shows that, in hormone-treated mice, DFMO produced a dramatic decrease in the levels of SF-1 and cytochrome P450scc mRNA and a moderate diminution in the levels of StAR and 3? HSD. The comparison of mRNAs at the diestrus stage between control and DFMO-treated mice at proestrus also showed a significant decrease of cytochrome P450scc, SF-1, and StAR mRNA (Fig. 9). No differences in the expression of 20-hydroxysteroid dehydrogenase, the enzyme that participates in the catabolism of progesterone in the luteal cell, between control and DFMO-treated animals were observed (results not shown).
FIG. 8. Influence of DFMO on mRNA expression of steroidogenic proteins in the ovaries of adult mice treated with gonadotropins. One group of adult female mice was injected with 10 IU of PMSG and, 52 h later, with 5 IU of hCG. Mice were killed 48 h after hCG injection, the ovaries were collected, total RNA was extracted, and mRNA expression was analyzed by semiquantitative RT-PCR. The other group received the same hormonal treatment but received an injection of DFMO (500 mg/kg, sc) 30 min before PMSG administration and DFMO (2% in the drinking water) until killing. Each group was formed by four animals. A, A representative band is presented for each amplified gene product. B, Bands were quantified and normalized with respect to the intensity of ?-actin band, and the results are given as mean ± SD. a, P < 0.01. Arom, Aromatase.
FIG. 9. Influence of the blockade of the preovulatory peak of ovarian ODC on mRNA expression of steroidogenic proteins at diestrus. Adult female mice were treated with DFMO (500 mg/kg, three injections in the evening of proestrus) or saline, and ovaries were obtained after killing the animals at the next diestrus. Total RNA was extracted, and mRNA expression was analyzed by semiquantitative RT-PCR. Each group was formed by four animals. A, A representative band is presented for each amplified gene product. B, Bands were quantified and normalized to the intensity of ?-actin, and the results are given as mean ± SD. a, P < 0.01. b, P< 0.05. Arom, Aromatase.
Discussion
The biochemical events that control the processes of folliculogenesis and ovulation in mammals are only partially understood. Follicular growth is controlled by intraovarian regulatory factors that act by autocrine, paracrine, and intracrine mechanisms and by endocrine factors such as the pituitary gonadotropins FSH and LH (1, 11, 30). Current evidence suggests that, although the preantral stages of committed follicles are dependent on the local regulators, with gonadotropins having a facilitatory role, these hormones are essential for the development of antral (tertiary and Graafian) follicles. It is well known that, in steroidogenic cells, gonadotropins stimulate the synthesis of steroid hormones through the intermediary cAMP, which up-regulates the expression of P450scc, the enzyme that catalyzes the first reaction of the steroidogenic cascade, and of StAR protein, which participates in the translocation of cholesterol from the outer to the inner mitochondrial membrane of steroidogenic cells (30, 31, 32). Apart from this role, gonadotropins stimulate the maturation and function of the gonads and the regulation of gametogenesis (33, 34). It is believed that, whereas FSH is required for the growth of preovulatory follicles and for the expression of LH receptors in granulosa cells, the preovulatory surge of LH acts on these cells to terminate the follicular program of gene expression, stimulating, at the same time, the induction of genes required for ovulation and luteinization (11, 35).
In spite of the important role of gonadotropins in the physiology of the ovary, the molecular mechanisms by which these hormones exert their actions are only partially known. Early reports demonstrated that, in the rat, ovarian ODC is under the control of LH, showing this enzyme to have a transitory rise only in the evening of proestrus (19). Further studies with granulosa cells isolated from porcine ovarian follicles suggested that cAMP may mediate gonadotropin stimulation of ODC (36). The rise of ovarian ODC induced by hCG was associated with the increase in the amount of ODC mRNA (37). Despite that the strategic positioning of the enzyme induction in the ovary suggested a possible association with the ovulatory process, experiments using DFMO did not support an essential role for ODC in the final stages of follicular maturation and ovulation, at least in the rat (38).
Our results show that increased ovarian ODC activity at different stages of follicular development plays an important role in the ovarian physiology of mice. In adult cycling females, the preovulatory rise of ODC that follows the LH surge at the evening of proestrus appears to be related to the acquisition of progesterone secretory capacity by the corpus luteum, which is necessary for the successful implantation and development of the ovum. The effect of DFMO seems to be the result of the blockade of the augmentation of ODC activity observed in the ovary rather than the consequence of the diminution of gonadotropin secretion that could occur by the inhibition of the rise of ODC in the anterior pituitary gland, which, in the rat, has been related to the secretion of LH and prolactin at proestrus (39, 40, 41). Our results also indicate that the effects produced by DFMO on progesterone secretion are only observed when this compound is administered in the evening of proestrus, when the increase in ovarian ODC activity takes place. The fact that DFMO treatment did not affect the acute effect that hCG produced on progesterone secretion but decreased its secretion by the corpus luteum suggests that the rise in ovarian ODC in the evening of proestrus may be related with the process of luteinization. In turn, it is known that the preovulatory surge of LH is required for ovulation and luteinization (11). Moreover, the fact that ODC blockade is more critical for progesterone secretion than for ovulation, together with the high responsiveness of the mouse ovary inducing ODC after LH or hCG treatment, indicate that the rise of ODC in certain types of ovarian cells may be critical for the differentiation process that leads to the formation of luteal cells.
The immunocytochemical analysis of ODC in the mouse ovary revealed that there was a specific increase of ODC protein in response to hCG, mainly in the theca and granulosa cells of the antral follicles, whereas granulosa cells in preantral follicles were not responsive to gonadotropins. This result is in agreement with a previous report that showed that the increase of ODC activity in the rat ovary treated with LH was higher in the Graafian follicles than in the whole ovary (42). Regarding the cellular localization of ODC in the rat ovary, although some studies showed that the enzyme was confined to thecal and interstitial cells (43, 44), others found that the enzyme was mainly present in the cytoplasm of granulosa cells (45). Our results indicate that, in the mouse ovary, the presence of ODC in the granulosa cells is related to follicle development. Its presence in granulosa and theca cells of preovulatory follicles would support the hypothesis that ODC induction in the evening of proestrus may be required for the luteinization of the theca and granulosa cells in the ovulating follicle. Although ODC is also expressed in the oocytes of the different types of follicles and is up-regulated by LH, its physiological relevance remains to be determined. In this regard, recent experiments have revealed the expression of gonadotropin receptors in murine oocytes, suggesting a potential role for oocyte maturation (46, 47).
The molecular and cellular events that lead to the formation of a functional corpus luteum are only partially understood. Although it is clear that the development of luteal steroidogenesis is critical for progesterone secretion by the corpus luteum (48, 49, 50, 51), it is also evident that luteal development is associated with a dramatic increase in the number of blood vessels (52, 53, 54). Our results about the influence of DFMO on progesterone values in plasma and ovary could be explained not only by the inhibition of progesterone biosynthesis in luteal cells, which would decrease the hormone values in both compartments, but also by a limited vascularization of the corpus luteum, which would generate a more pronounced decrease of the hormone in the vasculature. The down-regulation produced by DFMO on cytochrome P450scc enzyme and StAR protein, which participate in key steps of the progesterone biosynthetic pathway, would explain the decrease in progesterone levels found in plasma and ovary of the animals treated with DFMO. Additionally, the dramatic decrease observed in the expression of SF-1 in mice treated with the polyamine inhibitor suggests that this change could be responsible for the effects observed in the levels of cytochrome P450scc and StAR mRNA because this transcription factor plays a central role in the expression of StAR and other genes encoding cytochrome P450 steroid hydroxylases (55). Our results suggest that the increase of polyamine levels that takes place during the evening of proestrus in the ovary of adult mice is critical for the development of a fully functional corpus luteum. In turn, increased polyamine biosynthesis is required in many processes of cell growth and differentiation (15, 16, 17, 18), and the corpus luteum is a transient endocrine gland that develops from the follicular cells that remain after ovulation. Although polyamines participate in the regulation of many proteins and genes, the molecular mechanisms by which they could mediate in the expression of steroidogenic proteins remain to be clarified. The possibility that the recently discovered polyamine-responsive element and the associated trans-acting proteins (56, 57) may have a role in the transcription of steroidogenic genes should be considered. On the other hand, the diminished vascularization of the corpus luteum observed in animals treated with DFMO is in agreement with the contention that ODC and polyamines may have an important role in angiogenesis (58, 59).
Our study also shows that ovarian ODC has a role not only in the functioning of the adult ovary but also in ovarian development because the inhibition of the induction of ovarian ODC found in the immature animals treated with gonadotropins was associated with the delay in ovarian growth and follicular progression. This result also corroborates the view that the effects produced by DFMO in ovarian physiology during this period are mainly due to the inhibition of ovarian ODC rather than to the decrease in the release of gonadotropins by the pituitary. Our results and those obtained with transgenic mice overexpressing ODC or spermidine/spermine acetyl transferase (24), a key enzyme in the polyamine retroconversion pathway, support the view that polyamines play a relevant role in the mammalian reproductive system.
In conclusion, the present experiments demonstrate that the elevated ODC values found in the ovary of immature and adult mice play a relevant function in ovarian physiology and that ODC/polyamines must be considered as important mediators of some of the effects of LH on follicular development and luteinization.
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Address all correspondence and requests for reprints to: Dr. Rafael Pe?afiel, Department of Biochemistry and Molecular Biology, Faculty of Medicine, Campus de Espinardo, 30100 Murcia, Spain. E-mail: rapegar@um.es.
Abstract
LH plays a relevant role in folliculogenesis, ovulation, and luteinization. Although ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis, is a target of LH in the ovary, the functional significance of ODC induction has remained elusive. Our study reveals that the blockade of the induction of ovarian ODC by means of the specific inhibitor -difluoromethylornithine (DFMO) affects folliculogenesis and luteinization. In immature female mice, DFMO was found to inhibit ovarian growth, the formation of Graafian follicles, and the secretion of progesterone and estradiol. In adult cycling females, the administration of DFMO on the evening/night of proestrus markedly decreased plasma progesterone levels at diestrus, which was associated to the decrease in the expression of steroidogenic factor 1, cytochrome cholesterol side chain cleavage enzyme, and steroidogenic acute regulatory protein in the ovary and to a reduced vascularization of the corpora lutea. These effects were not reverted by the administration of gonadotropins or prolactin. ODC immunoreactivity was also stimulated by LH in theca and granulosa cells of antral follicles but not in preantral follicles. Overall, these experiments demonstrate that elevated ODC values found in the ovary of immature and adult mice play a relevant function in ovarian physiology and that ODC/polyamines must be considered as important mediators of some of the effects of LH on follicular development and luteinization.
Introduction
FOLLICLE DEVELOPMENT IS a complex and dynamic process requiring the coordinate interactions of multiple intragonadal and extragonadal factors (1, 2). Pituitary hormones as well as growth factors and steroids derived from the gonads play key functions in regulating specific aspects of folliculogenesis. Current evidence suggests that, although preantral follicles are dependent on local regulators with gonadotropins having a facilitatory role, antral follicles require gonadotropins as essential factors for their development and locally produced hormones and growth factors play a facilitatory role (3, 4, 5, 6, 7, 8, 9). In rodents, ovulation and luteinization are initiated by the pituitary surge of LH that takes place at the evening of proestrus (1, 10, 11). The biochemical mechanisms that participate in the cytological, histological, and functional changes that gonadotropins produce in the ovary are complex and only partially understood. It is widely accepted that gonadotropins either directly or indirectly regulate the expression of many proteins in the ovary, including growth factors, enzymes, and transcription factors that may impact multiple signaling cascades (12, 13, 14).
Polyamines are ubiquitous polycationic substances that play an important role in cell growth, differentiation, and gene expression (15, 16, 17, 18). Early studies demonstrated that ornithine decarboxylase (ODC), a key enzyme in the biosynthetic pathway of polyamines, was induced in the rat ovary in response to the administration of human chorionic gonadotropin (hCG) (19, 20, 21) and that, in cycling adult females, there was a marked increase of ovarian ODC activity that followed the preovulatory surge of LH (19). The functional significance of ODC induction in the ovary has remained elusive. In the last decade, several transgenic mouse models for studying the role of polyamines in normal, hypertrophic, and neoplastic growth have been developed (22). The high frequency of infertile animals observed in these transgenic mice suggested that polyamine regulation may be an important factor for the correct development of reproductive tissues (23, 24, 25). Moreover, we recently found that the blockade of ovarian ODC induction by treating adult cycling female mice with -difluoromethylornithine (DFMO), a potent and specific inhibitor of ODC (26), produced a marked fall in the levels of plasma progesterone at diestrus, suggesting that the preovulatory rise of ovarian ODC may be critical for the process of luteinization (27). On the basis of these considerations, the present study was undertaken to explore further the role of ODC in ovarian physiology. In this work, we have studied the influence of gonadotropins on the induction of ODC by determining enzyme activity and immunolocalization of the ODC protein in the mouse ovary at different stages of follicular development as well as the effect of ODC blockade in folliculogenesis and steroid hormone secretion in both normal cycling adult female mice and immature animals primed with pregnant mare serum gonadotropin (PMSG) and hCG. Our results indicate that ODC is required for both the development of antral follicles and the luteinization of granulosa cells in Graafian follicles, suggesting the implication of this enzyme in the signaling pathway promoted by LH in the ovary.
Materials and Methods
Animals and treatments
Adult and immature Swiss CD1 female mice were used in these experiments. Animals were fed standard chow (UAR A03; Panlab, Barcelona, Spain) and water ad libitum. Animals were maintained at 22 C ambient temperature and 50% relative humidity under a controlled 12-h light, 12-h dark cycle (lights on at 0800 h). Estrous cycles were monitored by daily vaginal smears, and only mice exhibiting at least two consecutive 4-d estrous cycles were included in the study. Blood samples were collected under light ether anesthesia by cardiac puncture at 0900–1000 h of proestrus, estrus, and diestrus (one puncture per animal). Plasma was obtained by centrifugation at 4 C and was kept frozen at –70 C until analysis. Animals were killed by cervical dislocation under ether anesthesia, and the ovaries were quickly removed, weighed, and processed. Procedures involving animals and their care were conducted in conformity with the institutional guidelines that are in compliance with national (RD 223/1988) and international laws and policies (European Economic Community Council Directive 86/009; National Institutes of Health Guide for the Care and Use of Laboratory Animals, 7th ed.).
For collecting ovulated eggs, oviducts were removed and placed in the well of a dish containing phosphate buffer (pH 7.4). To collect oocyte-cumulus complexes, the ampullary region of the oviduct was identified and torn open with fine steel tweezers under a dissecting microscope. The oocyte-cumulus complexes were isolated, and the cumulus cells were removed by digestion for 5 min with sheep testicular hyaluronidase (1 mg/ml). The oocytes were identified and counted.
The ODC inhibitor DFMO (Illex Products Inc., San Antonio, TX) was given to adult mice under different schedules. For chronic studies, DFMO was injected (500 mg/kg, sc) at the day of proestrus, estrus, or diestrus I during the afternoon/evening (see Results), and mice were killed at diestrus II (0900–1000 h). In acute experiments, DFMO (500 mg/kg, sc) was given 30 min before hormone administration. In immature animals, a group of 24-d-old female mice was treated with PMSG (10 IU, ip). Fifty-two hours after PMSG, the mice were injected with hCG (5 IU, im) and killed 48 h later. Another group received the same treatment, but DFMO (500 mg/kg, sc) was given 30 min before PMSG injection and was also in the drinking water (2% solution) during the whole period. Control animals were injected with vehicle. To study the influence of gonadotropins on ovarian ODC induction, adult female mice were injected with 5 IU of LH, FSH, or hCG and killed 5 h after the injection. To study the influence of pituitary hormones on the effect of DFMO on progesterone secretion, mice were treated with LH (6 IU, im), FSH (6 IU, im), or prolactin (20 IU, sc) at 2000 h of proestrus. PMSG, LH, and prolactin were obtained from Sigma Chemical Co. (St. Louis, MO); hCG and FSH were supplied by Serono (Madrid, Spain).
Enzymatic measurements
For enzyme determination, ovaries were homogenized with the aid of a Polytron homogenizer (Kinematica GmbH, Kriens/Luzern, Switzerland) in buffer containing 25 mM Tris (pH 7.2), 2 mM dithiothreitol, 0.1 mM pyridoxal phosphate, 0.1 mM EDTA, and 0.25 M sucrose. The extract was centrifuged at 20,000 x g for 20 min, and ODC activity was determined in the supernatant. ODC activity was determined basically as described by Russell and Snyder (28) by measuring 14CO2 release from L-[1-14C] ornithine. The incubation mixture contained 20 mM Tris (pH 7.2), 0.1 mM pyridoxal phosphate, 0.1 mM EDTA, 2 mM dithiothreitol, and 0.4 mM L-[1-14C] ornithine (NEN Life Science Products, Boston, MA; specific activity, 2.6 Ci/mol) in a total volume of 62.5 μl. The samples were incubated at 37 C for 30 min, and the reaction was stopped by adding 0.5 ml of 2 M citric acid. Activity was expressed as nanomoles of CO2 produced per hour and per gram of wet weight.
Polyamine analysis
For polyamine analysis, the dansylation method of Seiler (29) was used. Dansylated polyamines were separated by HPLC using a Lichrosorb 10-RP-18 column (4.6 x 250 mm; Merck, Darmstadt, Germany) and acetonitrile-water mixtures (running from 70:30 to 96:4 ratio during 25 min of analysis) as mobile phase. 1,6-Hexanediamine was used as internal standard. Detection of the derivatives was achieved using a fluorescence detector, with a 340-nm excitation filter and a 435-nm emission filter.
RT-PCR
Total RNA was extracted from tissues with the RNAqueous-Midi Kit (Ambion Inc., Austin, TX) and purified following the manufacturer’s specifications. Total RNA was reverse transcribed using oligo(dT)18 as primer and MMLV reverse transcriptase (Ambion Inc). Products were amplified by means of Taqpolymerase (Sigma) using specific primer pairs within the linear range for each gene product. Amplified products were resolved by electrophoresis in 2% agarose gel containing 40 mM Tris/acetate and 1 mM EDTA (pH 8.0) in a horizontal slab gel apparatus using 1x Tris/acetate/EDTA buffer. The gel was stained with ethidium bromide (0.2 μg/ml for 15 min), followed by destaining in water, and photographed by UV transillumination using a Gel Doc system camera (Bio-Rad, Hercules, CA); the bands were quantified using the Multi-Analyst PC software for Bio-Rad. Primers (Sigma-Genosys, Suffolk, UK) were as follows: mouse ?-actin (accession no. NM_007393), 606-1142: forward, 5'-tgcgtctggacctggctg; reverse, 5'-ctgctggaaggtggacag; mouse aromatase (accession no. D00658), 1022–1614: forward, 5'-cctgaaggagatccacactg; reverse, 5'-tggtttccatgtaattacgg; mouse 3?-hydroxysteroid dehydrogenase 1 (3? HSD; accession no. M58567), 361-1050: forward, 5'-tgttgtcatccacactgctg; reverse, 5'-ggacgtagcaggaagctcac; mouse cytochrome cholesterol side chain cleavage enzyme (P450scc; accession no. NM_019779), 129-1250: forward, 5'-gtacctctactagcagtcct; reverse, 5'-aagtcttggctggaatcttg; mouse steroidogenic factor 1 (SF-1; accession no. AF511594), 441-1070: forward, 5'-actacatgttaccccctagc; reverse, 5'-tgtgcagcagggagccagcc; and mouse steroidogenic acute regulatory protein (StAR; accession no. GI:1236242), 251–905: forward, 5'-ctcaactggaagcaacactc; reverse, 5'-gggctggcttccaggcgctt.
Analytical methods
Progesterone and estradiol were determined by ELISA using the Enzymun-Test kit supplied by Boehringer Mannheim Immunodiagnostics (Mannheim, Germany). Concentrations of progesterone and estradiol in plasma were measured in duplicate. Ovarian progesterone was determined after homogenization of ovaries using a Polytron (Brinkmann Instruments, Westbury, NY) in ice-cold ethanol (1:20 wt/vol). The extract was centrifuged at 10,000 x g for 20 min, the supernatant was diluted in 50% ethanol containing 0.9% NaCl, and progesterone was measured in duplicate. The intraassay variation and sensitivity were approximately 10% and 5 pg/tube, respectively.
Histology and cytochemistry
Ovaries from the different experimental groups were removed and fixed in 10% formalin in PBS (0.01 M PBS; pH 7.4) for 10 h. After washing in PBS, samples were processed routinely and cut in 5-μm paraffin sections. Sections were stained with hematoxylin and eosin, lectin histochemistry, and immunocytochemistry.
For lectin histochemistry, tissue sections from paraffin blocks were deparaffinized in xylene and hydrated in a graded ethanol series. Endogenous peroxidase activity was destroyed by treatment with 0.3% hydrogen peroxide in PBS for 30 min. Sections were washed in three 5-min changes of PBS and then incubated in a moist chamber for 2 h at room temperature in horseradish peroxidase-conjugated lectin from Dolichus biflorus (12 μg/ml), a specific marker for endothelial cells. After washing in PBS, the peroxidase activity was visualized with PBS containing 0.05% 3,3-diaminobenzidine tetrahydrochloride and 0.015% hydrogen peroxide. Sections were counterstained with hematoxylin, dehydrated, cleared, and mounted in DPX medium (BDH Chemicals, Poole, UK).
For immunocytochemistry, a two-step method was applied. After blocking endogenous peroxidase activity in the same way that was described for lectin histochemistry, sections were incubated with normal goat serum (1:30) for 1 h and then with rabbit polyclonal antibody to ODC (Euro-Diagnostica, Malm?, Sweden; 1:100) overnight at 4 C. After washing in PBS, sections were incubated with peroxidase-conjugated goat antirabbit Ig (Chemicon International Inc., Temecula, CA; 1:100) for 1 h and developed with 3,3-diaminobenzidine tetrahydrochloride and 0.015% hydrogen peroxide. After immunostaining, sections were counterstained with hematoxylin. In the control, anti-ODC antibody was substituted by PBS. Similar results were obtained using a rabbit polyclonal antibody to ODC obtained from Affiniti (Exeter, UK).
Image analysis
Density of blood vessels in corpora lutea was performed with a computer-assisted image analysis system, using MIP-4.5 software (Consulting Image Digital, Barcelona, Spain). A square field (134-μm side) was superimposed upon the captured image to be used as reference area. The area of blood vessels included in this square was used for estimating the percentage of blood vessels. Thirty random fields were evaluated in each experimental group, and the mean and SEM were calculated.
Statistics
Results are given as the mean ± SD. The significance of differences observed was assessed by ANOVA, followed by the post hoc Newman-Keuls multiple range test. P < 0.05 was considered statistically significant.
Results
Effects of the inhibition of ODC activity on plasma steroid levels in adult female mice
Figure 1 shows the values of ODC activity in the ovary of immature mice and adult females at different stages of the estrous cycle. In adult cycling females, there was a marked increase in ovarian ODC activity in the evening of proestrus that was associated with the rise in putrescine and spermidine concentrations (from 85 ± 11 to 158 ± 39 nmol/g for putrescine and 716 ± 104 to 929 ± 42 nmol/g for spermidine; P < 0.05). When the rise in ODC activity at the evening of proestrus was blocked by administration of DFMO, a specific inhibitor of ODC (26), no significant changes either in the estrous cycle or in the number of ova found in the oviducts the next morning were observed (data not shown). The influence of the inhibition of ODC by DFMO, given in the evening of proestrus, on plasma concentration of progesterone and estradiol at the different days of the estrous cycle is shown in Fig. 2. In agreement with results in rats reported by others, there was a transient increase in plasma progesterone concentration in the evening of proestrus and a sustained rise of progesterone values during diestrus in control animals. Our results showed that the treatment with DFMO did not affect plasma progesterone values at proestrus or estrus but produced a marked decrease of this hormone during diestrus (25% of control values). This effect of the ODC inhibitor on plasma progesterone at diestrus was not observed when DFMO was given in the evening of estrus or metaestrus (data not shown). DFMO treatment also produced a moderate fall in plasma estradiol at diestrus. Because the analysis of progesterone concentration in the ovaries at diestrus in both control and DFMO-treated animals showed a clear decrease of 27% (data not shown), which was considerably smaller than the fall observed in plasma, we tested the possible influence of DFMO treatment in ovarian angiogenesis by using a histochemical analysis of blood vessel density by means of lectins that react and stain vascular endothelial cells. This analysis revealed a significant decrease (35%) of the density of blood vessels in the corpora lutea from ovaries of DFMO-treated mice relative to controls (Fig. 3).
FIG. 1. Changes in ovarian ODC activity as a function of age. Immature and peripubertal mice were killed in the morning of diestrus when possible. Adult animals were killed in the morning of estrus (E), diestrus (DE), and proestrus (PE) and between 1800 and 2300 h of proestrus (PE*). Ovaries were collected at the time indicated, and homogenized and ODC activity was measured by a radiometric method. Results are the mean ± SD of four to eight animals per point.
FIG. 2. Changes in progesterone and estradiol levels during the estrous cycle produced by treatment of adult female mice with DFMO at proestrus. DFMO (500 mg/kg, sc) was injected into adult female at 1200, 1600, and 2000 h of proestrus, and progesterone was measured the next estrus, diestrus, and proestrus (0900–1000 h). Blood was collected by cardiac puncture, and steroid hormones were determined by ELISA. Results are the mean ± SD of six to eight animals per group. a, P < 0.001 vs. control group.
FIG. 3. Blood vessel staining of corpus luteum in adult female mice at diestrus. Lectin from Dolichus biflorus was used to specifically label blood vessels in corpus luteum. A, Control mice. B, Mice treated with DFMO at proestrus (500 mg/kg at 1200, 1600, and 2000 h, sc) and killed on the diestrus morning. Note the decrease in the density of vessels in DFMO-treated samples. The percentage of stained blood vessels were 8.0 ± 0.5% in controls vs. 5.2 ± 0.3% in DFMO-treated mice (P < 0.01).
Regulation of ovarian ODC by gonadotropins and their influence on the effect of DFMO on plasma steroid levels in adult female mice
To explore the effects of gonadotropins on ovarian ODC activity, female mice were treated with LH, FSH, or both, and enzyme activity was determined 5 h after hormone administration. Figure 4 shows that FSH produced a small increase in ODC activity, whereas LH and hCG increased the activity of the decarboxylase by about 10-fold. The simultaneous administration of LH and FSH produced additive effects. To determine whether the effect produced by DFMO treatment on plasma progesterone levels could be directly related to the blockade of ovarian ODC activity in the evening of proestrus or be the consequence of the alteration of the periovulatory surge of gonadotropins or prolactin, female mice treated with DFMO on the evening of proestrus received a simultaneous administration of pituitary hormones, and plasma progesterone was determined the next diestrus. Table 1 shows that neither LH plus FSH nor prolactin administration were able to prevent the decrease in plasma progesterone produced by DFMO during diestrus. Figure 5 also shows the effect of DFMO on the acute changes produced by hCG administration in the morning of proestrus on both ovarian ODC activity and plasma steroid concentrations, measured 5 h after hormone administration. Although DFMO completely abolished the remarkable rise of ovarian ODC activity, this compound did not affect the changes in plasma progesterone induced by the gonadotropin. Interestingly, hCG produced a marked decrease in plasma estradiol levels that was totally prevented by DFMO administration.
FIG. 4. Influence of gonadotropins on ovarian ODC activity. LH, FSH, and hCG (6 IU each, im) were given to adult female mice in the morning of diestrus, and animals were killed 5 h later. Ovaries were collected and homogenized, and ODC activity was measured by a radiometric method. Results are the mean ± SD of five to six animals per group. a, P < 0.001 vs. control; b, P < 0.05 vs. control; c, P < 0.05 vs. LH.
TABLE 1. Effect of DFMO on diestrus plasma progesterone levels and influence of gonadotropins or prolactin administration
FIG. 5. Influence of DFMO on ovarian ODC activity and plasma steroid levels after acute hCG treatment. Adult female mice were treated in the morning of proestrus with 10 IU of hCG, and animals were killed 5 h after hormone administration. DFMO (500 mg/kg, sc) was given 30 min before hCG administration. Control animals were injected with vehicle. Blood was obtained by cardiac puncture, and steroid hormones were determined by ELISA. Ovaries were collected at the time indicated and homogenized, and ODC activity was measured by a radiometric method. P, Progesterone; E2, estradiol. Results are the mean ± SD of five to six animals per group. a, P < 0.001 vs. control and hCG+DFMO; b, P< 0.05 vs. control; c, P< 0.05 vs. control and hCG+DFMO.
Influence of ODC in ovarian development
Figure 1 shows the variation of ovarian ODC activity along the postnatal development. The activity, which was low before weaning, increased during the peripubertal period and fell again in the mature animals except in the evening of proestrus. To study the influence of ovarian ODC activity in ovarian development, two groups of 24-d-old female mice were primed with PMSG and hCG, and the influence of DFMO, which was given to one group, was determined by comparison with control mice and with mice receiving the hormone treatment alone. Ovarian ODC activity raised from 7.2 ± 1.4 nmol/h/g in controls to 134.5 ± 5.2 nmol/h/g in the hormone-primed mice. In the DFMO-treated group, ODC activity was lower than 1 nmol/h/g. Figure 6A shows three representative sections of ovaries from control and treated mice. DFMO slightly diminished (28%) the increase of the ovarian size elicited by the hormone treatment but almost abolished the formation of antral follicles. The analysis of progesterone and estradiol in the plasma of mice under the different experimental conditions revealed that DFMO inhibited the marked increase in ovarian progesterone promoted by the hormones and also diminished the modest increase in ovarian estradiol (Fig. 6B).
FIG. 6. A, Influence of DFMO in the growth of the ovary of immature mice. The 24-d-old female mice were injected with 10 IU of PMSG to stimulate follicular growth, followed 52 h later with 5 IU of hCG to stimulate ovulation and luteinization; the mice were killed 48 h after hCG administration. a, Control animals. Mainly preantral small follicles were observed; note the small size of the ovary. b, Hormone-stimulated animals. An increase of antral follicles (Af) was shown. c, Hormone-stimulated animals that received an injection of DFMO (500 mg/kg, sc) 30 min before PMSG administration and DFMO (2% in the drinking water) until killing. A regression of the maturation process was detected. Scale bar, 500 μm. B, Influence of DFMO on plasma steroid hormones of immature mice. Plasma progesterone and estradiol were determined by ELISA. Results are the mean ± SD of four to six animals per group. a, P < 0.001 vs. other groups.
Immunocytochemical analysis of ODC in the mouse ovary
Cellular localization of ODC protein in the ovary was studied by immunocytochemical techniques using antibodies directed against mouse ODC. Figure 7A shows a negative control of the technique obtained when the ovarian section was incubated with the second antibody alone. When the two antibodies were used, a light and diffuse immunoreactivity was found in the granulosa cells of antral follicles and in the luteal cells of control ovaries (Fig. 7, B and C). The treatment with hCG produced a marked increase in ODC immunoreactivity in the oocyte and in the cytosol of theca and granulosa cells of antral and ovulatory follicles (Fig. 7, D and F). Granulosa cells from preantral follicles failed to express ODC in response to hCG (Fig. 7G). In the oocyte, immunoreactivity was localized in the cytoplasm and was absent in the nucleus and in the zona pellucida (Fig. 7, D and F).
FIG. 7. Localization of ODC in the ovary of adult mice. A, Negative control of the technique. Section was incubated only with secondary antibody; no reaction was observed. B, Control ovary. A slight labeling of ODC was evident in the cytoplasm of oocyte and granulosa cells of antral follicles. C, Corpus luteum in control ovary. Luteal cells were slightly immunoreactive. D–F, Antral follicles of hCG-treated animals. hCG (10 IU) was given in the morning, and ovaries were obtained 5 h after hCG administration. An increased immunoreactivity was observed in the theca and granulosa cells and in the cytoplasm of oocyte of antral follicles. No reaction was detected in zona pellucida and nucleus of oocyte. G, Preantral follicle of hCG-treated mice at proestrus. ODC reaction in primary follicles was only restricted to cytoplasm of oocyte. No labeling was observed in granulosa cells. O, Oocyte; G, granulosa; T, theca; arrowhead, zona pellucida. Scale bar, 22 μm.
Effect of DFMO on the levels of mRNAs of steroidogenic proteins in the ovary
The levels of mRNAs of SF-1, StAR, P450scc, 3? HSD, aromatase, and ?-actin were analyzed by means of semiquantitative RT-PCR in the ovaries of adult female mice primed with PMSG and hCG that had received either saline or DFMO. Figure 8 shows that, in hormone-treated mice, DFMO produced a dramatic decrease in the levels of SF-1 and cytochrome P450scc mRNA and a moderate diminution in the levels of StAR and 3? HSD. The comparison of mRNAs at the diestrus stage between control and DFMO-treated mice at proestrus also showed a significant decrease of cytochrome P450scc, SF-1, and StAR mRNA (Fig. 9). No differences in the expression of 20-hydroxysteroid dehydrogenase, the enzyme that participates in the catabolism of progesterone in the luteal cell, between control and DFMO-treated animals were observed (results not shown).
FIG. 8. Influence of DFMO on mRNA expression of steroidogenic proteins in the ovaries of adult mice treated with gonadotropins. One group of adult female mice was injected with 10 IU of PMSG and, 52 h later, with 5 IU of hCG. Mice were killed 48 h after hCG injection, the ovaries were collected, total RNA was extracted, and mRNA expression was analyzed by semiquantitative RT-PCR. The other group received the same hormonal treatment but received an injection of DFMO (500 mg/kg, sc) 30 min before PMSG administration and DFMO (2% in the drinking water) until killing. Each group was formed by four animals. A, A representative band is presented for each amplified gene product. B, Bands were quantified and normalized with respect to the intensity of ?-actin band, and the results are given as mean ± SD. a, P < 0.01. Arom, Aromatase.
FIG. 9. Influence of the blockade of the preovulatory peak of ovarian ODC on mRNA expression of steroidogenic proteins at diestrus. Adult female mice were treated with DFMO (500 mg/kg, three injections in the evening of proestrus) or saline, and ovaries were obtained after killing the animals at the next diestrus. Total RNA was extracted, and mRNA expression was analyzed by semiquantitative RT-PCR. Each group was formed by four animals. A, A representative band is presented for each amplified gene product. B, Bands were quantified and normalized to the intensity of ?-actin, and the results are given as mean ± SD. a, P < 0.01. b, P< 0.05. Arom, Aromatase.
Discussion
The biochemical events that control the processes of folliculogenesis and ovulation in mammals are only partially understood. Follicular growth is controlled by intraovarian regulatory factors that act by autocrine, paracrine, and intracrine mechanisms and by endocrine factors such as the pituitary gonadotropins FSH and LH (1, 11, 30). Current evidence suggests that, although the preantral stages of committed follicles are dependent on the local regulators, with gonadotropins having a facilitatory role, these hormones are essential for the development of antral (tertiary and Graafian) follicles. It is well known that, in steroidogenic cells, gonadotropins stimulate the synthesis of steroid hormones through the intermediary cAMP, which up-regulates the expression of P450scc, the enzyme that catalyzes the first reaction of the steroidogenic cascade, and of StAR protein, which participates in the translocation of cholesterol from the outer to the inner mitochondrial membrane of steroidogenic cells (30, 31, 32). Apart from this role, gonadotropins stimulate the maturation and function of the gonads and the regulation of gametogenesis (33, 34). It is believed that, whereas FSH is required for the growth of preovulatory follicles and for the expression of LH receptors in granulosa cells, the preovulatory surge of LH acts on these cells to terminate the follicular program of gene expression, stimulating, at the same time, the induction of genes required for ovulation and luteinization (11, 35).
In spite of the important role of gonadotropins in the physiology of the ovary, the molecular mechanisms by which these hormones exert their actions are only partially known. Early reports demonstrated that, in the rat, ovarian ODC is under the control of LH, showing this enzyme to have a transitory rise only in the evening of proestrus (19). Further studies with granulosa cells isolated from porcine ovarian follicles suggested that cAMP may mediate gonadotropin stimulation of ODC (36). The rise of ovarian ODC induced by hCG was associated with the increase in the amount of ODC mRNA (37). Despite that the strategic positioning of the enzyme induction in the ovary suggested a possible association with the ovulatory process, experiments using DFMO did not support an essential role for ODC in the final stages of follicular maturation and ovulation, at least in the rat (38).
Our results show that increased ovarian ODC activity at different stages of follicular development plays an important role in the ovarian physiology of mice. In adult cycling females, the preovulatory rise of ODC that follows the LH surge at the evening of proestrus appears to be related to the acquisition of progesterone secretory capacity by the corpus luteum, which is necessary for the successful implantation and development of the ovum. The effect of DFMO seems to be the result of the blockade of the augmentation of ODC activity observed in the ovary rather than the consequence of the diminution of gonadotropin secretion that could occur by the inhibition of the rise of ODC in the anterior pituitary gland, which, in the rat, has been related to the secretion of LH and prolactin at proestrus (39, 40, 41). Our results also indicate that the effects produced by DFMO on progesterone secretion are only observed when this compound is administered in the evening of proestrus, when the increase in ovarian ODC activity takes place. The fact that DFMO treatment did not affect the acute effect that hCG produced on progesterone secretion but decreased its secretion by the corpus luteum suggests that the rise in ovarian ODC in the evening of proestrus may be related with the process of luteinization. In turn, it is known that the preovulatory surge of LH is required for ovulation and luteinization (11). Moreover, the fact that ODC blockade is more critical for progesterone secretion than for ovulation, together with the high responsiveness of the mouse ovary inducing ODC after LH or hCG treatment, indicate that the rise of ODC in certain types of ovarian cells may be critical for the differentiation process that leads to the formation of luteal cells.
The immunocytochemical analysis of ODC in the mouse ovary revealed that there was a specific increase of ODC protein in response to hCG, mainly in the theca and granulosa cells of the antral follicles, whereas granulosa cells in preantral follicles were not responsive to gonadotropins. This result is in agreement with a previous report that showed that the increase of ODC activity in the rat ovary treated with LH was higher in the Graafian follicles than in the whole ovary (42). Regarding the cellular localization of ODC in the rat ovary, although some studies showed that the enzyme was confined to thecal and interstitial cells (43, 44), others found that the enzyme was mainly present in the cytoplasm of granulosa cells (45). Our results indicate that, in the mouse ovary, the presence of ODC in the granulosa cells is related to follicle development. Its presence in granulosa and theca cells of preovulatory follicles would support the hypothesis that ODC induction in the evening of proestrus may be required for the luteinization of the theca and granulosa cells in the ovulating follicle. Although ODC is also expressed in the oocytes of the different types of follicles and is up-regulated by LH, its physiological relevance remains to be determined. In this regard, recent experiments have revealed the expression of gonadotropin receptors in murine oocytes, suggesting a potential role for oocyte maturation (46, 47).
The molecular and cellular events that lead to the formation of a functional corpus luteum are only partially understood. Although it is clear that the development of luteal steroidogenesis is critical for progesterone secretion by the corpus luteum (48, 49, 50, 51), it is also evident that luteal development is associated with a dramatic increase in the number of blood vessels (52, 53, 54). Our results about the influence of DFMO on progesterone values in plasma and ovary could be explained not only by the inhibition of progesterone biosynthesis in luteal cells, which would decrease the hormone values in both compartments, but also by a limited vascularization of the corpus luteum, which would generate a more pronounced decrease of the hormone in the vasculature. The down-regulation produced by DFMO on cytochrome P450scc enzyme and StAR protein, which participate in key steps of the progesterone biosynthetic pathway, would explain the decrease in progesterone levels found in plasma and ovary of the animals treated with DFMO. Additionally, the dramatic decrease observed in the expression of SF-1 in mice treated with the polyamine inhibitor suggests that this change could be responsible for the effects observed in the levels of cytochrome P450scc and StAR mRNA because this transcription factor plays a central role in the expression of StAR and other genes encoding cytochrome P450 steroid hydroxylases (55). Our results suggest that the increase of polyamine levels that takes place during the evening of proestrus in the ovary of adult mice is critical for the development of a fully functional corpus luteum. In turn, increased polyamine biosynthesis is required in many processes of cell growth and differentiation (15, 16, 17, 18), and the corpus luteum is a transient endocrine gland that develops from the follicular cells that remain after ovulation. Although polyamines participate in the regulation of many proteins and genes, the molecular mechanisms by which they could mediate in the expression of steroidogenic proteins remain to be clarified. The possibility that the recently discovered polyamine-responsive element and the associated trans-acting proteins (56, 57) may have a role in the transcription of steroidogenic genes should be considered. On the other hand, the diminished vascularization of the corpus luteum observed in animals treated with DFMO is in agreement with the contention that ODC and polyamines may have an important role in angiogenesis (58, 59).
Our study also shows that ovarian ODC has a role not only in the functioning of the adult ovary but also in ovarian development because the inhibition of the induction of ovarian ODC found in the immature animals treated with gonadotropins was associated with the delay in ovarian growth and follicular progression. This result also corroborates the view that the effects produced by DFMO in ovarian physiology during this period are mainly due to the inhibition of ovarian ODC rather than to the decrease in the release of gonadotropins by the pituitary. Our results and those obtained with transgenic mice overexpressing ODC or spermidine/spermine acetyl transferase (24), a key enzyme in the polyamine retroconversion pathway, support the view that polyamines play a relevant role in the mammalian reproductive system.
In conclusion, the present experiments demonstrate that the elevated ODC values found in the ovary of immature and adult mice play a relevant function in ovarian physiology and that ODC/polyamines must be considered as important mediators of some of the effects of LH on follicular development and luteinization.
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