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Regular Article |
a Reproductive Medicine Unit, Department of Obstetrics and Gynaecology, The University of Adelaide, The Queen Elizabeth Hospital, Woodville, South Australia 5011, Australia
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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cytokines, leptin, leptin receptor, oocyte development
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Leptin was originally thought to be produced exclusively by adipose tissue [1]. More recently, however, leptin mRNA and its protein product have been found using reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence in human cumulus and granulosa cells [7]. With RT-PCR, leptin receptor mRNA has been found in the human ovary, in thecal and granulosa cells [8, 9]. Mutations in either the ob gene or the leptin receptor gene (db) result in obesity as well as infertility [1, 10]. The administration of leptin to leptin-deficient mice has the ability to restore normal weight as well as reproductive function [11].
Leptin is found in follicular fluid in the ovary, and the addition of leptin to isolated ovarian cells such as theca and granulosa has the ability to affect steroidogenesis, indicating that this protein has direct effects on the ovary [12–16]. We have shown that the administration of leptin to rats and the treatment of rat ovaries in vitro with leptin both decrease ovulation [17].
Leptin also affects the oocyte. The addition of leptin to pig oocytes in vitro advances the onset of germinal vesicle breakdown (GVB) and reduces cumulus cell coupling [18]. Leptin activates signal transducer and activator of transcription 3 (STAT3) in the mouse oocyte, indicating a function in the regulation of transcription [19]. Leptin activates phosphodiesterase 3B (PDE3B) and hence decreases cAMP levels in rat pancreatic cells [20] and hepatocytes [21]. PDE3B is also present in the oocyte [22] and decreases cAMP levels in the oocyte to signal the initiation of GVB, leading to oocyte maturation [23]. Therefore, leptin-induced activation of oocyte PDE3B could initiate maturation through decreased oocyte cAMP levels.
The present experiments were undertaken to localize expression of leptin and its receptor in whole tissue sections of the mouse ovary. We also aimed to determine the effect of leptin on mouse oocyte maturation using several different in vitro oocyte maturation models, and to relate these effects to possible changes in oocyte cAMP levels.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sexually immature SV129 female mice (27–33 days of age) were used throughout this study. The animals were bred and housed at The Queen Elizabeth Hospital animal house at 24°C on a 14L:10D illumination cycle with water and pelleted food available ad libitum. The animal ethics committees of both The Queen Elizabeth Hospital and the University of Adelaide approved all experiments, and the animals were handled in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. Mice were injected i.p. with 5 IU eCG (Intervet, Boxmeer, Holland) in 0.1 ml of PBS (Gibco BRL, Life Technologies, Grand Island, NY) with 0.1% BSA (fraction V; Sigma Aldrich Chemical Co., St. Louis, MO) and, 48 h later, were either killed (preovulatory) or injected i.p. with 5 IU hCG (Pregnyl; N.V. Organon, Oss, The Netherlands) in 0.1 ml of PBS with 0.1% BSA and killed 24 h later (postovulatory).
Immunohistochemistry
Both preovulatory and postovulatory mice were used, and at each time point, both ovaries and the hypothalamus were collected and frozen in Jung Tissue Freezing Medium (Leica Instruments GmbH, Nussloch, Germany) and stored at -80°C. These tissues were then stained using immunohistochemistry to determine the sites of expression of both leptin and its receptor protein within a complete tissue section. Six-micrometer serial tissue sections were cut using a -20°C Leica cryostat (Leica Instruments GmbH, Nussloch, Germany). These sections were then brought to room temperature and fixed with 96% alcohol at 4°C for 10 min. They were then incubated with primary antibody; either Ob-Y (Santa Cruz Biotechnology, Santa Cruz, CA) or anti-leptin receptor antibody (Affinity Bioreagents Inc., Golden, CO) diluted 1:100 in PBS containing 10% normal mouse serum (NMS) and 1% BSA (PBS-BSA-NMS) for a period of 16 h in a humidified container at 4°C. The primary antibody was then washed off in 3 successive PBS washes, followed by a PBS-1% BSA wash, and incubated with biotinylated goat anti-rabbit secondary antibody (Vector Laboratories, Burlingame, CA) diluted 1:100 in PBS-BSA-NMS for 2 h at 4°C. This antibody was then washed off in PBS and PBS-1% BSA, and the sections were incubated with avidin-horseradish-peroxidase (DAKO Corporation, Carpinteria, CA) diluted 1:400 in PBS-BSA-NMS for 40 min at 4°C. After the PBS washes, the enzyme was visualized using Sigma-Fast DAB tablets (Sigma Aldrich) diluted in water and applied for 10 min at room temperature. The sections were then counterstained with hematoxylin, dehydrated, and mounted in DPX (both from BDH Laboratory Supplies, Poole, England). Two types of controls were included. For negative controls, primary antibody was omitted and replaced with PBS-BSA-NMS. For preabsorption controls, primary antibodies were preincubated for 24 h at 4°C at a ratio of 1:100 with either leptin (Diagnostic Systems Laboratories, Inc., Webster, TX) or leptin receptor peptide (Affinity Bioreagents), and immunohistochemistry was performed.
Oocyte Preparation
Preovulatory mouse ovaries were removed and placed into Hepes-buffered tissue culture medium 199 (TCM 199) (ICN Biochemicals Inc., Costa Mesa, CA) containing 2 mM sodium bicarbonate, 2 mM sodium pyruvate, 25 mM Hepes, (all from Sigma Aldrich), and 1% penicillin-streptomycin solution (CSL Biosciences, Victoria, Australia). The media were also supplemented with 0.3% BSA, and PDE inhibitor: either 200 µM 3-isobutyl-1-methylxanthine (IBMX) or 2 µM milrinone (MR) (both from Sigma Aldrich).
For collection of isolated oocytes, cumulus-oocyte complexes (COCs) were recovered by puncturing large antral follicles with sterile 29-gauge insulin needles (Becton Dickinson, Franklin Lakes, NJ) to release the oocytes into the media. Denuded oocytes (DOs) were prepared by carefully passing COCs through a sterile mouth-controlled, finely drawn Pasteur pipette to remove the cumulus cells. For collection of follicle-enclosed oocytes, large preovulatory follicles (>400 µm in diameter) were dissected out from the ovary using sterile watchmakers forceps under a dissecting microscope, and rinsed in Hepes-buffered TCM supplemented with 0.3% BSA.
Oocyte Maturation
Isolated oocytes, cumulus-enclosed or denuded, were washed 3 times in IBMX/MR-free media and then washed once in bicarbonate buffered TCM 199 containing 25 mM sodium bicarbonate, 2 mM sodium pyruvate, 1% penicillin-streptomycin solution, and 2.5 µM phenol red (Sigma Aldrich). The media were also supplemented with 0.3% BSA, before being placed into culture. Oocytes were cultured in four-well dishes (Nunc Brand Products, Roskilde, Denmark) at a density of approximately 25 oocytes per well in 250 µl bicarbonate-buffered TCM 199 (control) or media containing combinations of one or more of the following: 100 mIU/ml recombinant human FSH (rhFSH) (N.V. Organon), 50 µM IBMX, 2 µM milrinone, and 10–1000 ng/ml human recombinant leptin. Oocytes were incubated at 37°C for 24 h in 5% CO2 in air. After incubation, the oocytes were assessed microscopically for the presence or absence of a GV, which indicates whether the oocyte has been maintained in meiotic arrest.
For culture of follicle-enclosed oocytes, 5 isolated follicles prepared as previously described were transferred to stoppered glass Vacutainers (Becton Dickinson) in 1 ml of alpha minimum essential media (MEM) (Trace Biosciences Pty. Ltd., NSW Australia) supplemented with 25 mM sodium bicarbonate and 0.5% heat-inactivated NMS (control). Treatment groups consisted of control media alone or containing either 1) 500 mIU/ml rhFSH and 1 µg/ml ovine LH (pLH-26 no. AFP-5551B, kindly provided by National Institute of Diabetes and Digestive Kidney Diseases, Bethesda, MD) or 2) 10–1000 ng/ml leptin. An incubation in FSH + LH was included as a positive control treatment to determine whether the follicles being used had the ability to respond to gonadotropin stimulation. The tubes were then gassed with a mixture of 95% O2 and 5% CO2 and placed in a 37°C water bath for 4 h. At the end of the incubation period, culture media were removed and frozen at -20°C for progesterone and estradiol assays. The follicles were pierced, and the oocytes were recovered and visually assessed for the presence or absence of a GV. Oocytes from dead or atretic follicles were omitted (5% of all follicles).
The results are presented as the percentage of oocytes undergoing GVB, which was determined by calculating the number of oocytes that underwent GVB as a percentage of the total number of oocytes.
Oocyte Culture for cAMP Analysis
COCs collected as described were cultured for an initial 1-h period (primary culture period) in bicarbonate-buffered TCM containing 0.3% BSA with 2 µM MR and 125 µM forskolin (FK) (Sigma Aldrich). The oocytes were then rinsed 3 times, denuded, and cultured for an additional hour (secondary culture period) in 250 µl bicarbonate-buffered TCM containing 0.3% BSA containing either 62.5 µM or 125 µM FK with or without 100 ng/ml leptin. At the end of the secondary culture period, the oocytes were removed in 40 µl media and transferred to 460 µl cold 100% ethanol and stored at -20°C for cAMP assay.
Cyclic AMP Assay
An RIA method described and validated previously [24] was used to determine cAMP levels in DO extracts. Briefly, samples were vortexed for 30 sec and then pelleted by centrifugation for 30 sec. The supernatant was removed (450 µl), dried, and then reconstituted in 220 µl of 50 mM sodium acetate pH 5.5 (assay buffer) and acetylated using 6.6 µl of a 2:1 (v:v) solution of triethylamine (AJAX Chemicals, Sydney, Australia) and acetic anhydride (BDH Laboratory Supplies). Samples were divided into 100-µl replicates and 125I-labeled cAMP (specific activity, 2175 Ci/mM) was added; the labeled cAMP was prepared by iodinating 2'-0-monosuccinyladenosine 3':5'-cyclic monophosphate tyrosyl methyl ester (Sigma Aldrich) using the chloramine T method [25]. Cyclic AMP antibody (as prepared by Reddoch et al. [24]) was added at a final dilution of 1:850, and the samples were covered and left overnight at 4°C. The following day, 1 ml of cold 100% ethanol was added, the tubes were centrifuged, the supernatant was removed, and the pellet was dried and counted using a gamma counter (LKB-Wallac; Perkin Elmer, Shelton, CT). Triplicate samples were produced to create a standard curve (0–800 fmol cAMP).
Steroid Analysis
Steroid levels in media collected from the follicle cultures were assayed with a chemiluminescence immunoassay system (Johnson & Johnson Vitros ECI; Orthoclinical Diagnostics, Amersham Pharmacia Biotech, Freiburg, Germany) specific for estradiol and progesterone. The sensitivities of the assays were 10 and 0.3 pmol/L for estradiol and progesterone, respectively, and the interassay coefficients of variation were <6% and <8%, respectively.
Statistics
Chi-square contingency tables were used to test independence and trends between meiotic status of oocytes in each group (i.e., GV or GVB) for different treatments in oocyte and follicle maturation. Progesterone and estradiol results were assessed by a one-way ANOVA with Tukey post hoc analysis. In all cases, P < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Leptin protein Staining for leptin protein in tissue sections from all ovaries was extremely dark in the oocyte of follicles of all sizes, indicating that there is a high level of immunoreactive leptin within these cells (Fig. 1A). Lighter protein staining was observed in the granulosa cells, stroma, and corpora lutea (Fig. 1C). Only marginal staining was observed in the thecal cell layer (Fig. 1A). Leptin protein staining in the hypothalamus was positive. Negative ovary controls are shown in Figure 1, B and D. Preabsorption controls showed no obvious positive staining.
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Leptin receptor The intensity of staining for the leptin receptor was highest in the thecal layer (Fig. 1E). Lighter staining for the receptor was observed in oocytes, granulosa cells, and stroma (Fig. 1E) as well as in corpora lutea (Fig. 1G). Hypothalamic staining was also positive for leptin receptor. Negative ovary controls are shown in Figure 1, F and H.
Preabsorption controls for leptin receptor showed reduced positive staining.
Oocyte Meiotic Maturation
Denuded oocytes DOs were examined to determine if leptin could act on the receptors observed on the oocyte and affect oocyte maturation directly. Both PDE inhibitors, IBMX and MR, were highly effective in preventing spontaneous oocyte maturation (Fig. 2). IBMX is a nonspecific PDE inhibitor, whereas MR is specific for PDE3, the isoform of the enzyme specific to oocytes [22]. Leptin by itself did not inhibit spontaneous meiotic resumption of DOs or reverse the inhibitory effects of IBMX or MR (Fig. 2).
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Cumulus-oocyte complexes Leptin did not affect the spontaneous resumption of meiosis in isolated COCs (Fig. 3). It also did not induce meiotic resumption of MR- or IBMX-treated oocytes, in contrast to FSH, which was able to overcome the effects of both inhibitors. Leptin also had no effect on FSH-induced maturation.
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Follicle-enclosed oocytes To determine whether leptin has the ability to affect the maturation of an oocyte within its normal follicle environment, preovulatory follicle-enclosed oocytes were cultured for 4 h, after which the oocytes were recovered to determine their meiotic status. In follicles cultured without added hormones, 96% of oocytes remained in the GV stage, illustrating the ability of the intact follicular milieu to maintain meiotic arrest under these in vitro conditions. Addition of FSH + LH was highly effective in overcoming this inhibition, causing GVB to occur in 83% of oocytes, consistent with the physiologic role of these gonadotropins as triggers of oocyte maturation in the preovulatory follicle in vivo. The addition of leptin to cultured follicles in the absence of gonadotropins resulted in increased GVB in oocytes at all concentrations tested when compared with control oocytes (Fig. 4), although the maximum level of meiotic resumption observed at the highest leptin concentration (36% GVB) fell considerably short of that induced by FSH + LH (83% GVB).
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After the 4-h culture period, as the follicles were removed and GV status was determined, culture media was stored and later assayed for progesterone and estradiol. Although increasing leptin concentrations tended to be associated with higher progesterone levels, at no concentration did leptin have any statistically significant effect on the follicle steroid production (Fig. 5). Steroid levels for the FSH + LH group were 12.3 and 4.2 pmol per follicle for progesterone and estradiol, respectively.
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Cyclic AMP
Experiments were conducted to investigate whether the ability of leptin to induce meiotic resumption in follicle-enclosed oocytes was the result of a stimulatory effect on oocyte PDE, which would reduce intraoocyte levels of cAMP. Oocytes were precultured for 1 h as intact COCs in the presence of MR and FK to increase initial cAMP levels within the COCs. FK increases adenylate cyclase activity nonspecifically, and MR acts to inhibit the oocyte-specific PDE3 and hence cAMP degradation, thereby maintaining high intracellular levels of cAMP within the oocytes. After this initial primary culture, the cumulus cells were removed, and the DOs were cultured for an additional 1-h secondary culture period in the presence of FK with or without added leptin but without MR. Although both 62.5 and 125 µM FK resulted in 5-fold increases in cAMP, the addition of leptin caused no significant reductions in oocyte cAMP concentration (Fig. 6), indicating a lack of effect of leptin in stimulating oocyte PDE activity.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Leptin mRNA is not present in the murine or human oocyte [7, 19], although its protein product is found in both [7, 27]. It has been suggested that leptin may enter the oocyte by endocytosis after production elsewhere [27]. The amount of leptin protein found in the oocyte has been suggested to increase as the oocyte matures from GV to the second metaphase (MII), indicating an influx of leptin into the oocyte at this time [19]. The physiologic function of this influx, if any, is unknown.
Leptin also has the ability to affect isolated ovarian cells in vitro. Both theca and granulosa cells contain leptin receptors, and the addition of leptin to isolated human and bovine granulosa cells can affect hCG-, LH-, and insulinlike growth factor I (IGF-I)-induced steroid production [9, 12, 13, 15]. In rat granulosa cells, leptin inhibits FSH- and IGF-I-stimulated estradiol production [16].
We have shown using immunohistochemistry that leptin protein is present in several ovarian cell types. The highest staining intensity was seen in the oocyte at all stages of follicular development, with lower intensities in the granulosa, stroma, theca, and corpora lutea. Other studies using immunofluorescence have shown the presence of leptin staining in human oocytes, granulosa cells, and cumulus cells [7]. Since leptin mRNA is also present in granulosa and theca cells, but not in the oocyte, our results suggest that the leptin protein detected at high levels was transported into the oocyte after synthesis in follicular somatic cells.
Both leptin receptor mRNA and protein are present in mouse GV and MII stage oocytes, suggesting that the oocyte is capable of responding to leptin produced locally within the follicle [19]. We have also shown that leptin receptor is present in most cells in the ovary, with the highest levels in the theca. Others have found leptin receptor protein in human stroma, granulosa cells, and thecal cells using RNase protection assays [9]. The high levels of receptor we observed in the thecal cell layer indicates that these cells may be more receptive to leptin treatment than other ovarian cells. Isolated thecal cells have been shown previously to respond to leptin administration in the cow, and in this animal, leptin increased insulin-induced proliferation but inhibited progesterone and androstenedione production [14]. Together, these findings suggest that leptin may be a paracrine regulatory molecule in the follicle, produced by different somatic cell types and transported to other sites where it has multiple essential regulatory functions. Regulation of oocyte maturation may be one such function.
Our studies of oocyte maturation indicate that leptin has no direct effects on spontaneous or induced meiotic resumption in oocytes cultured in isolation. Leptin had no effect on spontaneous maturation and was unable to affect the inhibition of oocyte maturation by the PDE inhibitors IBMX and MR in either DOs or COCs. In COCs, leptin was unable to affect induced maturation when FSH was added to overcome the IBMX and MR inhibition.
Galeati et al. [18] demonstrated that leptin induces an acceleration of oocyte maturation in isolated pig oocytes, whereby twice as many oocytes treated with leptin undergo meiotic resumption after 24 h of culture compared with control oocytes. It is difficult, however, to study advances in maturation in the isolated mouse oocyte, as spontaneous in vitro maturation begins within 1 h of removal from the follicular environment. The only way to show an induction of maturation in the isolated mouse oocyte is by overcoming an inhibitor of oocyte maturation. In this study, MR and IBMX inhibited oocyte PDE3B. If leptin acts on the PDE3B enzyme within the oocyte, such an action could not be detected in the presence of these inhibitors, thus making interpretation of such studies difficult.
The possibility that leptin may influence oocyte maturation indirectly through actions on follicular somatic cells was examined by culturing intact follicles. Follicle-enclosed oocytes do not undergo spontaneous maturation when cultured in vitro without added hormones or inhibitors, but maturation can be induced by addition of the gonadotropic hormones FSH and LH. Intact follicles therefore provide an alternate model for studying the control of in vitro oocyte maturation from isolated DOs or COCs. When preovulatory follicles were cultured for 4 h with and without leptin, significantly more oocytes were able to resume meiosis at all leptin treatment concentrations than in control follicles. Leptin may have induced maturation in the oocyte via actions on other cells within the follicle and not on the actual COCs. We suggest that thecal cells may be a target for leptin activity, as our immunohistochemistry results show the highest level of receptors within this cell layer, and previous studies have shown that leptin affects both granulosa and theca cells when cultured in vitro [12–16].
We found that leptin failed to affect cAMP in cultured oocytes. It is possible that leptin acts on the PDE3B within the oocyte to affect cAMP levels, as leptin is a known activator of PDE3B [20]. However, our failure to find any effect of leptin on oocyte cAMP levels when adenylate cyclase was stimulated by FK indicates that leptin's ability to advance oocyte maturation is probably not the result of its reducing cAMP levels through stimulation of oocyte PDE enzyme activity. Another factor, possibly produced by thecal or granulosa cells, must act in conjunction with leptin to procure the entire reduction in cAMP that is implicated in maturation induction. However, thecal cells inhibit oocyte maturation when cultured in vitro [28], possibly by maintaining high cAMP levels in the oocyte. This high cAMP level in response to follicular cells has also been seen in pig oocytes; pig oocytes cultured with follicular cells undergo an initial increase in cAMP levels [29]. Therefore, a more likely alternative for the stimulatory action of leptin on follicle-enclosed oocyte maturation may be through stimulation of a factor(s) of follicular somatic cell origin that can override the inhibitory action of cAMP. Leptin may indirectly promote the production of meiosis-activating sterol (MAS) via action on follicular cells. MAS is produced by cumulus cells in response to FSH stimulation [30], and its production may be enhanced by the presence of thecal cells.
It is also possible that leptin is involved in other pathways within the oocyte. In rat pancreatic B cells, leptin has been shown to activate the mitogen-activated protein (MAP) kinase pathway inducing STAT3 phosphorylation [31]. This may imply that leptin is involved in a similar pathway and hence control of gene transcription in the oocyte. Members of the MAP kinase pathway have been shown to play a role in meiotic resumption of Xenopus oocytes [32].
In conclusion, our results suggest that leptin does have direct effects on the ovary, possibly via thecal cells, and that these actions on the ovary may act to control stages of oocyte maturation. Clearly, the role of follicular somatic cells in regulation of oocyte maturation is complex, and further studies are under way to elucidate the mechanism by which leptin influences their regulatory effects on the oocyte.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This research was funded by the National Health and Medical Research Council of Australia. 
2 Correspondence: Robert J. Norman, Reproductive Medicine Unit, Obstetrics and Gynaecology, The University of Adelaide, The Queen Elizabeth Hospital, Woodville Rd., Woodville, SA 5011, Australia. FAX: 61 08 8222 7521; robert.norman{at}adelaide.edu.au
Accepted: January 4, 2002.
Received: June 29, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The mean ± SEM is shown for each treatment group, with at least 30 oocytes examined in six replicate experiments. Treatment with FSH + LH resulted in 12.3 and 4.2 pmol per follicle of progesterone and estradiol, respectively