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db mice is not mediated by intraovarian defective leptin signaling
The impairment of reproduction in db/db mice is not mediated by intraovarian defective leptin signaling Yuehui Zhang, M.D., Ph.D.,a,b Min Hu, M.D.,a Hongxia Ma, M.D., Ph.D.,c Junwei Qu, M.D., Ph.D.,d Yong Wang, Ph.D.,a,e Lihui Hou, M.D.,a Li Liu, M.D., Ph.D.,a and Xiao-Ke Wu, M.D., Ph.D.a,e a
Department of Obstetrics and Gynecology, Key Laboratory and Unit of Infertility in Chinese Medicine, First Affiliated Hospital, and Postdoctoral Research Station, Heilongjiang University of Chinese Medicine, Harbin; c Department of Chinese Medicine, First Affiliated Hospital, Guangzhou Medical College, Guangzhou; d Department of Gynecologic Oncology, Jiangsu Institute of Cancer Research, and e Jiangsu Key Laboratory of Molecular Medicine, Medical School, Nanjing University, Nanjing, People's Republic of China b
Objective: To demonstrate whether leptin modulates reproduction by a direct effect within the ovary. Design: Animal model. Setting: National Key Laboratory of Infertility. Animal(s): Adult female db/db mice. Intervention(s): Adult littermate wild-type (WT) and diabetic (db) leptin receptor (LR) mutant female mice were matched for the allograft of the ovary to construct new genotypic models, respectively. WT mouse received only one ovary from a WT or a db/db mouse (WT Ov-WT, WT Ov-db), and db/db mouse received one ovary from a WT or a db/db mouse (db Ov-WT, db Ov-db). WT and db/db mice received one ovary from a WT mouse and another ovary from a db/db mouse (WT Ov-WT/db, db Ov-WT/db) or received two ovaries all from a WT mouse (db Ov-WT/WT). Main Outcome Measure(s): Hormones, lipids, and reverse transcription polymerase chain reaction. Result(s): Both WT Ov-WT and WT Ov-db mice presented normal cycles, comparable serum E2 and FSH levels, and ovarian expressions of the Star, Cyp17, and Cyp19 mRNA, even with different ovary genotypes. In WT Ov-WT/db with hMG stimulation, db ovaries with LR mutation expressed higher Star, Cyp17, Cyp19, Jak2, Stat3, and Pias3 mRNA than in the basal state, whereas WT ovaries with intact LR expressed higher Star, Cyp17, and Cyp19 but divergently lower Jak2, Stat3, and Pias3 levels. Conclusion(s): We confirmed that impairment of reproduction in intact db/db mice is not mediated by intraovary intact/defective leptin signaling even in face of a divergent modulation by gonadotropins. (Fertil SterilÒ 2012;97:1183–91. Ó2012 by American Society for Reproductive Medicine.) Key Words: Db/db mice, leptin receptor, reproduction, ovary, leptin signaling, PCOS
L
eptin, which is secreted by the adipose tissue, suppresses feeding and promotes energy expenditure through a variety of neuroendocrine and autonomic mechanisms. Leptin regulates several physiologic processes, including inflammation, angiogenesis, hematopoiesis, and immune function. It also affects reproduction (1–4) during oocyte maturation (5, 6), follicle rupture, corpus luteum formation (7, 8),
embryo implantation, and pregnancy (9). Additional effects of leptin are also reported, including inhibition of glucocorticoids and the enhancement of thyroxine and reproductive hormone concentrations in human and mice (1, 10–12). It also acts as an important facilitator of the early phases of human puberty (13). Accumulating evidencesuggests that leptin abundance or deficiency contributes to the
Received May 16, 2011; revised and accepted January 26, 2012; published online February 15, 2012. Y.Z. has nothing to disclose. M.H. has nothing to disclose. H.M. has nothing to disclose. J.Q. has nothing to disclose. Y.W. has nothing to disclose. L.H. has nothing to disclose. L.L. has nothing to disclose. X.-K.W. has nothing to disclose. Yuehui Zhang, Min Hu, and Hongxia Ma contributed equally to this work. Reprint requests: Xiao-Ke Wu, M.D., Ph.D., Department of Obstetrics and Gynecology, National Key Laboratory, Unit of Infertility and Trial Base, First Affiliated Hospital, Heilongjiang University of Chinese Medicine, Harbin 150040, People’s Republic of China. (E-mail: xiaokewu2002@vip. sina.com) or Li Liu, M.D., Ph.D. (E-mail: [email protected]). Fertility and Sterility® Vol. 97, No. 5, May 2012 0015-0282/$36.00 Copyright ©2012 American Society for Reproductive Medicine, Published by Elsevier Inc. doi:10.1016/j.fertnstert.2012.01.126 VOL. 97 NO. 5 / MAY 2012
pathogenesis of reproductive abnormalities at both the central and the gonadal levels (3, 14). High circulating leptin levels commonly seen in obesity and the underlying leptin resistance lead to reproductive dysfunction. Approximately 50% of women with polycystic ovary syndrome are overweight or obese (15), and the impact of obesity on the hyperandrogenic state is possibly linked with leptin (16–18). Low leptin levels are directly responsible for the neuroendocrine dysfunctions associated with anorexia nervosa (19). Animal studies and clinical observations in humans have shown that obesity caused by either a mutation in the leptin or the leptin receptor (LR) is associated with reproductive dysfunction/hypothalamic 1183
ORIGINAL ARTICLE: REPRODUCTIVE BIOLOGY hypogonadism (14). Subjects with congenital leptin deficiency and/or loss of leptin function due to mutations in either leptin or its receptor demonstrate clinical evidence of hypogonadotropic hypogonadism and the reduced expression of secondary sexual characteristics. Additional disturbances include primary or secondary amenorrhea (14). The reproductive abnormalities seen in either leptin-deficient humans or mice can be corrected by administration of leptin in replacement doses, supporting the causative role of leptin in the pathophysiology of hypogonadism-induced infertility (2, 14). The effects of leptin are mediated through the activation of the LR, which exists in at least six isoforms as a member of the class I cytokine receptor family as a result of differential splicing (20). Only the long form associates with the Janus kinase 2 (Jak2) tyrosine kinase to mediate intracellular signaling and to account for all of the functions of leptin (20–22). The long isoform LR is expressed abundantly in the hypothalamic arcuate, ventromedial, and dorsomedial nuclei (23). Most effects of leptin resulting from actions in the central nervous system (CNS), particularly the hypothalamus (24–26), are achieved by regulating the release of gonadotropins after the stimulation of the GnRH pulsatility in arcuate hypothalamic neurons (27). Leptin may also directly stimulate LH and FSH release from the pituitary gland (28). In fact, LR has been found in human granulosa and theca cells as well as in mouse stromal tissues (29–31). In addition, the LR has also been demonstrated in the follicular cells, oocytes, and corpus luteum of rodents (20, 31–33). Leptin has also been found in follicular fluid (29, 30, 34–36). In conclusion, these findings suggest that leptin can exert a direct effect on the ovary. Although there is some evidence that leptin may modulate reproduction by a direct effect at the ovarian level or by an indirect effect mainly from hypothalamus pituitary glands outside ovaries, it has not been demonstrated which one is the key factor. In this study, we aimed to pinpoint the key target of leptin on reproduction. Db/db mice, which lack a functional LR, tend to develop obesity, impaired growth, infertility, and diabetes in both males and females (20, 22, 37). Female db/db mice have a utero-ovarian lipoinvolution (38–40) to induce sterility and premature cytoinvolution, culminating in reproductive incompetence (38, 39). We chose db/db mice, through the reciprocal ovarian transfer technique, to elucidate whether leptin may have a direct effect on reproduction at the level of the ovary or and indirect effect outside the ovary.
and D). In experiment 2, female homozygous mice (db/db, n ¼ 10) and female WT mice (n ¼ 10) were used for two ovary transplantations with either intact or defective LR to evaluate their reproductive behaviors at a genotypic host with same overall endometabolic conditions (Fig. 1B and E). In experiment 3, another group of female homozygous mice (db/db n ¼ 14) and female WT mice (n ¼ 24) were used for two ovary transplantations with intact and/or defective LR to differentiate their hMG-stimulated mRNA expressions of steroidogenic enzymes and leptin signaling components (Fig. 1C and F). All mice were fed ad libitum with a standard diet and maintained in a temperature- and light-controlled room (22–24 C with 55%–65% relative humidity, 12/12 hours light/dark). The study was approved by the Animal Ethics Committee at Heilongjiang University of Chinese Medicine. Animals were cared for in accordance with the principles of the Guide to the Care and Use of Experimental Animals.
MATERIALS AND METHODS Mice and Experimental Protocols
Reproductive/Metabolic Evaluations in Experiment 1
Adult female db/db mice and littermate wild-type control (WT) mice on 129/C57BL/6 background (Jackson Laboratory, University of Pennsylvania Medical Center) were used. Mice were genotyped by polymerase chain reaction (PCR) analysis of tail-tip DNA at the age of 4 weeks. In experiment 1, female homozygous mice (db/db n ¼ 26) and female WT mice (n ¼ 26) were used for single ovary transplantation to evaluate the contribution of one ovary with intact/defective LR to overall endometablic and reproductive conditions (Fig. 1A
Five to six weeks after ovarian transplantation and after a 12hour fast, mice in diestrus were anaesthetized with ether. Their blood glucose levels in tail blood were measured with a blood glucose meter (Roche) before the mice were killed by decapitation. Blood from the constructed mice of four genotypic groups was collected and centrifuged, and serum was stored at 20 C to measure estrogen (E2), FSH, glucose, insulin, and low-density lipoprotein cholesterol (LDL-C). The ovaries were excised, cleaned of fat, weighed, and frozen at
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Ovary Transplantation At 12 weeks of age, db/db and WT mice received reciprocal ovary transplantation as shown in Figure 1 (41). Experiment 1: To determine whether defective leptin signaling within local ovary contributes to the impairment of reproduction and metabolism in db/db mice, we performed reciprocal ovary transplantation between db/db and WT mice. After removal of both ovaries, each WT mouse received one ovary from a WT mouse (WT Ov-WT) or a db/db mouse (WT Ov-db), and each db/db mouse received an ovary from a WT mouse (db Ov-WT) or a db/db mouse (db Ov-db), thus producing four genotypic mice with deficient LR in body host and/or ovary (WT Ov-WT, WT Ov-db, db Ov-WT, db Ov-db). Experiment 2: After removal of both ovaries, each WT mouse received two ovaries, one from a WT mouse and another from the db/db mouse (WT Ov-WT/db), and each db/db mouse received two ovaries, one from a WT mouse and another from the db/db mouse (db Ov-WT/db). Experiment 3: After removal of both ovaries, each WT mouse received two ovaries, one from a WT mouse and another from the db/db mouse (WT Ov-WT/db), and each db/db mouse received two ovaries both from a WT mouse (db Ov-WT/WT). Two weeks after transplantation for recovery, the stage of cyclicity was determined by microscopic analysis of the predominant cell type in vaginal smears obtained daily (42) during two cycles in experiments 1, 2, and 3, followed by the experiment as described below.
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FIGURE 1
(A) Flowchart illustrating the experiment 1 design and the timing of the interventions. (B) Flowchart illustrating the experiment 2 design and the timing of the interventions. (C) Flowchart illustrating the experiment 3 design and the timing of the interventions. (D) Flowchart illustrating the experiment 1 ovarian transplantation and four genotypic mice models. (E) Flowchart illustrating the experiment 2 ovarian transplantation and two genotypic models. (F) Flowchart illustrating the experiment 3 ovarian transplantation and two genotypic models. Zhang. Leptin signaling within ovary. Fertil Steril 2012.
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ORIGINAL ARTICLE: REPRODUCTIVE BIOLOGY 80 C. Inguinal fat and parametrical fat depots were dissected and weighed.
10%, and 80 pg/mL, respectively, for insulin; 2.1%, 4.0%, and 0.06 mmol/L, respectively, for TC; 2.6%, 4.2%, and 0.04 mmol/L, respectively, for LDL-C.
Ovary Morphology/Ovulation in Experiment 2 To evaluate the reproductive phenotypes in different genotypic ovaries within the same genotypic host, we compared ovarian morphology between db and WT ovaries within the same host WT mouse and in the same db mouse. The ovaries from WT Ov-WT/db and db Ov-WT/db mice after 4 weeks of ovarian transplantation (WT Ov-WT/db, n ¼ 3; db Ov-WT/db, n ¼ 3) were longitudinally and serially sectioned at 5 mm; every sixth section and five serial sections per ovary were mounted on a glass slide, stained with hematoxylin and eosin, and analyzed under a conventional birefringence microscope by two persons blinded to the origin of the sections. Four weeks after ovarian transplantation, WT Ov-WT/db mice and WT male were mated to see whether oocytes released from db ovaries were comparable to these from WT ovaries within WT mice in experiment 2. WT Ov-WT/db mice were examined for vaginal embolus at 8:00 A.M. everyday. Mice were euthanized after finding vaginal embolus, and the fallopian tubes were quickly collected. The time required for oocyte transport after ovulation was established by direct observation of the cumulus-oocyte complex using a microscope to see through the tubal wall as described (43). Oocytes ovulated from db ovaries and WT ovaries in the same host were counted by two persons blinded to the genotype of the ovaries.
Ovary Stimulation by hMG in Experiment 3 Four to five weeks after ovarian transplantation, mice from each group had two IM injections with 0.2 mL of GnRH analogue (GnRH-a; 675 pg/g) for a medical castration, with an interval of 5 days. Vaginal smears over the course of 10 days demonstrated disrupted cyclicity in all mice with the constant diestrus phase. Ten days after the last GnRH-a injection, after a 12-hour fast, mice from each group were randomly injected IP with 0.2 mL of hMG (0.75 IU/g) or with saline as baseline. Four hours later, mice in all groups were anesthetized with ether and were killed by decapitation. Blood was collected and centrifuged, and serum was stored at 20 C to measure E2, FSH, glucose, insulin, total cholesterol (TC), and LDL-C. The ovaries were excised, cleaned of fat, weighed, and frozen at 80 C. Uterus, inguinal fat, and parametrical fat depots were dissected and weighed.
Measurement of Reproductive Hormones, Insulin, and the Lipid Profile Serum concentrations of E2 and FSH (E-601 kits, Roche Diagnostics) were measured with electrochemiluminescence, using a Roche cobas 6000 analyzer. Serum insulin concentrations were determined with an insulin radioimmunoassay kit (1606-0606; Beifang Biotechnology Research Institute). Concentrations of TC and LDL-C were determined enzymatically with a Hitachi 7600 automatic biochemical analyzer. The intra- and interassay coefficients of variation and sensitivity were 1.8%, 3.0%, and 18.4 pmol/L, respectively, for E2; 3.6%, 5.6%, and 11.2 pmol/L, respectively, for FSH; 10%, 1186
RT-PCR Total RNA was extracted from the ovaries with Trizol reagents (Invitrogen). Single cDNA was synthesized from RNA using random hexamers and the M-MLV reverse transcriptase (Invitrogen). PCR reactions were performed with a thermal cycler (Eppendorf). The sense and antisense primers used for amplification were as follows: 50 -TTGTGCCGACTTCCCTAC-30 and 50 -TTGGACGAGTAAGACCCA-30 for a 415-bp steroidogenic acute regulatory protein (Star) fragment; 50 -CTCTGTGC TGAGCTGGAT-30 and 50 -ACCCTTGGGGTTATAACA-30 for a 385-bp 17alpha-hydroxylase (Cyp17) fragment; 50 -ACTTCC CTAAGCCCAATG-30 and 50 -GGCGTGCTAGAGGATAAT-30 for a 375-bp aromatase (Cyp19) fragment; 50 -CATACTCGG TCACTCACAATGCT-30 and 50 -GCCAATGAGAACATGCGAC TTCT-30 for a 557-bp leptin long-form receptor (Ob-Rb or LR) fragment; 50 -AGTTCTACCGAAGGACAT-30 and 50 -GAGC GAGTTAAGTAGACC-30 for a 310-bp Janus Kinases-2 (Jak2) fragment; 50 -GTCTGGCTAGACAATATCATCG-30 and 5-AGT GTTACGACTGCGACA-30 for a 589-bp Signal Transducer and Activator of Transcription-3 (Stat3) fragment; 50 -GTG GCTACTACAAGTCTCCG-30 and 50 -CTTCGACCCTCTTCTTAC TCT-30 for a 394-bp protein inhibitor of activated stat3 (Pias3) fragment; 50 -TTCACGGCTGCCAACATC-30 and 50 -GC TCCAGTAGAATCCGCTCT-30 for a 345-bp suppressor of cytokine signaling-3 (Socs3) fragment; and 50 -ACCACAGTC CATGCCATCAC-30 and 50 -TCCACCACCCTGTTGCTGTA-30 for a 452-bp GAPDH fragment. The amplification was followed by 35 cycles of denaturation at 95 C for 30 seconds, annealing at 52 C (Star), 56 C (Cyp17), 54 C (Ob-Rb, Stat3 and Pias3), 51 C (Jak2), 59 C (Socs3), or 53 C (Cyp19 and Gapdh) for 30 seconds, extension at 72 C for 30 seconds, and telo-extension at 72 C for 10 minutes. The samples were stored at 4 C until analysis. The PCR products were electrophoresed on 2% agarose gels. The intensity of ethidium bromide luminescence was measured with a laser densitometer (Bio-Rad). The computational method is as follows: intensity of objective gene area/(Gapdh intensity area).
Statistical Analysis Values are expressed as mean SEM. Differences among groups were tested by analysis of variance (ANOVA) with Bonferroni adjustments. P< .05 was considered statistically significant. All statistical evaluations were performed with the SPSS software (version 17.0, SPSS).
RESULTS Phenotypic and Molecular Evaluation of Four Genotypic Mice at Experiment 1 Two weeks after ovarian transplantation, both the WT Ov-WT and the WT Ov-db mice showed normal cycles, while the db Ov-WT mice and the db Ov-db mice had absent cycles. Reduced ovarian weight was found in the db Ov-WT and the db Ov-db mice versus the WT Ov-WT and the WT Ov-db VOL. 97 NO. 5 / MAY 2012
Fertility and Sterility® mice (P< .01). The body weight and the parametrial and inguinal fat depot of the db Ov-WT and db Ov-db mice were greater than that of the WT Ov-WT and the WT Ov-db mice (P< .01; data not shown). Glucose, LDL-C, cholesterol, and insulin levels were higher in the db Ov-WT and the db Ov-db mice than in the WT Ov-db and the WT Ov-WT mice (P< .01). In contrast, serum E2 and FSH were lower in the db Ov-WT and the db Ov-db mice than in the WT Ov-db and the WT OvWT mice (P< .05–.01; data not shown). Furthermore, the ovarian expressions of the Star, Cyp17, and Cyp19 mRNA were lower in the db Ov-WT and the db Ov-db mice than in the WT Ov-db and the WT Ov-WT mice (Fig. 2). There were no significant differences in above-indicated reproductive/metabolic index between WT Ov-WT and WT Ov-db mice or between the db Ov-WT and the db Ov-db mice.
Phenotypic and Molecular Evaluation of Two Genotypic Mice at Experiments 2 and 3 Two weeks after ovarian transplantation, the WT Ov-WT/db mice had normal cycles and ovarian weight, while the db Ov-WT/db mice displayed absent cycles and decreased ovarian weight. Histological examination demonstrated equivalent ovarian morphology between the db and WT ovaries in the same WT mice. Developing follicles and corpus luteum could be observed. (Fig. 3A, panels a and b). Similar numbers of corpus luteum (3.50 1.12 vs. 4.00 1.32; P>.05) and oocytes ovulated (2.80 1.07 vs. 3.00 1.14, n ¼ 7, P>.05) were found in the db and WT ovaries within the same host WT mice. Histological examination demonstrated equivalent ovarian morphology between the db and WT ovaries in the same db mice, of which ovarian volume were 2/3 of normal size, primary follicles and atretic follicles could be observed, and corpus luteum was absent. (Fig. 3A, panels c and d). Two weeks after ovarian transplantation, the WT Ov-WT/ db mice had normal cycles, while the db Ov-WT/WT mice
displayed absent cycles. All mice displayed absent cycles after an IM injection of GnRH-a. The ovarian and uterine weights were lower in the db Ov-WT/WT mice than in the WT OvWT/db mice (P< .05, while body weight and the weight of parametrial fat and inguinal fat were higher in the db OvWT/WT mice than in the WT Ov-WT/db mice (P< .05). The concentrations of glucose, insulin, cholesterol, and LDL-C were higher in the db Ov-WT/WT than in the WT Ov-db/WT mice (P< .05; Table 1). Serum E2 and FSH concentrations at baseline were comparable between WT Ov-WT/db and db Ov-WT/WT mice after 21 days of an IM GnRH-a injection (E2, 35.34 2.92 vs. 33.63 2.11; FSH, 19.42 2.66 vs. 18.76 2.44; WT Ov-WT/db, n ¼ 12; db Ov-WT/WT, n ¼ 7). With hMG stimulation, serum E2 and FSH were still comparable between WT Ov-WT/db and db Ov-WT/WT mice (E2, 78.23 3.41 vs. 75.71 2.71; FSH, 87.33 3.26 vs. 84.57 3.46; WT Ov-WT/db, n ¼ 12; db Ov-WT/WT, n ¼ 7) and 2- to 4-fold higher than these at baseline state. At baseline, the ovarian expressions of the Star, Cyp17, and Cyp19 mRNA were comparable, while the Ob-Rb, Jak2, Stat3, and Pias3 mRNA were lower in db ovaries than in WT ovaries from the WT Ov-WT/db mice under GnRHa down-regulated castration (Fig. 3B). With an IP hMG stimulation 4 hours thereafter, the Star, Cyp17, and Cyp19 expressions remained comparable, but the Jak2, Stat3, and Pias3 expressions were higher in db ovaries than in WT ovaries from the WT Ov-WT/db mice (Fig. 3B). In db ovaries from WT Ov-WT/db mice, expressions of the Star, Cyp17, Cyp19, Jak2, Stat3, and Pias3 mRNA were higher and the expression of Socs3 mRNA was lower with the hMG stimulation than in the basal state. In contrast, in WT ovaries from WT Ov-WT, the Ob-Rb, Jak2, Stat3, Pias3, and Socs3 mRNA expressions were lower while the ovarian expressions of the Star, Cyp17, and Cyp19 mRNA remained higher with the hMG stimulation than in the basal state (Fig. 3B). In db Ov-WT/
FIGURE 2
Experiment 1: Relative ovarian mRNA expressions of Star, Cyp17, Cyp19, and Ob-Rb at baseline in the WT Ov-db (n ¼ 8), WT Ov-WT (n ¼ 8), db OvWT (n ¼ 8), and db Ov-db (n ¼ 8). Values are mean SEM. *P<.01 vs. WT Ov-db; #P<.01 vs. WT Ov-WT; +P<.01 vs. db Ov-WT (one-way ANOVA followed by Bonferroni post hoc test). Zhang. Leptin signaling within ovary. Fertil Steril 2012.
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FIGURE 3
(A) Experiment 2: Ovarian morphology in WT Ov-WT/db mice and db Ov-WT/db mice. Panel a: db ovaries in WT mice. Panel b: WT ovaries in WT mice. Panel c: db ovaries in db mice. Panel d: WT ovaries in db mice (magnification, 4). (B) Experiment 3: Relative ovarian mRNA expressions of Star, Cyp17, Cyp19, Ob-Rb, Jak2, Stat3, Pias3, and Socs3 at baseline and with IP hMG injections in the WT Ov-db/WT mice (n ¼ 12). Values are mean SEM. *P<.01 vs. WT Ov-WT; #P<.01 vs. WT Ov-db or WT Ov-WT (one-way ANOVA followed by Bonferroni post hoc test). (C) Experiment 3: Relative ovarian mRNA expressions of Star, Cyp17, Cyp19, Ob-Rb, Jak2, Stat3, Pias3, and Socs3 at baseline and with IP hMG injections in db Ov-WT/WT (n ¼ 7). Values are mean SEM. *P<.01 vs. saline group (one-way ANOVA followed by Bonferroni post hoc test). Zhang. Leptin signaling within ovary. Fertil Steril 2012.
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TABLE 1 Body weight and weight of dissected ovaries, uterus, and individual fat depots and insulin, glucose, and lipids in two genotypic mice (experiment 3).
Body weight, g Ovary, mg Uterus, mg Parametrial fat, mg Inguinal fat, mg Glucose, mmol/L Insulin, pg/mL Cholesterol, mmol/L LDL-C, mmol/L
WT Ov-WT/db (n [ 12)
db Ov-WT/WT (n [ 7)
21.95 1.45 21.60 1.38 47.82 3.21 161.36 9.10 267.75 25.80 6.04 0.57 1,033.40 26.97 3.85 0.29 1.34 0.19
52.67 2.33b 14.78 0.52a 26.02 1.31b 863.35 33.18a 845.40 63.15a 21.82 2.15a 4,152.6 97.23a 7.80 0.46a 3.29 0.22a
Note: Values are mean SEM. WT ¼ wildtype; db ¼ Ob-Rb knockout. a P< .05 vs. WT Ov-WT/knockout (one-way ANOVA followed by Bonferroni post hoc test). b P< .01 vs. WT Ov-WT/knockout (one-way ANOVA followed by Bonferroni post hoc test). Zhang. Leptin signaling within ovary. Fertil Steril 2012.
WT mice, the ovarian expressions of the Star, Cyp17, and Cyp19 mRNA were higher under the hMG stimulation mice than in the basal state. In addition, in these mice, the levels of the Ob-Rb, Stat3, Pias3, and Socs3 mRNA were lower under the hMG stimulation than in the basal state, while the Jak2 mRNA did not differ (Fig. 3C).
DISCUSSION In this study, we produced four novel genotypic model mice by ovary transplantation, which contained a construction of db Ov-WT with LR mutant (LR) host body but LR intact (LRþ) ovary, a construction of WT Ov-db with LRþ body but LR ovary, a construction of WT Ov-db/WT with LRþ body but LR/LRþ ovary, and a construction of db Ov-db/WT with LR body but LR/LRþ ovary. Thus we can differentiate the contribution of leptin pathway, respectively, within ovary versus outside ovary to reproductive abnormalities. Some studies showed that the ovary may be a site of leptin action. Leptin mRNA and protein were detected in human granulosa and theca cells, follicular fluid, oocytes, and corpus luteum (44–47) as well as in these tissues in mice (41, 48). The effects of leptin were mediated through the activation of the receptor of the long (Ob-Rb) form, and the Ob-Rb has been found in human and mouse granulose and theca cells and stromal tissues (29–31). In addition, the LR has also been found in follicular cells, oocytes, and corpus luteum of rodents (20, 31–33). All of these findings suggest that leptin may directly act on the mouse ovary to modulate reproduction. However, in our study, in the 2-week recovery after transplantation, WT Ov-db mice with mutant LR ovary displayed a normal estrus cycle and normal hormone levels of E2 and FSH, similar to the WT Ov-WT mice with intact LR ovary. In contrast, the db Ov-WT mice with intact LR ovary displayed absent cycles and lower hormone levels of E2 and FSH, similar to the db Ov-db mice. The ovarian histological images show equivalent ovarian morphology between db and WT ovaries in the same WT mice. The ovarian mRNA expressions of Star, Cyp17, and Cyp19 were lower in the db Ov-WT and db Ov-db mice than in the WT Ov-db and WT Ov-WT mice. The function of db ovaries with mutant LR VOL. 97 NO. 5 / MAY 2012
were recovered within the WT body, while the function of WT ovaries with intact LR were impaired within the db body, suggesting that lacking LR in a local ovary did not contribute to ovarian impairment and expressing LR in a local ovary did not maintain the normal ovarian function. Furthermore, the ovarian histology show equivalent ovarian morphology between db and WT ovaries in the same WT mice and similar numbers of oocytes from db ovaries and WT ovaries in the same WT mice, indicating the ovarian function of db ovaries were recovered within the WT body even in case of LR deficiency. All of these results confirm that leptin signaling affects the ovaries either through a gainof-function or loss-of-function mechanism outside the ovary, possibly at endometabolic or CNS levels. Some studies have suggested that exposure to the chronic influences of the nonhomeostatic, hypercaloric, metabolic conditions promotes ovarian lipoatrophy (49) and organelle dissolution (49, 50). The db/db mice have a hyperglycemic, hyperinsulinemic, and hypertriglyceridemic endometabolic pathology (51, 52). The expressed diabetes obesity syndrome and induced tissue disruption may compromise the integrity of the reproductive tract cellular architecture (50). But in this study, in the WT Ov-WT/db mice, the db ovaries and WT ovaries lie in the same body. After blocking the pituitary function, the ovarian mRNA expressions of Star, Cyp17, and Cyp19 were not different in the db ovaries and the WT ovaries at the same normal internal environment, which did not have a hyperglycemic, hyperinsulinemic, or hypertriglyceridemic endometabolic state, indicating that an endometabolic state per se as a peripheral factor outside the ovary was not a contributor to ovarian impairment in the db/db mice. On the other hand, the levels of glucose, LDL-C, and insulin were higher in the db Ov-WT and db Ov-db mice than in the WT Ov-db and WT Ov-WT mice. WT mice receiving ovaries from either the db/db mice or the WT mice did not develop overall glucoselipid metabolism or systemic hyperinsulinemia and insulin resistance, which suggests that ovarian defective leptin signaling alone did not affect systemic endometabolic pathology in db mice. Some studies reported that many of the effects of leptin resulted from actions in the CNS, particularly in the hypothalamus, a site of high Ob-Rb mRNA expression (24–26). Leptin, which directly or indirectly regulates levels of circulating hormones by acting on neurons (53, 54), may also directly stimulate the LH and FSH release from the pituitary gland (28). Leptin promotes the secretion of GnRH in hypothalamus, and it also promotes FSH and LH hormone secretion in pituitary (33, 55–57). In our study, the pituitary function was initially suppressed by the GnRH-a treatment. With hMG stimulation, the ovarian mRNA expressions of Star, Cyp17, and Cyp19 were increased in db ovaries or WT ovaries from WT Ov-WT/db mice or db Ov-WT/WT mice, although the db OvWT/WT mice had a hyperglycemic, hyperinsulinemic, and hypertriglyceridemic endometabolic state. These data indicate that hMG plays a key role in the regulation of ovarian function independent of the Ob-Rb expression within the ovary, which may imply that leptin may regulate the ovarian function via directly stimulating LH and FSH release from the pituitary gland. In support of our findings, De Luca et al. found it possible to 1189
ORIGINAL ARTICLE: REPRODUCTIVE BIOLOGY restore fertility in female db/db mice after their hypothalamus LR was reconstructed by the SYN-LEPR-B transgene, pointing to an important role of CNS for leptin regulation of sexual development and/or reproduction (58). Interestingly, the ovarian mRNA expressions of Ob-Rb, Jak2, Stat3, Pias3, and Socs3 were lower with hMG stimulation than in the basal state in the WT ovaries with intact LR in the WT Ov-WT/db mice, similar to those in the db Ov-WT/WT mice. In contrast, the ovarian expressions of the Star, Cyp17, Cyp19, Jak2, Stat3, and Pias3 mRNA were higher under the hMG stimulation than in the basal state in db ovaries with mutant LR in WT Ov-db WT/ mice. These differences imply that the db ovary may exhibit an intrinsically alternated response to gonadotropin during leptin signaling. Thus, we confirm that gonadotropins play a significantly divergent (inhibitory/stimulatory) role in leptin signaling components dependent on intact or mutant LR within the ovary. In summary, female db/db homozygous mice and female WT mice were used for construction of novel genotypic models by reciprocal ovary transplantations with intact/ defective LR, with an aim to differentiate the contribution of leptin signaling within versus outside the ovary to reproductive impairments in db/db mice. We confirmed that ovarian LR deficiency did not contribute to ovarian impairment or to systemic endometabolic pathology in db mice. Furthermore, LR intact/deficient ovaries by hMG stimulation show similarly increased mRNA expressions of steroidogenic enzymes but significantly divergent expressions of leptin signaling components within both of WT and db bodies, suggesting that ovary impairment in db mice may be rescued by gonadotropin treatment.
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Department of Obstetrics and Gynecology, Key Laboratory and Unit of Infertility in Chinese Medicine, First Affiliated Hospital, and Postdoctoral Research Station, Heilongjiang University of Chinese Medicine, Harbin; c Department of Chinese Medicine, First Affiliated Hospital, Guangzhou Medical College, Guangzhou; d Department of Gynecologic Oncology, Jiangsu Institute of Cancer Research, and e Jiangsu Key Laboratory of Molecular Medicine, Medical School, Nanjing University, Nanjing, People's Republic of China b
Objective: To demonstrate whether leptin modulates reproduction by a direct effect within the ovary. Design: Animal model. Setting: National Key Laboratory of Infertility. Animal(s): Adult female db/db mice. Intervention(s): Adult littermate wild-type (WT) and diabetic (db) leptin receptor (LR) mutant female mice were matched for the allograft of the ovary to construct new genotypic models, respectively. WT mouse received only one ovary from a WT or a db/db mouse (WT Ov-WT, WT Ov-db), and db/db mouse received one ovary from a WT or a db/db mouse (db Ov-WT, db Ov-db). WT and db/db mice received one ovary from a WT mouse and another ovary from a db/db mouse (WT Ov-WT/db, db Ov-WT/db) or received two ovaries all from a WT mouse (db Ov-WT/WT). Main Outcome Measure(s): Hormones, lipids, and reverse transcription polymerase chain reaction. Result(s): Both WT Ov-WT and WT Ov-db mice presented normal cycles, comparable serum E2 and FSH levels, and ovarian expressions of the Star, Cyp17, and Cyp19 mRNA, even with different ovary genotypes. In WT Ov-WT/db with hMG stimulation, db ovaries with LR mutation expressed higher Star, Cyp17, Cyp19, Jak2, Stat3, and Pias3 mRNA than in the basal state, whereas WT ovaries with intact LR expressed higher Star, Cyp17, and Cyp19 but divergently lower Jak2, Stat3, and Pias3 levels. Conclusion(s): We confirmed that impairment of reproduction in intact db/db mice is not mediated by intraovary intact/defective leptin signaling even in face of a divergent modulation by gonadotropins. (Fertil SterilÒ 2012;97:1183–91. Ó2012 by American Society for Reproductive Medicine.) Key Words: Db/db mice, leptin receptor, reproduction, ovary, leptin signaling, PCOS
L
eptin, which is secreted by the adipose tissue, suppresses feeding and promotes energy expenditure through a variety of neuroendocrine and autonomic mechanisms. Leptin regulates several physiologic processes, including inflammation, angiogenesis, hematopoiesis, and immune function. It also affects reproduction (1–4) during oocyte maturation (5, 6), follicle rupture, corpus luteum formation (7, 8),
embryo implantation, and pregnancy (9). Additional effects of leptin are also reported, including inhibition of glucocorticoids and the enhancement of thyroxine and reproductive hormone concentrations in human and mice (1, 10–12). It also acts as an important facilitator of the early phases of human puberty (13). Accumulating evidencesuggests that leptin abundance or deficiency contributes to the
Received May 16, 2011; revised and accepted January 26, 2012; published online February 15, 2012. Y.Z. has nothing to disclose. M.H. has nothing to disclose. H.M. has nothing to disclose. J.Q. has nothing to disclose. Y.W. has nothing to disclose. L.H. has nothing to disclose. L.L. has nothing to disclose. X.-K.W. has nothing to disclose. Yuehui Zhang, Min Hu, and Hongxia Ma contributed equally to this work. Reprint requests: Xiao-Ke Wu, M.D., Ph.D., Department of Obstetrics and Gynecology, National Key Laboratory, Unit of Infertility and Trial Base, First Affiliated Hospital, Heilongjiang University of Chinese Medicine, Harbin 150040, People’s Republic of China. (E-mail: xiaokewu2002@vip. sina.com) or Li Liu, M.D., Ph.D. (E-mail: [email protected]). Fertility and Sterility® Vol. 97, No. 5, May 2012 0015-0282/$36.00 Copyright ©2012 American Society for Reproductive Medicine, Published by Elsevier Inc. doi:10.1016/j.fertnstert.2012.01.126 VOL. 97 NO. 5 / MAY 2012
pathogenesis of reproductive abnormalities at both the central and the gonadal levels (3, 14). High circulating leptin levels commonly seen in obesity and the underlying leptin resistance lead to reproductive dysfunction. Approximately 50% of women with polycystic ovary syndrome are overweight or obese (15), and the impact of obesity on the hyperandrogenic state is possibly linked with leptin (16–18). Low leptin levels are directly responsible for the neuroendocrine dysfunctions associated with anorexia nervosa (19). Animal studies and clinical observations in humans have shown that obesity caused by either a mutation in the leptin or the leptin receptor (LR) is associated with reproductive dysfunction/hypothalamic 1183
ORIGINAL ARTICLE: REPRODUCTIVE BIOLOGY hypogonadism (14). Subjects with congenital leptin deficiency and/or loss of leptin function due to mutations in either leptin or its receptor demonstrate clinical evidence of hypogonadotropic hypogonadism and the reduced expression of secondary sexual characteristics. Additional disturbances include primary or secondary amenorrhea (14). The reproductive abnormalities seen in either leptin-deficient humans or mice can be corrected by administration of leptin in replacement doses, supporting the causative role of leptin in the pathophysiology of hypogonadism-induced infertility (2, 14). The effects of leptin are mediated through the activation of the LR, which exists in at least six isoforms as a member of the class I cytokine receptor family as a result of differential splicing (20). Only the long form associates with the Janus kinase 2 (Jak2) tyrosine kinase to mediate intracellular signaling and to account for all of the functions of leptin (20–22). The long isoform LR is expressed abundantly in the hypothalamic arcuate, ventromedial, and dorsomedial nuclei (23). Most effects of leptin resulting from actions in the central nervous system (CNS), particularly the hypothalamus (24–26), are achieved by regulating the release of gonadotropins after the stimulation of the GnRH pulsatility in arcuate hypothalamic neurons (27). Leptin may also directly stimulate LH and FSH release from the pituitary gland (28). In fact, LR has been found in human granulosa and theca cells as well as in mouse stromal tissues (29–31). In addition, the LR has also been demonstrated in the follicular cells, oocytes, and corpus luteum of rodents (20, 31–33). Leptin has also been found in follicular fluid (29, 30, 34–36). In conclusion, these findings suggest that leptin can exert a direct effect on the ovary. Although there is some evidence that leptin may modulate reproduction by a direct effect at the ovarian level or by an indirect effect mainly from hypothalamus pituitary glands outside ovaries, it has not been demonstrated which one is the key factor. In this study, we aimed to pinpoint the key target of leptin on reproduction. Db/db mice, which lack a functional LR, tend to develop obesity, impaired growth, infertility, and diabetes in both males and females (20, 22, 37). Female db/db mice have a utero-ovarian lipoinvolution (38–40) to induce sterility and premature cytoinvolution, culminating in reproductive incompetence (38, 39). We chose db/db mice, through the reciprocal ovarian transfer technique, to elucidate whether leptin may have a direct effect on reproduction at the level of the ovary or and indirect effect outside the ovary.
and D). In experiment 2, female homozygous mice (db/db, n ¼ 10) and female WT mice (n ¼ 10) were used for two ovary transplantations with either intact or defective LR to evaluate their reproductive behaviors at a genotypic host with same overall endometabolic conditions (Fig. 1B and E). In experiment 3, another group of female homozygous mice (db/db n ¼ 14) and female WT mice (n ¼ 24) were used for two ovary transplantations with intact and/or defective LR to differentiate their hMG-stimulated mRNA expressions of steroidogenic enzymes and leptin signaling components (Fig. 1C and F). All mice were fed ad libitum with a standard diet and maintained in a temperature- and light-controlled room (22–24 C with 55%–65% relative humidity, 12/12 hours light/dark). The study was approved by the Animal Ethics Committee at Heilongjiang University of Chinese Medicine. Animals were cared for in accordance with the principles of the Guide to the Care and Use of Experimental Animals.
MATERIALS AND METHODS Mice and Experimental Protocols
Reproductive/Metabolic Evaluations in Experiment 1
Adult female db/db mice and littermate wild-type control (WT) mice on 129/C57BL/6 background (Jackson Laboratory, University of Pennsylvania Medical Center) were used. Mice were genotyped by polymerase chain reaction (PCR) analysis of tail-tip DNA at the age of 4 weeks. In experiment 1, female homozygous mice (db/db n ¼ 26) and female WT mice (n ¼ 26) were used for single ovary transplantation to evaluate the contribution of one ovary with intact/defective LR to overall endometablic and reproductive conditions (Fig. 1A
Five to six weeks after ovarian transplantation and after a 12hour fast, mice in diestrus were anaesthetized with ether. Their blood glucose levels in tail blood were measured with a blood glucose meter (Roche) before the mice were killed by decapitation. Blood from the constructed mice of four genotypic groups was collected and centrifuged, and serum was stored at 20 C to measure estrogen (E2), FSH, glucose, insulin, and low-density lipoprotein cholesterol (LDL-C). The ovaries were excised, cleaned of fat, weighed, and frozen at
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Ovary Transplantation At 12 weeks of age, db/db and WT mice received reciprocal ovary transplantation as shown in Figure 1 (41). Experiment 1: To determine whether defective leptin signaling within local ovary contributes to the impairment of reproduction and metabolism in db/db mice, we performed reciprocal ovary transplantation between db/db and WT mice. After removal of both ovaries, each WT mouse received one ovary from a WT mouse (WT Ov-WT) or a db/db mouse (WT Ov-db), and each db/db mouse received an ovary from a WT mouse (db Ov-WT) or a db/db mouse (db Ov-db), thus producing four genotypic mice with deficient LR in body host and/or ovary (WT Ov-WT, WT Ov-db, db Ov-WT, db Ov-db). Experiment 2: After removal of both ovaries, each WT mouse received two ovaries, one from a WT mouse and another from the db/db mouse (WT Ov-WT/db), and each db/db mouse received two ovaries, one from a WT mouse and another from the db/db mouse (db Ov-WT/db). Experiment 3: After removal of both ovaries, each WT mouse received two ovaries, one from a WT mouse and another from the db/db mouse (WT Ov-WT/db), and each db/db mouse received two ovaries both from a WT mouse (db Ov-WT/WT). Two weeks after transplantation for recovery, the stage of cyclicity was determined by microscopic analysis of the predominant cell type in vaginal smears obtained daily (42) during two cycles in experiments 1, 2, and 3, followed by the experiment as described below.
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FIGURE 1
(A) Flowchart illustrating the experiment 1 design and the timing of the interventions. (B) Flowchart illustrating the experiment 2 design and the timing of the interventions. (C) Flowchart illustrating the experiment 3 design and the timing of the interventions. (D) Flowchart illustrating the experiment 1 ovarian transplantation and four genotypic mice models. (E) Flowchart illustrating the experiment 2 ovarian transplantation and two genotypic models. (F) Flowchart illustrating the experiment 3 ovarian transplantation and two genotypic models. Zhang. Leptin signaling within ovary. Fertil Steril 2012.
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ORIGINAL ARTICLE: REPRODUCTIVE BIOLOGY 80 C. Inguinal fat and parametrical fat depots were dissected and weighed.
10%, and 80 pg/mL, respectively, for insulin; 2.1%, 4.0%, and 0.06 mmol/L, respectively, for TC; 2.6%, 4.2%, and 0.04 mmol/L, respectively, for LDL-C.
Ovary Morphology/Ovulation in Experiment 2 To evaluate the reproductive phenotypes in different genotypic ovaries within the same genotypic host, we compared ovarian morphology between db and WT ovaries within the same host WT mouse and in the same db mouse. The ovaries from WT Ov-WT/db and db Ov-WT/db mice after 4 weeks of ovarian transplantation (WT Ov-WT/db, n ¼ 3; db Ov-WT/db, n ¼ 3) were longitudinally and serially sectioned at 5 mm; every sixth section and five serial sections per ovary were mounted on a glass slide, stained with hematoxylin and eosin, and analyzed under a conventional birefringence microscope by two persons blinded to the origin of the sections. Four weeks after ovarian transplantation, WT Ov-WT/db mice and WT male were mated to see whether oocytes released from db ovaries were comparable to these from WT ovaries within WT mice in experiment 2. WT Ov-WT/db mice were examined for vaginal embolus at 8:00 A.M. everyday. Mice were euthanized after finding vaginal embolus, and the fallopian tubes were quickly collected. The time required for oocyte transport after ovulation was established by direct observation of the cumulus-oocyte complex using a microscope to see through the tubal wall as described (43). Oocytes ovulated from db ovaries and WT ovaries in the same host were counted by two persons blinded to the genotype of the ovaries.
Ovary Stimulation by hMG in Experiment 3 Four to five weeks after ovarian transplantation, mice from each group had two IM injections with 0.2 mL of GnRH analogue (GnRH-a; 675 pg/g) for a medical castration, with an interval of 5 days. Vaginal smears over the course of 10 days demonstrated disrupted cyclicity in all mice with the constant diestrus phase. Ten days after the last GnRH-a injection, after a 12-hour fast, mice from each group were randomly injected IP with 0.2 mL of hMG (0.75 IU/g) or with saline as baseline. Four hours later, mice in all groups were anesthetized with ether and were killed by decapitation. Blood was collected and centrifuged, and serum was stored at 20 C to measure E2, FSH, glucose, insulin, total cholesterol (TC), and LDL-C. The ovaries were excised, cleaned of fat, weighed, and frozen at 80 C. Uterus, inguinal fat, and parametrical fat depots were dissected and weighed.
Measurement of Reproductive Hormones, Insulin, and the Lipid Profile Serum concentrations of E2 and FSH (E-601 kits, Roche Diagnostics) were measured with electrochemiluminescence, using a Roche cobas 6000 analyzer. Serum insulin concentrations were determined with an insulin radioimmunoassay kit (1606-0606; Beifang Biotechnology Research Institute). Concentrations of TC and LDL-C were determined enzymatically with a Hitachi 7600 automatic biochemical analyzer. The intra- and interassay coefficients of variation and sensitivity were 1.8%, 3.0%, and 18.4 pmol/L, respectively, for E2; 3.6%, 5.6%, and 11.2 pmol/L, respectively, for FSH; 10%, 1186
RT-PCR Total RNA was extracted from the ovaries with Trizol reagents (Invitrogen). Single cDNA was synthesized from RNA using random hexamers and the M-MLV reverse transcriptase (Invitrogen). PCR reactions were performed with a thermal cycler (Eppendorf). The sense and antisense primers used for amplification were as follows: 50 -TTGTGCCGACTTCCCTAC-30 and 50 -TTGGACGAGTAAGACCCA-30 for a 415-bp steroidogenic acute regulatory protein (Star) fragment; 50 -CTCTGTGC TGAGCTGGAT-30 and 50 -ACCCTTGGGGTTATAACA-30 for a 385-bp 17alpha-hydroxylase (Cyp17) fragment; 50 -ACTTCC CTAAGCCCAATG-30 and 50 -GGCGTGCTAGAGGATAAT-30 for a 375-bp aromatase (Cyp19) fragment; 50 -CATACTCGG TCACTCACAATGCT-30 and 50 -GCCAATGAGAACATGCGAC TTCT-30 for a 557-bp leptin long-form receptor (Ob-Rb or LR) fragment; 50 -AGTTCTACCGAAGGACAT-30 and 50 -GAGC GAGTTAAGTAGACC-30 for a 310-bp Janus Kinases-2 (Jak2) fragment; 50 -GTCTGGCTAGACAATATCATCG-30 and 5-AGT GTTACGACTGCGACA-30 for a 589-bp Signal Transducer and Activator of Transcription-3 (Stat3) fragment; 50 -GTG GCTACTACAAGTCTCCG-30 and 50 -CTTCGACCCTCTTCTTAC TCT-30 for a 394-bp protein inhibitor of activated stat3 (Pias3) fragment; 50 -TTCACGGCTGCCAACATC-30 and 50 -GC TCCAGTAGAATCCGCTCT-30 for a 345-bp suppressor of cytokine signaling-3 (Socs3) fragment; and 50 -ACCACAGTC CATGCCATCAC-30 and 50 -TCCACCACCCTGTTGCTGTA-30 for a 452-bp GAPDH fragment. The amplification was followed by 35 cycles of denaturation at 95 C for 30 seconds, annealing at 52 C (Star), 56 C (Cyp17), 54 C (Ob-Rb, Stat3 and Pias3), 51 C (Jak2), 59 C (Socs3), or 53 C (Cyp19 and Gapdh) for 30 seconds, extension at 72 C for 30 seconds, and telo-extension at 72 C for 10 minutes. The samples were stored at 4 C until analysis. The PCR products were electrophoresed on 2% agarose gels. The intensity of ethidium bromide luminescence was measured with a laser densitometer (Bio-Rad). The computational method is as follows: intensity of objective gene area/(Gapdh intensity area).
Statistical Analysis Values are expressed as mean SEM. Differences among groups were tested by analysis of variance (ANOVA) with Bonferroni adjustments. P< .05 was considered statistically significant. All statistical evaluations were performed with the SPSS software (version 17.0, SPSS).
RESULTS Phenotypic and Molecular Evaluation of Four Genotypic Mice at Experiment 1 Two weeks after ovarian transplantation, both the WT Ov-WT and the WT Ov-db mice showed normal cycles, while the db Ov-WT mice and the db Ov-db mice had absent cycles. Reduced ovarian weight was found in the db Ov-WT and the db Ov-db mice versus the WT Ov-WT and the WT Ov-db VOL. 97 NO. 5 / MAY 2012
Fertility and Sterility® mice (P< .01). The body weight and the parametrial and inguinal fat depot of the db Ov-WT and db Ov-db mice were greater than that of the WT Ov-WT and the WT Ov-db mice (P< .01; data not shown). Glucose, LDL-C, cholesterol, and insulin levels were higher in the db Ov-WT and the db Ov-db mice than in the WT Ov-db and the WT Ov-WT mice (P< .01). In contrast, serum E2 and FSH were lower in the db Ov-WT and the db Ov-db mice than in the WT Ov-db and the WT OvWT mice (P< .05–.01; data not shown). Furthermore, the ovarian expressions of the Star, Cyp17, and Cyp19 mRNA were lower in the db Ov-WT and the db Ov-db mice than in the WT Ov-db and the WT Ov-WT mice (Fig. 2). There were no significant differences in above-indicated reproductive/metabolic index between WT Ov-WT and WT Ov-db mice or between the db Ov-WT and the db Ov-db mice.
Phenotypic and Molecular Evaluation of Two Genotypic Mice at Experiments 2 and 3 Two weeks after ovarian transplantation, the WT Ov-WT/db mice had normal cycles and ovarian weight, while the db Ov-WT/db mice displayed absent cycles and decreased ovarian weight. Histological examination demonstrated equivalent ovarian morphology between the db and WT ovaries in the same WT mice. Developing follicles and corpus luteum could be observed. (Fig. 3A, panels a and b). Similar numbers of corpus luteum (3.50 1.12 vs. 4.00 1.32; P>.05) and oocytes ovulated (2.80 1.07 vs. 3.00 1.14, n ¼ 7, P>.05) were found in the db and WT ovaries within the same host WT mice. Histological examination demonstrated equivalent ovarian morphology between the db and WT ovaries in the same db mice, of which ovarian volume were 2/3 of normal size, primary follicles and atretic follicles could be observed, and corpus luteum was absent. (Fig. 3A, panels c and d). Two weeks after ovarian transplantation, the WT Ov-WT/ db mice had normal cycles, while the db Ov-WT/WT mice
displayed absent cycles. All mice displayed absent cycles after an IM injection of GnRH-a. The ovarian and uterine weights were lower in the db Ov-WT/WT mice than in the WT OvWT/db mice (P< .05, while body weight and the weight of parametrial fat and inguinal fat were higher in the db OvWT/WT mice than in the WT Ov-WT/db mice (P< .05). The concentrations of glucose, insulin, cholesterol, and LDL-C were higher in the db Ov-WT/WT than in the WT Ov-db/WT mice (P< .05; Table 1). Serum E2 and FSH concentrations at baseline were comparable between WT Ov-WT/db and db Ov-WT/WT mice after 21 days of an IM GnRH-a injection (E2, 35.34 2.92 vs. 33.63 2.11; FSH, 19.42 2.66 vs. 18.76 2.44; WT Ov-WT/db, n ¼ 12; db Ov-WT/WT, n ¼ 7). With hMG stimulation, serum E2 and FSH were still comparable between WT Ov-WT/db and db Ov-WT/WT mice (E2, 78.23 3.41 vs. 75.71 2.71; FSH, 87.33 3.26 vs. 84.57 3.46; WT Ov-WT/db, n ¼ 12; db Ov-WT/WT, n ¼ 7) and 2- to 4-fold higher than these at baseline state. At baseline, the ovarian expressions of the Star, Cyp17, and Cyp19 mRNA were comparable, while the Ob-Rb, Jak2, Stat3, and Pias3 mRNA were lower in db ovaries than in WT ovaries from the WT Ov-WT/db mice under GnRHa down-regulated castration (Fig. 3B). With an IP hMG stimulation 4 hours thereafter, the Star, Cyp17, and Cyp19 expressions remained comparable, but the Jak2, Stat3, and Pias3 expressions were higher in db ovaries than in WT ovaries from the WT Ov-WT/db mice (Fig. 3B). In db ovaries from WT Ov-WT/db mice, expressions of the Star, Cyp17, Cyp19, Jak2, Stat3, and Pias3 mRNA were higher and the expression of Socs3 mRNA was lower with the hMG stimulation than in the basal state. In contrast, in WT ovaries from WT Ov-WT, the Ob-Rb, Jak2, Stat3, Pias3, and Socs3 mRNA expressions were lower while the ovarian expressions of the Star, Cyp17, and Cyp19 mRNA remained higher with the hMG stimulation than in the basal state (Fig. 3B). In db Ov-WT/
FIGURE 2
Experiment 1: Relative ovarian mRNA expressions of Star, Cyp17, Cyp19, and Ob-Rb at baseline in the WT Ov-db (n ¼ 8), WT Ov-WT (n ¼ 8), db OvWT (n ¼ 8), and db Ov-db (n ¼ 8). Values are mean SEM. *P<.01 vs. WT Ov-db; #P<.01 vs. WT Ov-WT; +P<.01 vs. db Ov-WT (one-way ANOVA followed by Bonferroni post hoc test). Zhang. Leptin signaling within ovary. Fertil Steril 2012.
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FIGURE 3
(A) Experiment 2: Ovarian morphology in WT Ov-WT/db mice and db Ov-WT/db mice. Panel a: db ovaries in WT mice. Panel b: WT ovaries in WT mice. Panel c: db ovaries in db mice. Panel d: WT ovaries in db mice (magnification, 4). (B) Experiment 3: Relative ovarian mRNA expressions of Star, Cyp17, Cyp19, Ob-Rb, Jak2, Stat3, Pias3, and Socs3 at baseline and with IP hMG injections in the WT Ov-db/WT mice (n ¼ 12). Values are mean SEM. *P<.01 vs. WT Ov-WT; #P<.01 vs. WT Ov-db or WT Ov-WT (one-way ANOVA followed by Bonferroni post hoc test). (C) Experiment 3: Relative ovarian mRNA expressions of Star, Cyp17, Cyp19, Ob-Rb, Jak2, Stat3, Pias3, and Socs3 at baseline and with IP hMG injections in db Ov-WT/WT (n ¼ 7). Values are mean SEM. *P<.01 vs. saline group (one-way ANOVA followed by Bonferroni post hoc test). Zhang. Leptin signaling within ovary. Fertil Steril 2012.
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TABLE 1 Body weight and weight of dissected ovaries, uterus, and individual fat depots and insulin, glucose, and lipids in two genotypic mice (experiment 3).
Body weight, g Ovary, mg Uterus, mg Parametrial fat, mg Inguinal fat, mg Glucose, mmol/L Insulin, pg/mL Cholesterol, mmol/L LDL-C, mmol/L
WT Ov-WT/db (n [ 12)
db Ov-WT/WT (n [ 7)
21.95 1.45 21.60 1.38 47.82 3.21 161.36 9.10 267.75 25.80 6.04 0.57 1,033.40 26.97 3.85 0.29 1.34 0.19
52.67 2.33b 14.78 0.52a 26.02 1.31b 863.35 33.18a 845.40 63.15a 21.82 2.15a 4,152.6 97.23a 7.80 0.46a 3.29 0.22a
Note: Values are mean SEM. WT ¼ wildtype; db ¼ Ob-Rb knockout. a P< .05 vs. WT Ov-WT/knockout (one-way ANOVA followed by Bonferroni post hoc test). b P< .01 vs. WT Ov-WT/knockout (one-way ANOVA followed by Bonferroni post hoc test). Zhang. Leptin signaling within ovary. Fertil Steril 2012.
WT mice, the ovarian expressions of the Star, Cyp17, and Cyp19 mRNA were higher under the hMG stimulation mice than in the basal state. In addition, in these mice, the levels of the Ob-Rb, Stat3, Pias3, and Socs3 mRNA were lower under the hMG stimulation than in the basal state, while the Jak2 mRNA did not differ (Fig. 3C).
DISCUSSION In this study, we produced four novel genotypic model mice by ovary transplantation, which contained a construction of db Ov-WT with LR mutant (LR) host body but LR intact (LRþ) ovary, a construction of WT Ov-db with LRþ body but LR ovary, a construction of WT Ov-db/WT with LRþ body but LR/LRþ ovary, and a construction of db Ov-db/WT with LR body but LR/LRþ ovary. Thus we can differentiate the contribution of leptin pathway, respectively, within ovary versus outside ovary to reproductive abnormalities. Some studies showed that the ovary may be a site of leptin action. Leptin mRNA and protein were detected in human granulosa and theca cells, follicular fluid, oocytes, and corpus luteum (44–47) as well as in these tissues in mice (41, 48). The effects of leptin were mediated through the activation of the receptor of the long (Ob-Rb) form, and the Ob-Rb has been found in human and mouse granulose and theca cells and stromal tissues (29–31). In addition, the LR has also been found in follicular cells, oocytes, and corpus luteum of rodents (20, 31–33). All of these findings suggest that leptin may directly act on the mouse ovary to modulate reproduction. However, in our study, in the 2-week recovery after transplantation, WT Ov-db mice with mutant LR ovary displayed a normal estrus cycle and normal hormone levels of E2 and FSH, similar to the WT Ov-WT mice with intact LR ovary. In contrast, the db Ov-WT mice with intact LR ovary displayed absent cycles and lower hormone levels of E2 and FSH, similar to the db Ov-db mice. The ovarian histological images show equivalent ovarian morphology between db and WT ovaries in the same WT mice. The ovarian mRNA expressions of Star, Cyp17, and Cyp19 were lower in the db Ov-WT and db Ov-db mice than in the WT Ov-db and WT Ov-WT mice. The function of db ovaries with mutant LR VOL. 97 NO. 5 / MAY 2012
were recovered within the WT body, while the function of WT ovaries with intact LR were impaired within the db body, suggesting that lacking LR in a local ovary did not contribute to ovarian impairment and expressing LR in a local ovary did not maintain the normal ovarian function. Furthermore, the ovarian histology show equivalent ovarian morphology between db and WT ovaries in the same WT mice and similar numbers of oocytes from db ovaries and WT ovaries in the same WT mice, indicating the ovarian function of db ovaries were recovered within the WT body even in case of LR deficiency. All of these results confirm that leptin signaling affects the ovaries either through a gainof-function or loss-of-function mechanism outside the ovary, possibly at endometabolic or CNS levels. Some studies have suggested that exposure to the chronic influences of the nonhomeostatic, hypercaloric, metabolic conditions promotes ovarian lipoatrophy (49) and organelle dissolution (49, 50). The db/db mice have a hyperglycemic, hyperinsulinemic, and hypertriglyceridemic endometabolic pathology (51, 52). The expressed diabetes obesity syndrome and induced tissue disruption may compromise the integrity of the reproductive tract cellular architecture (50). But in this study, in the WT Ov-WT/db mice, the db ovaries and WT ovaries lie in the same body. After blocking the pituitary function, the ovarian mRNA expressions of Star, Cyp17, and Cyp19 were not different in the db ovaries and the WT ovaries at the same normal internal environment, which did not have a hyperglycemic, hyperinsulinemic, or hypertriglyceridemic endometabolic state, indicating that an endometabolic state per se as a peripheral factor outside the ovary was not a contributor to ovarian impairment in the db/db mice. On the other hand, the levels of glucose, LDL-C, and insulin were higher in the db Ov-WT and db Ov-db mice than in the WT Ov-db and WT Ov-WT mice. WT mice receiving ovaries from either the db/db mice or the WT mice did not develop overall glucoselipid metabolism or systemic hyperinsulinemia and insulin resistance, which suggests that ovarian defective leptin signaling alone did not affect systemic endometabolic pathology in db mice. Some studies reported that many of the effects of leptin resulted from actions in the CNS, particularly in the hypothalamus, a site of high Ob-Rb mRNA expression (24–26). Leptin, which directly or indirectly regulates levels of circulating hormones by acting on neurons (53, 54), may also directly stimulate the LH and FSH release from the pituitary gland (28). Leptin promotes the secretion of GnRH in hypothalamus, and it also promotes FSH and LH hormone secretion in pituitary (33, 55–57). In our study, the pituitary function was initially suppressed by the GnRH-a treatment. With hMG stimulation, the ovarian mRNA expressions of Star, Cyp17, and Cyp19 were increased in db ovaries or WT ovaries from WT Ov-WT/db mice or db Ov-WT/WT mice, although the db OvWT/WT mice had a hyperglycemic, hyperinsulinemic, and hypertriglyceridemic endometabolic state. These data indicate that hMG plays a key role in the regulation of ovarian function independent of the Ob-Rb expression within the ovary, which may imply that leptin may regulate the ovarian function via directly stimulating LH and FSH release from the pituitary gland. In support of our findings, De Luca et al. found it possible to 1189
ORIGINAL ARTICLE: REPRODUCTIVE BIOLOGY restore fertility in female db/db mice after their hypothalamus LR was reconstructed by the SYN-LEPR-B transgene, pointing to an important role of CNS for leptin regulation of sexual development and/or reproduction (58). Interestingly, the ovarian mRNA expressions of Ob-Rb, Jak2, Stat3, Pias3, and Socs3 were lower with hMG stimulation than in the basal state in the WT ovaries with intact LR in the WT Ov-WT/db mice, similar to those in the db Ov-WT/WT mice. In contrast, the ovarian expressions of the Star, Cyp17, Cyp19, Jak2, Stat3, and Pias3 mRNA were higher under the hMG stimulation than in the basal state in db ovaries with mutant LR in WT Ov-db WT/ mice. These differences imply that the db ovary may exhibit an intrinsically alternated response to gonadotropin during leptin signaling. Thus, we confirm that gonadotropins play a significantly divergent (inhibitory/stimulatory) role in leptin signaling components dependent on intact or mutant LR within the ovary. In summary, female db/db homozygous mice and female WT mice were used for construction of novel genotypic models by reciprocal ovary transplantations with intact/ defective LR, with an aim to differentiate the contribution of leptin signaling within versus outside the ovary to reproductive impairments in db/db mice. We confirmed that ovarian LR deficiency did not contribute to ovarian impairment or to systemic endometabolic pathology in db mice. Furthermore, LR intact/deficient ovaries by hMG stimulation show similarly increased mRNA expressions of steroidogenic enzymes but significantly divergent expressions of leptin signaling components within both of WT and db bodies, suggesting that ovary impairment in db mice may be rescued by gonadotropin treatment.
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