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BOR - Papers in Press, published online ahead of print June 23, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.033035
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BIOLOGY OF REPRODUCTION 71, 1446–1452 (2004)
DOI: 10.1095/biolreprod.104.033035
© 2004 by the Society for the Study of Reproduction, Inc.

Ovary

Direct Effects of Leptin on Mouse Reproductive Function: Regulation of Follicular, Oocyte, and Embryo Development

Jason E. Swain2,5, Rodney L. Dunn4, Daniel McConnell5, Janis Gonzalez-Martinez6, and Gary D. Smith1,5,4,3,2

Departments of Molecular and Integrative Physiology,2 Obstetrics Gynecology,3 Urology,4 Reproductive Sciences Program,5 University of Michigan, Ann Arbor, Michigan 48109-0617 Caribbean Primate Research Center,6 Sabana Seca, Puerto Rico 00952-1053


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Because body condition can affect reproduction, research has focused on the role of leptin, a body condition signal, in regulation of reproductive function. Objectives of this study were to determine if leptin supplementation directly affects 1) ovarian follicle growth and function, 2) oocyte maturation, or 3) preimplantation embryo development. Follicles cultured in the presence of recombinant mouse leptin resulted in a significant decrease in rate of follicle, but not oocyte, growth in a dose-dependent manner, with higher doses of leptin inhibiting growth. Leptin was also found to significantly increase stimulated progesterone, estradiol, and testosterone production/secretion by cultured follicles in a dose-dependent manner, with higher concentrations of leptin significantly increasing steroidogenesis. Culture of fully grown cumulus-enclosed germinal vesicle-intact (GV) mouse oocytes in the presence of increasing concentrations of leptin (0, 12.5, 25, 50, 100 ng/ml) had no effect on germinal vesicle breakdown (GVBD) or development to metaphase II (MII). Similarly, fully grown denuded oocytes showed no difference in GVBD at any concentration of leptin. However, maturation of denuded oocytes with 100 ng/ml leptin resulted in significantly reduced development to MII compared with oocytes matured with 0 or 12.5 ng/ml leptin. Culture of one-cell mouse embryos in increasing concentrations of leptin had no effect on cleavage or blastomere degeneration at 24 h of culture. Exposure of embryos for the first 96 h of development to increasing concentrations of leptin did not significantly affect total or expanded blastocyst development or hatching of blastocysts from zona pellucida. These results indicate leptin directly enhances insulin and gonadotropin-stimulated ovarian steroidogenesis, compromises denuded oocyte maturation, yet has no direct effect on preimplantation embryo development.

embryo, follicle, leptin, oocyte development, steroid hormones


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A product of the obese gene [1], leptin is a 16-kDa protein hormone, produced primarily by adipocytes, that acts on the hypothalamus to regulate metabolism and fat deposition [2, 3]. Systemic leptin concentrations are elevated at times of food ingestion and in individuals with increased body fat [4–9]. Due to the long-known relationship between nutritional status and reproductive efficiency, recent research has focused on the role of leptin in regulation of reproductive function.

Leptin from adipose tissue may signal the reproductive system to indicate whether adequate energy stores are available for normal reproduction [10]. Research along these lines indicates leptin may be important in controlling the hypothalamic-pituitary-gonadal axis and thus influence states of reproductive transitions, such as puberty [11–14]. Evidence has also emerged indicating a potential direct role for leptin in regulation of mammalian ovarian function as well as oocyte and preimplantation embryo development. Leptin receptors have been identified in theca and granulosa cells [15–17], cumulus cells [18], oocytes [18–20], and embryos [21]. In addition, the ovary may be a site of leptin synthesis. Leptin and/or its mRNA have been identified in ovaries [17, 18, 20], oocytes [18, 20, 22], and preimplantation embryos [22, 23]. Last, follicular and uterine fluids contain leptin [4, 16, 21, 24], suggesting its availability to act on follicle, oocyte, and preimplantation embryo leptin receptors.

Direct effects of leptin on regulation of ovarian steroidogenesis have been investigated in numerous cell culture systems and species. Many of these studies indicate leptin attenuates gonadotropin or growth factor-stimulated steroidogenesis in isolated theca [15, 25] or granulosa cells [15, 26–28]. Additionally, a biphasic effect of leptin was observed in pig granulosa cells, where physiological levels of leptin were stimulatory to activity of steroidogenic acute regulatory protein (StAR) and steroidogenesis, while higher levels resulted in inhibition of StAR expression and estrogen production [29]. Leptin also increases granulosa cell aromatase activity [30]. It is important to note that all of these studies utilized single-cell suspensions of granulosa or theca cells and examined the short-term effects of leptin, conditions that are quite removed from normal ovarian physiological conditions.

Because oocytes and embryos contain leptin receptors, recent studies have focused on potential direct effects of leptin on oocyte maturation and early embryo development. Culturing preovulatory mouse follicles in the presence of leptin increased subsequent rates of oocyte germinal vesicle breakdown (GVBD) [20]. However, no effect of leptin was observed on rat oocyte meiosis in vitro [31]. Interestingly, reports on leptin's direct effects on embryo development are contradictory. Leptin exposure during culture of two-cell mouse embryos compromised blastocyst development and hatching [32]. Conversely, treatment of two-cell embryos with increasing concentrations of leptin increased development to the blastocyst, expanded blastocyst and hatched blastocyst stages, while increasing the total blastocyst cell number [21]. However, studies have not assessed effects of leptin on the one- to two-cell transition, which includes zygotic genome activation (ZGA), a developmentally sensitive transition state. To further confuse the issue, a significant positive correlation was found between elevated leptin:body mass index (BMI) ratios and inferior quality of human embryos, resulting in lower implantation and live birth rate [33]. With such little research done in this area and with conflicting findings such as these, it is apparent that further studies are required to assess leptin's direct role in the regulation of oocyte meiotic maturation and early preimplantation embryo development.

Taken together, these findings suggest leptin may act directly on the mouse ovary to modulate function. Leptin may also be directly regulating oocyte maturation and preimplantation embryo development. This research examines the direct effects of leptin on regulating mouse follicular growth and steroidogenesis, oocyte maturation and interaction with cumulus cells, and preimplantation embryonic development.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
All procedures described within were reviewed and approved by The University Committee on Use and Care of Animals at the University of Michigan and were performed in accordance with the Guiding Principles for the Care and Use of Laboratory Animals.

Animals

All experiments were performed using follicles, oocytes, or embryos collected from CF1 mice (Harlan, Indianapolis, IN). In regard to embryo culture studies, this strain was purposely selected due to the increased sensitivity to perturbations and reduced robustness in preimplantation embryo development [34].

Follicle Collection/Culture

For follicle culture experiments, preantral follicles were collected from 13-day-old mice by microdissecting ovaries in HEPES-buffered Human Tubal Fluid medium (HTFH; [35]; Irvine Scientific, Santa Ana, CA) supplemented with 10 µg/ml transferrin (Sigma, St. Louis, MO), 5 µg/ml insulin (Sigma), 50 µg/ml ascorbic acid (Sigma), and 2 ng/ml selenium (Sigma). Isolated secondary follicles, with an intact basal lamina and surrounding theca/interstitial cells, were placed into culture medium consisting of {alpha}-MEM (Gibco, Carlsbad, CA) supplemented with 5% fetal bovine serum (Gibco); 4 mM hypotaurine; 2 mM glutamine; 5 µg/ml insulin; 10 µg/ml transferrin; 2 ng/ml selenium; 50 µg/ml ascorbic acid; 50 µg/ml gentamicin; 10 ng/ml eCG; 1 ng/ml hCG; and either 0, 50, 100, or 200 ng/ml mouse recombinant leptin (R & D Systems, Minneapolis, MN). The low dose of leptin was selected to mimic leptin levels observed in the obese state. Supraphysiological doses of leptin at 100 and 200 ng/ml were selected based on previous studies examining isolated granulosa and theca cells, some of which include doses of up to 1000 ng/ml leptin [25, 27, 29]. In order to assess follicle/oocyte growth, individual follicles were cultured in 50-µl drops covered in oil at 37°C in 5% CO2 in air in a humidified incubator. Media and leptin were replaced every other day for 9 days by removing and replacing 40 µl of media.

Follicles and oocytes were measured at 200x on an inverted microscope using a SPOT camera and image analysis software (Diagnostic Instruments, Sterling Heights, MI). Diameters were calculated by taking the average of two measurements, aligned at right angles to each other, passing through the center of the oocyte or follicle.

To assess steroid hormone production/secretion, follicles were cultured in groups of 50/ml in medium supplemented with 0, 50, 100, or 200 ng/ ml leptin. Media and leptin were replaced every other day by removing 900 µl of media and replacing it with 900 µl media supplemented with 10 ng/ml eCG, 1 ng/ml hCG, and fresh leptin.

Hormone Assays

The progesterone assay was a competitive chemiluminesence immunoassay run on the automated analyzer ACS-180 (Bayer Corp., Tarrytown, NY) with offline incubation. Assays were run using two antibodies, a rabbit antiprogesterone labeled with dimethylacridiniumester (DMAE) and an anti-rabbit IgG covalently coupled to PMP. The reporting range for this assay is 0.1–40 ng/ml. Progesterone standards were confirmed by gas chromatography-mass spectrometry (GC-MS). The testosterone assay was a competitive binding chemiluminesence assay using testosterone labeled with DMAE, a polyclonal rabbit antitestosterone antibody and a monoclonal mouse anti-rabbit antibody, which was coupled to superparamagnetic particles (PMP) modified to run on the ACS-180 analyzer. The reporting range for the testosterone assay was 10–100 ng/dl (actual assay range, 2– 478 ng/dl). The ACS testosterone assay was standardized analytically and confirmed by GC-MS. Estradiol levels in media collected from group follicle culture were measured using an ultrasensitive chemiluminesence competitive binding immunoassay [36] modified to run on an ACS-180. The reporting range for the estradiol assay was 1–200 pg/ml. The ACS Estradiol-6-Master Curve standards used in the assay were manufactured and evaluated by GC-MS. Interassay coefficients of variation for progesterone, testosterone, and estradiol assays were 7.3%, 21.6%, and 11.5%, respectively. Intraassay coefficients of variation for progesterone, testosterone, and estradiol were 2.4%, 3.6%, and 2.9%, respectively.

Oocyte Collection/Maturation

For oocyte maturation studies, ovaries were collected from 18- to 22-day-old mice following injection of 10 IU eCG (Sigma). Fully grown germinal vesicle-intact oocytes (GV) were isolated by manual rupturing of antral ovarian follicles in HTFH + 0.3% (w/v) polyvinylpyrrolidine (Sigma). Oocytes were either stripped of cumulus cells through repeat mouth pipetting using hand-pulled pipettes or left with surrounding cumulus cells intact. Oocytes were then distributed between treatments of 0, 12.5, 25, 50, and 100 ng/ml recombinant mouse leptin in human tubal fluid medium (Irvine Scientific) supplemented with 0.3% bovine serum albumin (HTF+BSA; FisherBiotech, Fair Lawn, NJ) and cultured in groups of 20–30/ml of medium at 37°C in 5% CO2 in air. Stage of nuclear maturation was assessed under 400x after 2 and 18 h of culture.

Embryo Collection/Culture

For embryo collection, 6- to 8-week-old mice were injected with 10 IU eCG followed 44 h later with an injection of 10 IU hCG and placed with a mature CF1 male mouse of known fertility. Presumptive zygotes were collected 18 h later by dissecting oviducts in HTFH medium supplemented with 0.1% (w/v) hyaluronidase to remove surrounding cumulus cells. Zygotes were randomly distributed between treatments of 0, 12.5, 25, 50, and 100 ng/ml leptin in Potassium Simplex Optimized Medium (KSOM) media with glucose and amino acids (Specialty Media, Phillipsburg, NJ) + 0.1% BSA (Serologicals, Kankakee, IL; KSOM+BSA) and cultured in groups of 20–30/ml of medium at 37°C in 5% CO2 in air. Media and leptin were replaced fresh every day. Embryo development was assessed every 24 h for 96 h.

Statistics

Oocyte maturation and embryo development data were analyzed using the Mantel-Haenszel chi-square test. Oocyte and follicular growth, as well as steroid hormone data, were assessed and overall differences determined using a mixed linear regression model (MLRM). The advantages of this approach are twofold. First, treatments utilized in this study caused a monotonic increase of various outcomes (i.e., the growth rate of the outcomes) and not just on values at particular timepoints, and this model tests for the treatment effect on linear growth rate. Performing separate analyses at individual timepoints would ignore this effect. Second, MLRM analyzes data from all timepoints for a particular outcome in a single analysis and thus has greater power to detect treatment differences than analyzing only data from a particular time point. While the main statistical endpoint from the MLRM is the treatment differences in the growth rate of the outcome, these models also produce estimates of the mean outcome value for each treatment/time point combination, along with 95% confidence intervals around the estimates. Differences in steroid production between treatments at a single time point or between timepoints for a single treatment were performed by comparison of confidence intervals. Oocyte maturation and embryo development experiments were performed in triplicate. Follicle and oocyte measurement experiments were performed in duplicate and follicular steroidogenesis experiments were repeated four times.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The effects of leptin supplementation on mouse follicular and oocyte growth over 9 days were determined microscopically by taking the average of two measurements at right angles to each other through the oocyte and follicle (Fig. 1). Supplementation of leptin on individual cultured mouse follicles resulted in an overall decrease in follicle growth over 9 days of culture in a dose-dependent manner, with higher concentrations of leptin inhibiting the rate of growth, P = 0.04 (Fig. 2a). Leptin did have a treatment effect on oocyte diameter (P = 0.03); however, rates of murine oocyte growth did not differ between leptin treatment groups (Fig. 2b).



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  		FIG. 1. Representative micrographs of an individual follicle and enclosed oocyte growth over 9 days of culture. Measurements for growth comparisons were averages of two measurements taken at right angles to one another, through the center of the follicle and oocyte. Follicle and oocyte measurements were taken within the basement membrane and zona pellucida, respectively. Original magnification x200



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  		FIG. 2. Growth rates of individually cultured (a) mouse follicles and (b) enclosed oocytes during 9 days of culture in increasing concentrations of mouse recombinant leptin. Increasing concentration of leptin inhibited rate of follicle growth, while it had no effect on rate of oocyte growth as assessed by MLRM, P < 0.05. Data reported is the mean ± SEM

The effect of leptin supplementation on murine in vitro follicular steroidogenesis was also assessed. Mixed linear regression models indicate an overall increase in production of progesterone (P = 0.03; Fig. 3a), testosterone (P = 0.004; Fig. 3b), and estradiol (P = 0.005; Fig. 3c) between treatments over time. Specifically, on Days 7 and 9 of culture, 200 ng/ml leptin resulted in significantly greater testosterone production compared with 0 and 50 ng/ml leptin, P < 0.05. Treatment with 200 ng/ml leptin also resulted in significantly greater production of estradiol compared with 0 ng/ml leptin on Day 7 and greater estradiol production compared with both 0 and 50 ng/ml leptin on Day 9. Concentrations of 100 ng/ml leptin showed no individual differences in steroid hormone production between treatments within a day. Thus, at 200 ng/ml leptin significantly increased mouse follicular progesterone, testosterone, and estradiol synthesis/secretion.



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  		FIG. 3. Production/secretion of steroid hormones (a) progesterone, (b) testosterone, and (c) estradiol from group cultured mouse follicles over 9 days in response to increasing concentrations of mouse recombinant leptin. Data were assessed by MLRM and were reported as the mean ± SEM

Oocyte maturation experiments were performed with or without surrounding cumulus cells to determine if effects of leptin on oocyte development are mediated through cumulus cell interactions. Maturation of cumulus cell-enclosed mouse oocytes in the presence of varying doses of leptin had no significant effect on GVBD at either 2 or 18 h of culture (Table 1). Similarly, metaphase II (MII) development was not significantly affected by leptin. Maturation of denuded mouse oocytes in the presence of leptin also had no significant effect on GVBD at either 2 or 18 h of culture. However, maturation of denuded oocytes in 100 ng/ml leptin resulted in significantly lower development to MII compared with control and 12.5 ng/ml leptin treatment, P < 0.05 (Table 1).


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  		TABLE 1. Germinal vesicle breakdown (GVBD) and metaphase II (MII) development after maturation of mouse oocytes in the presence of in creasing concentrations of mouse recombinant leptin

To assess the effects of leptin supplementation on mouse embryo development, one-cell mouse embryos were cultured in varying concentrations of leptin for 96 h, with development assessed and media replaced every 24 h (0 ng/ml, n = 79; 12.5 ng/ml, n = 79; 25 ng/ml, n = 80; 50 ng/ml, n = 80; or 100 ng/ml, n = 79). No significant differences were observed in normal cleavage at 24 h after culture in leptin (0 ng/ml, 57%; 12.5 ng/ml, 56%; 25 ng/ ml, 61%; 50 ng/ml, 65%; 100 ng/ml, 54%). In addition, rates of degeneration were not significantly different between treatment groups (0 ng/ml, 8%; 12.5 ng/ml, 7%; 25 ng/ml, 8%; 50 ng/ml, 7%; 100 ng/ml, 13%). Similarly, after 96 h of culture, no differences were observed in development to total blastocyst (0 ng/ml, 38%; 12.5 ng/ml, 39%; 25 ng/ml, 42%; 50 ng/ml, 43%; 100 ng/ml, 27%), expanded blastocyst (0 ng/ml, 15%; 12.5 ng/ml, 6%; 25 ng/ml, 13%; 50 ng/ml, 8%; 100 ng/ml, 7%), or hatched blastocyst (0 ng/ ml, 13%; 12.5 ng/ml, 13%; 25 ng/ml, 10%; 50 ng/ml, 10%; 100 ng/ml, 7%).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have determined that leptin increases insulin and gonadotropin-stimulated follicular progesterone, testosterone, and estradiol production in a dose-dependent manner, with increasing concentrations of leptin increasing steroidogenesis. Indeed, it has been reported leptin can stimulate aromatase activity and estrogen production in granulosa cells [30] and leptin, at 10 ng/ml, can stimulate progesterone production by pig granulosa cells [29]. Furthermore, injection of rats with physiological doses of leptin increases testosterone and progesterone production in the ovary [37], and administration of leptin elevated StAR levels, suggesting increased steroidogenic capacity [29]. Conversely, other studies indicate elevated leptin levels are inhibitory to insulin or growth factor-stimulated theca and granulosa cell steroidogenesis [15, 25–28]. These studies utilized single, dispersed culture systems of purified theca or granulosa cells and did not take into account various autocrine and paracrine factors possibly acting within the follicle. Supporting this, leptin was found to inhibit isolated granulosa cell estrogen production; however, no effect was observed on whole rat follicles [31]. Thus, it is evident the model system utilized is an important factor in understanding leptin's influence on follicular function and steroidogenesis.

Contradictory findings exist with respect to leptin's effects on steroidogenesis. As mentioned, some of this can be explained by differences in cell systems utilized for analysis. Our study, conducted with isolated mouse follicles, indicates an increase in insulin and gonadotropin-augmented steroid hormone production in response to increasing levels of leptin. However, a previous report utilizing in vitro-cultured mouse follicles suggests leptin is inhibitory to FSH+IGF-1- and FSH+GH-stimulated estrogen production [38]. Caution must be taken when comparing these studies. Culture media and conditions were significantly different between these respective studies; most apparent was the addition of fresh media and leptin every day and the addition of insulin and serum in our culture media. It was previously shown that preculture of cells with serum was necessary for stimulation of steroidogenesis by leptin [30]. Additionally, gonadotropins were absent during the first day of culture in our study. Thus, culture conditions may be an important consideration when determining the effect of leptin on ovarian steroidogenesis. Furthermore, previous studies have only examined leptin's effects on steroidogenesis for relatively short periods of time. Cell culture studies showing an inhibitory effect of leptin on steroidogenesis often only examine hormone production for up to 4 days. These reports indicate leptin does not alter basal steroidogenesis, yet inhibits augmented steroid production [39]. A previous report on follicular steroidogenesis indicated leptin had no effect on progesterone or estradiol production [31]. However, follicles in the study were only cultured for 4 h. Our study indicates changes in steroid hormone production did not become apparent until extended periods of culture, beyond 4 days. Visual assessment indicated no differences in atresia or early luteinization between treatment groups that might account for this difference. Therefore, extended culture periods may be required to elicit or recognize leptin's stimulatory effect on follicular steroid production.

A clinically relevant question concerning this observed increase in follicular steroidogenesis in response to increased leptin concentrations can be raised when considering the relationship between obesity and reproduction. Obesity is an extremely common clinical characteristic in women affected by polycystic ovarian syndrome (PCOS). In 1990, the National Institutes of Health defined the diagnostic criteria for PCOS as presence of hyperandrogenism and chronic oligo-anovulation, with the exclusion of hyperandrogenism caused by adult-onset congenital adrenal hyperplasia, hyperprolactinemia, and androgen-secreting neoplasms [40]. Approximately 50% of women afflicted with PCOS are overweight or obese [41], and a history of weight gain frequently precedes symptoms of PCOS. Numerous investigations have focused on the impact of obesity on the hyperandrogenic state in women with PCOS and a possible link with leptin [42–45]. Obese PCOS women have significantly worsened hyperandrogenism compared with their normal-weight PCOS counterparts [46], and a positive correlation has been demonstrated between body fat mass and circulating androgens [41, 47]. Obese women that are placed on a low caloric diet and demonstrate a significant reduction in body mass index also have a significant reduction in free testosterone and androstenedione [48]. It is well documented that adipocytes secrete leptin [2, 3] and that body mass index and serum leptin levels are highly positively correlated [4, 8]. Undoubtedly, numerous factors contribute to the pathophysiology of hyperandrogenism in obese PCOS women. However, our findings of leptin significantly stimulating in vitro follicle production/ secretion of testosterone raise the question of whether serum leptin levels are a contributing factor to hyperandrogenism in obese PCOS patients. Further studies are required to determine the leptin contribution to hyperandrogenism and the mechanism by which this adipose-derived protein influences ovarian steroid production.

These studies have demonstrated that leptin inhibits mouse follicular growth rate but increases steroidogenesis. This inhibition is in agreement with a previous report of increased levels of leptin being inhibitory to growth in preantral follicles isolated from adult and prepubertal mice thought to be a result of a block in the cAMP pathway [38]. Alternatively, increased steroid hormone levels, seen in our examination of follicular steroidogenesis, may result in decreased follicle growth. Elevated androgen levels cause follicular atresia in rodents and enhance granulosa cell apoptosis [49, 50]. Androgens also prevent induction of LH receptor expression and appear to act at multiple levels to inhibit ovarian follicular growth and development [51]. However, contradictory information exists as to the stimulatory or inhibitory effects of steroid hormones on ovarian follicles [52]. Because this is a follicle culture system, without hypothalamic/pituitary feedback, the physiological relevance of the correlation is uncertain.

Although leptin and its receptor are found within the oocyte, it has been suggested this protein hormone is not required for normal oocyte development. Oocytes from ob/ ob leptin-deficient mice are able to undergo normal fertilization and embryo development, resulting in pregnancy and live birth, following transplant to a normal surrogate [53]. Additionally, treatment of ob/ob females with exogenous gonadotropins allows for successful ovulation, fertilization, and embryo development [54], suggesting the sterility defect in these obese mice is not at the level of the oocyte. Supporting this notion, in vitro maturation of isolated cumulus-enclosed mouse oocytes in the presence of leptin had no effect on nuclear maturation endpoints examined in our study. Also, oocytes isolated from mouse follicles cultured in vitro for 9 days in the presence of varying concentrations of leptin had no effect on GVBD or MII following in vitro maturation (data not shown). Similarly, rat oocyte meiotic resumption was not affected by the presence of leptin [31]. However, leptin has been reported to increase the rate of GVBD in mouse oocytes cultured within preovulatory follicles [20]. Again, these apparent contradictions may be due to differences in culture systems utilized. However, isolated denuded oocytes showed a significant decrease in development to MII in the highest dose of leptin. This may be due to a lack of critical interactions with surrounding cumulus cells, resulting in compromised developmental competence [55]. Furthermore, although not examined in this study, leptin may still be exerting some unobserved effect on oocyte cytoplasmic maturation.

Few previous studies have reported on leptin's effects on in vitro embryo development. We have shown addition of leptin to one-cell mouse embryos at varying doses had no effect on subsequent cleavage or cell degeneration at 24 h. Furthermore, no difference in blastocyst, expanded blastocyst, or hatched blastocyst numbers were observed in increasing concentrations of leptin. However, previous reports have found positive and negative effects of leptin on preimplantation embryo development. It has been reported that increasing doses of leptin during mouse embryo culture enhances development to various blastocyst stages [21]. Conversely, an elevated leptin:BMI has been found to be predictive of inferior quality embryos [33]. Thus, experimental conditions may be important in assessment of leptin's role in preimplantation embryo development. Additionally, it has been previously suggested that leptin, along with STAT3, may be important factors in establishment of cell polarity and delineation of the inner cell mass and trophectoderm in preimplantation embryos [22]. Therefore, although leptin does not appear to be affecting embryo cleavage or subsequent blastocyst development, it may influence intrablastocyst cell distribution and subsequent postimplantation embryo development.

The purpose of this research was to determine direct effects of leptin on mouse reproductive function and attempt to reconcile conflicting results found in the literature. We have shown leptin does indeed have a direct effect on the follicle, thereby increasing augmented follicular steroid hormone production. However, it does not appear that leptin directly affects oocyte maturation of oocyte-cumulous complexes or preimplantation embryo development. This research may have potential clinical implications in explaining and treating infertility associated with aberrant androgen levels in obese human females and those experiencing PCOS.


   ACKNOWLEDGMENTS
 
The authors wish to thank Russel Possley for his assistance in measuring steroid hormone levels and Dr. Carrie Smith for her review of this manuscript.


   FOOTNOTES
 
1 Correspondence: Gary D. Smith, University of Michigan, 6428 Medical Sciences Building I, 150 W. Medical Center Drive, Ann Arbor, MI 48109-0617. FAX: 734 647 1006; smithgd{at}umich.edu Back

Received: 9 June 2004.

First decision: 10 June 2004.

Accepted: 10 June 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Zhang Y, Proenca R, Maffei M. Positional cloning of the mouse obese gene and its human homologue. Nature 1994 372:425-432[CrossRef][Medline]
  2. Hamilton B, Paglia D, Dwan A, Beital M. Increased obese mRNA expression in omental fat cells from massively obese humans. Nature Med 1995 1:953-956[CrossRef][Medline]
  3. Lonnqvist F, Arner P, Nordfors L, Schalling M. Overexpression of the obese gene in adipose tissues of human obese subjects. Nat Med 1995 1:950-953[CrossRef][Medline]
  4. Butzow TL, Moilanen JM, Lehtovirta M, Tuomi T, Hovatta O, Siegber R, Nilsson CG, Apter D. Serum and follicular fluid leptin during in vitro fertilization: relationship among leptin increase, body fat mass, and reduced ovarian response. J Clin Endocrinol Metab 1999 84:3135-3139[Abstract/Free Full Text]
  5. Considine RV, Sinha MK, Heiman ML, Kriauciunas A, Stephens TW, Nyce MR, Ohannesian JP, Marco CC, McKee LJ, Bauer TL, Caro JF. Serum immunoreactive leptin concentration in normal weight and obese humans. New Eng J Med 1996 334:292-295[Abstract/Free Full Text]
  6. Dagago-Jack S, Fanelli C, Paramore D, Brothers J, Landt M. Plasma leptin and insulin relationship in obese and non-obese humans. Diabetes 1996 45:695-698[Abstract]
  7. Klein S, Coppack SW, Mohamed AV, Landt M. Adipose tissue leptin production and plasma leptin kinetics in humans. Diabetes 1996 45:984-987[Abstract]
  8. Maffei M, Halaas J, Ravussin E, Pratley RE, Lee GH, Zhang Y, Fei H, Kim S, Lallone R, Ranganathan S. Leptin levels in human and rodent: measurement of plasma leptin and ob RNA in obese and weight reduced subjects. Nat Med 1995 1:1155-1161[CrossRef][Medline]
  9. Mantzoros XS, Dunaif A, Flier JS. Leptin concentrations in the polycystic ovarian syndrome. J Clin Endocrinol Metab 1997 82:1687-1691[Abstract/Free Full Text]
  10. Tataranni PA, Monroe MB, Dueck CA. Adiposity, plasma leptin concentration and reproductive function in active and sedentary females. Int J Obes Relat Metab Disord 1997 21:818-821[CrossRef][Medline]
  11. Moschos S, Chan JL, Mantzoros CS. Leptin and reproduction: a review. Fertil Steril 2002 77:433-444[CrossRef][Medline]
  12. Rene-Gonzalez R, Simon C, Caballero Campo-P, Norman R, Chardonnens D, Devoto L, Bischof P. Leptin and reproduction. Hum Reprod Update 2000 6:290-300[Abstract/Free Full Text]
  13. Smith GD, Jackson LM, Foster DL. Leptin regulation of reproductive function and fertility. Theriogenology 2002 57:73-86[CrossRef][Medline]
  14. Spicer LJ. Leptin: a possible metabolic signal affecting reproduction. Domest Anim Endocrinol 1991 21:251-270
  15. Agarwal SK, Vogel K, Weitsman SR, Magoffin DA. Leptin antagonizes the insulin like growth factor I augmentation of steroidogenesis in granulosa and theca cells of the human ovary. J Clin Endo Metab 1999 84:1072-1076[Abstract/Free Full Text]
  16. Karlsson C, Lindell K, Svensson E, Bergh C, Lind P, Billig H, Carlsson LMS, Carlsson B. Expression of functional leptin receptors in the human ovary. J Clin Endocrinol Metab 1997 82:4144-4148[Abstract/Free Full Text]
  17. Loffler S, August G, Kohler U, Spanel-Borowski K. Evidence of leptin expression in normal and polycystic human ovaries. Mol Hum Reprod 2001 7:1143-1149[Abstract/Free Full Text]
  18. Cioffi JA, Van Blerkom J, Antczak M, Shafer A, Wittner S, Snidgrass HR. The expression of leptin and its receptors in pre-ovulatory human follicles. Mol Hum Reprod 1997 3:467-472[Abstract/Free Full Text]
  19. Matsuoka T, Tahara M, Yokoi T, Masumotoa N, Takeda T, Yamaguchi M, Tasaka K, Kurachi H, Murata Y. Tyrosine phosphorylation of STAT3 by leptin though leptin receptor in mouse metaphase 2 stage oocyte. Biochem Biophys Res Comm 1999 256:480-484[CrossRef][Medline]
  20. Ryan NK, Woodhouse CM, Van der Hoek KH, Gilchrist RB, Armstrong DT, Norman RJ. Expression of leptin and its receptor in the murine ovary: possible role in the regulation of oocyte maturation. Biol Reprod 2002 66:1548-1554[Abstract/Free Full Text]
  21. Kawamura K, Sato N, Fukuda J, Kodama H, Kumagai J, Tanikawa H, Nakamura A, Tanaka T. Leptin promotes the development of mouse preimplantation embryos in vitro. Endocrinology 2002 143:1922-1931[Abstract/Free Full Text]
  22. Antczak M, Van Blerkom J. Oocyte influences on early development; the regulatory protein leptin and STAT3 are polarized in mouse and human oocytes and differentially distributed within the cells of the preimplantation stage embryo. Mol Hum Reprod 1997 3:1067-1086[Abstract/Free Full Text]
  23. Gonzalez RR, Caballero-Campo P, Jasper M, Mercader A, Devoto L, Pellicer A, Simon C. Leptin and leptin receptor are expressed in the human endometrium and endometrial leptin secretion is regulated by the human blastocyst. J Clin Endocrinol Metab 2000 85:4883-4888[Abstract/Free Full Text]
  24. Barroso G, Barrionuevo M, Rao P, Graham L, Danforth D, Huey S, Abuhamad A, Oehninger S. Vascular endothelial growth factor, nitric oxide, and leptin follicular fluid levels correlate negatively with embryo quality in IVF patients. Fertil Steril 1999 72:1024-1026[CrossRef][Medline]
  25. Spicer LJ, Francisco CC. Adipose obese gene product, leptin, inhibits bovine ovarian thecal cell steroidogenesis. Biol Reprod 1998 58:207-212[Abstract/Free Full Text]
  26. Brannian JD, Zhao Y, McElroy M. Leptin inhibits gonadotropin stimulated granulosa cell progesterone production by antagonizing insulin action. Hum Reprod 1999 14:1445-1448[Abstract/Free Full Text]
  27. Spicer LJ, Francisco CC. The adipose obese gene product, leptin: evidence of a direct inhibitory role in ovarian function. Endocrinology 1997 138:3374-3379[Abstract/Free Full Text]
  28. Zachow RJ, Magoffin DA. 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 1997 138:847-850[Abstract/Free Full Text]
  29. Ruiz-Cortez AT, Martel-Kennes Y, Gevery NY, Downey BR, Palin MF, Murphy BD. Biphasic effects of leptin in porcine granulosa cells. Biol Reprod 2003 68:789-796[Abstract/Free Full Text]
  30. Kitawaki J, Kusiuki I, Koshiba H, Tsukamoto K, Honjo H. Leptin directly stimulates aromatase activity in human luteinized granulosa cells. Mol Hum Reprod 1999 5:708-713[Abstract/Free Full Text]
  31. Duggal PS, Ryan NK, Van der Hoek KH, Ritter LJ, Armstrong DT, Magoffin DA, Norman RJ. Effects of leptin administration and feed restriction on thecal leucocytes in the preovulatory rat ovary and the effects of leptin on meiotic maturation, granulosa cell proliferation, steroid hormone and PGE2 release in culture rat ovarian follicles. Reproduction 2002 123:891-898[Abstract]
  32. Fedorcsák P, Storeng R. Effects of leptin and leukemia inhibitory factor on preimplantation development and STAT3 signaling of mouse embryos in vitro. Biol Reprod 2003 69:1531-1538[Abstract/Free Full Text]
  33. Brannian JD, Schmidt SM, Kreger DO, Hansen KA. Baseline non-fasting serum leptin concentration to body mass index ratio is predictive to IVF outcomes. Hum Reprod 2001 16:1819-1826[Abstract/Free Full Text]
  34. Dandekar PV, Glass RH. Development of mouse embryos in vitro is affected by strain and culture medium. Gam Res 1987 17:279-285
  35. Quinn P, Kerin J, Warnes G. Improved pregnancy rate in human in vitro fertilization with the use of a medium based on the composition of human tubal fluid. Fertil Steril 1985 44:493-498[Medline]
  36. England BG, Parsons GH, Possley RM, McConnell DS, Midgley AR. Ultrasensitive semiautomated chemiluminescent immunoassay for estradiol. Clin Chem 2002 48:1584-1586[Free Full Text]
  37. Cannady WE, Brann DW, Mahesh VB. The potential role of periovarian fat and leptin in initiation of puberty in the immature rat. Int J Obes Relat Metab Disord 2000 (suppl 2) 146-147
  38. Kikuchi N, Andohg K, Abe Y, Yamada K, Mizunuma H, Ibuki Y. Inhibitory action of leptin on early follicular growth differs in immature and adult female mice. Biol Reprod 2001 65:66-71[Abstract/Free Full Text]
  39. Spicer LJ, Chamberlain CS, Francisco CC. Ovarian action of leptin: effects on insulin like growth factor-I-stimulated function of granulose and thecal cells. Endocr J 2000 12:53-59
  40. Goudas VT, Dumesic DA. Polycystic ovarian syndrome. Endocrinol Metab Clin N Am 1997 26:893-912[CrossRef][Medline]
  41. Pasquali R, Casimirri F. The impact of obesity on hyperandrogenism and polycystic ovary syndrome in premenopausal women. Clin Endocrinol 1993 39:1-16[Medline]
  42. Brzechffa PR, Jakimiuk AJ, Agarwal SK, Weitsman SR, Buyalos RP, Magoffin DA. Serum immunoreactive leptin concentrations in women with polycystic ovary syndrome. J Clin Endocrinol Metab 1996 81:4166-4169[Abstract/Free Full Text]
  43. El Orabi H, Ghalia AA, Khalifa A, Mahfouz H, El Shalkani A, Shoieb N. Serum leptin as an additional possible pathogenic factor in polycystic ovary syndrome. Clin Biochem 1999 32:71-75[CrossRef][Medline]
  44. Jacobs HS, Conway GS. Leptin, polycystic ovaries and polycystic ovary syndrome. Hum Reprod Update 1999 5:166-171[Abstract/Free Full Text]
  45. Panidis D, Roussa D, Kourtis A, Tsimas V, Papathanasiou K, Makedos G. Serum leptin levels in normal weight and overweight women with polycystic ovary syndrome. Clin Exp Obstet Gynecol 2003 30:207-210[Medline]
  46. Kiddy DS, Sharp PS, White DM, Scanlon MF, Mason HD, Bray CS, Polson DW, Reed MJ, Franks S. Differences in clinical and endocrine features between obese and non-obese subjects with polycystic ovary syndrome: an analysis of 263 consecutive cases. Clin Endocrinol 1990 32:213-220[Medline]
  47. Poretsky L, Cataldo NA, Rosenwaks Z, Giudice LC. The insulin-related ovarian regulatory system in health and disease. Endocr Rev 1999 20:535-582[Abstract/Free Full Text]
  48. Craves JC, Fimbel S, Lejeune H, Cugnardey N, Dechaud H, Pugeat M. Effects of diet and metformin administration on sex-hormone-binding globulin, androgens, and insulin in hirsute and obese women. J Clin Endocrinol Metab 1995 80:2057-2062[Abstract]
  49. Hillier SG, Ross GT. Effects of exogenous testosterone on ovarian weight, follicular morphology and intraovarian progesterone concentration in estrogen primed hypohysectomized immature female rats. Biol Reprod 1979 20:261-268[Abstract]
  50. Billig H, Furuta I, Hsueh JW. Estrogens inhibit and androgens enhance granulosa cell apoptosis. Endocrinology 1993 133:2204-2212[Abstract]
  51. Farookhi R. Effects of aromatizable and nonaromatizable androgen treatments on luteinizing hormone receptors and ovulation induction in immature rat. Biol Reprod 1985 33:363-369[Abstract]
  52. Murray A, Spears N. Follicular development in vitro. Sem Reprod Med 2000 18:109-121
  53. Runner MN, Gates A. Sterile obese mothers. J Hered 1954 45:51-55[Abstract/Free Full Text]
  54. Smithberg M, Runner MN. Pregnancy induced in genetically sterile mice. J Hered 1957 48:97-100[Free Full Text]
  55. Buccione R, Schroeder AC, Eppig JJ. Interactions between somatic cells and germ cells throughout mammalian oogenesis. Biol Reprod 1990 43:543-547[Abstract]



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