ARTICLE |
Correspondence to: María A. Burrell, Dept. of Histology and Pathology, University of Navarra, C/Irunlarrea 1, 31008 Pamplona, Spain. E-mail: mburrell@unav.es
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Summary |
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Leptin is a hormone originally identified in adipocytes. It is involved in the regulation of fat deposition and energy expenditure and in other functions, such as reproduction. The presence of leptin has been reported in several reproductive organs. However, few studies have addressed its expression in the ovary. Moreover, the existing information is not consistent with regard to the particular cell types responsible for leptin expression. In this work we studied the distribution of leptin in the rat ovary by immunohistochemistry (IHC) and in situ hybridization (ISH). Leptin staining was found in steroid-producing cells: thecal, luteal, and interstitial cells. The strongest signal with both techniques was found in the cytoplasm of oocytes. A weak reaction for leptin mRNA was detected in granulosa of all growing follicles, although leptin protein was found only in the mature follicle. Western blotting analysis detects a strongly reactive 16-kD band, giving further support to the presence of leptin in the rat ovary. Variations in this immunoreactive band were found throughout the estrous cycle. Localization of leptin in the ovary may contribute to a better understanding of female reproductive function.
(J Histochem Cytochem 51:12691277, 2003)
Key Words: leptin, ovary, estrous cycle, oocyte, follicular cells, corpora lutea, interstitial gland, immunohistochemistry, Western blotting, in situ hybridization
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Introduction |
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LEPTIN, the product of the ob gene, is a protein originally identified in adipocytes, which plays an important role in the regulation of food intake and energy balance (
Leptin has been directly related to reproductive performance. The relationship between fat stores and the reproductive axis has been studied in mice with a homozygous mutation for the ob gene, which developed obesity and sterility (
In the ovary, several authors have reported the relevance of leptin in the regulation of reproductive processes. The expression of Ob-R in this organ has been mainly related to a role of leptin in steroidogenesis (
Leptin protein has been identified in mouse and human oocytes and in preimplantation stage embryos, and appears to have a critical role in early mammalian development (
The aim of the present work was to identify, by a combination of immunohistochemical (IHC) and in situ hybridization (ISH) methods, which cell types in the rat ovary are responsible for leptin production. Furthermore, we analyzed possible variations in leptin expression during the estrous cycle by Western blotting.
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Materials and Methods |
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Animals
All experiments were performed under protocols approved by the Ethical Committee of the University of Navarra. Twenty mature female Wistar rats were used throughout this study. The animals were maintained under conventional conditions, 12-hr light/12-hr dark schedule and at a temperature of 2123C, with water and pellet food available ad libitum. To determine the estrous cycle phase, vaginal smears were collected daily (between 0900 and 1000 hr) for three consecutive cycles, and only rats with consistent 4-day cycles were used. Five animals of each stage (proestrus, estrus, metestrus, and diestrus) were sacrificed by decapitation between 1200 and 1300 hr. In addition, four adult Swiss mice were used for a comparative analysis. The genital tract of the rodents was exposed by abdominal incision and the ovaries were removed by dissection. After careful removal of periovarian fat, they were fixed for 24 hr in Bouin solution, dehydrated in ethanol, and embedded in paraffin for IHC analysis. Three rat ovaries of each stage were immediately frozen in liquid N2 and stored at 80C for subsequent protein isolation.
Two additional rats, whose cycle phase was unknown, were used for ISH. Animals were decapitated and their ovaries were fixed in 10% neutral formalin (pH 7.4) for 24 hr, dehydrated, and embedded in paraffin.
Immunohistochemical Methods
Paraffin sections (5 µm thick) were placed on slides coated with Vectabond (SP-1800; Vector Laboratories, Burlingame, CA). Sections were deparaffinized with xylene and rehydrated with graded ethanol. Endogenous peroxidase activity was blocked with 3% H2O2 for 10 min. Slides were washed with distilled water for 5 min, placed in 0.01 M citrate buffer (pH 6.0), and heated in a microwave oven for 15 min at maximal power (750 W) and for 15 min at medium power (375 W) for antigen retrieval. Background blocking was performed with 1:20 normal goat serum (Dako; Glostrup, Denmark) in 0.05 M Tris-HCl buffer, 0.5 M NaCl, pH 7.6 (TBS) before incubation with specific antiserum. The tissue sections were incubated overnight at 4C with a rabbit polyclonal antibody specific for human leptin (Y-20 sc-843; Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:200 in TBS. The detection system used was the Envision method (Dako) consisting of a goat anti-rabbit Ig secondary antibody coupled to a peroxidase labeled dextran polymer. The sections were incubated with this reagent for 30 min at room temperature and successively washed in TBS for 5 min and in 0.1 M sodium acetate/acetic acid buffer, pH 6.0, for 5 min. The peroxidase activity was revealed using 0.03% 3,3'-diaminobenzidine (DAB; Sigma, St Louis, MO) in 0.1 M sodium acetate/acetic acid buffer, pH 6.0, containing 2.5% nickel ammonium sulfate, 0.2% ß-D-glucose, 0.04% ammonium chloride, and 0.01% glucose oxidase (
Stomach samples were used as positive controls. In addition, the specificity of the antiserum was tested by absorption of optimally diluted antiserum with the entire peptide (1 nmol of antigen/ml) (catalogue no. 300-27; PeproTech EC, London, UK).
Preparation of Tissue Homogenates and Protein Assay
Ovaries were crushed in a metal cylinder cooled to -80C. Tissue fragments were transferred into a lysis buffer containing 10 mM pH 7.4 Tris (Sigma), 150 mM NaCl (PANREAC; Barcelona, Spain), 1% Triton X-100 (Sigma), 1% deoxicholate (Sigma), 0.1% sodium dodecyl sulfate (Sigma), 5 mM ethylene diamine tetraacetic acid (EDTA; Sigma), and a cocktail of protease inhibitors (Complete Mini EDTA-free; Roche, Mannheim, Germany). After homogenization, samples were centrifuged at 16,060 x g at 4C for 15 min, supernatants were recovered, and the protein content was measured spectrophotometrically using the BCA protein assay kit (Pierce; Rockford, IL).
Western Blotting Analysis
Tissue protein extracts (approximately 100 µg per sample) were electrophoretically separated under reduced conditions using NuPAGE 412% Bis-Tris gels (Invitrogen; Paisley, UK). Recombinant mouse leptin (16 kD; PeproTech EC) was loaded as positive control. Unstained Standard Mark12 (Invitrogen) was used as the molecular weight standard. Proteins were then electrotransferred to 0.2 µm nitrocellulose membranes (BIORAD; Munich, Germany) and the immunoblots were subsequently blocked for 2 hr on an orbital shaker at RT with PBS (pH 7.7) containing 0.1% Tween-20 (Fluka Chemica; Buchs, Switzerland) and 5% nonfat dry milk. The membranes were incubated overnight at 4C with a polyclonal antiserum against leptin (Y-20 sc-843; Santa Cruz Biotechnology) diluted 1:1000 or a monoclonal antibody against ß-actin (Sigma) diluted 1:5000, which was used as an internal control for equal loading. After rinsing the membranes with PBS/0.1% Tween-20, they were incubated in HRP-conjugated anti-rabbit or anti-mouse secondary antibodies (Amersham; Aylesbury, UK) diluted 1:4000 in PBS/Tween/milk for 30 min at RT. After additional rinses in PBS/Tween, the membranes were developed using the Western blotting Lumi-light Plus reagent (Roche). Blots were then exposed to high-performance chemiluminescence film (Hyperfilm ECL, Amersham). Finally, membranes were stained with Coomasie Blue (Serva Blue R tablets; Serva, Heidelberg, Germany) according to the manufacturer's instructions, to visualize protein standards.
Probes
A 405-bp leptin cDNA was generated as an RT-PCR product of the rat leptin mRNA (GenBank accession no.
D45862) using the following primers: 5'-CCAGGATGACACCAAAACCC-3' sense and 5'-TCCAACTGTTGAAGAATGTCC-3' antisense. The PCR product was purified and ligated into the EcoRI site of the pCRII vector (Invitrogen) and used to generate riboprobes. The plasmid was linearized either with Hind III to create a template for antisense probe production or with EcoRV to create a template for the sense probe (Boehringer Mannheim; Mannheim, Germany). Digoxigenin (DIG)-labeled antisense and sense probes were synthesized with T7 and SP6 RNA polymerases, respectively, using a DIG RNA (SP6/T7) labeling kit (Boehringer Mannheim). Probe transcription was carried out at 37C for 2 hr. Riboprobes were precipitated with ethanol at -20C and resuspended in 20 µl of H2O treated with diethilpirocarbonate (DEPC; Sigma) containing 40 U of RNasin (Promega; Madison, WI).
In Situ Hybridization
We followed the protocol used by
Hybridization of each section was performed for 20 hr at 43C in a moist chamber, in a total volume of 20 µl of hybridization buffer [50% formamide, 5 x SSC, 10% dextran sulfate, 5 x Denhardt's solution, 2% sodium dodecyl sulfate (SDS), and diethyl pyrocarbonate-treated water], supplied with 40 ng/ml riboprobe. Hybridization was followed by four washes in 2 x SSC/0.1% SDS at RT, two washes in 0.1 x SSC/0.1% SDS at 48C, and brief rinses in 2 x SSC. Next, the samples were incubated in 2 x SSC containing 25 µg/ml RNase (Boehringer Mannheim) at 37C for 15 min, and additional rinses in 2 x SSC were performed. Visualization of DIG was carried out using a monoclonal antibody coupled to alkaline phosphatase (anti-DIG-AP Fab fragments; Boehringer Mannheim), diluted 1:500, for 2 hr at RT. Nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl-phosphate (Boehringer Mannheim) were used as substrates for the alkaline phosphatase activity. Slides were finally mounted in glycerolPBS.
Controls included use of the sense probe, different dilutions of the antisense probe, omission of the probe, and hybridization of stomach samples as a positive control.
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Results |
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IHC Localization of Leptin
Strong leptin immunoreactivity in oocytes, theca cells, corpora lutea, and interstitial gland (Fig 1A1G, Fig 2A, and Fig 2B) was detected in the different stages of the rat ovary (Fig 1A and Fig 1B) and in the mouse ovary (Fig 1C). A positive reaction was also observed in endothelial cells of some blood vessels, mainly arterioles (Fig 1H). The stroma of both the cortex and the medulla was negative. Absorption control confirmed the specificity of the leptin antiserum (Fig 2C and Fig 2D).
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Follicular Stages. Immunostaining for leptin was extremely dark in the oocytes of primordial (Fig 1D), primary (Fig 1D and Fig 1E), secondary (Fig 1F), and antral (Fig 1G) follicles, appearing in the cytoplasm but not in the nucleus. Leptin positivity was also observed in theca cells of pre-antral follicles (Fig 1E and Fig 1F). Granulosa of all follicular stages was negative (Fig 1D and Fig 1F) except in the mature Graafian follicle, in which a weak reaction appeared in some cells in the periphery and in the cumulus oophorus (Fig 1G). The antral follicles also displayed strong leptin immunoreactivity in the concentrically arranged layers of the inner theca (Fig 1G).
Corpora Lutea. The number and distribution of leptin-positive cells in the corpora lutea depended on its stage of maturation. After ovulation, positive cells were mainly localized in the periphery and only on one side of the corpus luteum. The number of positive cells increased as the corpora lutea matured (Fig 2A). In luteal cells the leptin immunostaining was observed throughout the cytoplasm, except in the many lipid droplets (Fig 2B). Leptin immunostaining was significantly less in the regressing corpora lutea, in which a few positive cells were observed among degenerating luteal cells.
Interstitial Gland. The interstitial gland, well developed in the mature rodent ovary, showed intense leptin immunoreactivity (Fig 1A1C and Fig 1F). The staining pattern was very similar to that of luteal cells. The immunoreaction appeared specifically in the cytoplasm, but not in the multitude of lipid droplets.
Western Blotting Detection of Leptin
Western blotting analysis using the Ob Y-20 antiserum showed that leptin was expressed in the rat ovary, with a positive 16-kD band in most of the samples (Fig 3). Differences in the intensity of the band from different ovaries were observed, depending on the estrous cycle stage. Our data indicate that leptin protein levels increase from estrus to metestrus and decrease in diestrus.
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Detection of Leptin mRNA by ISH
ISH studies agreed with the IHC results for almost all elements of the rat ovary, except for granulosa cells. The optimal staining signal was obtained by microwave heating of tissue sections in citrate buffer (pH 6.0) before the ISH procedure. Hybridization was also found in gastric chief cells of the stomach used as a positive control. When the sense probe was used, no stain was detected either in the ovary (Fig 4B) or in the stomach cells. Leptin mRNA was found in oocytes, theca, corpora lutea, and interstitial gland of the rat ovary (Fig 4A, Fig 4C, and Fig 4D), all of which were also positive for the leptin protein (Table 1). Granulosa cells of early follicular stages were the only cell type positive with ISH (Fig 4A, Fig 4C, and Fig 4D) and negative in sections treated with IHC techniques (Table 1). As occurred for leptin protein, the stroma of both the cortex and the medulla was negative for leptin mRNA (Fig 4A).
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Discussion |
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In this study we examined the expression of the leptin gene and the distribution of its protein in the different preovulatory and postovulatory elements of the rodent ovary. Furthermore, we show the changes that take place in ovarian leptin content throughout the estrous cycle.
Leptin is a hormone involved in regulation of energy balance (
Our results add further weight to the observation that the ovary is an important source of leptin. This is the first report showing in situ leptin mRNA expression in the ovary. It is important to highlight the coincident pattern of staining obtained with IHC and ISH techniques. Both leptin mRNA and protein have been detected in thecal, luteal, and interstitial cells, which are the steroid-producing cell types in the ovary. Moreover, the strongest signal for both techniques appear in the oocytes throughout follicular development. The granulosa cells are the unique element that shows some discrepancy in IHC and ISH results. In early follicular stages these cells show the leptin mRNA but not the protein, which appears only in mature follicles. The degree of immunostaining obtained by IHC and ISH methods in the steroid-producing cells of the rat ovary depends on their maturation stage. Leptin staining varies in follicular cells throughout follicle development and in luteal cells during corpora lutea formation and regression.
Our results show, for the first time, high leptin expression in the oocyte by a combination of IHC and ISH techniques. However, all previous reports had detected leptin protein but not the mRNA in human and mouse oocytes (
In this study we detected the production of leptin in the postovulatory corpus luteum by IHC and ISH. Our findings coincide with previous reports that have detected leptin mRNA by RT-PCR (
As mentioned above, histological studies about leptin expression in ovary are scarce and are sometimes divergent in their observations. The discrepancies found in previous reports and in our study may be due to the different species studied (human, rat, and mouse) and/or to the different antibodies and processing methods (fixation and immunocytochemical techniques) used for the detection of leptin. To clarify some of these differences, in the present study we provide evidence of parallel results of leptin protein distribution in rat and mouse ovaries.
In the present work, Western blotting analysis has confirmed that the leptin antiserum detected a band of the predicted size (16 kD). We have found changes in the intensity of the bands from different ovary samples. Our data indicate a progressive increase in the levels of the hormone from proestrus to metestrus and a decrease in diestrus. These variations would be in agreement with the reciprocal interactions between leptin and estrogens reported previously. On the one hand, estrogens increase in vivo leptin production in rats and humans (
The direct role of leptin in the ovary remains to be fully elucidated. A better knowledge of the histological localization of this hormone in the ovary may be of particular interest from the functional point of view. The present study suggests that corpora lutea and interstitial gland may represent critical sources for the interpretation of leptin fluctuations in rat serum. Further studies are required to clarify the role of ovarian leptin in the complex network of actions of this hormone.
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Acknowledgments |
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Supported by the PIUNA (University of Navarra) and the Spanish Ministry of Science and Technology (project no. BCM2000-1137).
We thank Prof E. Rodríguez (Department of Histology and Pathology, Facultad de Medicina, Universidad Austral de Chile) for helpful suggestions, M.T. Sabata and A. Urbiola for technical assistance, and K. Pfeiffer for English revision.
Received for publication September 23, 2002; accepted May 27, 2003.
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