Expression of leptin and its receptor in the murine ovary: possible role in the regulation of oocyte maturation

Leptin is a product of the ob gene that is produced primarily by adipose tissue. Leptin and its receptors are found within the ovary, but it is unclear what function this hormone has in the ovary. Using immunohistochemistry, we determined that leptin is found in most cell types in the murine ovary, with the highest staining levels observed in the oocyte. Leptin receptor was also expressed in all of the main ovarian cell types, with the thecal cell layer exhibiting the highest staining levels. Leptin administration did not affect spontaneous or induced maturation of either isolated denuded oocytes or cumulus-oocyte complexes, but it did significantly increase the rate of meiotic resumption in preovulatory follicle-enclosed oocytes (P < 0.01). Measurements of cAMP within oocytes cultured with leptin showed that this enhanced ability to resume meiosis does not occur via activation of phosphodiesterase 3B and subsequent cAMP reduction. These results provide evidence that leptin affects oocyte maturation when the oocyte is cultured within its normal follicular environment. It is suggested that leptin may induce the production of another factor, possibly from thecal cells, that directly or indirectly acts on the oocyte to initiate germinal vesicle breakdown in this species.     

Leptin accelerates pronuclear formation following intracytoplasmic sperm injection of porcine oocytes: Possible role for MAP kinase inactivation

Leptin, a multifunctional hormone, is present in mammalian oocytes and follicular fluids and cumulus cells. While leptin modulates oocyte maturation in vitro which seems to result in enhancement of embryo development, it is unclear whether leptin treatment of oocytes affects cytoplasmic maturation and fertilization processes. In order to gain a better understanding of the role of leptin during oocyte maturation, we examined microtubule and microfilament assembly following oocyte maturation and blastocyst formation, mitogen-activated protein kinase (MAPK) activity, and pronuclear formation following parthenogenetic stimuli or intracytoplasmic sperm injection (ICSI) in leptin-treated oocytes. Addition of 10 or 100 ng/ml leptin during oocyte maturation did not increase the proportion of metaphase II oocytes, but enhanced development to blastocyst stage by day 7 (P < 0.01) after parthenogenetic activation (PA), accompanied by increased cell number. However there was no effect on the number of apoptotic cells in blastocysts. Following maturation in the presence of leptin, there were more oocytes with normal spindle formation. MAPK activity decreased more rapidly, and pronuclear formation was accelerated after parthenogenetic activation or ICSI of leptin-treated oocytes. These results suggested that exogeneous leptin enhanced spindle assembly and accelerated pronuclear formation following fertilization, possibly via the MAPK pathway.     

Functional expression of the short isoform of the murine leptin receptor Ob-Rc (muB1.219) inXenopus laevis oocytes

Leptin, a hormone mainly secreted by the adipose tissue, acts on the hypothalamus to regulate food intake and thermogenesis. Six leptin receptor isoforms have been identified and localized in different tissues. While it is clear that leptin action in the brain occurs by binding to the long receptor isoform, several studies have shown that the short isoforms could be involved in the transcellular transport of the hormone from the blood to the brain. Based on these works, we decided to investigate whether the murine short leptin receptor isoform Ob-Rc (muB1.219) could transport leptin when expressed in Xenopus laevis oocytes. MuB1.219 cRNA was injected into the oocytes and functional studies were performed by incubating the oocytes in the presence of 2.5 nM [125I]-leptin, under different conditions. Results showed that leptin binding to the injected oocytes was four to eight-fold higher than the binding to the non-injected oocytes. This was blocked by 250 nM of non-radiolabeled leptin, suggesting that the binding was specific. Leptin internalization was observed from 30 min incubation onwards. Coexpression of the human Na+/glucose cotransporter and the leptin receptor showed that leptin increased sugar uptake into the oocytes. These results demonstrate that the short leptin receptor Ob-Rc is able to mediate binding and internalization of the hormone when expressed in oocytes and that it may perform intracellular signaling.     

Direct effects of leptin on mouse reproductive function: regulation of follicular, oocyte, and embryo development

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.     

Involvement of the metabolic hormones leptin, ghrelin, obestatin, IGF-I and of MAP kinase in control of porcine oocyte maturation

The general aim of our in vitro experiments was to study the role of the metabolic hormones leptin, ghrelin, obestatin and IGF-I and mitogen-activated protein kinase (MAPK)-dependent intracellular mechanisms in the control of nuclear maturation of porcine oocytes. For this purpose, porcine oocytes were isolated from the ovary and cultured in the presence of leptin, ghrelin, obestatin, IGF-I, MAPK blocker PD98059 and the combinations of hormones with PD98059. Proportions of matured oocytes (at metaphase II of meiosis, determined by DAPI staining) and of oocytes containing MAPK/ERK1-2 (determined by immunocytochemistry) were measured before and after culture. It was observed that the majority of oocytes isolated from the ovary before culture were immature and did not contain visible MAPK, but some oocytes were mature, and the majority of these oocytes contained MAPK. Incubation of oocytes resulted in a significant increase in the proportion of matured oocytes and in the percentage of oocytes containing MAPK in both the matured and not matured groups. Addition of IGF-I to the culture medium increased the proportion of matured oocytes, addition of leptin decreased it, and ghrelin and obestatin did not oocyte maturation. Addition of hormones did not affect the expression of MAPK in either immature or mature oocytes. PD98059, when given alone, suppressed the maturation and accumulation of MAPK in both mature and immature oocytes. When given together with hormones, PD98059 was able to reduce the stimulatory effect of IGF-I, to invert the inhibitory action of leptin to stimulatory and to induce the stimulatory action of ghrelin and obestatin on meiosis. IGF-I, ghrelin and obestatin, but not leptin, when given together with PD98059, increased the accumulation of MAPK in both immature and mature oocytes. Association of nuclear maturation and expression of MAPK in oocytes before, but not after culture, as well as the prevention of oocyte maturation by MAPK blocker suggests the involvement of MAPK-dependent intracellular mechanisms in the promotion of reinitiation, but not completion of meiosis. The effect of hormonal additions on meiosis of oocytes suggests that IGF-I is a stimulator, leptin can be an inhibitor, while ghrelin and obestatin probably do not control oocyte maturation. The ability of PD98059 to modify the effect of hormones on oocyte maturation and on MAPK expression suggests possible interference of hormones and MAPK-dependent intracellular mechanisms in oocytes. However, no influence of hormones on MAPK and lack of association between action of hormones and PD98059 on MAPK and meiosis suggest that MAPK is probably not a mediator of effect of IGF-I, leptin, ghrelin and obestatin on porcine oocyte nuclear maturation.     

Leptin stimulates both JAK2-dependent and JAK2-independent signaling pathways

Leptin controls body weight by activating the long form of the leptin receptor (LEPRb). Janus kinase 2 (JAK2) is associated with LEPRb and autophosphorylates in response to leptin. JAK2 also phosphorylates LEPRb, STAT3, and multiple other downstream molecules. Surprisingly, here we show that JAK2 is not required for leptin stimulation of STAT3 phosphorylation. Leptin time- and dose-dependently stimulated tyrosine phosphorylation of STAT3 in both human and mouse JAK2-null cells. Leptin also increased the viability of JAK2-null cells. Overexpression of c-Src or Fyn, two Src family members, promoted STAT3 phosphorylation, whereas inhibition of the endogenous Src family members by either pharmacological inhibitors or dominant negative Src(K298M) decreased the ability of leptin to stimulate the phosphorylation of STAT3 and ERK1/2. Leptin also stimulated tyrosine phosphorylation of kinase-inactive JAK2(K882E) in JAK2-null cells. Overexpression of JAK2(K882E) enhanced the ability of leptin to stimulate STAT3 phosphorylation in JAK2-null cells. Tyr1138 in LEPRb was required for leptin-stimulated phosphorylation of STAT3 but not JAK2(K882E). These data suggest that leptin stimulates non-JAK2 tyrosine kinase(s), including the Src family members, which phosphorylate JAK2, STAT3, and other molecules downstream of LEPRb. JAK2 mediates leptin signaling by both phosphorylating its substrates and forming a signaling complex as a scaffolding/adaptor protein. The non-JAK2 kinase(s) and JAK2 may act coordinately and synergistically to mediate leptin response.     

Expression of leptin receptor isoforms and effects of leptin on the proliferation and hormonal secretion in human pituitary adenomas

OBJECTIVE: To pursue whether leptin regulates anterior pituitary cells, we studied the ex vivo expression of several isoforms of the leptin receptor (OB-R) as well as the in vitro effects of leptin administration in human pituitary adenomas. METHODS: OB-R mRNA expression and in vitro response to leptin were studied in 39 pituitary macroadenomas. RESULTS: All 4 OB-R subtypes were expressed in most adenomas. The expression was significantly more pronounced in GH-secreting adenomas as compared to non-functioning tumor cells (p < 0.05). Leptin administration in vitro did not significantly influence cell proliferation or the secretion of GH, FSH, LH or alpha-subunit. CONCLUSIONS: (1) Several isoforms of the OB-R, including the signal transducing full-length receptor, are expressed in most human pituitary adenomas. (2) This expression ex vivo is not associated with significant effects of leptin in vitro.     

Roles for leptin receptor/STAT3-dependent and -independent signals in the regulation of glucose homeostasis

Leptin activates the long form of the leptin receptor (LRb) to control feeding and neuroendocrine function and thus regulate adiposity. While adiposity influences insulin sensitivity, leptin also regulates glucose homeostasis independently of energy balance. Disruption of the LRb/STAT3 signal in s/s mice results in hyperphagia, neuroendocrine dysfunction, and obesity similar to LRb null db/db mice. Insulin resistance and glucose intolerance are improved in s/s compared to db/db animals, however, suggesting that LRb/STAT3-independent signals may contribute to the regulation of glucose homeostasis by leptin. Indeed, caloric restriction normalized glycemic control in s/s animals, but db/db mice of similar weight and adiposity remained hyperglycemic. These differences in glucose homeostasis were not attributable to differences in insulin production between s/s and db/db animals but rather to decreased insulin resistance in s/s mice. Thus, in addition to LRb/STAT3-mediated adiposity signals, non-LRb/STAT3 leptin signals mediate an important adiposity-independent role in promoting glycemic control.