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From the Clinical Research Centers |
Departments of Obstetrics/Gynecology (M.C.H., J.S.O., K.F.S., W.S.), Physiology (M.C.H., W.S.), and Anatomy (M.C.H.) and the Interdisciplinary Program in Molecular and Cellular Biology (M.C.H., J.S.O., A.E.G.), Tulane University School of Medicine, New Orleans, Louisiana 70112-2699; Tulane Regional Primate Research Center (M.C.H.), Covington, Louisiana 70433-8915; and the Department of Obstetrics/Gynecology, Texas Tech University Health Sciences Center (V.D.C., T.G.), Amarillo, Texas 79106-1797
Address all correspondence and requests for reprints to: Michael C. Henson, Ph.D., Department of Obstetrics/Gynecology, Tulane University School of Medicine, 1430 Tulane Avenue, New Orleans, Louisiana 70112-2699. E-mail: michael.henson{at}tulane.edu
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184
days), and leptin mRNA was quantitated by competitive RT-PCR. Finally,
in situ hybridization was employed to localize
transcripts to specific placental cell types. Results determined that
maternal leptin levels (mean ± SEM), which were
dramatically greater (P < 0.01) than those in
nonpregnant cycling baboons (1.4 ± 0.1 ng/mL), increased
(P < 0.005) with gestational age from 63.6 ±
10.4 ng/mL on day 60 of gestation to 157.8 ± 16.1 near term.
Levels declined to those found in cycling baboons by 15 days
postdelivery. In contrast to maternal leptin concentrations, placental
leptin mRNA decreased (P < 0.02) with advancing
pregnancy, as transcript abundance declined approximately 8-fold from
early to late gestation. Maternal peripheral leptin concentrations were
positively correlated (r = 0.66; P < 0.001)
whereas placental leptin mRNA levels were negatively correlated (r
= -0.64; P < 0.01) with gestational age.
Expression of leptin mRNA transcripts, as evidenced by RT-PCR in
villous tissue, was localized principally within syncytiotrophoblast by
in situ hybridization. In summary, changes in maternal peripheral leptin concentrations and placental leptin mRNA abundance that occur commensurate with advancing gestational age may imply evolving roles for the polypeptide with advancing primate pregnancy. In this capacity, localization of leptin transcripts within the baboon syncytiotrophoblast suggests the potential for autocrine or paracrine interactions within this endocrinologically active tissue. Finally, both the similarities in leptin ontogeny in baboon and human pregnancy and the singular enhancement of maternal leptin levels inherent throughout baboon gestation emphasize the potential of this nonhuman primate model for the study of leptin action in the maternal-fetoplacental unit.
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Leptin is also linked to a variety of reproductive processes. Therefore, although female mice that are genotypically homozygous for the ob gene are obese, leptin deficient, and sterile, treatment with exogenous leptin readily restores fertility. However, simple restoration of normal body weight via food restriction alone had no appreciable effect (4). In similar experiments, leptin-treated ob/ob females exhibited elevated LH levels, increased ovarian and uterine weights, and stimulated ovarian and uterine histology compared to untreated controls. Leptin-treated ob/ob males evidenced enhanced FSH levels, increased testicular and seminal vesicle weights, and elevated sperm counts (5). Leptin-treated, genetically normal, prepuberal females reached reproductive maturity at a dramatically earlier age than did untreated controls and evidenced accelerated maturation of the reproductive tract (6). Therefore, it has been hypothesized that the polypeptide may serve as either a primary signal initiating puberty or a permissive regulator of sexual maturation (7). Also linking leptin with reproduction, leptin receptor messenger ribonucleic acid (mRNA) transcripts are expressed in the ovary, uterus, testis, hypothalamus, and anterior pituitary, as well as in granulosa cells in the rat (8), whereas in women undergoing in vitro fertilization, leptin mRNA and leptin protein have been determined in both granulosa and cumulus cells (9).
Intriguingly, women exhibit significantly higher peripheral serum leptin levels than men, even when matched in regard to adipose mass (10). During pregnancy, maternal concentrations rise with advancing gestation (11) and until parturition are significantly higher than postpartum (12). In addition to leptin produced in adipose tissue, a significant amount of the polypeptide measured in the maternal periphery may originate in the placenta, and the expression of both leptin (13, 14, 15) and leptin receptor (16, 17) transcripts has been detected in villous tissue. Immunohistochemical staining of term human placenta has localized leptin (14) and leptin receptor protein (17) to trophoblast cells. Recently, we reported the presence of mRNA transcripts for leptin and two leptin receptor isoforms within human syncytiotrophoblast by in situ hybridization and, using, competitive RT-PCR, determined a diminution in leptin transcript abundance commensurate with advancing gestation (18).
Although a number of possible roles for leptin in perinatal development and the endocrinology of pregnancy have been proposed, little has been definitively determined with regard to leptin ontogeny with advancing human pregnancy. Thus, further characterization of a well validated nonhuman primate model (19) from which blood and tissue samples may be collected throughout gestation may be efficacious. The objectives of the current study, therefore, were to 1) quantitate serum leptin concentrations in both pregnant as well as normally cycling and postpartum nonpregnant baboons, 2) determine the presence of mRNA transcripts for leptin in placental villous tissue throughout gestation, 3) evaluate changes in leptin levels and placental transcription relative to gestational age, and 4) localize mRNA transcripts for leptin within specific cell types of the placenta.
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Materials and Methods |
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Animals were maintained and used strictly in accordance with
USDA regulations and the Guide for the Care and Use of Laboratory
Animals (NIH Publication 86–23). The experimental protocol was
approved by the institutional care and use committee of the Tulane
Regional Primate Research Center (Covington, LA). Thus, 15 female
baboons (Papio sp), weighing 14–17 kg, were individually
housed in stainless steel cages as previously described (19, 20, 21). A
12-h light photoperiod (0600–1800 h) was maintained in air-conditioned
rooms, and animals received a primate maintenance ration with fresh
fruit daily and water ad libitum. Females were quartered
with males for mating in indoor/outdoor enclosures for 4–5 days,
coinciding with the estimated occurrence of ovulation, as determined by
daily menstrual cycle records and visible turgescence of external sex
skin. Blood samples were obtained from nonpregnant baboons and from
pregnant baboons at regular intervals from days 60–160 of gestation
(term = 184 days). Therefore, at 0800–0900 h a 6-mL blood sample
was withdrawn from an antecubital vein via a 21-gauge needle after
brief restraint and sedation with an im injection of ketamine HCl
(10–15 mg/kg BW; Ketalar, Fort Dodge Pharmaceuticals, Fort Dodge, IA).
The extended sample collection period during gestation (100 days) was
deemed necessary to establish a reliable baseline profile for maternal
peripheral leptin.
Baboon placental tissue was obtained upon cesarean delivery, as previously described, under isofluorane anesthesia after sedation with ketamine HCl/atropine (21), early in gestation (days 60–62; n = 5), at midgestation (days 98–102; n = 5), or late in gestation (days 159–167; n = 5). Thus, immediately after laparotomy, a hysterotomy was performed, and the fetus and placenta were delivered. As we have previously described (22), samples of placental villous tissue were collected from the maternal surface, from areas equidistant between the cord attachment and the outer rim of the placental disc. Tissue was then flash-frozen in liquid nitrogen for storage at -80 C. Tissue samples were also fixed (Histochoice, Amresco, Solon, OH) for in situ hybridization.
Leptin RIA
Serum samples were stored at -20 C until leptin concentrations were determined by RIA (Linco Research, Inc., St. Charles, MO), as we have previously described (23), employing an antibody against human leptin. Inter- and intraassay coefficients of variation (n = 5) for leptin were 8.7% and less than 5.0%, respectively, with a sensitivity of 0.5 ng/mL. The human assay was validated for the baboon using a pregnant serum pool, where serial dilutions yielded concentrations that were linear and parallel to those in human serum.
RNA extraction and RT-PCR
Total RNA was isolated from placental villous tissue using
TRIzol reagent (Life Technologies, Grand Island, NY)
according to the method of Chomczynski and Sacchi (24) and Chirgwin
et al. (25) as adapted for use in our laboratory (18).
Oligonucleotide primers were synthesized (Midland Reagent Co., Midland,
TX) according to the sequences for leptin (18) and the constitutively
expressed housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) (21). PCR primer sequences and product sizes are as
follows: leptin (235-bp product): 5'-primer,
5'-GACTTCATTCCTGGGCTCCACC-3'; 3'-primer, 5'-CCTGAAGCTTCCAGGACACC-3';
and GAPDH (240-bp product): 5'-primer, 5'-TGATGACATCAAGAAGGTGGTGAAG-3';
3'-primer, 5'-TCCTTGGAGGCCATGTAGGCCAT-3'. Sequences for oligonucleotide
primers for leptin exhibit approximately 50% GC content and were
designed to be RNA specific by spanning the junction of two exons.
Oligonucleotides anneal to specific gene sequences (as referenced by
GenBank accession numbers) at the following nucleotide positions:
leptin (U18915): 5'-primer, 181–202; 3'-primer, 397–416; and GAPDH
(M33197): 5'-primer, 825–849; 3'-primer, 1042–1064. Sequence analysis
of PCR products (Biotech Core, Palo Alto, CA) confirmed the specificity
of this primer pair to leptin mRNA. Complimentary DNAs (cDNAs) were
synthesized from 2 µg total RNA using the SuperScript kit (Life Technologies), and PCR was performed in a Temp-Tronic
Thermocycler (Barnstead/Thermolyne, Dubuque, IA) as previously reported
in our laboratory (18, 21). Conditions for PCR were: leptin, 40 cycles
for amplification, denaturation at 95 C for 30 s, hybridization at
55 C for 30 s, extension at 72 C for 60 s; and GAPDH, 24
cycles for amplification, denaturation at 94 C for 30 s,
hybridization at 58 C for 60 s, extension at 72 C for 60 s.
PCR products were visualized in 2% agarose gels containing ethidium
bromide. PCR reactions were accompanied by controls, including 1) GAPDH
as a positive cDNA synthesis control, 2) RNA that had not been
transcribed into cDNA as a genomic DNA contamination control, and 3)
sterile water blanks as a reagent control. After semiquantitative
assessment of mRNA transcripts by comparison of leptin PCR products
with GAPDH (21, 24), quantitative competitive RT-PCR was performed
using the PCR MIMIC Construction Kit (CLONTECH Laboratories, Inc., Palo Alto, CA), as we have previously described (18).
Thus, products were resolved by 2% agarose gel electrophoresis, and
band intensities were analyzed using the Alpha Imager 2000 digital
analysis system (Alpha Inotech, San Leandro, CA). Final concentrations
were expressed as attomoles per µg RNA (1 attomole = 6
x 105 molecules).
In situ hybridization
Paraffin-embedded tissue sections (6 µm) were floated onto the surface of SuperFrost Plus microscope slides (Labcraft, Fisher Scientific, Norwalk, GA) and fixed overnight at 37 C. Just before analysis, slides were baked (1 h, 65 C), deparaffinized, and in situ hybridization for leptin was performed using a 5'-biotinylated oligonucleotide (Midland Reagent Co.), as we have previously reported (18). Thus, the antisense (3') oligonucleotide primer used for PCR was used as the hybridization probe, and the sense strand (5') primer was used as a negative control for the specificity of hybridization. Hybridization was detected using the In Situ Hybridization and Detection Kit (Life Technologies). Sections were mounted for photomicroscopy with Histomount (Zymed, San Francisco, CA).
Statistical analysis
Standard least squares ANOVAs using the general linear models procedure, followed by mean separation according to the Student- Newman-Keuls method of the Statistical Analysis System (26), was employed to assess the effects of gestational stage on measured parameters. Pearson product-moment correlation coefficients were also generated. Significant differences were understood to exist when P < 0.05.
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, the mean ± SEM
peripheral serum leptin concentration in pregnant baboons (n = 5)
between days 60–160 of gestation was 125.2 ± 11.4 ng/mL compared
to 1.4 ± 0.1 ng/mL in nonpregnant animals in the luteal phase of
the menstrual cycle (n = 6) and 1.8 ± 0.1 ng/mL in
nonpregnant animals 15–18 days after cesarean delivery (n = 4).
Therefore, although leptin concentrations in nonpregnant (cycling
vs. postpartum) animals were similar (P >
0.05), concentrations of the peptide in pregnant animals were
dramatically higher (P < 0.01) than those in either
cycling or postpartum baboons. Further, to obtain a reliable
gestational profile for leptin, maternal blood samples were collected
at intervals throughout pregnancy, and serum levels were determined
(Fig. 2). Thus, maternal leptin
concentrations increased approximately 2.5-fold (P <
0.005) between day 60 (63.6 ± 10.4 ng/mL) and day 160 (157.8
± 16.1 ng/mL) of gestation. In addition, subsequent to cesarean
delivery in both early and late gestation, maternal blood samples were
collected for 15–18 days postpartum, and serum leptin were
quantitated. After both early pregnancy delivery (days 58 and 64;
n = 2) and near term delivery (days 162 and 165; n = 2),
maternal leptin levels plummeted (P < 0.005) within 15
days, to levels comparable to those found in nonpregnant females (Fig. 3).
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, A and B. Although expression of GAPDH
mRNA transcripts was similar at all stages of pregnancy, the abundance
of leptin transcripts appeared to decline with respect to increasing
gestational age. This potential decrease in leptin mRNA prompted
competitive quantitative assessment of leptin transcripts by the PCR
MIMIC method. With respect to this quantitative analysis, leptin mRNA
transcript abundance declined from 0.068 ± 0.021 attomoles/µg
RNA early in gestation to 0.035 ± 0.011 at midgestation to
0.009 ± 0.003 near term (Fig. 5A).
Thus, leptin transcripts in placental villous tissue decreased
approximately 8-fold between early and late gestation
(P < 0.02). Commensurately, maternal serum leptin
levels increased slightly less than 3-fold between early (63.6 ±
10.4 ng/mL), mid (98.0 ± 10.0), and late (157.8 ± 16.1)
gestation (Fig. 5B), and in late pregnancy, therefore, these levels
were significantly greater (P < 0.005) than at either
early or midgestation. Maternal leptin concentrations, measured in
early, mid, and late gestation, were positively correlated (r =
0.66; P < 0.001) and placental leptin mRNA levels were
negatively correlated (r = -0.64; P < 0.01) with
advancing gestational age. Maternal leptin concentrations were also
correlated (r = 0.76; P < 0.01) with maternal
body weight, although not with fetal birth weight (r = 0.11;
P > 0.7), as measured at the time of cesarean delivery
in late gestation.
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, multinucleated syncytiotrophoblasts
formed a continuous surface covering of placental villi (black
arrowhead) in tissue collected in early pregnancy (day 60), as
determined by hematoxylin and eosin staining. Mononucleated
cytotrophoblasts (white arrowhead), however, were relatively
few in number. Specific in situ hybridization to leptin
(Fig. 6B) determined transcript expression to be predominantly
localized in trophoblast cells, almost exclusively to that layer of
syncytiotrophoblasts encasing placental villi. The specificity of
hybridization was demonstrated by use of the 5'-sense strand primer for
leptin as a negative control (Fig. 6C). Similarly, no hybridization was
determined in the absence of probe (Fig. 6D).
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Discussion |
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In the present study, we have determined that in the baboon, a nonhuman primate previously shown to be an excellent model for the study of human pregnancy (19, 20, 21, 34, 35), leptin profiles are similar to those in humans, in that maternal concentrations are enhanced during pregnancy and increase with advancing gestational age. Also, as we have previously demonstrated in human placental villous tissue (18), the expression of leptin transcripts has been localized to syncytiotrophoblast by in situ hybridization. The baboon differs from the human in one respect, however, in that the increase in leptin concentrations determined in pregnant baboons over those in nonpregnant baboons is substantially greater than the increase noted during human pregnancy. A further similarity involves the progressive decline in placental leptin mRNA transcript abundance during baboon pregnancy, which is reminiscent of our previous investigation (18) of human placental villous tissue collected both early (7–14 weeks) in gestation and at term (38 weeks). In that study, the abundance of placental leptin mRNA, quantitated by competitive RT-PCR, declined (P < 0.005) over 100-fold between early pregnancy and term. Similarly, Masuzaki et al. (13) and Yura et al. (36) had previously suggested a dramatic decline semiquantitatively via Northern analysis. In the current study, placental leptin transcript abundance declined approximately 8-fold between days 60–160 of baboon gestation, a period that is roughly equivalent to weeks 13–33 of human pregnancy. Although the potential for species differences must be acknowledged when comparing differences in leptin transcript abundance between the human and the baboon, it should also be considered that samples collected in very early human pregnancy (7–8 weeks), for which transcript abundance was exceedingly high, and those collected at human term (38 weeks), for which mRNAs were quite low, were not directly matched by samples collected in the baboon. Therefore, collection of baboon villous tissue earlier in pregnancy, after the time of the luteal-placental shift on approximately day 25 (34) and nearer term, on approximately day 184 (19, 20), may be necessary for accurate ontogenetic comparisons between species. In view of the current comparison, however, we propose that a significant increase in maternal leptin concentrations with advancing gestation combined with a general decline in leptin mRNA abundance in the syncytiotrophoblast are common features of human and baboon pregnancy.
It is evident that increases in maternal leptin levels throughout human and baboon pregnancy do not directly reflect concomitant decreases in placental leptin mRNA. Therefore, we might postulate that 1) a decline in placental leptin transcript abundance is simply not predictive of the amount of leptin protein secreted by the primate placenta; 2) the increasing mass of the syncytiotrophoblast, which increases with advancing gestation along with placental size, may be solely responsible for increases in maternal peripheral leptin concentrations; 3) the decidua and fetal membranes may function as leptin-producing tissues, contributing leptin in increasing amounts, commensurate with increasing gestational age; 4) an enhanced contribution by maternal adipose stores, stimulated by the elevated levels of estrogen or other hormones that are typical of advancing primate pregnancy, might be at least partially responsible for increased serum leptin concentrations; or 5) the increase in maternal leptin concentrations with advancing gestational age might be attributed to the action of a leptin-binding protein expressed in greater abundance, or perhaps exclusively, during pregnancy. Certainly, a variety of factors may contribute to the disparity between maternal leptin levels and placental transcript abundance with advancing gestation, although postulates 4 and 5 above deserve special consideration. Therefore, although controversial, the potential effect of estrogen may be of particular interest (37), in that estrogen has been proposed to regulate serum leptin concentrations in both humans and rats. Thus, serum leptin levels were dramatically higher in cycling women than in postmenopausal women, ostensibly in response to available estrogen (38), although recent work in our laboratory indicated no significant differences in leptin levels in cycling and postmenopausal women (23). However, in vitro, leptin secretion by adipocytes was dramatically enhanced when they were cocultured with either estradiol or hCG (39). In rats, ovariectomy acted to diminish ob gene expression in sc and retroperitoneal white adipose tissue and caused a peripheral decline in serum leptin levels, but enhanced expression in mesenteric white adipose tissue (38). Administration of exogenous estradiol reversed all of the effects of ovariectomy. Although our current work has dealt specifically with the characterization of leptin-related mechanisms within the placenta, ongoing efforts in our laboratory are addressing the potential contributions of varied maternal adipose depots to maternal leptin level throughout primate pregnancy. Finally, the potential significance of leptin-binding proteins should also be seriously considered in future investigations, as proteins with definite leptin binding capacities have been identified in nonpregnant human serum (40, 41). In this regard, a circulating form of the leptin receptor, which is a product of the placenta (42), has been shown to be enhanced in late gestation in the mouse (43). Similarly, it has been proposed that high levels of soluble leptin receptor may be of significance in the development of leptin resistance during pregnancy (44).
In summary, we have quantitated leptin in maternal serum by RIA and demonstrated the presence of leptin mRNA transcripts in the placental syncytiotrophoblast by RT-PCR and in situ hybridization in the baboon. We have further determined in this species both an increase in maternal peripheral leptin levels and a decline in placental leptin mRNA that are commensurate with advancing gestation. We have also reported that the increase in peripheral leptin concentrations, inherent in baboon pregnancy, is dramatically greater than that observed during human pregnancy and that levels decline rapidly after delivery of the conceptus. These constitute the first such observations in a nonhuman primate and, because of similarities in leptin ontogeny with that of human gestation, suggest the baboon to be a viable model for the study of leptin action in pregnancy.
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Acknowledgments |
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Footnotes |
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Received December 9, 1998.
Revised March 2, 1999.
Accepted March 29, 1999.
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) and 64 (•) of pregnancy, and in late gestation
(B), days 162 (
) and 165 (
) of pregnancy. CDEL, Cesarean
delivery.
