- Email: [email protected]
Leptin regulation of reproductive function and fertility
ELSEWER
LEPTIN REGULATION
OF REPRODUCTIVE FUNCTION AND FERTILITY
G. D. Smith’X3.4,5,L. M. Jackson’, and D. L. Foster’,*,’ Departments of Obstetrics and Gynecology’, Biology’, Physiology3, and Urology4; Reproductive Sciences Program’, University of Michigan, Ann Arbor, Michigan, USA 48109-0617 ABSTRACT Leptin, a 16-KD protein secreted primarily by adipose tissue, was first discovered in the search for a satiety signal. When administered into the brain, leptin depresses appetite. Interestingly, hyperphagic, obese, transgenic mice with leptin deficiency were noted to be reproductively incompetent, and administration of leptin restored their fertility. These pivotal observations led to numerous studies on the site of action of leptin within the hypothalamohypophyseal-gonadal axis, and a variety of models have been used ranging from the prepubertal condition to fasting suppression of reproductive hormones. The preponderance of studies thus far has focused on how leptin serves as a metabolic signal of energy balance within the neuroendocrine system, particularly as a regulator of GnRH/LH secretion. Less research has been conducted with other components of the reproductive system, but local effects of leptin have been demonstrated in the gonads where hyperleptinemia suppresses steroidogenesis and potentially affects gamete maturation. This presentation will review the major concepts for the role of leptin in the modulation of fertility and will consider the potential use of leptin in assisted reproductive technology and embryo transfer. 0 2001 by Elsevier Science Inc. Key words: leptin, neuroendocrine, ovary, gametes, embryo
INTRODUCTION Leptin is a 16-kD protein product of the obese gene (ob) produced primarily by adipose tissue; it is believed to play an important role in the regulation of food intake and body weight (68). As a satiety hormone, leptin concentrations can change in response to caloric intake and can suppress appetite, increase metabolic rate, and regulate weight gain and fat deposition (13, 26, 40, 78). Studies in humans demonstrate a positive correlation between the release and synthesis of leptin and body mass index or percentage of body fat (27, 54) and peripheral leptin concentrations decrease in response to fasting or dietary restriction (2, 61, 65). In addition, numerous reports suggest that leptin is an important regulator of reproductive function and provides a signaling link between nutritional status and reproduction. This review will focus on accepted and developing theories of how leptin is involved in mediating reproduction and
Acknowledgments: Works presented in this review and presentation were supported by NIH Grants HD35125OlAl (GDS), HD18394 (DLF). Postdoctoral training support (L.M. Jackson) provided by HD07048 (Training Grant, Reproductive Sciences Program, University of Michigan). Correspondence: Dr. Gary D. Smith, 6428 Medical Sciences Building I, 1301 E. Catherine St., Ann Arbor, MI 481090617. Phone: (734) 764-4134; Fax: (734) 647-1006; E-mail: [email protected]. Theriogenology 57:73-66. 0 2001 Elsevier Science
2002 Inc.
0093-691W02/$-.see front matter PII: SOO93-691X(01)00656-6
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fertility. As a means to this end, information will be discussed regarding: 1) leptin, its receptors and mechanism of intracellular signal transduction; 2) the role of leptin in reproductive neuroendocrinology; 3) direct effects of leptin on gonadal function; and 4) leptin in the gamete and preimplantation embryo. We will conclude with a section addressing possible future applications of leptin monitoring or treatment to aid in assisted reproductive practices and/or modulation of fertility. Leptin, Its Receptors, and Mechanism of Action Two mutant mouse strains, oblob and dbldb, demonstrate the importance of leptin in regulating appetite, metabolism, and body composition. The absence of the complete protein (ob/ob) or the leptin receptor (db/db) leads to hyperphagia and subsequent obesity (24). However, examination of the human ob gene found that the gene encoding for leptin is normal in most cases of obesity (25, 54, 59, 69). A missense mutation in the leptin gene has been identified in two families in which individuals homozygous for the mutation exhibit low circulating leptin concentrations, hyperphagia, and severe obesity. Treatment of children with this congenital defect with recombinant human leptin restores leptin concentrations to normal levels, reduces body weight, and alleviates other metabolic irregularities (32). Similarly, defects in the human leptin receptor are rare (23), and, thus, morbid obesity in humans differs from rodent models and is not readily explained by alterations in a single gene. Other aspects of leptin physiology currently under investigation for their role in appetite disorders and obesity include central leptin resistance caused by impaired transport across the blood-brain barrier (16) and interactions between circulating leptin and its binding protein (65). The leptin receptor is a member of the class I cytokine receptor family and exists in at least five isoforms arising from mRNA splice variants (52, 71). The single long form of the receptor (Ob-Rb) includes a long (302 amino acids) intracellular domain involved in activating the Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway (8). One short form (Ob-Ra) has a short (31 amino acids) intracellular domain capable of activating the mitogen-activated protein (MAP) kinase pathway (9, 74). Ob-Rb is the predominant receptor expressed within the brain, particularly in the hypothalamus (3 1, 58), and Ob-Ra is found principally in peripheral tissues such as liver, pancreas, gonads, and skeletal muscle (44, 47, 52). Nevertheless, Ob-Ra is present in the choroid plexus (53) and neural capillaries (39), where it is involved with leptin transport between blood and cerebrospinal fluid, and across the blood-brain barrier. Another isoform, Ob-Re, lacks both transmembrane and cytoplasmic domains, is soluble, and serves as a circulating leptin-binding protein (50). Multiple forms of the leptin receptor and their distribution outside the central nervous system suggest that the role of leptin extends beyond its function as a satiety factor. In addition to regulating appetite and energy intake, leptin affects several metabolic and neuroendocrine mechanisms to control or partition energy expenditure (34,73). Role of Leptin in Reproductive Neuroendocrinology Historical Association of Fatness and Fertility. The link between leptin and reproduction has multiple and seemingly unrelated origins in studies of human puberty, the effects of energy balance on human reproductive function, optimizing fertility by increasing feed in domestic
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species, and infertility in a mutant mouse model. The earlier demographic studies of Frisch (35) sought to explain the initiation of reproductive activity in women and demonstrated that a certain degree of fatness is associated with menarche as well as with maintenance of menstrual cyclicity. This work was of clinical use, but failed to provide a mechanism for how the brain detects how fat the body is and then uses that information to increase gonadotropin secretion to stimulate the ovaries. The simple fact that there was nothing yet identified that was secreted by fat and could serve as a signal to the brain was a practical constraint, but Frisch suggested an indirect link by proposing that fatness enhanced fertility through increased conversion of androgens to estrogens by adipose tissue (36). Given the negative feedback effects of estrogen on GnRH and LH secretion (except during the preovulatory surge), this was never considered to be a viable mechanistic explanation in terms of neuroendocrine control. Nonetheless, this work provided a historical background for the current interest in fat and fertility and the possibility that fat produces something that the brain can monitor to assess somatic energetics. During the several years when mouse mutants were used to study metabolism and appetite behavior, most reproductive scientists paid little attention to the fact that the ob/ob and db/db mutants were infertile. This changed dramatically following the report that leptin injections into adult ob/ob mice restored their reproductive capacity (18), a finding that provided a novel mechanistic bridge between fatness and fertility. Since this discovery, the possibility that leptin serves as a molecular intermediary to link metabolism and reproduction has attracted the attention of many reproductive scientists. Neuroendocrine Actions of Leutin on GnRH Secretion. To date, only a handful of studies have focused on the neuroendocrine control of GnRH secretion by leptin in a physiological Barash et al. (5) found that, in ob/ob mice, leptin administration increases basal LH setting. levels. In wild-type mice, fasting suppressed peripheral leptin concentrations, basal LH concentrations, and estrous cyclicity, and such deficits could be prevented by exogenous leptin (2). Intravenous infusion of leptin restored both LH and FSH secretion on the second day of a 2d fast in peripubertal male rhesus monkeys (33). Peripheral leptin treatment is likely to produce central modulation of GnRH secretion based on our recent findings that leptin can alter LH pulse frequency (60). In the adult female rat during a 48-hr fast, both leptin concentrations and LH pulse frequency are decreased. More importantly, this fasting-induced reduction of LH secretion is prevented by peripheral administration of leptin. This study was repeated in the sheep with similar results (61; Figure 1). High frequency (-hourly) LH pulses were measured in fed sheep, and LH pulse frequency was decreased after a 72-hr fast. Leptin treatment during the fast prevented a reduction in LH pulse frequency. Leptin could act within the brain to influence GnRH secretion during fasting. When central leptin action is blunted in the fed rat by intracerebroventricular (icv) treatment with leptin antibody, estrous cyclicity and pulsatile LH secretion cease (17). In vitro, GnRH release from hypothalamic explants in leptin-free medium is lower than in the presence of leptin (75). However, a cautious evaluation as to the central action of leptin is needed, because in vivo, icv leptin treatment only partially restores basal LH secretion in the fasted rat (46). In chronically feed-restricted (not fasted) sheep, LH secretion is increased after 3 d of icv leptin treatment (43). Interestingly, central administration of leptin suppressed appetite in feed-restricted, but not ad libitum-fed sheep, and leptin did not alter LH secretion in well-fed animals.
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Figure 1. Representative LH profiles from castrated, estradiol-treated male sheep before and after (shaded panels) a 72-hr fast. Leptin administered during the fast to the four animals shown on the right prevented the decrease in LH pulse frequency that occurred in vehicle-treated animals on the left. Figure redrawn from (61). Peripheral (19) and central (4 1) administration of leptin also prevents the hypogonadotropic delay in puberty caused by undernutrition. However, only two studies have advanced puberty, as determined by vaginal opening, using exogenous leptin (2, 18); thus, the question of whether leptin is the metabolic key to neuroendocrine puberty remains unanswered. Peripubertal changes in leptin concentrations have been reported in rats (62), monkeys (70), and humans (56), but other studies in some of these same species have failed to find a correlation between leptin and puberty (37, 63). These disparities could be due to species-specific differences in leptin physiology (including the relevance of critical fat reserves), blood sampling frequency, the anorexigenic effects of leptin, and the inability to identify or thoroughly examine critical early prepubertal stages encompassing initial changes in GnRH secretion. We have recently reported that exogenous leptin stimulates LH pulse frequency in well-nourished, prepubertal lambs, although this effect appears to depend on degree of somatic development. Leptin treatment did not increase pulse frequency in small lambs not yet producing LH pulses in the presence of steroid negative feedback (45). Thus, leptin is not the sole metabolic signal controlling the pubertal increase in GnRH secretion. Leptin may act as a permissive signal to increase GnRH secretion only after the pulse generator has been sensitized to energy balance by other developmental or metabolic cues. Internretation of Neuroendocrine Studies. Our current understanding of how leptin regulates reproductive neuroendocrinology is limited by the fact that leptin probably does not act
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directly on GnRH neurons based on the paucity of leptin receptors on these cells (33, 42). In addition, leptin has widespread endocrine and metabolic effects outside the brain. Thus, conclusions about the role of leptin in hypothalamic-hypophyseal function must include consideration of central versus peripheral effects and nutritional status. Finally, metabolic signaling must be integrated with other external factors, such as photoperiod, social stimuli, and stress. The relative importance of these other endogenous and exogenous factors depends on where leptin acts within the brain (Figure 2) and whether central effects of leptin are regulated by the hormone’s peripheral actions. Leptin receptors are co-localized in the arcuate and ventromedial nuclei of the hypothalamus with neuropeptides such as galanin, endogenous opioids, and neuropeptide Y (NPY). These peptides regulate both appetite and LH secretion and, thus, could mediate leptin’s effects on GnRH secretion relative to energy balance. Leptin could also act indirectly on the hypothalamus by regulating glucose availability through its actions on glucose transport molecules or by peripheral mechanisms such as decreasing insulin release (49) and increasing insulin sensitivity (7, 20). Our results in prepubertal male lambs (45) provide support for an indirect or convergent action of leptin, incorporating other developmentally dependent metabolic signals (e.g., insulin or glucose).
presynaptic
Figure 2.
Possible sites of action for leptin within the reproductive neuroendocrine system. Neuropeptide Y (NPY), pro-opiomelanocortin (POMC), and galanin (GAL) neurons in the arcuate (ARC) and ventromedial hypothalamic (VMH) nuclei contain leptin receptors, and these neuropeptides have been shown to regulate GnRH secretion. Thus, they are potential mechanistic links between leptin, regulation of appetite, and reproduction.
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Direct Effects of Leptin on Gonadal Function As is the case in all initial endocrine studies, one must ask the question of responsiveness of the tissue of interest to the ligand of interest. In this respect, studies have focused on the presence/absence of leptin receptors in mammalian gonads. Using reverse transcription polymerase chain reaction (RT-PCR), leptin receptor transcript presence has been demonstrated in rat ovarian tissue and isolated granulosa cells (77). In similar RT-PCR approaches, transcripts for the leptin receptor long form, which has known signal-transducing capabilities, have been found in human granulosa, theta (1, 47), and Leydig cells (15). In addition, ‘251-leptin displays concentration-dependent binding to isolated bovine granulosa and theta cells (66, 67). Thus, granulosa and theta cells, as well as Leydig cells, contain the ability to respond to leptin. The next question that arises is whether leptin is spatially available to interact with ovarian granulosa and theta cells. As might be expected, follicular fluid, which is derived from serum, has been found to contain leptin (1, 6, 12, 21, 47, 56). Contradictory data exist regarding whether the ovary is capable of producing leptin. Using RT-PCR and immunolocalization, both leptin transcript and protein were identified in human granulosa and cumulus cells (21). Conversely, leptin transcript was not identified by RT-PCR in human granulosa, theta, or interstitial cells (47). Mean leptin concentrations within serum and follicular fluid are not significantly different (1, 12, 47) suggesting that leptin is neither sequestered in, nor excluded from, follicular fluid. There is no evidence for intra-testicular production of leptin. However, a small amount of serum-born leptin has been demonstrated to cross the blood-testis barrier by a passive, nonsaturable process (4). Interestingly, identification of very low expression of alternatively spiced leptin receptor isoforms could be achieved with an RNase protection assay approach, even though the investigators were unable to detect the leptin receptor in the testis by in situ hybridization. In vitro experiments performed on theta, granulosa, and luteinized granulosa cells demonstrate that leptin can have a direct inhibitory influence on steroidogenesis in rodent, bovine, and primate models (1, 10, 47, 66, 67, 76). Although leptin does not appear to alter basal steroidogenesis, it does inhibit various mechanisms of augmented or stimulated steroidogenesis. Specifically, leptin can inhibit insulin-induced progesterone and estradiol production, as well as insulin-induced progesterone and androstenedione production from bovine granulosa and theta cells, respectively (66, 67). In the rodent model, leptin impairs hormonally stimulated release of estradiol from rat granulosa cells (76). Leptin can significantly reduce estradiol production by human granulosa cells in response to LH (47). Similarly, leptin treatments of human granulosa cells caused a dose-dependent inhibition of insulin-like growth factor I augmentation of follicle In addition, human luteinized stimulating hormone-stimulated estradiol production (1). granulosa cells display a leptin time- and dose-dependent inhibition of human chorionic gonadotropin-stimulated progesterone production (10). These observations demonstrate that high leptin concentrations commonly found in obese women have the potential to compromise normal ovarian steroidogenesis. Considering the central role gonadal steroids play in regulating hypothalamic/pituitary hormones, in particular preovulatory LH release, one can appreciate how elevated serum and/or follicular fluid levels of leptin might compromise reproduction in the form of anovulation.
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elevated leptin may disrupt normal hypothalamic/pituitaryry/gonadal Although communication, a recent report suggests that elevated leptin can also directly inhibit ovulation (28). Intraperitoneal administration of leptin caused a significant reduction in ovulation rate in response to exogenous gonadotropins in comparison with non-leptin treated and weight loss controls. This effect of leptin was not a result of reduced follicular growth as suggested by equivalent representation of follicular stages between leptin treatment and controls. These results were confirmed in a perfused rat ovarian model, suggesting that leptin might have a direct effect on ovulation at the ovarian level. Collectively, the ability of elevated leptin concentrations to compromise gonadal steroid production and potentially interrupt normal oocyte maturation and/or gonadal-steroid positive feedback on preovulatory gonadotropin secretion, or to block the ovulatory process, are inviting correlates in beginning to explain reduced fertility and anovulation, which can occur in obese states (22). In the Leydig cells, leptin can inhibit LH/hCG-stimulated testosterone and androstenedione production and result in a concomitant elevation in steroidogenic precursor metabolites (15). These results are in agreement with the ability of leptin to inhibit testosterone production from adult rat testicular slices (72). Collectively, the results in the male suggest that leptin can modulate gonadotropin-stimulated steroidogenesis, which is analogous to its perceived role in the female. Leptin in the Gametes and Preimplantation
Embryo
A possible role of leptin in the regulation of gamete and preimplantation embryo development has been suggested in both rodents and humans. Although the transcript for leptin is not identifiable, the transcript and protein representing the functional long form of the leptin receptor is present in germinal vesicle-intact and metaphase II oocytes (57). The oocyte leptin receptor appears to be functional, as suggested by tyrosine phosphorylation of STAT3 in response to leptin (57). Leptin protein has been immunolocalized to metaphase II human oocytes (21); however, the means by which leptin gains access into the oocyte cytoplasm is not fully understood. It has been suggested that leptin from granulosa/cumulus cells may enter the oocyte by way of receptor-mediated events, endocytosis, or by a presently unidentified means. Currently, the functional importance of leptin at the oocyte level is yet to be reported. The functional leptin receptor has been identified in mouse male germ cells and appears to have both age- and stage-specific intra-germ cell locations (30). In 5-d-old mice, functional leptin receptor was expressed primarily in type A spermatogonia, whereas in the 20 to 30-d-old and adult mouse, leptin receptor was localized to the spermatocyte. The authors speculate that leptin may have a direct role in regulating proliferation and differentiation of male germ cells. Such a developmental role could partially explain infertility observed in leptin-deficient mice. However, future research is needed to support or refute this hypothesis. Currently, there is but one report of the presence of leptin in preimplantation embryos. Both mouse and human embryos display leptin and its intracellular second messenger protein STAT3 (3). However, whether mammalian preimplantation embryos contain functional leptin receptors and, thus, the link between leptin and STAT3, have not been reported. The immunolocalization and trafficking of leptin and STAT3 in the embryo from the two-cell to blastocyst stage has given rise to the idea that their polarity are important factors contributing to
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identity and fate of individual blastomeres. Such unique protein domains or intracellular/intercellular concentration gradients have been suggested to be important for early mammalian development, such as determining the animal pole (38) and/or establishing inner cell mass and trophectoderm (29). Although this is an interesting speculation that warrants further investigation, currently, direct evidence to suggest such a role for leptin is limited. Leptin Monitoring or Treatment and Modulation of Fertility Considering the numerous reports generated regarding leptin and possible regulation of reproduction at the neuroendocrine axis, gonadal, and gamete/embryo levels, it appears that exciting times are ahead in determining the functional importance of leptin and how this information might be used to regulate or predict fertility. As recently suggested by Caprio et al. (14), it is quite possible that leptin plays a dual role in regulating reproduction (Figure 3). Low leptin levels may negatively influence the neuroendocrine regulation of reproduction with a threshold level being permissive to normal reproduction. Conversely, elevated leptin levels may negatively influence normal ovarian function and/or embryo development and viability. In addition, these thresholds are most likely flexible and, thus, can be pushed in one direction or the other based on responsiveness of the neuroendocrine system andfor gonads to exogenous leptin. This plasticity may be a result of changing leptin receptor numbers and/or altered second messenger systems. Polycystic ovarian syndrome (PCOS) is one of the most prevalent reproductive abnormalities in premenopausal women and is an example of an area currently studied with regard to leptin and reproduction. Combining the information that many women with PCOS are obese and that Ieptin is involved with animal and human obesity has given way to the speculation that leptin is involved with the pathophysiology of PCOS. Potential contribution of leptin to PCOS has been suggested from reports demonstrating that leptin levels are elevated in a cohort of women with PCOS compared with women without the syndrome (11). However, subsequent studies have been unable to confirm this relationship (5 1, 55, 64). Another prominent symptom of PCOS is insulin resistance and hyperinsulinemia. These parameters also have been investigated in relation to leptin and PCOS, and current data are conflicting (48, 55, 64). These conflicts demonstrate the necessity for further investigations into the possible role of leptin and/or leptin mediators in the pathogenesis of PCOS. As a mediator of fertility, one can begin to appreciate that future efforts may focus on prepubertal administration of leptin to accelerate sexual maturation in domestic livestock species. In addition, administration of exogenous leptins may potentiate shortening of postpartum anestrus intervals in those species that experience this source of infertility. Unsuccessful attempts have been made to correlate follicular fluid leptin levels to embryo development in human-assisted reproduction technology cycles (21). Interestingly, it was found that a postovulatory rise in serum leptin concentrations was associated with implantation potential in women undergoing infertility treatment (21). This observation, coupled with the suggestion that leptin is important in regulating preimplantation embryo development, raises possibilities for
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Leptin Levels Plasticity due to 4 or t Leptin Responsiveness
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Figure 3. Theoretical proposal whereby low levels of leptin during significant negative (Neg.) energy balance can be non-supportive of normal reproductive capacity through actions at the neuroendocrine level. In addition, significantly elevated leptin levels may also adversely influence reproductive capacity through actions at the gonadal or embryo level. Increased (e) or decreased (+) responsiveness of the leptin system could generate plasticity in the influence of leptin on reproductive capacity. Adapted from ( 14). future investigations into the exogenous administration of leptin, following IVF, in an attempt to improve implantation rates. Although these speculations may hold great promise, the feasibility of such options currently requires testing in model systems. REFERENCES 1.
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37. Garcia-Mayor RV, Andrade M, Rios M, Lage M, Diequez C, Casaneuva FF. Serum leptin levels in normal children: relationship to age, gender, body mass index, pituitary-gonadal hormones and pubertal stage. J Clin Endocrinol Metab 1997;82:2849-2855. 38. Gardner R. The early blastocyst is bilaterally symmetrical and its axis of symmetry is aligned with the animal-vegetal axis of the zygote in the mouse. Development 1997;124:289-301. 39. Golden PL, Maccagnan TJ, Pardridge WM. Human blood-brain barrier leptin receptor. J Clin Invest 1997;99: 14-l 8. 40. Granowitz E. Transforming growth factor-beta enhances and pro-inflammatory cytokines inhibit ob gene expression in 3T3-LI adipocytes. Biochem Biophys Res Commun 1997;240:382-385. 41. Gruaz NM, Laloui M. Pierroz DD, Englaro P, Sizonenko PC, Blum WF, Aubert ML. Chronic administration of leptin into the lateral ventricle induces sexual maturation in severely food-restricted female rats. J Neuroendocrinol 1998;10:627-633. 42. H&kansson ML, Brown H, Ghilardi N, Skoda RC, Meister B. Leptin receptor immunoreactivity in chemically defined target neurons of the hypothalamus. J Neurosci 1998; 18:559-572. 43. Henry BA, Goding JW, Tilbrook AJ, Dunshea FR, Clarke IJ. Intracerebroventricular infusion of leptin elevates the secretion of luteinizing hormone without affecting food intake in long-term food-restricted sheep, but increases growth hormone irrespective of bodyweight. J Endocrinol2001;168:67-77. 44. Hoggard N, Mercer JG, Rayner DV, Moar K, Trayhurm P, Williams LM. Localization of leptin receptor mRNA splice variants in murine peripheral tissues by RT-PCR and in situ hybridization. Biochem Biophys Res Commun 1997;232:383-387. 45. Jackson LM, Jaffe CA, Pelt J, Foster DL. Leptin regulation of LH secretion in prepubertal sheep. Biol Reprod 2001; 64(Suppl 1): 189 abstr. 46. Kalra SP, Xu B, Dube MG, Moldawer LL, Marin D, Kalra PS. Leptin and ciliary neurotropic factor (CNTF) inhibit fasting-induced suppression of luteinizing hormone release in rats: Role of neuropeptide Y. Neurosci Lett 1998;240:45-49. 47. 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. 48. Krassas GE, Kaltsas TT, Pontikides N, Jacobs H, Blum W, Messinis I. Leptin levels in women with polycystic ovary syndrome before and after treatment with d&oxide. Eur J Endocrinol 1998; 139: 184-l 89. 49. Kulkami RN, Wang ZL, Wang RM, Hurley JD, Smith DM, Ghatei MA, Withers DJ, Gardiner JV, Bailey CJ, Bloom SR. Leptin rapidly suppresses insulin release from insulinoma cells, rat and human islets and, in vivo, in mice. J Clin Invest 1997;100:2729-36. 50. Lahlou M, Clement K, Care1 JC, Vaisse C, Lotton C, LeBihan Y, Basdevant A, Lebouc Y, Froguel P, Roger M, Guy-Grand B. Soluble leptin receptor in serum of subjects with complete resistance to leptin: Relation to fat mass. Diabetes 2000;49: 1347-l 352. 5 I. Laughlin GA, Morales AJ, Yen SS. Serum leptin levels in women with polycystic ovary J Clin Endocrinol Metab syndrome: The role of insulin resistance/hyperinsulinemia. 1997;82: 1692-I 696. 52. Lee GH, Proenca R, Montez JM, Carroll KM, Darvishzadeh JG, Lee JI, Friedman JM. Abnormal splicing of the leptin receptor in diabetic mice. Nature 1996;379:632-635.
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53. Lynn RB, Cao G-Y, Considine RV, Hyde TM, Caro JF. Autoradiographic localization of leptin binding in the choroid plexus of ob/ob and db/db mice. Biochem Biophys Res Commun 1996;219:884-889. 54. 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-l 161. 55. Mantzoros CS, Dunaif A, Flier JS. Leptin concentrations in the polycystic ovary syndrome. J Clin Endocrinol Metab 1997;82: 1687- 1691. 56. Mantzoros CS, Pleit JS, Rogol AD. A longitudinal assessment of hormonal and physical alterations during normal puberty in boys. V. Rising leptin levels may signal the onset of puberty. J Clin Endocrinol Metab 1997;82: 1066-1070. 57. Matsuoka T, Tahara M, Yokoi T, Masumoto N, Takeda T, Yamaguchi M, Tasaka K, Kurachi H, Murata Y. Tyrosine Phosphorylation of STAT3 by leptin through leptin receptor in mouse metaphase 2 stage oocyte. Biochem Biophys Res Comm 1999;256:480484. 58. Mercer JG, Hoggard N, Williams LM, Lawrence DB, Hannah LT, Morgan PJ, Trayhum P. Coexpression of leptin receptor and preproneuropeptide Y mRNA in arcuate nucleus of mouse hypothalamus. J Neuroendocrinol 1996;8:733-735. 59. Montague CT, Farooqi IS, Whitehead JP, Soos MA Rau H, Wareham NJ, Sewter CP, Digby JE, Mohammed SN, Hurst JA, Cheetham CH, Earley AR, Barnett AH, Prins JB, O’Rahilly S. Congenital leptin deficiency is associated with severe early-onset obesity in humans. Nature 1997;387:903-908. 60. Nagatani S, Guthikonda P, Thompson RC, Tsukamura H, Maeda K-I, Foster DL. Evidence for G&I-I regulation by leptin: Leptin administration prevents reduced pulsatile LH secretion during fasting. Neuroendocrinology 1998;67:370-376. 61. Nagatani S, Zeng Y, Keisler DH, Foster DL, Jaffe CA. Leptin regulates pulsatile luteinizing hormone and growth hormone secretion in the sheep. Endocrinology 2000;141:3965-3975. 62. Nagatani S, Guthikonda P, Foster DL. Appearance of a nocturnal peak of leptin secretion in the prepubertal rat. Hotm Behav 2000;37:345-352. 63. Plant TM, Dun-ant AR. Circulating leptin does not appear to provide a signal for triggering the initiation of puberty in the male rhesus monkey (Macaca mulatta). Endocrinology 1998; 139:2284-2286. 64. Rouru J, Anttila L, Koskinen P, Penttila TA, Irjala K, Huupponen R, Koulu M. Serum leptin concentrations in women with polycystic ovary syndrome. J Clin Endocrinol Metab 1997; 82:1697-1700. 65. Sinha MK, Opentonaova I, Ihannesian JP, Kolaczynski JW, Heiman ML, Hale J, Becker GW, Bowsher RR, Stephens TW, Caro JF. Evidence of free and bound leptin in human circulation - studies in lean and obese subjects and during short-term fasting. J Clin Invest 1996; 98:1277-1282. 66. Spicer LJ, Francisco CC. The adipose obese gene product, leptin: evidence of a direct inhibitory role in ovarian function. Endocrinology 1997;138:3374-3379. 67. Spicer LJ, Francisco CC. Adipose obese gene product, leptin, inhibits bovine ovarian thecal cell steroidogenesis. Biol Reprod 1998;58:207-212. 68. Stephens TW, Basinski M, Bristow PK, Bue-Valleskey JM, Burgett SG, Craft L, Hale J, Hoffmann J, Hsiung HM, Kriauciunas A, MacKellar W, Rosteck, Jr. PR, Schoner B, Smith
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D, Tinsley FC, Zhang XY, Heiman M. The role of neuropeptide Y in the antiobesity action of the obese gene product. Nature 1995;377:530-532. Strobe1 A, Issad T, Camoin L, Ozata M, Strosberg AD. A leptin missense mutation associated with hypogonadism and morbid obesity. Nat Genet 1998; 18:2 13-2 15. Suter KJ, Pohl CR, Wilson ME. Circulating concentrations of nocturnal leptin, growth hormone, and insulin-like growth factor-I increase before the onset of puberty in agonadal male monkeys: potential signals for the initiation of puberty. J Clin Endocrinol Metab 2000;85:808-814. Tartaglia LA, Dembski M, Weng X, Deng N, Culpepper J, Devos R, Richards GJ, Campfield LA, Clark FT, Deeds J. Identification and expression cloning of a leptin receptor, OB-R. Cell 1995;83:1263-1271. Tena-Sempere M, Pinilla L, Gonzalez LC, Dieguez C, Casanueva FF, Aguilar E. Leptin inhibits testosterone secretion from adult rat testis in vitro. J Endocrinol 1999; 16 1:2 1 l-2 18. Wauters M, Considine RV, Van Gaal, LF. Human leptin: from an adipocyte hormone to an endocrine mediator. Eur J Endocrinol2000;143:293-311. Yamashita T, Marakami T, Otani S, Kuwajima M, Shima K. Leptin receptor signal transduction: OBRa and OBRb offi type. Biochem Biophys Res Comm 1998;246:752-759. Yu WH, Kimura M, Walczewdka A, Karanth S, McCann SM. Role of leptin in hypothalamic pituitary function. Proc Nat1 Acad Sci USA 1997;94: 1023-l 028. 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-17P production by rat ovarian granulosa cells. Endocrinology 1997;138:847-850. Zamorano PL, Mahesh VB, De Sevilla LM, Chorich LP, Bhat GK, Brann DW. Expression and localization of the leptin receptor in endocrine and neuroendocrine tissues of the rat. Neuroendocrinology 1997;65:223-228. Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM. Positional cloning of the mouse obese gene and its human homologue. Nature 1994;372:425-432.
LEPTIN REGULATION
OF REPRODUCTIVE FUNCTION AND FERTILITY
G. D. Smith’X3.4,5,L. M. Jackson’, and D. L. Foster’,*,’ Departments of Obstetrics and Gynecology’, Biology’, Physiology3, and Urology4; Reproductive Sciences Program’, University of Michigan, Ann Arbor, Michigan, USA 48109-0617 ABSTRACT Leptin, a 16-KD protein secreted primarily by adipose tissue, was first discovered in the search for a satiety signal. When administered into the brain, leptin depresses appetite. Interestingly, hyperphagic, obese, transgenic mice with leptin deficiency were noted to be reproductively incompetent, and administration of leptin restored their fertility. These pivotal observations led to numerous studies on the site of action of leptin within the hypothalamohypophyseal-gonadal axis, and a variety of models have been used ranging from the prepubertal condition to fasting suppression of reproductive hormones. The preponderance of studies thus far has focused on how leptin serves as a metabolic signal of energy balance within the neuroendocrine system, particularly as a regulator of GnRH/LH secretion. Less research has been conducted with other components of the reproductive system, but local effects of leptin have been demonstrated in the gonads where hyperleptinemia suppresses steroidogenesis and potentially affects gamete maturation. This presentation will review the major concepts for the role of leptin in the modulation of fertility and will consider the potential use of leptin in assisted reproductive technology and embryo transfer. 0 2001 by Elsevier Science Inc. Key words: leptin, neuroendocrine, ovary, gametes, embryo
INTRODUCTION Leptin is a 16-kD protein product of the obese gene (ob) produced primarily by adipose tissue; it is believed to play an important role in the regulation of food intake and body weight (68). As a satiety hormone, leptin concentrations can change in response to caloric intake and can suppress appetite, increase metabolic rate, and regulate weight gain and fat deposition (13, 26, 40, 78). Studies in humans demonstrate a positive correlation between the release and synthesis of leptin and body mass index or percentage of body fat (27, 54) and peripheral leptin concentrations decrease in response to fasting or dietary restriction (2, 61, 65). In addition, numerous reports suggest that leptin is an important regulator of reproductive function and provides a signaling link between nutritional status and reproduction. This review will focus on accepted and developing theories of how leptin is involved in mediating reproduction and
Acknowledgments: Works presented in this review and presentation were supported by NIH Grants HD35125OlAl (GDS), HD18394 (DLF). Postdoctoral training support (L.M. Jackson) provided by HD07048 (Training Grant, Reproductive Sciences Program, University of Michigan). Correspondence: Dr. Gary D. Smith, 6428 Medical Sciences Building I, 1301 E. Catherine St., Ann Arbor, MI 481090617. Phone: (734) 764-4134; Fax: (734) 647-1006; E-mail: [email protected]. Theriogenology 57:73-66. 0 2001 Elsevier Science
2002 Inc.
0093-691W02/$-.see front matter PII: SOO93-691X(01)00656-6
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fertility. As a means to this end, information will be discussed regarding: 1) leptin, its receptors and mechanism of intracellular signal transduction; 2) the role of leptin in reproductive neuroendocrinology; 3) direct effects of leptin on gonadal function; and 4) leptin in the gamete and preimplantation embryo. We will conclude with a section addressing possible future applications of leptin monitoring or treatment to aid in assisted reproductive practices and/or modulation of fertility. Leptin, Its Receptors, and Mechanism of Action Two mutant mouse strains, oblob and dbldb, demonstrate the importance of leptin in regulating appetite, metabolism, and body composition. The absence of the complete protein (ob/ob) or the leptin receptor (db/db) leads to hyperphagia and subsequent obesity (24). However, examination of the human ob gene found that the gene encoding for leptin is normal in most cases of obesity (25, 54, 59, 69). A missense mutation in the leptin gene has been identified in two families in which individuals homozygous for the mutation exhibit low circulating leptin concentrations, hyperphagia, and severe obesity. Treatment of children with this congenital defect with recombinant human leptin restores leptin concentrations to normal levels, reduces body weight, and alleviates other metabolic irregularities (32). Similarly, defects in the human leptin receptor are rare (23), and, thus, morbid obesity in humans differs from rodent models and is not readily explained by alterations in a single gene. Other aspects of leptin physiology currently under investigation for their role in appetite disorders and obesity include central leptin resistance caused by impaired transport across the blood-brain barrier (16) and interactions between circulating leptin and its binding protein (65). The leptin receptor is a member of the class I cytokine receptor family and exists in at least five isoforms arising from mRNA splice variants (52, 71). The single long form of the receptor (Ob-Rb) includes a long (302 amino acids) intracellular domain involved in activating the Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway (8). One short form (Ob-Ra) has a short (31 amino acids) intracellular domain capable of activating the mitogen-activated protein (MAP) kinase pathway (9, 74). Ob-Rb is the predominant receptor expressed within the brain, particularly in the hypothalamus (3 1, 58), and Ob-Ra is found principally in peripheral tissues such as liver, pancreas, gonads, and skeletal muscle (44, 47, 52). Nevertheless, Ob-Ra is present in the choroid plexus (53) and neural capillaries (39), where it is involved with leptin transport between blood and cerebrospinal fluid, and across the blood-brain barrier. Another isoform, Ob-Re, lacks both transmembrane and cytoplasmic domains, is soluble, and serves as a circulating leptin-binding protein (50). Multiple forms of the leptin receptor and their distribution outside the central nervous system suggest that the role of leptin extends beyond its function as a satiety factor. In addition to regulating appetite and energy intake, leptin affects several metabolic and neuroendocrine mechanisms to control or partition energy expenditure (34,73). Role of Leptin in Reproductive Neuroendocrinology Historical Association of Fatness and Fertility. The link between leptin and reproduction has multiple and seemingly unrelated origins in studies of human puberty, the effects of energy balance on human reproductive function, optimizing fertility by increasing feed in domestic
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species, and infertility in a mutant mouse model. The earlier demographic studies of Frisch (35) sought to explain the initiation of reproductive activity in women and demonstrated that a certain degree of fatness is associated with menarche as well as with maintenance of menstrual cyclicity. This work was of clinical use, but failed to provide a mechanism for how the brain detects how fat the body is and then uses that information to increase gonadotropin secretion to stimulate the ovaries. The simple fact that there was nothing yet identified that was secreted by fat and could serve as a signal to the brain was a practical constraint, but Frisch suggested an indirect link by proposing that fatness enhanced fertility through increased conversion of androgens to estrogens by adipose tissue (36). Given the negative feedback effects of estrogen on GnRH and LH secretion (except during the preovulatory surge), this was never considered to be a viable mechanistic explanation in terms of neuroendocrine control. Nonetheless, this work provided a historical background for the current interest in fat and fertility and the possibility that fat produces something that the brain can monitor to assess somatic energetics. During the several years when mouse mutants were used to study metabolism and appetite behavior, most reproductive scientists paid little attention to the fact that the ob/ob and db/db mutants were infertile. This changed dramatically following the report that leptin injections into adult ob/ob mice restored their reproductive capacity (18), a finding that provided a novel mechanistic bridge between fatness and fertility. Since this discovery, the possibility that leptin serves as a molecular intermediary to link metabolism and reproduction has attracted the attention of many reproductive scientists. Neuroendocrine Actions of Leutin on GnRH Secretion. To date, only a handful of studies have focused on the neuroendocrine control of GnRH secretion by leptin in a physiological Barash et al. (5) found that, in ob/ob mice, leptin administration increases basal LH setting. levels. In wild-type mice, fasting suppressed peripheral leptin concentrations, basal LH concentrations, and estrous cyclicity, and such deficits could be prevented by exogenous leptin (2). Intravenous infusion of leptin restored both LH and FSH secretion on the second day of a 2d fast in peripubertal male rhesus monkeys (33). Peripheral leptin treatment is likely to produce central modulation of GnRH secretion based on our recent findings that leptin can alter LH pulse frequency (60). In the adult female rat during a 48-hr fast, both leptin concentrations and LH pulse frequency are decreased. More importantly, this fasting-induced reduction of LH secretion is prevented by peripheral administration of leptin. This study was repeated in the sheep with similar results (61; Figure 1). High frequency (-hourly) LH pulses were measured in fed sheep, and LH pulse frequency was decreased after a 72-hr fast. Leptin treatment during the fast prevented a reduction in LH pulse frequency. Leptin could act within the brain to influence GnRH secretion during fasting. When central leptin action is blunted in the fed rat by intracerebroventricular (icv) treatment with leptin antibody, estrous cyclicity and pulsatile LH secretion cease (17). In vitro, GnRH release from hypothalamic explants in leptin-free medium is lower than in the presence of leptin (75). However, a cautious evaluation as to the central action of leptin is needed, because in vivo, icv leptin treatment only partially restores basal LH secretion in the fasted rat (46). In chronically feed-restricted (not fasted) sheep, LH secretion is increased after 3 d of icv leptin treatment (43). Interestingly, central administration of leptin suppressed appetite in feed-restricted, but not ad libitum-fed sheep, and leptin did not alter LH secretion in well-fed animals.
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Fast+vehicle 60
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Figure 1. Representative LH profiles from castrated, estradiol-treated male sheep before and after (shaded panels) a 72-hr fast. Leptin administered during the fast to the four animals shown on the right prevented the decrease in LH pulse frequency that occurred in vehicle-treated animals on the left. Figure redrawn from (61). Peripheral (19) and central (4 1) administration of leptin also prevents the hypogonadotropic delay in puberty caused by undernutrition. However, only two studies have advanced puberty, as determined by vaginal opening, using exogenous leptin (2, 18); thus, the question of whether leptin is the metabolic key to neuroendocrine puberty remains unanswered. Peripubertal changes in leptin concentrations have been reported in rats (62), monkeys (70), and humans (56), but other studies in some of these same species have failed to find a correlation between leptin and puberty (37, 63). These disparities could be due to species-specific differences in leptin physiology (including the relevance of critical fat reserves), blood sampling frequency, the anorexigenic effects of leptin, and the inability to identify or thoroughly examine critical early prepubertal stages encompassing initial changes in GnRH secretion. We have recently reported that exogenous leptin stimulates LH pulse frequency in well-nourished, prepubertal lambs, although this effect appears to depend on degree of somatic development. Leptin treatment did not increase pulse frequency in small lambs not yet producing LH pulses in the presence of steroid negative feedback (45). Thus, leptin is not the sole metabolic signal controlling the pubertal increase in GnRH secretion. Leptin may act as a permissive signal to increase GnRH secretion only after the pulse generator has been sensitized to energy balance by other developmental or metabolic cues. Internretation of Neuroendocrine Studies. Our current understanding of how leptin regulates reproductive neuroendocrinology is limited by the fact that leptin probably does not act
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directly on GnRH neurons based on the paucity of leptin receptors on these cells (33, 42). In addition, leptin has widespread endocrine and metabolic effects outside the brain. Thus, conclusions about the role of leptin in hypothalamic-hypophyseal function must include consideration of central versus peripheral effects and nutritional status. Finally, metabolic signaling must be integrated with other external factors, such as photoperiod, social stimuli, and stress. The relative importance of these other endogenous and exogenous factors depends on where leptin acts within the brain (Figure 2) and whether central effects of leptin are regulated by the hormone’s peripheral actions. Leptin receptors are co-localized in the arcuate and ventromedial nuclei of the hypothalamus with neuropeptides such as galanin, endogenous opioids, and neuropeptide Y (NPY). These peptides regulate both appetite and LH secretion and, thus, could mediate leptin’s effects on GnRH secretion relative to energy balance. Leptin could also act indirectly on the hypothalamus by regulating glucose availability through its actions on glucose transport molecules or by peripheral mechanisms such as decreasing insulin release (49) and increasing insulin sensitivity (7, 20). Our results in prepubertal male lambs (45) provide support for an indirect or convergent action of leptin, incorporating other developmentally dependent metabolic signals (e.g., insulin or glucose).
presynaptic
Figure 2.
Possible sites of action for leptin within the reproductive neuroendocrine system. Neuropeptide Y (NPY), pro-opiomelanocortin (POMC), and galanin (GAL) neurons in the arcuate (ARC) and ventromedial hypothalamic (VMH) nuclei contain leptin receptors, and these neuropeptides have been shown to regulate GnRH secretion. Thus, they are potential mechanistic links between leptin, regulation of appetite, and reproduction.
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Direct Effects of Leptin on Gonadal Function As is the case in all initial endocrine studies, one must ask the question of responsiveness of the tissue of interest to the ligand of interest. In this respect, studies have focused on the presence/absence of leptin receptors in mammalian gonads. Using reverse transcription polymerase chain reaction (RT-PCR), leptin receptor transcript presence has been demonstrated in rat ovarian tissue and isolated granulosa cells (77). In similar RT-PCR approaches, transcripts for the leptin receptor long form, which has known signal-transducing capabilities, have been found in human granulosa, theta (1, 47), and Leydig cells (15). In addition, ‘251-leptin displays concentration-dependent binding to isolated bovine granulosa and theta cells (66, 67). Thus, granulosa and theta cells, as well as Leydig cells, contain the ability to respond to leptin. The next question that arises is whether leptin is spatially available to interact with ovarian granulosa and theta cells. As might be expected, follicular fluid, which is derived from serum, has been found to contain leptin (1, 6, 12, 21, 47, 56). Contradictory data exist regarding whether the ovary is capable of producing leptin. Using RT-PCR and immunolocalization, both leptin transcript and protein were identified in human granulosa and cumulus cells (21). Conversely, leptin transcript was not identified by RT-PCR in human granulosa, theta, or interstitial cells (47). Mean leptin concentrations within serum and follicular fluid are not significantly different (1, 12, 47) suggesting that leptin is neither sequestered in, nor excluded from, follicular fluid. There is no evidence for intra-testicular production of leptin. However, a small amount of serum-born leptin has been demonstrated to cross the blood-testis barrier by a passive, nonsaturable process (4). Interestingly, identification of very low expression of alternatively spiced leptin receptor isoforms could be achieved with an RNase protection assay approach, even though the investigators were unable to detect the leptin receptor in the testis by in situ hybridization. In vitro experiments performed on theta, granulosa, and luteinized granulosa cells demonstrate that leptin can have a direct inhibitory influence on steroidogenesis in rodent, bovine, and primate models (1, 10, 47, 66, 67, 76). Although leptin does not appear to alter basal steroidogenesis, it does inhibit various mechanisms of augmented or stimulated steroidogenesis. Specifically, leptin can inhibit insulin-induced progesterone and estradiol production, as well as insulin-induced progesterone and androstenedione production from bovine granulosa and theta cells, respectively (66, 67). In the rodent model, leptin impairs hormonally stimulated release of estradiol from rat granulosa cells (76). Leptin can significantly reduce estradiol production by human granulosa cells in response to LH (47). Similarly, leptin treatments of human granulosa cells caused a dose-dependent inhibition of insulin-like growth factor I augmentation of follicle In addition, human luteinized stimulating hormone-stimulated estradiol production (1). granulosa cells display a leptin time- and dose-dependent inhibition of human chorionic gonadotropin-stimulated progesterone production (10). These observations demonstrate that high leptin concentrations commonly found in obese women have the potential to compromise normal ovarian steroidogenesis. Considering the central role gonadal steroids play in regulating hypothalamic/pituitary hormones, in particular preovulatory LH release, one can appreciate how elevated serum and/or follicular fluid levels of leptin might compromise reproduction in the form of anovulation.
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elevated leptin may disrupt normal hypothalamic/pituitaryry/gonadal Although communication, a recent report suggests that elevated leptin can also directly inhibit ovulation (28). Intraperitoneal administration of leptin caused a significant reduction in ovulation rate in response to exogenous gonadotropins in comparison with non-leptin treated and weight loss controls. This effect of leptin was not a result of reduced follicular growth as suggested by equivalent representation of follicular stages between leptin treatment and controls. These results were confirmed in a perfused rat ovarian model, suggesting that leptin might have a direct effect on ovulation at the ovarian level. Collectively, the ability of elevated leptin concentrations to compromise gonadal steroid production and potentially interrupt normal oocyte maturation and/or gonadal-steroid positive feedback on preovulatory gonadotropin secretion, or to block the ovulatory process, are inviting correlates in beginning to explain reduced fertility and anovulation, which can occur in obese states (22). In the Leydig cells, leptin can inhibit LH/hCG-stimulated testosterone and androstenedione production and result in a concomitant elevation in steroidogenic precursor metabolites (15). These results are in agreement with the ability of leptin to inhibit testosterone production from adult rat testicular slices (72). Collectively, the results in the male suggest that leptin can modulate gonadotropin-stimulated steroidogenesis, which is analogous to its perceived role in the female. Leptin in the Gametes and Preimplantation
Embryo
A possible role of leptin in the regulation of gamete and preimplantation embryo development has been suggested in both rodents and humans. Although the transcript for leptin is not identifiable, the transcript and protein representing the functional long form of the leptin receptor is present in germinal vesicle-intact and metaphase II oocytes (57). The oocyte leptin receptor appears to be functional, as suggested by tyrosine phosphorylation of STAT3 in response to leptin (57). Leptin protein has been immunolocalized to metaphase II human oocytes (21); however, the means by which leptin gains access into the oocyte cytoplasm is not fully understood. It has been suggested that leptin from granulosa/cumulus cells may enter the oocyte by way of receptor-mediated events, endocytosis, or by a presently unidentified means. Currently, the functional importance of leptin at the oocyte level is yet to be reported. The functional leptin receptor has been identified in mouse male germ cells and appears to have both age- and stage-specific intra-germ cell locations (30). In 5-d-old mice, functional leptin receptor was expressed primarily in type A spermatogonia, whereas in the 20 to 30-d-old and adult mouse, leptin receptor was localized to the spermatocyte. The authors speculate that leptin may have a direct role in regulating proliferation and differentiation of male germ cells. Such a developmental role could partially explain infertility observed in leptin-deficient mice. However, future research is needed to support or refute this hypothesis. Currently, there is but one report of the presence of leptin in preimplantation embryos. Both mouse and human embryos display leptin and its intracellular second messenger protein STAT3 (3). However, whether mammalian preimplantation embryos contain functional leptin receptors and, thus, the link between leptin and STAT3, have not been reported. The immunolocalization and trafficking of leptin and STAT3 in the embryo from the two-cell to blastocyst stage has given rise to the idea that their polarity are important factors contributing to
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identity and fate of individual blastomeres. Such unique protein domains or intracellular/intercellular concentration gradients have been suggested to be important for early mammalian development, such as determining the animal pole (38) and/or establishing inner cell mass and trophectoderm (29). Although this is an interesting speculation that warrants further investigation, currently, direct evidence to suggest such a role for leptin is limited. Leptin Monitoring or Treatment and Modulation of Fertility Considering the numerous reports generated regarding leptin and possible regulation of reproduction at the neuroendocrine axis, gonadal, and gamete/embryo levels, it appears that exciting times are ahead in determining the functional importance of leptin and how this information might be used to regulate or predict fertility. As recently suggested by Caprio et al. (14), it is quite possible that leptin plays a dual role in regulating reproduction (Figure 3). Low leptin levels may negatively influence the neuroendocrine regulation of reproduction with a threshold level being permissive to normal reproduction. Conversely, elevated leptin levels may negatively influence normal ovarian function and/or embryo development and viability. In addition, these thresholds are most likely flexible and, thus, can be pushed in one direction or the other based on responsiveness of the neuroendocrine system andfor gonads to exogenous leptin. This plasticity may be a result of changing leptin receptor numbers and/or altered second messenger systems. Polycystic ovarian syndrome (PCOS) is one of the most prevalent reproductive abnormalities in premenopausal women and is an example of an area currently studied with regard to leptin and reproduction. Combining the information that many women with PCOS are obese and that Ieptin is involved with animal and human obesity has given way to the speculation that leptin is involved with the pathophysiology of PCOS. Potential contribution of leptin to PCOS has been suggested from reports demonstrating that leptin levels are elevated in a cohort of women with PCOS compared with women without the syndrome (11). However, subsequent studies have been unable to confirm this relationship (5 1, 55, 64). Another prominent symptom of PCOS is insulin resistance and hyperinsulinemia. These parameters also have been investigated in relation to leptin and PCOS, and current data are conflicting (48, 55, 64). These conflicts demonstrate the necessity for further investigations into the possible role of leptin and/or leptin mediators in the pathogenesis of PCOS. As a mediator of fertility, one can begin to appreciate that future efforts may focus on prepubertal administration of leptin to accelerate sexual maturation in domestic livestock species. In addition, administration of exogenous leptins may potentiate shortening of postpartum anestrus intervals in those species that experience this source of infertility. Unsuccessful attempts have been made to correlate follicular fluid leptin levels to embryo development in human-assisted reproduction technology cycles (21). Interestingly, it was found that a postovulatory rise in serum leptin concentrations was associated with implantation potential in women undergoing infertility treatment (21). This observation, coupled with the suggestion that leptin is important in regulating preimplantation embryo development, raises possibilities for
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Leptin Levels Plasticity due to 4 or t Leptin Responsiveness
f@@J@J:gy
Obese @Wte GonadallEmbryo Regulation
Neuroendocrine Regulation
Figure 3. Theoretical proposal whereby low levels of leptin during significant negative (Neg.) energy balance can be non-supportive of normal reproductive capacity through actions at the neuroendocrine level. In addition, significantly elevated leptin levels may also adversely influence reproductive capacity through actions at the gonadal or embryo level. Increased (e) or decreased (+) responsiveness of the leptin system could generate plasticity in the influence of leptin on reproductive capacity. Adapted from ( 14). future investigations into the exogenous administration of leptin, following IVF, in an attempt to improve implantation rates. Although these speculations may hold great promise, the feasibility of such options currently requires testing in model systems. REFERENCES 1.
2.
Agarwal SK, Vogel K, Weitsman SR, Magoflin DA. Leptin antagonizes the insulin-like growth factor-I augmentation of steroidogenesis in granulosa and theta cells of the human ovary. J Clin Endocrinol Metab 1999;84: 1072- 1076. Ahima RS, Prabakaran D, Mantzoros C, Qu D, Lowell B, Maratos-Flier E, Flier JS. Role of leptin in the neuroendocrine response to fasting. Nature 1996;382:250-252.
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