Journal of Histochemistry and Cytochemistry, Vol. 51, 1269-1277, October 2003, Copyright © 2003, The Histochemical Society, Inc.


ARTICLE

Leptin Expression in the Rat Ovary Depends on Estrous Cycle

Marta Archancoa, Francisco J. Muruzábala, Diana Llopiza, Mercedes Garayoaa, Javier Gómez–Ambrosib, Gema Frühbeckb,c, and María A. Burrella
a Department of Histology and Pathology, Clínica Universitaria de Navarra, Pamplona, Spain
b Metabolic Research Laboratory, Clínica Universitaria de Navarra, Pamplona, Spain
c University of Navarra, Pamplona, Spain, and Department of Endocrinology, Clínica Universitaria de Navarra, Pamplona, Spain

Correspondence to: María A. Burrell, Dept. of Histology and Pathology, University of Navarra, C/Irunlarrea 1, 31008 Pamplona, Spain. E-mail: mburrell@unav.es


  Summary
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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 51:1269–1277, 2003)

Key Words: leptin, ovary, estrous cycle, oocyte, follicular cells, corpora lutea, interstitial gland, immunohistochemistry, Western blotting, in situ hybridization


  Introduction
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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 (Zhang et al. 1994 ). More recently, leptin has also been detected in a variety of tissues such as placenta (Cioffi et al. 1996 ), stomach (Bado et al. 1998 ), skeletal muscle (Wang et al. 1998 ), mammary glands (Smith-Kirwin et al. 1998 ), brain, and pituitary (Morash et al. 1999 ). This wide pattern of expression suggests very different functions of leptin in the organism, including reproduction.

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 (Zhang et al. 1994 ). After treatment with human recombinant leptin, these ob/ob mice recovered fertility (Chehab et al. 1996 ). Furthermore, different central and peripheral effects of this molecule have been described in reproductive organs, providing new insights into the physiology of reproduction. The main function of leptin is to act at various levels of the hypothalamic–pituitary–gonadal (HPG) axis via endocrine, paracrine, and/or autocrine pathways (Caprio et al. 2001 ). The long form of the leptin receptor (Ob-R) has been localized in mouse (Vaisse et al. 1996 ) and rat (Elmquist et al. 1998 ) hypothalamus and in rat pituitary gland (Sone et al. 2001 ). It has been suggested that leptin has a facilitatory effect on the central networks that regulate pituitary gonadotropin secretion (Ahima et al. 1996 ; Finn et al. 1998 ; Pinilla et al. 1999 ). In addition, this hormone appears to be involved in the regulation of growth and differentiation of pituitary cells (Jin et al. 2000 ). Other researchers have identified the Ob-R in peripheral tissues of male and female rats such as testis, ovary, uterus (Zamorano et al. 1997 ), and adrenal gland (Bornstein et al. 1997 ).

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 (Cioffi et al. 1996 , Cioffi et al. 1997 ; Karlsson et al. 1997 ; Spicer and Francisco 1997 ). In the human ovary, Ob-R mRNA has been found in the theca and granulosa cells (Karlsson et al. 1997 ) and appears to have an inhibitory effect on estradiol production. There is controversy about the interaction between leptin and steroid hormones. Several authors have reported that leptin suppresses ovarian steroid synthesis in different species (Karlsson et al. 1997 ; Spicer and Francisco 1997 ; Zachow and Magoffin 1997 ; Agarwal et al. 1999 ; Barkan et al. 1999 ; Caprio et al. 2001 ). On the other hand, one study has demonstrated that leptin has a stimulatory effect on estrogen production and, consequently, on aromatase activity in human luteinized granulosa cells (Kitawaki et al. 1999 ). More recently, Duggal et al. 2002 have reported variations in ovarian leptin mRNA at different times of the rat estrous cycle.

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 (Antczak and Van Blerkom 1997 ). In addition, both leptin and its receptor have been localized in cultured ovarian cells from the granulosa, cumulus cells of preantral follicles, and mature oocytes from in vitro-fertilized women (Cioffi et al. 1997 ). More recently, leptin expression has been detected in histological sections of normal and polycystic human ovaries (Loffler et al. 2001 ) and in murine ovary (Ryan et al. 2002 ), although there is no total agreement on the exact cellular distribution of this expression at each follicular stage. Loffler et al. 2001 detected leptin-like immunoreactivity in the developing corpus luteum and also in oocytes, granulosa, and theca, but not in all stages. On the other hand, Ryan et al. 2002 observed strong immunostaining in oocytes and weaker staining in granulosa, stroma, theca, and corpora lutea.

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.


  Materials and Methods
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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 21–23C, 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 (Shu et al. 1988 ). The reaction was stopped by a wash in TBS. Finally the sections were dehydrated and mounted with DPX.

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 4–12% 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 Garcia et al. 1998 with minor modifications. Sections (5 µm thick) of formalin-fixed ovaries were mounted on slides coated with ProbeON Plus (Fisher Biotech; Pittsburgh, PA), deparaffinized, rehydrated, and permeabilized in 0.2% Triton–PBS for 15 min. Rat tissues were either microwave-preheated for 10 min at 750 W in 0.01 M citrate buffer (pH 6.0) or digested for 30 min at 37C with 20 µg/ml proteinase K (Sigma) in 0.1 M Tris/0.05 M EDTA, pH 8.0. Tissues were acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine, pH 8, washed in DEPC–H2O, and air-dried at RT.

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 glycerol–PBS.

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.


  Results
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

IHC Localization of Leptin
Strong leptin immunoreactivity in oocytes, theca cells, corpora lutea, and interstitial gland (Fig 1A–1G, 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).



View larger version (173K):
[in this window]
[in a new window]
 
Figure 1. Immunohistochemical detection of leptin in the rodent ovary. Panoramic views of rat ovaries from diestrous (A) and estrous (B) stages immunostained for leptin. (C) A mouse ovary showing a similar staining pattern for leptin. Bars = 200 µm. (D) Detail of one primordial follicle (arrow) and two primary unilaminar (arrowheads) follicles, all of them showing strong leptin immunoreactivity in the oocyte cytoplasm. (E) In the primary multilaminar follicles, leptin immunostaining appears in the oocyte and in the differentiating thecal cells (arrows). Bars = 20 µm. (F) A pre-antral follicle exhibits leptin immunolabeling in the oocyte and the more developed theca (arrowheads). Intense staining is also observed in the interstitial gland (asterisks). (G) An intact antral follicle shows a leptin immunostain similar to the previous stages. Furthermore, some cells in the periphery of the granulosa (arrowheads) and in the cumulus oophorus (arrow) stain faintly. Bars = 80 µm. (H) The endothelium of some blood vessels is positive for leptin. Bar = 20 µm.



View larger version (190K):
[in this window]
[in a new window]
 
Figure 2. (A) A corpus luteum in the rat ovary is immunolabeled for leptin. The labeling appears in some secretory cells shown in detail in B. (C,D) Absorption control for the Y-20 sc-843 antiserum. The immunostaining for leptin in a corpus luteum (C) disappeared in the contiguous section (D) when the antiserum was preabsorbed with 1 nmol ml-1 of the entire leptin peptide. Bars: A,C,D = 80 µm; B = 10 µm.

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 1A–1C 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.



View larger version (42K):
[in this window]
[in a new window]
 
Figure 3. Representative Western blotting analysis of leptin (A) and ß-actin (B) in rat ovary samples (100 µg of protein). (A) Recombinant mouse leptin was used as control (L). Note the differences in intensity of the 16-kD band in the different samples (P, Proestrus; E, estrus; M, metestrus; D, diestrus). (B) ß-Actin staining shows equivalent sample loading.

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).



View larger version (195K):
[in this window]
[in a new window]
 
Figure 4. Detection of leptin mRNA in the rodent ovary by ISH. (A) Panoramic view of the rat ovary stained with ISH for leptin. (B) Staining disappeared in the consecutive section treated with the sense riboprobe. Bars = 200 µm. (C) Strong staining is observed in the secretory cells of a corpus luteum (CL) and in the oocyte (asterisk) of a pre-antral follicle. (D) Labeling also appears in thecal (arrowheads) and granulosa cells (G) of all the follicles. Faint reaction is detected in the interstitial gland (asterisks). Bars = 40 µm.


 
View this table:
[in this window]
[in a new window]
 
Table 1. Distribution of leptin mRNA (ISH) and protein (IHC) stains in the rat ovarya


  Discussion
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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 (Zhang et al. 1994 ) but also in very different functions such as hematopoiesis (Cioffi et al. 1996 ), the immune response (Loffreda et al. 1998 ), and reproduction (Caprio et al. 2001 ). With regard to reproductive function, several reports have shown that this hormone has significant effects on the different cell types of the ovary (Spicer and Francisco 1997 ; Zachow and Magoffin 1997 ; Duggal et al. 2000 ; Spicer et al. 2000 ; Caprio et al. 2001 ; Loffler et al. 2001 ; Ryan et al. 2002 ). Initial studies have reported abundant expression of the leptin receptor in ovary (Cioffi et al. 1997 ; Karlsson et al. 1997 ; Spicer and Francisco 1997 ). In vitro studies on rodent and bovine ovary demonstrated the inhibitory effect of leptin on secretion of steroids (Spicer and Francisco 1997 , Spicer and Francisco 1998 ; Zachow and Magoffin 1997 ) and ovulation (Duggal et al. 2000 ). Leptin and its receptors have been investigated in normal and polycystic human ovaries (Cioffi et al. 1997 ; Loffler et al. 2001 ) and in murine ovary (Ryan et al. 2002 ). These reports agree in the fact that oocytes and corpora lutea are two main sites of leptin expression. However, there is controversy about other possible sites of leptin production in the ovary, in particular, the granulosa and thecal layers.

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 (Antczak and Van Blerkom 1997 ; Cioffi et al. 1997 ; Loffler et al. 2001 ; Ryan et al. 2002 ). It has been suggested that leptin plays a critical role in early mammalian development (Antczak and Van Blerkom 1997 ). Regarding the granulosa cells, leptin protein (by IHC) and mRNA (by RT-PCR) have been detected in human cultured cells (Cioffi et al. 1997 ) and in the intact ovary (Loffler et al. 2001 ) although there is controversy about the follicular stage in which they appear. In another report, Ryan et al. 2002 found slight reactivity in the granulosa of murine ovary but did not specify the follicular stage. Our results are in only partial agreement with the previous observations. We have found that leptin mRNA is expressed in granulosa cells of all growing follicles but that the leptin protein appeared only in mature ones. The signal has been detected in scattered granulosa peripheral cells and in the cumulus oophorus. In the latter, leptin expression has been previously reported by Cioffi et al. 1997 . We suggest that the discrepancy in our IHC and ISH findings can be related to the changes that take place in granulosa cells throughout maturation of follicles. These cells may express leptin mRNA from the beginning of follicular development but its translation may not occur until their differentiation in luteal cells after ovulation. Finally, the theca is another important source of leptin in the preovulatory follicles. We have detected strong staining in the thecal layer by both IHC and ISH. However, other authors have reported only weak positivity for leptin protein in thecal cells of human mature follicles (Loffler et al. 2001 ) and mouse ovary (Ryan et al. 2002 ).

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 (Loffler et al. 2001 ) and protein by IHC (Loffler et al. 2001 ; Ryan et al. 2002 ). We found that the number of luteal cells containing leptin protein increases during maturation and decreases during regression of the corpora lutea. Immunoreactivity for leptin appears throughout the cytoplasm of these steroid-secreting cells. Loffler et al. 2001 also reported variations in leptin mRNA and protein in the corpus luteum progression. However, contrary to our findings, these authors reported that the greatest amounts of leptin are found in the developing corpus luteum, whereas lesser amounts were found in the secretory phase. This contradiction may be explained by the fact that they obtained the stain not in the steroid-producing cells but in fibroblast-like cells derived from the thecal layer. Concerning the well-developed interstitial gland in rodent ovary, our results show strong positivity with IHC and weaker positivity with ISH. The only report of leptin in murine ovary did not refer to this gland (Ryan et al. 2002 ).

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 (Shimizu et al. 1997 ) and, on the other hand, there is an inhibitory effect of leptin in ovarian steroidogenesis (Caprio et al. 2001 ). A recent report indicates significant variations in Ob-R gene expression in the rat ovary during the estrous cycle, apparently in response to the changing hormonal environment (Duggal et al. 2002 ). These authors showed that the mRNA content for the long and short isoforms of the Ob-R is lower in proestrus and diestrus II than in estrus and diestrus I (metestrus), thus modulating ovarian sensitivity to the leptin protein. The high Ob-R gene expression in estrus and metestrus may increase the action of leptin protein on the corpora lutea (Duggal et al. 2002 ). Therefore, both leptin and its receptors respond to cyclical changes throughout the estrous cycle. Although several authors have reported unchanged circulating serum leptin concentrations throughout the estrous cycle in rodents (Chehab et al. 1997 ; Bennett et al. 1999 ), it is difficult to establish the quantitative contribution of leptin secreted by the different ovarian cell types to the serum leptin concentrations.

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.


  Acknowledgments

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.


  Literature Cited
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Agarwal SK, Vogel K, Weitsman SR, Magoffin DA (1999) Leptin antagonizes the insulin-like growth factor-I augmentation of steroidogenesis in granulosa and theca cells of the human ovary. J Clin Endocrinol Metab 84:1072-1076[Abstract/Free Full Text]

Ahima RS, Prabakaran D, Mantzoros C, Qu D, Lowell B, Maratos–Flier E, Flier JS (1996) Role of leptin in the neuroendocrine response to fasting. Nature 382:250-252[Medline]

Antczak M, Van Blerkom J (1997) Oocyte influences on early development: the regulatory proteins leptin and STAT3 are polarized in mouse and human oocytes and differentially distributed within the cells of the preimplantation stage embryo. Mol Hum Reprod 3:1067-1086[Abstract]

Bado A, Levasseur S, Attoub S, Kermorgant S, Laigneau JP, Bortoluzzi MN, Moizo L et al. (1998) The stomach is a source of leptin. Nature 394:790-793[Medline]

Barkan D, Jia H, Dantes A, Vardimon L, Amsterdam A, Rubinstein M (1999) Leptin modulates the glucocorticoid-induced ovarian steroidogenesis. Endocrinology 140:1731-1738[Abstract/Free Full Text]

Bennett PA, Lindell K, Wilson C, Carlsson LM, Carlsson B, Robinson IC (1999) Cyclical variations in the abundance of leptin receptors, but not in circulating leptin, correlate with NPY expression during the oestrous cycle. Neuroendocrinology 69:417-423[Medline]

Bornstein SR, Uhlmann K, Haidan A, Ehrhart–Bornstein M, Scherbaum WA (1997) Evidence for a novel peripheral action of leptin as a metabolic signal to the adrenal gland: leptin inhibits cortisol release directly. Diabetes 46:1235-1238[Abstract]

Caprio M, Fabbrini E, Isidori AM, Aversa A, Fabbri A (2001) Leptin in reproduction. Trends Endocrinol Metab 12:65-72[Medline]

Chehab FF, Lim ME, Lu R (1996) Correction of the sterility defect in homozygous obese female mice by treatment with the human recombinant leptin. Nature Genet 12:318-320[Medline]

Chehab FF, Mounzih K, Lu R, Lim ME (1997) Early onset of reproductive function in normal female mice treated with leptin. Science 275:88-90[Abstract/Free Full Text]

Cioffi JA, Shafer AW, Zupancic TJ, Smith–Gbur J, Mikhail A, Platika D, Snodgrass HR (1996) Novel B219/OB receptor isoforms: possible role of leptin in hematopoiesis and reproduction. Nature Med 2:585-589[Medline]

Cioffi JA, Van Blerkom J, Antczak M, Shafer A, Wittmer S, Snodgrass HR (1997) The expression of leptin and its receptors in pre-ovulatory human follicles. Mol Hum Reprod 3:467-472[Abstract]

Duggal PS, Van Der Hoek KH, Milner CR, Ryan NK, Armstrong DT, Magoffin DA, Norman RJ (2000) The in vivo and in vitro effects of exogenous leptin on ovulation in the rat. Endocrinology 141:1971-1976[Abstract/Free Full Text]

Duggal PS, Weitsman SR, Magoffin DA, Norman RJ (2002) Expression of the long (OB-RB) and short (OB-RA) forms of the leptin receptor throughout the oestrous cycle in the mature rat ovary. Reproduction 123:899-905[Abstract/Free Full Text]

Elmquist JK, Bjorbaek C, Ahima RS, Flier JS, Saper CB (1998) Distributions of leptin receptor mRNA isoforms in the rat brain. J Comp Neurol 395:535-547[Medline]

Finn PD, Cunningham MJ, Pau KY, Spies HG, Clifton DK, Steiner RA (1998) The stimulatory effect of leptin on the neuroendocrine reproductive axis of the monkey. Endocrinology 139:4652-4662[Abstract/Free Full Text]

García C, Montuenga LM, Medina JF, Prieto J (1998) In situ detection of AE2 anion-exchanger mRNA in the human liver. Cell Tissue Res 291:481-488[Medline]

Jin L, Zhang S, Burguera BG, Couce ME, Osamura RY, Kulig E, Lloyd RV (2000) Leptin and leptin receptor expression in rat and mouse pituitary cells. Endocrinology 141:333-339[Abstract/Free Full Text]

Karlsson C, Lindell K, Svensson E, Bergh C, Lind P, Billig H, Carlsson LM et al. (1997) Expression of functional leptin receptors in the human ovary. J Clin Endocrinol Metab 82:4144-4148[Abstract/Free Full Text]

Kitawaki J, Kusuki I, Koshiba H, Tsukamoto K, Honjo H (1999) Leptin directly stimulates aromatase activity in human luteinized granulosa cells. Mol Hum Reprod 5:708-713[Abstract/Free Full Text]

Löffler S, Aust G, Kohler U, Spanel–Borowski K (2001) Evidence of leptin expression in normal and polycystic human ovaries. Mol Hum Reprod 7:1143-1149[Abstract/Free Full Text]

Loffreda S, Yang SQ, Lin HZ, Karp CL, Brengman ML, Wang DJ, Klein AS et al. (1998) Leptin regulates proinflammatory immune responses. FASEB J 12:57-65[Abstract/Free Full Text]

Morash B, Li A, Murphy PR, Wilkinson M, Ur E (1999) Leptin gene expression in the brain and pituitary gland. Endocrinology 140:5995-5998[Abstract/Free Full Text]

Pinilla L, Seoane LM, González L, Carro E, Aguilar E, Casanueva FF, Diéguez C (1999) Regulation of serum leptin levels by gonadal function in rats. Eur J Endocrinol 140:468-473[Medline]

Ryan NK, Woodhouse CM, Van Der Hoek KH, Gilchrist RB, Armstrong DT, Norman RJ (2002) Expression of leptin and its receptor in the murine ovary: possible role in the regulation of oocyte maturation. Biol Reprod 66:1548-1554[Abstract/Free Full Text]

Shimizu H, Shimomura Y, Nakanishi Y, Futawatari T, Ohtani K, Sato N, Mori M (1997) Estrogen increases in vivo leptin production in rats and human subjects. J Endocrinol 154:285-292[Abstract]

Shu SY, Ju G, Fan LZ (1988) The glucose oxidase-DAB-nickel method in peroxidase histochemistry of the nervous system. Neurosci Lett 85:169-171[Medline]

Smith–Kirwin SM, O'Connor DM, De Johnston J, Lancey ED, Hassink SG, Funanage VL (1998) Leptin expression in human mammary epithelial cells and breast milk. J Clin Endocrinol Metab 83:1810-1813[Abstract/Free Full Text]

Sone M, Nagata H, Takekoshi S, Osamura RY (2001) Expression and localization of leptin receptor in the normal rat pituitary gland. Cell Tissue Res 305:351-356[Medline]

Spicer LJ, Chamberlain CS, Francisco CC (2000) Ovarian action of leptin: effects on insulin-like growth factor-I-stimulated function of granulosa and thecal cells. Endocrine Rev 12:53-59

Spicer LJ, Francisco CC (1997) The adipose obese gene product, leptin: evidence of a direct inhibitory role in ovarian function. Endocrinology 138:3374-3379[Abstract/Free Full Text]

Spicer LJ, Francisco CC (1998) Adipose obese gene product, leptin, inhibits bovine ovarian thecal cell steroidogenesis. Biol Reprod 58:207-212[Abstract]

Vaisse C, Halaas JL, Horvath CM, Darnell JE, Jr, Stoffel M, Friedman JM (1996) Leptin activation of Stat3 in the hypothalamus of wild-type and ob/ob mice but not db/db mice. Nature Genet 14:95-97[Medline]

Wang J, Liu R, Hawkins M, Barzilai N, Rossetti L (1998) A nutrient-sensing pathway regulates leptin gene expression in muscle and fat. Nature 393:684-688[Medline]

Zachow RJ, Magoffin DA (1997) Direct intraovarian effects of leptin: impairment of the synergistic action of insulin-like growth factor-I on follicle-stimulating hormone-dependent estradiol-17 beta production by rat ovarian granulosa cells. Endocrinology 138:847-850[Abstract/Free Full Text]

Zamorano PL, Mahesh VB, De Sevilla LM, Chorich LP, Bhat GK, Brann DW (1997) Expression and localization of the leptin receptor in endocrine and neuroendocrine tissues of the rat. Neuroendocrinology 65:223-228[Medline]

Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM (1994) Positional cloning of the mouse obese gene and its human homologue. Nature 372:425-432[Medline]





This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Archanco, M.
Articles by Burrell, M. A.
Articles citing this Article
PubMed
PubMed Citation
Articles by Archanco, M.
Articles by Burrell, M. A.


Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]