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Original Studies |
Department of Obstetrics and Gynecology, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan
Address all correspondence and requests for reprints to: Jo Kitawaki, M.D., Ph.D., Department of Obstetrics and Gynecology, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kamigyo-ku, Kyoto 602-8566, Japan. E-mail: kitawaki{at}koto.kpu-m.ac.jp
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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In addition to the action on energy metabolism, leptin appears to influence various reproductive functions. Injecting leptin into ob/ob mice that are infertile and with low levels of gonadotropin increases the weight of the uterus and ovaries and the number of follicles (7), resulting in restoration of fertility (8). Administering leptin treatment to normal female mice accelerates puberty (9), and in humans higher leptin levels have been shown to relate to the earlier onset of menarche (10). These actions of leptin are considered to be mediated mainly through brain OB-R. In contrast, the mRNA and protein of leptin and OB-R mRNA are also expressed in peripheral reproductive tissue, including granulosa cells (11, 12, 13), cumulus cells (11, 12) of human preovulatory follicles, oocytes and embryos (14, 15), and human placental trophoblasts (16, 17, 18, 19). These findings suggest that leptin plays a physiological role in early development. To investigate the role of leptin in implantation, we examined the expression of OB-R and leptin in human endometrium.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Endometrial biopsy specimens were obtained for diagnostic purposes from patients scheduled for laparotomy or laparoscopy at the Department of Obstetrics and Gynecology, Kyoto Prefectural University of Medicine. This study protocol was approved by the Kyoto Prefectural University of Medicine institutional review board, and informed consent was obtained from each patient. All patients were of reproductive age with normal menstrual cycles. The patients were not receiving any endocrine therapy, such as GnRH analog, danazol, or pseudopregnancy therapy. Endometrial specimens were classified into disease groups as described previously (20). Endometriosis, adenomyosis, and/or leiomyomas were diagnosed by histological examination using excised uteri or laparoscopy. Endometria obtained from patients with cervical cancer in situ but showing no other gynecological disease were defined as disease free. The following cases were excluded from the study: malignant neoplasms other than cervical carcinoma in situ, ovarian neoplasms, pelvic inflammation, and pregnancy. Fifty patients met the criteria for enrollment. The mean ages and body mass index (BMI) were 38.9 ± 6.1 (mean ± SD, 24–48 yr) and 21.5 ± 2.3 (16.9–26.4), respectively. There was no significant difference in the mean ages or BMI among the groups with endometriosis, adenomyosis, and leiomyomas and the disease-free group, except that the mean age of the group with leiomyomas (42.1 ± 3.9) was higher than those of the endometriosis (P < 0.01) and disease-free (P < 0.01) groups. Endometrial dating was performed using basal body temperature, transvaginal ultrasound sonography, and the criteria of Noyes et al. (21). Decidual tissue (n = 5) and chorionic tissue were collected from patients undergoing elective termination of pregnancy between 6 and 9 weeks gestation due to medical reasons other than gynecological. Fresh tissue samples were divided into two portions; one was frozen immediately at -80 C and stored for total RNA extraction, and the other was fixed with 4% paraformaldehyde for histological diagnosis.
RNA isolation and RT-PCR
Total RNA was extracted using Trizol (Life Technologies, Inc., Gaithersburg, MD), and the first strand complementary DNA (cDNA) synthesis from total RNA was catalyzed by Superscript II RT (Life Technologies, Inc.) using oligo(deoxythymidine)12–18, as previously described (22). The resulting first strand cDNA was used for PCR amplification with the following primers: 5'-GTCAGAAGATGTGGGAAA-3' (forward; nucleotides 2266–2283) and 5'-GTGCCCAGGAACAATTCTT-3' (reverse; nucleotides 2846–2828) (6) for all of the splice variants of human OB-R (OB-RT), 5'-TTGTGCCAGTAATTATTTCCTCTT-3' (forward; nucleotides 2727–2750) and 5'-CTGATCAGCGTGGCGTATTT-3' (reverse; nucleotides 3165–3146) (23) for OB-RL, 5'-ATGCATTGGGGAACCCTGTGCGG-3' (forward; nucleotides 1–23) and 5'-TGAGGTCCAGCTGCCACAGCATG-3' (reverse; nucleotides 468–490) for human ob (17), and the human glyceraldehyde-3-phosphate dehydrogenase (G3PDH) amplimer set for G3PDH (CLONTECH Laboratories, Inc., Palo Alto, CA). The PCR mixture comprised 1 µL first strand cDNA, 0.2 µmol/L of each of the primers mentioned above, 0.2 mmol/L deoxy-NTP, and 1 µL AdvanTaq DNA polymerase (CLONTECH Laboratories, Inc.) in a total volume of 50 µL PCR buffer (CLONTECH Laboratories, Inc.). After an initial denaturation at 95 C for 1 min, PCR was carried out at 95 C for 30 s; at 62 C (for OB-RT), 60 C (for OB-RL and leptin), or 55 C (for G3PDH) for 30 s; and at 68 C for 30 s. The PCR reactions were run at 35 cycles for positive gene expression.
The relative change in mRNA expression was assessed as previously described (24) with modifications. Duplicate aliquots of first strand cDNA synthesized from control endometrial specimens were subjected to PCR amplification for 16–32 cycles at 2-cycle intervals. The PCR products for OB-RT, OB-RL, and G3PDH were electrophoresed in 2% agarose gel and stained with ethidium bromide. Gels were photographed, and the intensity of each band was measured with a densitometer. The intensity increased exponentially with up to 26 PCR cycles for OB-RT and OB-RL and 22 cycles for G3PDH, and eventually reached a plateau. As the regression lines, which were determined by the linear portion of the curves, were parallel, we therefore set the PCR amplification at 26 cycles for OB-RT and OB-RL and 22 cycles for G3PDH. Each endometrial sample cDNA was PCR amplified for 22, 24, 26, and 28 cycles to confirm the exponential increase. The intensity of each transcript relative to that of G3PDH was calculated to compare the initial mRNA level.
Northern blot analysis
Twenty micrograms of total RNA were electrophoresed in a 1%
agarose/formaldehyde gel, transferred to a nylon membrane (Hybond
N+, Amersham Pharmacia Biotech,
Piscataway, NJ) by capillary blotting, and UV cross-linked. Membranes
were prehybridized for 1 h at 65 C in 0.5 mol/L
Na2HPO4/H3PO4
buffer (pH 7.2) containing 1 mmol/L ethylenediamine tetraacetate and
7% SDS. The radiolabeled probes for OB-RL
and ob were derived from the amplified cDNA fragments
produced in the RT-PCR of chorionic tissues. DNA bands were excised
from the agarose gel and extracted using a NucleoTrap DNA purification
kit (CLONTECH Laboratories, Inc.). Aliquots of the DNA
products were sequenced by the dye terminator method using a model 100
DNA analyzer (PE Applied Biosystems, Foster City, CA), and
the sequences were confirmed to be equal to those reported in the
GenBank databank (U43168 and U18915 for
OB-RL and ob, respectively). The
probes were radiolabeled with
[
-32P]deoxy-CTP using a Random Primer Plus
extension labeling system (NEN Life Science Products,
Boston, MA). After hybridization for 24 h at 60 C, membranes were
washed three times for 5 min each time at 65 C, followed by a wash for
15 min at 65 C in 0.04 mol/L
Na2HPO4/H3PO4
buffer (pH 7.2) containing 1% SDS. The hybridized signal was analyzed
using a bioimaging analyzer (BAS 2000, Fujix, Tokyo, Japan).
Western blot analysis
Approximately 0.1 g wet wt endometrial tissue was homogenized, solubilized in 0.3 mL lysis buffer (phosphate-buffered saline, pH 7.4, containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and 0.57 mmol/L phenylmethylsulfonylfluoride), and centrifuged at 15,000 x g for 20 min at 4 C. The resulting supernatant was mixed with sample buffer and electrophoresed under reducing conditions in 7.5% and 15% SDS-polyacrylamide gel for OB-RL and leptin, respectively. Prestained protein markers (Bio-Rad Laboratories, Inc., Hercules, CA) were used as standards. Proteins were electrotransferred to a polyvinylidene difluoride membrane (Bio-Rad Laboratories, Inc.). Membranes were incubated with goat antihuman OB-R antibody (C-20; 1:1000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or rabbit antihuman leptin antibody (A-20; 1:1000; Santa Cruz Biotechnology, Inc.). As the former was raised against a peptide corresponding to 20 amino acids mapping at the carboxyl-terminus of human OB-RL, it recognizes only OB-RL, but not the short forms of OB-R. Membranes were washed and incubated with a second antibody conjugated with horseradish peroxidase (1:1000; Santa Cruz Biotechnology, Inc.). Immunoreactions were detected with enhanced chemiluminescence using ECL Western blotting detection reagents (Amersham Pharmacia Biotech, Arlington Heights, IL) according to the manufacturer’s instructions. Membranes were exposed to Kodak X-Omat film (Eastman Kodak Co., Rochester, NY). Negative controls were carried out by incubating the membrane with either nonimmunized serum or only the second antibodies. No bands were detected.
Statistics
Differences in ages and BMI among disease groups were analyzed
with the one-factor ANOVA, and multiple comparisons were performed
using Scheffé’s procedure. Differences in the frequency of
transcript expression among genes and among disease groups were
analyzed with 
2 test. Pearson’s correlation
coefficient was used to evaluate the relationship between the abundance
of mRNA expression and the BMI values of the patients.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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In Northern blot analyses OB-RL mRNA
was detected as a doublet of 6.4 and 4.5 kb in endometrial specimens of
various phases throughout the menstrual cycle, whereas ob
mRNA was not detected (Fig. 1
). In
Western blot analyses OB-RL protein was detected
at 230 kDa, whereas leptin protein was not detected (Fig. 2).
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). Three cases (6%)
were positive for OB-RT but negative for
OB-RL transcripts (case B in Fig. 3).
Neither OB-RT nor
OB-RL was detected in 5 cases (10%), of
which 2 cases were from the early proliferative phase, 1 case was from
the midproliferative phase, and 2 cases were from the late secretory
phase (case C in Fig. 3). In all 5 cases of decidual tissue of early
pregnancy (6–9 weeks gestation), OB-RT and
OB-RL transcripts were detected (Fig. 3).
The sequences of these PCR products were confirmed by direct sequencing
(data not shown). In contrast, ob transcript was not
detected in any of the endometrial or decidual specimens (Fig. 3).
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The change in the amounts of OB-RT and
OB-RL mRNA expression during the menstrual
cycle was analyzed using the semiquantitative RT-PCR method (Fig. 4). OB-RT and
OB-RL mRNA expression were mostly parallel
throughout the menstrual cycle, suggesting that
OB-RL was predominant among the
OB-R isoforms in the human endometrium.
OB-RT and OB-RL
mRNA expression was low in the early proliferative phase and increased
gradually during the proliferative phase toward ovulation. The
expression was greatest in the early secretory phase and declined
during the mid- and late secretory phases toward menstruation (Fig. 4).
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, and the
frequency of expression was similar between
OB-RT and OB-RL
transcripts and between the endometrial samples of patients with
endometriosis, adenomyosis, and leiomyomas and in disease-free
endometria. The quantity of mRNA expression showed no difference among
the disease groups, nor was there any significant relationship between
the abundance of OB-RT or
OB-RL mRNA expression and the BMI values of
the patients (r = 0.001; P = 0.8401 for
OB-RT; r = 0.002; P =
0.7755 for OB-RL; graphs not shown).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Leptin and OB-R are expressed in various reproductive tissues. In the human ovary, the mRNA and protein of leptin are expressed in preovulatory granulosa and cumulus cells (11, 12), and the mRNA of OB-R isoforms are expressed in luteinized granulosa cells (11, 13) and cumulus cells (11). The addition of leptin results in increased estrogen-producing activity in cultured granulosa-luteal cells (24). Leptin protein and STAT3 are also expressed in polarized domains in oocytes, and after fertilization, these domains become differentially distributed between the inner cell mass and the trophoblast of the blastocyst (14). Eight regulatory proteins, including leptin and STAT3, are affected by the fragmentation process in the preimplantation stage of human embryos (15). The mRNA and protein of OB-R are also present in mouse metaphase 2 and germinal vesicle stage oocytes (25). As shown in this study, in the endometrium, OB-R mRNA expression peaks in the early secretory phase as the endometrium prepares for acceptance of a fertilized egg. The variation in mRNA abundance during the menstrual cycle may suggest that the expression of these genes is regulated by ovarian steroids. After conception, leptin (16, 17) and OB-R (18) transcripts are expressed in human placental trophoblasts. The content of leptin mRNA expression in villous tissue is greater in early gestation and declines as gestation advances (19). In addition to chorionic expression, as shown in the present study, the decidual tissue of early gestation expresses OB-R. In the local environment of the endometrium, leptin may play a specific role, in concert with other growth factors and cytokines, in acceptance of the fertilized egg and in early human development.
It is important to examine human endometrial specimens as distinguished by their original disease. The mRNA and protein of aromatase cytochrome P450 (P450arom), the enzyme responsible for estrogen biosynthesis, are expressed in the eutopic endometria of patients with endometriosis, adenomyosis, and/or leiomyomas, whereas P450arom is not detected in the endometrium of disease-free uterus, indicating that although histologically the eutopic endometria of patients with benign uterine diseases resemble the endometria of disease-free uterus, the estrogen metabolism is remarkably different (20, 22). Thus, we strictly defined only those samples obtained from normal menstruating women with cervical cancer in situ but showing no other gynecological disease as disease-free endometria. There is no evidence that the endometria of patients with cervical cancer in situ are different from those of normal uterus. We evaluated the leptin and OB-R expression discriminating them from endometria obtained from patients with benign uterine disease. Nevertheless, the incidence and abundance of OB-RT and OB-RL mRNA expression were comparable in the benign uterine disease and disease-free groups.
The present study demonstrated both mRNA and protein expression of OB-R and the cyclic change in the mRNA level; however, the cyclic change in the amount of protein or receptor binding has not been shown. The present study suggests no relationship between the abundance of OB-R mRNA expression and BMI at least within the normal range; however, the present study did not include endometria of patients who were excessively slender or obese. We did not examine the difference in OB-R expression in endometria of patients with fertility and infertility. Some researchers have suggested that uterine receptivity might be adversely affected by endometriosis (26, 27, 28). Further studies are needed to examine OB-R expression in the endometria of pathological conditions. The accumulation of such studies will contribute to knowledge of the physiological role of leptin in implantation and early human development.
Received August 25, 1999.
Revised December 8, 1999.
Accepted January 11, 2000.
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