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BOR - Papers in Press, published online ahead of print October 5, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.044545
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BIOLOGY OF REPRODUCTION 74, 75–87 (2006)
DOI: 10.1095/biolreprod.105.044545
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

Expression and Regulation of the SCD2 Desaturase in the Rat Ovary

Céline Moreau 2, Pascal Froment 3, Lucie Tosca 2, Virginie Moreau 2, and Joëlle Dupont 1 2

Unité de Physiologie de la Reproduction et des Comportements,2 Institut National de la Recherche Agronomique, 37380 Nouzilly, France Laboratory of Molecular Cancer Biology,3 9052 Ghent, Belgium

ABSTRACT

Despite the significant role of the lipid reserve in cell structure and function, very few studies have provided detailed descriptions of unsaturated fatty acid synthesis in the ovary. In the present study, we have shown by RT-PCR, Northern blot, and Western blot analyses the mRNA and protein expression of SCD2 (stearoyl-coenzyme A desaturase 2; also named delta 9 desaturase) in rat ovary. We also have localized Scd2 mRNA by in situ hybridization, mainly in granulosa cells of antral follicles, cumulus oophorus, and corpus luteum. Interestingly, either no or very weak SCD2 expression was observed in primordial follicles and oocytes. After eCG injection for 24 h in immature rats (age, 22 days), the level of SCD2 expression and SCD activity in ovary was increased by approximately fourfold (P < 0.05), and the response was further increased 48 h after hCG treatment. As expected, eCG/hCG treatment increased expression of the steroidogenesis enzymes (CYP11A1 and HSD3B) and STAR. We also found a decrease in the SCD2 expression and SCD activity in the corpus luteum at Days 10 and 15 compared to Day 3 of gestation, paralleled by a decrease in the expression of the steroidogenesis enzymes and STAR. To investigate the molecular mechanisms involved in the regulation of SCD2 expression in ovary, we performed primary culture of rat granulosa cells. We observed that both insulin-like growth factor 1 (IGF1) (7.5 x 10–8g/ml) and FSH (350 x 10–8g/ml) increased SCD2 expression and SCD activity by approximately threefold. Using specific inhibitors, we demonstrated that the MAPK3/MAP1 and PIK3R1/AKT pathways are involved in the IGF1- and FSH-induced SCD2 expression, respectively. The SCD2 is expressed and active in rat ovary, and it may be involved in the regulation of follicular growth and/or the oocyte maturation.

follicle, follicle-stimulating hormone, granulosa cells, insulin-like growth factor receptor, ovary

INTRODUCTION

Stearoyl-coenzyme A desaturase (SCD), also named delta 9 desaturase, is a microsomal, rate-limiting enzyme in the biosynthesis of monounsaturated fatty acids from saturated fatty acids [1, 2]. Its preferred substrates are palmitoyl- and oleoyl-coenzyme A. The roles of monounsatured fatty acids are multiple and crucial in living organisms. Indeed, palmitoleic and oleic acids are the major monounsaturated fatty acids in membrane phospholipids, triglycerides, and cholesterol esters [3]. Apart from being components of lipids, monounsaturated acids have been implicated as mediators in signal transduction, apoptosis, and differentiation of neurons and other cell types [4]. It also has been suggested that unsaturated fatty acids play a role in the regulation of reproduction by influencing energy homeostasis [5]. Also, testicular and ovarian cells and, more precisely, male and female germ cells are rich in unsaturated fatty acids, with different fatty acids dominating depending on the species. In the male, the distribution of the unsaturated fatty acids plays an important role in the membrane fluidity necessary to the spermatozoa motility [6]. In the female, oocytes of all mammals contain an endogenous lipid reserve. The lipid, however, is species-specific in terms of its apparent abundance and utilization. In immature cattle, pig, sheep [7], and rabbit oocytes [8] with intact zona pellucida, both palmitic acid and oleic acid account for more than approximately half the fatty acid reserves. These same fatty acids also are prominent in the oocytes of frogs [9] and toads [10].

A number of Scd genes have been isolated in several species, including rat [11], mouse [12–15], hamster [16], sheep [17], goat [18], pig [19] and human [20, 21]. In rodents, four Scd genes and four isoforms (SCD1 to SCD4) have been identified and characterized [12–15]. Furthermore, these SCD isoforms are differentially distributed in tissues. The SCD1 and SCD2 are the main isoforms expressed in liver and brain, respectively [12, 13]. The SCD3 is abundant in the harderian gland [3] and skin [14]. The SCD4 is expressed predominantly in the heart [15]. In rodents, data have been reported regarding the hormonal regulation of SCD1 expression and activity, mainly in the liver, whereas few studies have been performed on the regulation of other SCD isoforms [22]. In rat Sertoli cells, Scd1 mRNA is downregulated by dexamethasone and insulin, whereas Scd2 is upregulated by both factors [23]. The delta 9 desaturases SCD1 and SCD2 have been studied in rat testis [23], but to our knowledge, their expression has never been investigated in the ovary of any species. At a transcriptional level, rodent Scd genes also are regulated by polyunsaturated fatty acids as well as transcription effectors, such as SREBPs and PPAR{gamma} [24–26]. In the ovary, all these transcription effectors have been identified. For example, our group in sheep [27] and others in rat [28] have shown that the fatty-acid receptor, PPAR{gamma}, was expressed in granulosa cells and that PPAR{gamma} ligands inhibited cell proliferation and increased progesterone secretion, suggesting a role of lipid metabolism in the ovary.

In the present study, we have identified the rat SCD2 and studied its regulation in term of mRNA and protein in vivo during follicular development and pregnancy in rat ovary. Furthermore, we have examined the molecular mechanisms by which insulin-like growth factor 1 (IGF1) and FSH increase SCD2 expression in vitro in rat primary granulosa cells.

MATERIALS AND METHODS

Hormones and Reagents

The eCG and hCG used for injections to animals were obtained from Intervet. Purified ovine FSH-20 (oFSH; lot no. AFP-7028D; 4453 IU/mg; FSH activity, 175-fold that of oFSH-S1) used for culture treatment was a gift from the National Institutes of Diabetes and Digestive and Kidney Diseases, National Hormone Pituitary Program, Bethesda, MD. Recombinant human IGF1 was from Sigma.

Antibodies

Rabbit polyclonal antibodies to phospho-AKT (Ser 473), AKT, phospho-ERK1/2 (Thr202/Tyr204), and phospho-p38 (Thr180/Tyr182) were purchased from New England Biolabs, Inc. Rabbit polyclonal antibodies to ERK2 (C14) and p38 (C20) as well as goat polyclonal antibodies to SCD2 (G15) were purchased from Santa Cruz Biotechnology. Monoclonal anti-tubulin-{alpha} antibodies were obtained from Oncogene Research. Rabbit polyclonal antibodies to CYP11A1 and STAR were generously provided by Dr. Dale Buchanan Hales (University of Illinois, Chicago, IL) and Dr. Van Luu-The (CHUL Research Center and Laval University, Quebec City, Quebec, Canada), respectively. All antibodies were used at 1:1000 dilution in Western blot analysis.

Animals

All procedures were approved by the Agricultural Agency and the Scientific Research Agency and were conducted in accordance with the Guidelines for Care and Use of Agricultural Animals in Agricultural Research and Teaching. Immature female rats of the Wistar strain were purchased from Janvier Laboratories. The rats were housed under controlled temperature and photoperiod (14L:10D photoperiod, lights-on from 0600–2000 h). The animals had ad libitum access to food and water. Ovaries were collected from immature (age, 21 days) rats after one treatment with 25 IU of eCG for 24 h to induce follicle growth. Some rats received a single intraperitoneal injection of 25 IU of hCG at 24 h post-eCG treatment to induce ovulation and luteinization, and ovaries were obtained at different intervals (6, 24, and 48 h after eCG treatment) for measurement by Northern blot, in situ hybridization, and Western blot analyses. Most of the rats had ovulated approximately 24 h after eCG/hCG treatment. To study the expression of SCD2 in the corpus luteum during pregnancy, another group of sexually mature female rats (age, 10–12 wk) was used. These rats were placed overnight for mating, and the next morning, rats positive for spermatozoa in vaginal smears were designated as being at Day 1 of gestation. Litters were born on Day 21 of pregnancy. Ovaries from rats in gestation (Days 3, 10, and 15) were collected, and corpora lutea were dissected for Northern and Western blot analyses.

Isolation and Culture of Rat Granulosa Cells

Immature female rats were injected subcutaneously with diethylstilbestrol (DES; 1 mg/day) every day for 3 d. On the third day of DES treatment, the animals were killed and the ovaries removed aseptically and transferred to culture medium. Granulosa cells were harvested by puncturing the follicles, allowing expulsion of the cells. Cells were recovered by centrifugation, washed with fresh medium, and counted in a hemocytometer. The culture medium used was McCoy 5A supplemented with 20 mmol/L of Hepes, 100 U/ml of penicillin, 100 mg/L of streptomycin, 3 mmol/L of L-glutamine, 0.1% BSA, 50 µg/L of insulin, 0.1 µmol/L of androstenedione, 5 mg/L of transferrin, 20 µg/L of selenium, and 5% fetal bovine serum (FBS). The cells were initially cultured for 48 h with no other treatment and then incubated in fresh FBS-free culture medium with or without test reagents for the appropriate time. All cultures were performed under a water-saturated atmosphere of 95% air/5% CO2 at 37°C.

RNA Isolation and RT-PCR

Total RNA was extracted from whole tissue (spleen, kidney, lung, muscle, heart, ovary, liver, and brain) or from cultured granulosa cells using Trizol reagent according to the manufacturer's procedure (Invitrogen). The RNA was quantified by measuring the absorbance at 260 nm. Samples were stored at –80°C until use. The RT-PCR was performed to assay the expression of Scd1 and Scd2 delta 9 desaturases in rat ovary, liver, and brain. The RT-PCRs were then performed as described previously [29]. Single-strand cDNAs were amplified with specific sets of primer pairs designed to amplify parts of the different Scd genes (Table 1). The PCR amplifications with RNA were performed in parallel as negative controls. The RT-PCR consumables were purchased from Sigma except for Moloney murine leukemia virus reverse transcriptase and RNase inhibitor (RNasin), which were from Promega. The pCRII-Scd2 antisense and sense constructs used for in situ hybridization were generated by inserting the fragment of Scd2 cDNA (630 bp) into pCRII-TOPO vector (TOPO TA Cloning kit; Invitrogen) and selecting a clone with the appropriate antisense or sense orientation.


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  		TABLE 1. Oligonucleotide primer sequences

In Situ Hybridization

Frozen ovaries were serially sectioned (thickness, 10 µm) with a cryostat to perform in situ hybridization experiments using [35S]UTP-labeled rat Scd2 cRNA as probe following a method described previously [27]. Prehybridization and hybridization were performed as described previously [27]. The sections were then dehydrated and air-dried, and slides were dipped in Kodak NTB2 emulsion (Integra Bioscience) and exposed at 4°C for 3 wk in a dessicated dark box. Slides were developed and counterstained with hematoxylin. Specificity of hybridization was assessed by comparing signal obtained with the cRNA antisense probe and the corresponding cRNA sense probe. Histological determination of the degree of atresia was performed on adjacent sections by the Feulgen method. As described previously [27], follicles were judged to be normal or atretic using classical histological criteria (normal: frequent mitosis, no pyknosis in granulosa cells; atretic: no mitosis, frequent pyknotic bodies in granulosa cells).

Microscopic Analysis of Autoradiography

Quantitative autoradiographic analysis of [35S]Scd2 expression was performed using a microscope-linked, PC-based image analyzer (SAMBA TM 2005; Alcatel TITN). Each section was analyzed with a 40x objective. Quantification of labeling was performed by measuring the area occupied by silver grains in a constant area (200 µm2). This quantification was performed on granulosa cells from follicles and corpora lutea. Labeling was estimated from 10 measurements on each follicle and corpus luteum. The area for the counting of grains was chosen totally at random. Specific binding was obtained by subtracting the values of labeling associated with nonspecific binding from the total binding values [27].

Northern Blot Analysis

Total RNAs from cells or tissue (20 µg) were separated by denaturing formaldehyde electrophoresis, then transferred to a nylon membrane by capillarity overnight and hybridized as described previously [29]. The Scd1 and Scd2 probes were obtained by RT-PCR using the primers indicated in Table 1. After high-stringency washings, autoradiography was performed at –70°C for 48 h using Kodak X-OMAT film with intensifying screens. Membrane-incorporated radioactivity also was quantified using a Storm apparatus (Molecular Dynamics). The integrity and quantification of different transcripts were assessed using the human RNA 18S probe as a control (Ambion).

Western Blot Analysis

Lysates of granulosa cells or tissue were solubilized and centrifuged as described previously [29]. Cell extracts were then submitted to electrophoresis on 10% (w/v) SDS-PAGE under reducing conditions and electrotransferred as described previously [29].The membranes were then incubated overnight at 4°C with appropriate antibodies (final dilution, 1:1000) in Tris-buffered saline (TBS; 2 mM Tris-HCl [pH 8] and 15 mM NaCl [pH 7.6]) containing 0.1% Tween 20 and 5% nonfat dry milk powder (NFDMP). After washing in TBS-0.1% Tween 20, nitrocellulose membranes were incubated for 2 h at room temperature with a horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (final dilution, 1:10,000; Diagnostic Pasteur) in TBS-0.1% Tween 20–5% NFDMP. After washing in TBS-0.1% Tween 20, the signal was detected by enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech). The films were analyzed and signals quantified with the software MacBas (Ver 2.52; Fuji PhotoFilm USA, Inc.). The results are expressed as the intensity signal in arbitrary units after normalization allowed by the presence of tubulin as an internal standard and correspond to the average of three independent experiments.

Preparation of Microsomes and SCD Assay

Microsomes were isolated from rat ovary, corpus luteum, liver as a positive control, and granulosa cells by differential centrifugation and then resuspended in a 0.1 M potassium phosphate buffer (pH 7.2). The SCD activity was determined at 23°C with 3 µM [14C]stearoyl-coenzyme A (Amersham), 2 mM NADH, and 150 µg of microsomal protein. After 5 min of incubation, 200 µl of 2.5 M KOH in 75% ethanol were added, and the reaction mixture was saponified at 85°C for 1 h. The samples were cooled and acidified with 280 µl of formic acid. Free fatty acids were extracted with 700 µl of hexane and separated on a 10% AgNO3-impregnated TLC plate using chloroform:methanol:acetic acid:H20 (90:8:1:0.8). The TLC plates were analyzed with a Storm PhosphorImager. Band intensities corresponding to saturated and monosaturated fatty acids were used to calculate the percentage of conversion and enzyme activity.

Progesterone Radioimmunoassay

The concentration of progesterone in the culture medium of granulosa cells was measured after 24 h of culture by a radioimmunoassay protocol as described previously [30] and adapted to measure steroids in cell culture media. The limit of detection of progesterone was 12 pg/tube (60 pg/well), and the intra- and interassay coefficients of variation were less than 10% and 11%, respectively. Results are expressed as the amount of steroids secreted for 48 h per 100 µg of protein.

Statistical Analysis

All experimental data are presented as the mean ± SD. One-way ANOVA was used to test differences in levels of SCD2. If ANOVA revealed significant effects of time of tissue collection or treatment, the means were compared by the Newman test, with P < 0.05 considered to be significant.

RESULTS

Expression and Localization of SCD2 Desaturase in Rat Ovary

The RT-PCR analysis first performed on RNA from rat ovary resulted in the amplification of two cDNAs corresponding to a fragment of the region of Scd1 (649 bp) and Scd2(630 bp) (Fig. 1A). As expected, Scd2 is expressed in rat brain but not in rat liver [12, 13]. As shown in Figure 1B, Northern blot analysis revealed that Scd2 and Scd1 mRNA are expressed as a main transcript (5.9 kb) in rat ovary; however, Scd1 is expressed at very low levels as compared to Scd2 (Fig. 1, A and B). The Scd1 blot in Figure 1B is the result of more than 2 days of exposure, compared to approximately 12 h for the Scd2 gene. Because of the strong Scd2 expression in ovary compared to that of Scd1, we focused the present study on this desaturase. In situ hydridization on ovarian sections showed that Scd2 mRNA was highly expressed in granulosa cells of antral follicles (Fig. 1C, a and b) and in the corpus luteum (Fig. 1C, f and g). The intensity of labeling was higher in antral follicle than in primordial follicle (data not shown). Furthermore, the cumulus oophorus was labeled as intensively as mural granulosa cells (Fig. 1C, a, b, d, and e). The oocyte, however, was not labeled, and a weak labeling was observed in theca cells (Fig. 1C, a and b). Healthy and atretic follicles expressed identical levels of Scd2 mRNA (data not shown). By immunoblotting on protein extracts, one 37-kDa band corresponding to SCD2 was detected in ovary, granulosa cells from large follicles obtained after eCG injection for 24 h, corpus luteum, and in brain but not in liver (Fig. 1D). As shown in Figure 1E, SCD activity was measured in rat ovary, granulosa cells, and corpus luteum. This activity was approximately twofold higher than those determined in liver.


Figure 1
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  		FIG. 1. Detection of Scd2 mRNA and SCD2 protein in the rat ovary. A) RT-PCR analysis of the delta 9 desaturase Scd1 and Scd2 mRNA in liver, brain, and ovary. B) Detection of the Scd1 and Scd2 mRNA transcripts by Northern blot analysis in different tissues. C) Localization of Scd2 mRNA in the rat ovary by in situ hybridization in large follicle (a and b), the cumulus oophorus of preovulatory follicle (d and e), and the corpus luteum (f and g). Bright-field (a, d, and f) and dark-field (b, e, and g) photomicrographs are shown; both b and c are dark-field photomicrographs of the same large follicles hybridized with antisense and sense [35S]Scd2 probe, respectively. CL, corpus luteum; Cu, cumulus oophorus; F, Follicle; G, granulosa cells; O, oocyte; pF, preovulatory follicle; T, theca cells. Arrows indicate different cell types or structures of the ovary. Original magnification x20 (a–c) and x40 (d–g). Bar = 100 µm. D andE) Detection of SCD2 protein (D) by immunoblotting and SCD activity (E; nmol min–1 mg protein–1) in rat ovary, granulosa cells from antral follicle obtained after injection with eCG for 24 h (GC), corpus luteum (CL), liver, and brain. Each value represents the mean ± SD (n =3). Tubulin is used as a loading control for immunoblotting. Each value represents the mean ± SD (n =3)

Hormone-Regulated Expression of SCD2 in Rat Ovary

To examine the hormonal regulation of SCD2 expression in rat ovaries more closely, Northern blot, in situ hybridization, and Western blot analyses were performed using ovaries isolated from eCG/hCG-treated rat. Results revealed that the Scd2 mRNA and SCD2 protein levels were very low in immature rat ovaries (Figs. 2, A and B, and 3, A and B), and they increased significantly in response to stimulation with eCG treatment (24 h) and more abundantly with hCG treatment (24 and 48 h). As shown in Figure 2B, quantification of in situ hybridization (10 measurements realized at random) showed that Scd2 mRNA was increased by fourfold (in granulosa cells of antral follicles) in response to eCG treatment (24 h) and by eightfold (in corpus luteum ) in response to eCG (24 h)/hCG (48 h) treatment as compared to immature ovaries. Similar results were obtained with the whole ovaries by Northern (Fig. 3A) and Western (Fig. 3B) blot analyses at the protein level. Furthermore, SCD activity using 18:0 as a substrate was increased by approximately fourfold in microsomes from ovaries of eCG-injected rats and by approximately eightfold in microsomes from ovaries of eCG (24 h)/hCG (48 h)-injected rats compared with ovaries from immature rats (Fig. 3C). As expected, eCG and hCG treatments also have increased significantly the expression of the steroidogenesis enzymes, such as CYP11A1 and HSD3B, and of the cholesterol carrier, STAR (data not shown).


Figure 2
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  		FIG. 2. Localization of Scd2 mRNA by in situ hybridization in rat ovary after eCG/hCG treatment. A) Localization of Scd2 mRNA in an immature ovary (a and b) and in an ovary from a rat treated with eCG for 24 h (c and d), with eCG for 24 h and hCG for 6 h (e and f), with hCG for 24 h (g and h), or with hCG for 48 h (i and j). Both k and l are dark-field and bright-field photomicrographs, respectively, of an ovary from rat treated with eCG for 24 h and then hCG for 24 h and hybridized with sense [35S]-Scd2 probe. CL, Corpus luteum; Cu, cumulus oophorus; F, follicle; G, granulosa cells; O, oocyte; T, theca cells. Arrows or arrowheads indicate different cell types or structures of the ovary. Original magnification x10. Bar = 100 µm. All rats had ovulated 24 h after eCG/hCG treatment. B) Quantification of labeling of [35S]Scd2 mRNA localized in granulosa of small and large follicles (immatures; eCG, and hCG for 6 and 24 h) and in corpus luteum (hCG for 48 h). Values are presented as the mean ± SD (n = 8 rats/treatment). Different letters indicate significant differences at P <0.05


Figure 3
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  		FIG. 3. Northern blot (A) and Western blot (B) analysis of Scd2 mRNA and SCD2 protein expression in rat ovary after eCG/hCG injection. For Northern blot analysis, the quantification of the radioactivity was realized using the Storm apparatus, and the ratio of Scd2 to 18S is indicated. For Western blot analysis, all extracts contained equal amounts of proteins, as confirmed by reprobing membrane with an antitubulin antibody. Blots were quantitated, and the ratios of SCD2 to tubulin were represented. C) SCD activity (nmol min–1 mg protein–1) in rat ovary after eCG/hCG treatment. Values are present as the mean ± SD (n = 8 rats/treatment). Different letters indicate a significant difference at P < 0.05

Expression of SCD2 in Rat Ovaries During Gestation

Because SCD2 is strongly expressed in the corpus luteum in response to the eCG/hCG (48 h) treatment, we determined whether the SCD2 expression and SCD activity was regulated in the corpus luteum during gestation. Thus, the levels of Scd2 mRNA and SCD2 protein were studied by Northern blot, in situ hydridization, and Western blot analyses in isolated corpus luteum of rat ovaries from Days 3, 10, and 15 of pregnancy, respectively. As shown in Figures 4, A and B, and 5A, the level of Scd2 mRNA was reduced by at least twofold between 3 and 10 or 15 days of gestation. The SCD2 protein level (Fig. 5B) and the SCD activity (Fig. 5C) were similarly regulated. As expected, we observed that the mRNA expression levels of Hsd3b, Cyp11A1, and Star were significantly reduced after 10 and 15 days of pregnancy (data not shown).


Figure 4
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  		FIG. 4. Localization of Scd2 mRNA by in situ hybridization in the corpus lutea during the pregnancy. A) Localization of Scd2 mRNA in the corpus lutea from an ovary of rat at Day 3 (a and b), 10 (c and d), and 15 (e and f) of gestation. Both g and h are dark-field and bright-field photomicrographs, respectively, of an ovary from rat at 3 days of gestation hybridized with sense [35S]Scd2 probe. CL, Corpus luteum; G, granulosa cells; O, oocyte. Original magnificaiton x10. Bar = 100 µm. B) Quantification of labeling of [35S]Scd2 mRNA localized in corpus lutea of ovaries at Day 3, 10, and 15 of gestation. Each value represents the mean ± SD (n = 8 rats/gestational stage). Different letters indicate significant differences at P < 0.05


Figure 5
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  		FIG. 5. A and B) Northern blot (A) and Western blot (B) analyses of Scd2 mRNA and SCD2 protein expression in the rat corpus luteum during the pregnancy. C) SCD activity (nmol min–1 mg protein–1) in corpus lutea from ovaries of a pregnant rat at Day 3, 10, and 15. The ratios of Scd2 mRNA to 18S and of SCD2 protein and tubulin are shown. Each value represents the mean ± SD (n = 8 rats/treatment). Different letters indicate a significant difference at P < 0.05

Effects of FSH and IGF1 on Expression for SCD2 in Granulosa Cell Cultures

The elevated levels of Scd2 mRNA in granulosa cells of antral follicles in response to eCG prompted us to test its regulation by two hormones involved in follicular development, FSH and IGF1. We used primary culture of immature granulosa cells from DES-primed rats. As shown in Figure 6, A and B, Scd2 mRNA and SCD2 protein were undetectable in untreated, serum-starved granulosa cells cultured for 24 h. Addition of IGF1 (7.5 x 10–8 g/ml) or FSH (350 x 10–8 g/ml) to the culture medium for 24 h induced the expression of Scd2 mRNA and SCD2 protein by approximately threefold compared to untreated cells (Fig. 6, A and B). Moreover, an additive effect was observed when both IGF1 and FSH were combined in the culture medium (Fig. 6, A and B). As shown in Figure 6C, IGF1 treatment increased later Scd2 mRNA by approximately two-and fourfold after 6 and 12 h of stimulation, respectively; this increase persisted until 24 h of stimulation. In contrast, the FSH treatment rapidly increased Scd2 mRNA (twofold after 1 h of stimulation), and the effect peaked at 12 h and then decreased after 24 h of stimulation (Fig. 6C). We also showed that IGF1 (7.5 x 10–8 g/ml) and FSH (350 x 10–8 g/ml) increased SCD activity by approximately twofold when they were added separately and by fourfold when they were combined in the culture medium (Fig. 6D). As expected, progesterone production and CYP11A1 and STAR protein expression were increased slightly by IGF1 and strongly by FSH, respectively, as measured in conditioned media at 24 h of culture. An additive effect also was observed when both hormones were combined (Fig. 6E).


Figure 6
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  		FIG. 6. Effect of FSH and IGF1 treatments on the Scd2 mRNA and SCD2 protein expressions in rat granulosa cells. Rat primary granulosa cells were serum-starved overnight and then treated with IGF1 (7.5 x 10–8 g/ml) or FSH (350 x 10–8 g/ml) separately or in combination for 24 h or with IGF1 or FSH for various times (1, 3, 6, 12, and 24h). For Northern blot analysis (A and C), the ratio of Scd2 to 18S is represented, and for Western blot analysis (B), the ratio of SCD2 to tubublin is represented. Also shown are the effect of FSH and IGF1 treatments on the SCD activity (nmol min–1 mg protein–1) in rat granulosa cells (E) as well as progesterone secretion and CYP11A1 and STAR expressions in granulosa cells in response to IGF1 and FSH treatments (F). Granulosa cells were serum-starved for 24 h and then cultured for 24 h in serum-free medium in the presence or absence of IGF1 (7.5 x 10–8 g/ml), FSH (350 x 10–8 g/ml), or a combination of both. The culture medium was analyzed for progesterone content by RIA, and cells were submitted for Western blot analysis. Values represent the mean ± SD of three experiments from different cultures. Different letters indicate a significant difference at P < 0.05

Intracellular Signaling Mechanism Involved in Scd2 mRNA and SCD2 Protein Increase in Response to IGF1 and FSH in Primary Rat Granulosa Cells

To investigate the molecular mechanisms involved in both IGF1- and FSH-induced SCD2 expression in rat primary granulosa cells, we first studied the effect of these two hormones on the activation of three main signaling pathways, the AKT and MAPK3/MAP1 and p38 pathways. As shown in Figure 7A, IGF1 treatment rapidly increased the phosphorylation of AKT, MAPK3/MAP1, and p38 (after 1 min of stimulation). The IGF1-induced MAPK3/MAP1 and p38 phosphorylation was transient; after 60 min, no more stimulation was observed. In contrast, the IGF1-induced AKT phosphorylation was maintained after 60 min of stimulation. As shown in Figure 7B, FSH treatment increased AKT, MAPK3/MAP1, and p38 rapidly, and all these effects persisted after 1 h of stimulation. To determine which signaling pathway was involved in the IGF1- and FSH-induced Scd2 mRNA and SCD2 protein expression in rat granulosa cells, we used specific inhibitors of the two pathways of the IGF1 receptor and FSH receptor: MAPKs [p38 (SB202190, 50 µM) and MAPK3/MAP1 (U0126, 10 µM),], and AKT (LY294002, 50 µM). We first checked that the dose of each inhibitor used was effective in the rat granulosa cells (data not shown). As shown in Figure 8, the U0126 inhibitor inhibited the IGF1-induced SCD2 expression (mRNA and protein), whereas the LY294002 inhibitor inhibited the SCD2 expression induced by FSH, suggesting that the MAPK3/MAP1 and AKT signaling pathways are involved in the increase in the SCD2 expression in response to IGF1 and FSH, respectively.


Figure 7
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  		FIG. 7. IGF1 and FSH activate MAPK3/ MAP1, p38, and AKT signaling pathways in rat granulosa cells. Rat primary granulosa cells were serum-starved overnight and then treated with IGF1 (A; 7.5 x 10–8 g/ml) or FSH (B; 350 x 10–8 g/ml) for indicated times. To determine the level of AKT, MAP1/MAPK3, and p38 phosphorylation, all the blots were stripped and reprobed with antibodies against AKT, MAP1, and p38 proteins, and the ratio of phosphorylated to total protein was determined and plotted as the ratio of stimulation as compared with the unstimulated state. Values are presented as the mean ± SD from three independent experiments


Figure 8
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  		FIG. 8. Effect of specific inhibitors on Scd2 mRNA (A) and SCD2 protein (B) expressions in response to FSH and IGF1 treatments in rat granulosa cells. Rat primary granulosa cells were serum-starved overnight and then preincubated for 60 min with the specific inhibitors LY294002 (50 µM), U0126 (10 µM), or SB202190 (50 µM) and stimulated or not with IGF1 (7.5 x 10–8 g/ml) or FSH (350 x 10–8 g/ml) for 24 h. The Scd2 mRNA and SCD2 protein expressions were then determined by Northern-blot (A) and Western-blot (B) analyses, respectively. Values are presented as the mean ± SD from three independent experiments. *P < 0.05

DISCUSSION

In the present paper, we have shown, to our knowledge for the first time, that Scd1 and Scd2 are expressed in ovary and, more precisely, in granulosa cells of large follicles, corpus luteum, and cumulus oophorus. Furthermore, we have observed that the ovaries from adult rats express far higher levels of Scd2 mRNA compared to those in liver, whereas the opposite distribution is seen for Scd1. In vivo, eCG treatments increase Scd2 mRNA and SCD2 protein expression in immature rat ovaries, and this is augmented when rats also receive hCG. We also have shown that Scd2 mRNA and SCD2 protein expressions in corpus luteum decrease from Day 3 through Days 10 and 15 of gestation. In vitro, both FSH and IGF1 stimulate SCD2 expression (protein and mRNA) in rat primary granulosa cells, and this effect is additive when these two hormones are combined. Interestingly, these variations of SCD2 expression are associated to variations with the expression of some proteins of steroidogenesis, including HSD3B, CYP11A1, and STAR. Using specific inhibitor of the AKT and MAPK3/MAP1 pathways, we have shown that FSH-induced SCD2 expression (protein and mRNA) is mediated through AKT, whereas induction of SCD2 (protein and mRNA) by IGF1 is mainly a result of MAPK3/MAP1 activation.

The fact that Scd2 is several-fold higher in expression than Scd1 in ovary suggests that Scd2 is responsible for the delta 9 desaturase activity in this organ. Using RT-PCR, we did not detect the presence of the Scd3 and Scd4 in rat ovary (data not shown). These results are in good agreement with those of Ntambi et al. [1], who described large differences in expression and regulation between the Scd genes. Interestingly, we have observed a high expression of Scd2 in granulosa cells and in the cumulus oophorus, which is a mass of granulosa cells surrounding the oocyte [31], but we failed to detect Scd2 expression in the rat oocyte, as determined by in situ hybridization and confirmed by RT-PCR (data not shown). It is well-known that the oocyte is rich in unsaturated fatty acid and, more precisely, in palmitic acid and oleic acid [7–10]. The origin of these fatty acids, however, is unclear. Cumulus cells that express a high level of Scd2 are involved in oocyte growth and maturation. Indeed, evidence has shown that cumulus cells provide nutrition for the oocyte and influence oocyte development in a paracrine fashion [32, 33]. Thus, monounsaturated acid formed in the cumulus cells from SCD2 activation could be transported to the germ cells. They could then not only constitute a source of nutrients but also play a vital role in modifying the physical properties and biological functions of membranes and have potent effects on cell-cell interaction. Furthermore, in most mammalian species, removal of the cumulus oophorus at the time of fertilization often leads to a drop in fertilization rates. Indeed, removal of cumulus cells before in vitro fertilization has decreased sperm penetration in cattle [34] and in pigs [35], whereas it did not affect fertilization rates in some mouse strains [36]. How cumulus cells interact with the oocyte or with spermatozoa to promote fertilization is unknown, but we can speculate that the composition in monounsaturated fatty acid in the membrane triglycerides plays a role in the capacitation and penetration of the spermatozoa into the oocyte.

Interestingly, we have observed a very weak Scd2 expression in the small follicle as compared to the preovulatory follicles. Thus, Scd2 mRNA and SCD2 protein expressions increase during follicular development. Expression of Scd2 also varies in the developing mouse brain [37]. In the present study, we have shown that in vivo eCG injection increases Scd2 mRNA and SCD2 protein expressions in the large follicle and that hCG treatment enhances these effects. The high expression (mRNA and protein) of SCD2 and high SCD activity after the administration of eCG suggests that this desaturase may play a role in follicular development. In vitro, during primary culture of bovine granulosa cells from large follicle, palmitic and oleic acid treatment for 48 h inhibits cell proliferation by inducing apoptosis [38]. Palmitoyl- and oleoyl-coenzyme are the main substrates of the delta 9 desaturase, SCDs. Thus, these latter results could suggest a positive relation between SCD2 expression and/or SCD activity and follicular atresia. In vivo, however, we have observed a similar labeling in all large follicles (atretic or healthy large antral follicles), suggesting that the Scd2 expression is not related to follicular health in rat ovary.

As expected, eCG and hCG administration increased the mRNA expression of the steroidogenesis enzymes involved in progesterone (Hsd3b, Cyp11a1, and the cholesterol carrier, Star) and estradiol production (P450 aromatase; data not shown). These results were paralleled by an increase in SCD2 expression and SCD activity. We also have shown that Scd2 mRNA and SCD2 protein expressions and SCD activity were decreased in the corpora lutea at Day 10 or 15 as compared to corpora lutea at Day 3 of gestation, and these results are concomitant with a reduction in the Hsd3b, Cyp11a1, and Star expression. In vitro, during primary culture of bovine granulosa cells from large follicle, palmitic and oleic acid treatment for 48 h increased estradiol production [38]. Thus, we can speculate that the monounsaturated fatty acids synthesized from SCD activation in ovary could participate in the regulation of ovarian steroidogenesis. Indeed, it is well-known that free fatty acids can stimulate ovarian NADPH-dependent enzymes, including aromatase, which converts androgens to estrogens, by reducing NADP+ to NADPH [39]. Also, the polyunsaturated fatty acid, arachidonic acid, is able to modify STAR expression and, consequently, to regulate the cholesterol transfer to the mitochondrial inner membrane, which is the rate-limiting step of steroidogenesis in mouse Leydig cells [40–42]. Furthermore, the metabolites of the lipoxygenase and epoxygenase pathways have been demonstrated to stimulate HSD3B and HSD17B activities and to enhance the synthesis of testicular steroid hormones [43]. Oleic and palmitic acids synthesized from SCD activation could regulate progesterone and estradiol synthesis by acting in some regions of the promoter of different molecules involved in the steroidogenesis.

To understand better the molecular mechanism involved in regulation of Scd2 mRNA and SCD2 protein expression in ovarian cells, we used rat primary immature granulosa cells that were stimulated with two major factors involved in follicular development, IGF1 and FSH. We observed that IGF1 and FSH treatment increased Scd2 expression. Regulation of the desaturases is a result of diverse mechanisms that include altered transcription of the genes [1, 44] and altered stability of the mRNAs [45]. Concerning the FSH effect on the SCD2 desaturase, the present results are in accordance with those of earlier published studies involving rat Sertoli cells. Indeed, Saether et al. [23] showed in these cells that delta 9 desaturase Scd2 mRNA expression was upregulated by FSH treatment. That same study also showed that Scd2 mRNA expression is increased in response to insulin treatment. In rat immature granulosa cells, IGF1 treatment increases Scd2 expression and SCD activity. Both IGF1 and, mainly, FSH also increased progesterone secretion and steroidogenesis. Using specific inhibitors of MAPK3/MAP1, p38, and PIK3R1/AKT, we have shown that IGF1 increased SCD2 expression (protein and mRNA) through the MAPK3/MAP1 pathway, whereas FSH increased SCD2 expression (protein and mRNA) through the AKT signaling pathway. These two signaling pathways have already been shown to be involved in granulosa cell proliferation and steroidogenesis [46–48].

In conclusion, SCD2, as compared to SCD1 {Delta}9-desaturase, is mainly expressed in rat ovary. The high expression in granulosa cells of cumulus and the low to absent expression in oocytes suggest a lipid transport from the cumulus to the oocyte, which contains unsaturated acid. In vivo, administration of eCG and hCG increases SCD2 (protein and mRNA) expression and SCD activity in the preovulatory follicle and the corpus luteum as compared to small follicles of immature ovary. Furthermore, in vitro, treatment with FSH and IGF1 induces SCD2 expression (protein and mRNA) and SCD activity in immature granulosa cells. Thus, we hypothesize that SCD2 is involved in follicular development.

ACKNOWLEDGMENTS

The authors thanks to M. Peloille for the sequencing, C. Cahier and J.C. Braguer for the animal care, and P. Monget for helpful discussions.

FOOTNOTES

1 Correspondence: Joëlle Dupont, Unité de Physiologie de la Reproduction et des Comportements, Institut National de la Recherche Agronomique, Nouzilly 37 380, France. FAX: 33 2 47 42 77 43; jdupont{at}tours.inra.fr Back

Received: 8 June 2005.

First decision: 8 July 2005.

Accepted: 30 September 2005.

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