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The Journal of Clinical Endocrinology & Metabolism Vol. 86, No. 6 2518-2524
Copyright © 2001 by The Endocrine Society

Original Articles: Hormones and Reproductive Health

Regulation of Leptin and Leptin Receptor in Baboon Pregnancy: Effects of Advancing Gestation and Fetectomy1

Jennifer S. O’Neil, Amy E. Green, Deborah E. Edwards, Kenneth F. Swan, Terry Gimpel, V. Daniel Castracane and Michael C. Henson

Departments of Obstetrics and Gynecology (J.S.O., A.E.G., D.E.E., K.F.S., M.C.H.), Physiology (M.C.H.), and Structural and Cellular Biology (M.C.H.) and Interdisciplinary Program in Molecular and Cellular Biology (J.S.O., A.E.G., D.E.E., M.C.H.), Tulane University Health Sciences Center, New Orleans, Louisiana 70112-2699; Tulane Regional Primate Research Center (M.C.H.), Covington, Louisiana 70433-8915; and Department of Obstetrics and Gynecology and Women’s Health Research Institute of Amarillo, Texas Tech University Health Sciences Center (T.G., V.D.C.), Amarillo, Texas 79106-1797

Address all correspondence and requests for reprints to: Michael C. Henson, Ph.D., Department of Obstetrics and Gynecology (SL11), Tulane University Health Sciences Center, 1430 Tulane Avenue, New Orleans, Louisiana 70112-2699. E-mail: michael.henson{at}tulane.edu

Abstract

Leptin, a product of both adipose tissue and the placental syncytiotrophoblast and a potential regulator of primate conceptus development, increases in the maternal circulation with advancing gestation. This increase may be potentiated by estrogens, which also increase as pregnancy progresses. In the present study adipose tissue was collected from nonpregnant (n = 5) baboons (Papio sp) and in baboons during early (days 58–62; n = 5), mid (days 98–102; n = 5), and late (days 158–162; n = 5) pregnancy (term, ~184 days). Additionally, placental estrogen production was inhibited in pregnant baboons by the removal of fetal androgen precursors via fetectomy at midgestation, with tissues collected from fetectomized (n = 5) baboons approximately 60 days later. Leptin, estrogens, and androgens were quantitated in maternal serum by RIA. Leptin (LEP) and leptin receptor (LEP-RL and LEP-RS isoforms) messenger ribonucleic acids (mRNAs) were quantitated by competitive RT-PCR, and leptin concentrations were determined by RIA in maternal adipose and placental villous tissues.

Although LEP transcript abundance in adipose tissues was unchanged as a result of pregnancy or with advancing gestation, the leptin protein level was higher (P < 0.02) in pregnant baboons in early gestation than in nonpregnant baboons and increased with gestational age (P < 0.04). Maternal serum estrogens (estradiol and estrone) and androgens (androstenedione and testosterone) were lower (P < 0.0001) in fetectomized baboons than in intact controls. Serum leptin concentrations were unchanged by fetectomy, but the abundance of LEP mRNA transcripts was lower (P < 0.003) in sc adipose tissue and 3-fold higher (P < 0.05) in placenta. Similarly, the leptin protein level declined (P < 0.05) in sc adipose tissue and increased (P < 0.05) in placenta in fetectomized baboons. Although LEP-RL mRNA levels were unchanged after fetectomy, placental LEP-RS transcript abundance was lower (P < 0.04) than in pregnancy-intact baboons matched for gestational age. Results suggest that both adipose tissue and the placenta may contribute to maternal hyperleptinemia during normal primate pregnancy. Furthermore, the withdrawal of placental steroids results in the enhanced placental leptin production that is commensurate with a decline in production by sc adipose tissue.

LEPTIN IS THE protein product of the LEP, formerly the obese (ob), gene. It was originally considered an exclusive product of adipocytes, responsible for body weight regulation (1). However, the presence of the leptin receptor in various tissues suggests that leptin elicits a wide range of biological responses in diverse cell types (2, 3, 4). Thus, the discovery of leptin production by placental trophoblast cells has implicated it as a potential regulator of primate conceptus growth and development (5). Consequently, LEP messenger ribonucleic acid (mRNA) transcripts (6) and leptin protein (7) have been identified in human trophoblast cells, and two isoforms of the leptin receptor, characterized by long (LEP-RL) and short (LEP-RS), formerly OB-RL/OB-RS, intracellular domains, have been identified in human (6) and nonhuman primate (8) placentas. Expression of both leptin and its receptor in the endocrinologically active syncytiotrophoblast supports the projected role of leptin as an autocrine regulator of placental development and/or hormone biosynthesis (9).

Independently of changes in maternal adiposity, maternal serum leptin levels are elevated during human pregnancy, but decline rapidly after delivery to prepregnancy levels (10). Although this decrease has been associated with the removal of the placenta (11, 12), we reported that placental LEP mRNA abundance declines with advancing gestation in both the human (6) and the nonhuman primate (12) and is not correlated with elevated serum leptin concentrations. Circulating estrogen concentrations rise with advancing primate gestation and play key roles in placental progesterone biosynthesis and fetal adrenal maturation (13, 14). Previous studies suggested that leptin production is similarly regulated by estrogen (11, 15). In this capacity, adipose tissue, an estrogen-responsive entity (16), may exhibit enhanced leptin production under the influence of elevated estrogen levels typical of pregnancy (11). Estrogen has been reported to enhance LEP mRNA expression and leptin secretion in rat adipocytes in a dose-dependent manner. Both were inhibited by a specific estrogen receptor antagonist (17). Correspondingly, mRNA levels in sc adipose tissue were enhanced in rats by estradiol treatment (18), whereas ovariectomy reduced serum leptin levels in both rodents (19) and humans (20). To date, however, the effect of estrogen on leptin production in primate pregnancy has not been investigated. Intriguingly, although peripheral estrogen levels increase with advancing gestational age in both human (6) and nonhuman primate (12) pregnancy, levels of placental leptin mRNA transcripts decline. Of course, the potential for divergent effects of estrogen in placenta and adipose tissue exist, as evidenced by the presence of a functional enhancer for the leptin gene in choriocarcinoma cells, an enhancer that is not present in adipocytes (21, 22). Therefore, to more fully elucidate leptin and leptin receptor dynamics in primate pregnancy, we employed the baboon (Papio sp), an established nonhuman primate model for human pregnancy (13, 14). Like the human, the baboon lacks the 17{alpha}-hydroxylase and 17–20 lyase enzyme systems necessary for the placental conversion of progestins to androgens and subsequently relies on androgen precursors from the fetal adrenal for estrogen biosynthesis. Thus, the surgical removal of the fetus, but not the placenta (fetectomy), at midgestation inhibits estrogen production by the syncytiotrophoblast and reduces maternal serum estradiol levels to near baseline levels (13, 14, 23). Therefore, the objectives of the current study were to 1) document the effects of pregnancy and gestational age on LEP expression and leptin protein in maternal sc and omental adipose depots; 2) investigate the role of estrogen, via fetectomy, in regulating LEP expression and leptin protein in maternal adipose tissue and the placental trophoblast; and 3) determine the effects of fetectomy on LEP-RL and LEP-RS mRNA transcript abundance in leptin-producing tissues.

Materials and Methods

Animals

Animals were maintained and used in accordance with USDA regulations and the Guide for the Care and Use of Laboratory Animals (NIH Publication 86–23). The protocol was approved by the institutional care and use committee of Tulane Regional Primate Research Center (Covington, LA). Female baboons (Papio sp) were housed individually in stainless steel cages, as we have previously described (12, 24). Twelve-hour light photoperiods (0600–1800 h) were maintained in air-conditioned rooms, and animals received a maintenance ration with fresh fruit daily and water ad libitum. Females were quartered with males for mating in indoor/outdoor enclosures at the anticipated time of ovulation, as determined by daily menstrual cycle history and visible turgescense of external sex skin. Blood samples were obtained from nonpregnant baboons in the luteal phase of the menstrual cycle and from pregnant and fetectomized baboons at regular intervals from days 80–160 of gestation (term, ~184 days). Thus, at 0800–0900 h a 6-mL blood sample was withdrawn from an antecubital vein after brief restraint and sedation with an im injection of ketamine HCl (10–15 mg/kg BW; Ketalar, Fort Dodge Pharmaceuticals, Fort Dodge, IA).

Collection of tissues

Placental villous tissue as well as sc and omental adipose tissues were obtained from pregnant baboons upon cesarean delivery, under isofluorane anesthesia, after sedation with ketamine HCl/atropine, as we have previously described (12). Surgeries were performed early in gestation (days 58–62; n = 5), at midgestation (days 98–102; n = 5), or late in gestation (days 158–162; n = 5). Additionally, five baboons were fetectomized on day 100 of gestation, also as we have previously described (23). Briefly, after laparotomy and hysterotomy, the fetus was exteriorized, the umbilical cord was severed midway between placental and fetal attachments, and the placenta was left in situ. On day 160 of gestation, fetectomized baboons were anesthetized, and placentas were retrieved after hysterotomy. In all animals (fetectomized and pregnancy-intact baboons matched for gestational age), samples of villous tissue were collected from the maternal surface, from areas equidistant between the cord attachment and the outer rim of the placental disc. As we have previously described (8), sc adipose tissue was sampled at the site of the initial abdominal incision, with omental adipose tissue collected from the peritoneal cavity. Subcutaneous adipose tissue was collected from nonpregnant baboons in the luteal phase of the menstrual cycle (n = 5). Thus, under isofluorane anesthesia, tissue was excised from the margins of a midline abdominal incision, which approximated that required for cesarean deliveries. All tissues were flash-frozen in liquid nitrogen and stored at -70 C for later analyses.

RNA extraction and RT-PCR

Total RNA was extracted from all tissues using TRIzol reagent (Life Technologies, Inc., Grand Island, NY), according to the methods described by Chomczynski and Sacchi (25) and Chirgwin et al. (26) as adapted in our laboratory (6, 24). All samples were treated with deoxyribonuclease (Life Technologies, Inc., Gaithersburg, MD) to eliminate DNA contamination and were reprecipitated with sodium acetate and 100% ethanol. Oligonucleotide primers, as previously used in our laboratory (8, 12), were synthesized (Midland Reagent Co., Midland TX) for LEP, LEP-RL, LEP-RS, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Sequence analysis of PCR products (Biotech Core, Palo Alto, CA) confirmed that primers specifically amplified regions of LEP and leptin receptor complimentary DNAs (cDNA). cDNAs were synthesized from 2 µg total RNA (placenta) or 1 µg total RNA (adipose tissues) using SuperScript Kit (Life Technologies, Inc.), and PCR was performed in a Temp-Tronic Thermocycler (Barnstead/Thermolyne, Dubuque, IA), as we have previously described (6).

The conditions for PCR were as follows: LEP, 30 cycles for amplification, denaturation at 94 C for 60 s, annealing at 55 C for 30 s, extension at 72 C for 90 s (12); LEP-RS, 32 cycles for amplification, denaturation at 94 C for 30 s, annealing at 51 C for 30 s, extension at 72 C for 40 s (8); LEP-RL, 30 cycles for amplification, denaturation at 97 C for 20 s, annealing at 60 C for 30 s, extension at 72 C for 60 s (6); and GAPDH, 24 cycles for amplification, denaturation at 94 C for 30 s, annealing at 58 C for 60 s, extension at 72 C for 60 s (6). PCR products were visualized under UV light on 2% agarose gels with ethidium bromide. PCR reactions were accompanied by the following controls: GAPDH affirmed consistent cDNA synthesis, sterile water blanks served as a reagent control, and RNA that had not been transcribed into cDNA was used as a control for DNA contamination. After semiquantitative assessment of mRNA transcripts by comparison of PCR products with GAPDH, quantitative competitive RT-PCR was performed using the PCR MIMIC methodologies we have previously described (6, 12). Thus, products were resolved by 2% agarose gel electrophoresis, and band intensities were analyzed using the Alpha Imager 2000 digital analysis system (Alpha Innotech, San Leandro, CA). Final concentrations were expressed as attomoles per µg RNA (1 attomole; ~6 x 105 molecules).

Homogenization of tissues for RIA

Tissues were weighed and homogenized with 2.5 vol homogenization buffer containing 40 mmol/L KH2PO4, 10 mmol/L sucrose, 50 mmol/L KCl, 30 mmol/L ethylenediamine tetraacetate, 2 mmol/L ethyleneglycol-bis-(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 25 µg/mL aprotinin, 25 µg/mL leupeptin, and 1 mmol/L phenylmethylsulfonylfluoride in a glass-Teflon tissue homogenizer (Glas-Col, Terre Haute, IN). The homogenate was centrifuged at 3000 rpm at 4 C for 15 min, and the supernatant was analyzed. Total cell protein was determined according to the method described by Bradford (27), and leptin concentrations were determined by RIA.

RIA

Serum samples and tissue homogenates were stored at -20 and -70 C, respectively, until leptin concentrations were determined by RIA (Diagnostics Systems Laboratories, Inc., Webster, TX), employing an antibody against human leptin. Inter- and intraassay coefficients of variation (n = 5) for leptin were less than 9.0% and 5.0%, respectively, with a sensitivity of 0.5 ng/mL. The human assay was validated for the baboon using a pregnancy serum pool, where serial dilutions yielded concentrations that were linear and parallel to those in human serum. Serum testosterone, androstenedione, and estrone were measured by solid phase RIA (Diagnostics Systems Laboratories, Inc.). The intraassay coefficients of variation were less than 5.0%, and the interassay coefficients of variation were 7.5%, 12.1%, and 10.7%, respectively. Serum estradiol was measured with an automated chemiluminescent assay (Immulite, Diagnostic Products, Los Angeles, CA) and the interassay coefficient of variation was 11.8%.

Statistical analysis

ANOVA, with standard post-hoc Student-Newman-Keuls tests, was employed using SigmaStat statistical analysis software, version 5.0 (SPSS, Inc., Chicago, IL), to establish statistical significance between groups. Significant differences were understood to exist when P < 0.05.

Results

LEP mRNA transcript abundance was determined in sc adipose tissue from nonpregnant baboons in the luteal phase of the menstrual cycle (n = 5) and in omental and sc adipose tissues from pregnant baboons at early (days 58–62; n = 5), mid (days 98–102; n = 5), and late (days 158–162; n = 5) gestation by competitive RT-PCR. As illustrated in Fig. 1Go, no differences (P > 0.05) were detected in LEP mRNA transcript abundance (mean ± SEM) with advancing gestation in either sc or omental adipose tissues, and no differences were detected in LEP mRNA levels in sc fat between cycling and pregnant baboons. However, as depicted in Fig. 2Go, leptin protein in sc adipose tissue was approximately 2.5-fold (P < 0.02) greater in early gestation (0.40 ± 0.03 ng leptin/µg total cell protein) than in nonpregnant cycling (0.16 ± 0.06 ng leptin/µg total cell protein) baboons. Furthermore, leptin increased with advancing gestation from 0.40 ± 0.03 in early gestation to 0.90 ± 0.13 ng leptin/µg total cell protein in late gestation (P < 0.04). A slight increase was also noted in omental adipose tissue, from 0.59 ± 0.14 in early gestation, to 0.70 ± 0.15 at midgestation, to 0.97 ± 0.10 ng leptin/µg total cell protein late in gestation, but this trend failed to reach the specified level of significance. Leptin concentrations in placental villous tissue were near the minimum detectable range of the RIA in early (0.04 ± 0.024), mid (0.02 ± 0.003), and late (0.09 ± 0.012 ng leptin/µg total cell protein) gestation and were statistically unchanged with advancing gestation.



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  		Figure 1. LEP mRNA transcript abundance (mean ± SEM) in sc adipose tissue from nonpregnant cycling baboons in the luteal phase and in sc and omental adipose tissue during early (n = 5), mid (n = 5), and late (n = 5) gestation.

 


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  		Figure 2. Leptin levels (mean ± SEM) in sc adipose tissue from nonpregnant baboons and in sc and omental adipose tissue during early (n = 5), mid (n = 5), and late (n = 5) baboon gestation. Different lowercase letters indicate significant differences (a and b, P < 0.02; a and c, P < 0.01).

 
Maternal serum estradiol, estrone, androstenedione, and testosterone levels were determined in nonpregnant cycling (n = 5), pregnancy-intact (n = 5), and fetectomized (n = 5) baboons from venous blood samples collected in the luteal phase and at intervals between days 100–160 gestation. As illustrated in Fig. 3AGo, estradiol concentrations were dramatically lower (P < 0.0001) in fetectomized (0.5 ± 0.04 ng/mL) and nonpregnant (0.065 ± 0.02 ng/mL) baboons than in pregnancy-intact baboons (3.5 ± 0.23 ng/mL). Serum estrone levels were also lower (P < 0.0001) in fetectomized (0.13 ± 0.01 ng/mL) and nonpregnant (0.043 ± 0.005 ng/mL) baboons than in pregnancy-intact baboons (0.9 ± 0.07 ng/mL). Similarly, androstenedione levels (Fig. 3BGo) were lower (P < 0.0001) in fetectomized and nonpregnant animals (1.3 ± 0.07 and 0.52 ± 0.12 pg/mL, respectively) than in pregnancy-intact animals (2.4 ± 0.14 pg/mL). Testosterone levels (Fig. 3BGo) were also lower (P < 0.0001) in fetectomized and nonpregnant animals (0.22 ± 0.009 and 0.21 ± 0.04 pg/mL, respectively) than in pregnancy-intact animals (0.44 ± 0.02 pg/mL). Maternal serum leptin levels were determined from days 80–160 (Fig. 4Go). No differences were detected in maternal leptin concentrations between pregnancy-intact and fetectomized baboons.



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  		Figure 3. Maternal serum estrogens (A) and androgens (B) in pregnancy-intact, fetectomized, and nonpregnant cycling baboons (mean ± SEM). Different lowercase letters indicate significant differences (A: a, b, and c, P < 0.0001; d, e, and f, P < 0.0001; B: a, b, and c, P < 0.0001; d and e, P < 0.0001).

 


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  		Figure 4. Maternal serum leptin concentrations (mean ± SEM) with advancing gestation in both pregnancy-intact (n = 5) and fetectomized (n = 5) baboons.

 
Specific mRNA transcripts were quantitated by competitive RT-PCR, and leptin protein was quantitated by RIA in omental adipose tissue, sc adipose tissue, and placental villous tissue from both pregnancy-intact (n = 5) and fetectomized (n = 5) baboons on day 160 (Fig. 5Go). In placental villous tissue (Fig. 5AGo), LEP mRNA levels were 3-fold higher (P < 0.05) in fetectomized (1.14 ± 0.3 attomoles/µg total RNA) than in pregnancy-intact (0.40 ± 0.1 attomoles/µg total RNA) baboons. Corresponding leptin levels were 3-fold higher (P < 0.05) in fetectomized animals (0.28 ± 0.08 ng leptin/µg total cell protein) compared with pregnancy-intact animals (0.09 ± 0.01 ng leptin/µg total cell protein). LEP mRNA levels were only slightly lower in omental adipose tissue in fetectomized animals (Fig. 5BGo). Leptin levels were similar in omental adipose tissue (0.60 ± 0.15 ng leptin/µg total cell protein in fetectomized baboons compared with 0.97 ± 0.10 ng leptin/µg total cell protein in pregnancy-intact baboons). In sc adipose tissue, LEP mRNA transcript abundance was lower (P < 0.003; 5.8 ± 2.6 vs. 49.9 ± 9.8 attomoles/µg total RNA) in fetectomized baboons (Fig. 5CGo). Leptin levels in fetectomized baboons (0.56 ± 0.12 ng leptin/µg total cell protein) were approximately half those (P < 0.05) in pregnancy-intact baboons (0.90 ± 0.13 ng leptin/µg total cell protein).



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  		Figure 5. LEP mRNA transcript abundance and leptin protein levels (mean ± SEM) upon placental retrieval (day 160) in placental villous tissue (A), omental adipose tissue (B), and sc adipose tissue (C). Different lowercase letters indicate significant differences (a and b, c and d, g and h, P < 0.05; e and f, P < 0.003).

 
Finally, mRNA transcripts for two leptin receptor isoforms, LEP-RL and LEP-RS, were quantitated in placental villous (n = 5), omental adipose (n = 3), and sc adipose (n = 3) tissues by competitive RT-PCR. As summarized in Table 1Go, no differences in LEP-RL expression were detected in any tissue as a result of fetectomy. However, LEP-RS transcript abundance was lower (P < 0.04) in placental villous tissue from fetectomized baboons (19.8 ± 8.5 attomoles/µg total RNA) than in that from pregnancy-intact baboons (62.3 ± 16.9 attomoles/µg total RNA).


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  		Table 1. Leptin receptor mRNA transcript abundance (attomoles per µg total RNA) in placental villous (n = 5), sc (n = 3), and omental (n = 3) adipose tissues in pregnancy-intact and fetectomized baboons

 
Discussion

During pregnancy, the maternal metabolism adapts to support a rapidly growing conceptus and prepare for lactation. Therefore, based on the principal function of leptin as a regulator of energy homeostasis, a decline in maternal leptin levels might be expected during pregnancy to facilitate optimal nutritional intake. Conversely, leptin concentrations are enhanced throughout gestation. Significant increases early in pregnancy, before increases in maternal body weight, suggest that factors other than increased adiposity influence production (28). It has been proposed that maternal hyperleptinemia may be due to the stimulatory effects of gestational hormones on maternal adipose tissue (11). Thus, estradiol treatment up-regulated LEP mRNA expression and protein secretion by adipocytes in vitro (29, 30) and in vivo (18). Expression in isolated rat adipocytes was dose dependently enhanced by estrogen treatment and inhibited by a specific estrogen receptor antagonist. Coincubation of the transcriptional inhibitor actinomycin D with estradiol prevented increases in mRNA transcripts, suggesting that estrogen activates transcription (17). However, LEP transcript abundance is not always predictive of an attendant effect on protein synthesis, which may instead be more dependent on posttranscriptional regulatory mechanisms. In this respect, it has been reported that serum leptin levels are not directly related to LEP mRNA levels (31) and that alterations in serum leptin concentrations may be more dependent on factors that induce or inhibit protein secretion (32). Indeed, discrepancies between LEP mRNA and leptin protein levels in the present study may imply that estrogen influences an undefined posttranscriptional regulatory mechanism(s), perhaps resulting in a decrease in leptin release or enhanced leptin storage by adipocytes.

Therefore, the effects of estrogen on the actual production of leptin protein are unclear. Based on both animal and in vitro studies, it has been suggested that estrogen replacement therapy might act to enhance peripheral serum leptin levels, which typically decline in postmenopausal women. It has also been proposed that normally cycling women receiving oral contraceptives could be expected to exhibit higher serum leptin concentrations than untreated peers. In this regard, Elbers et al. (33) and Lavoie et al. (34) reported an increase in serum leptin in estrogen-treated postmenopausal women, although Haffern et al. (35) and Kohrt et al. (36) both failed to detect increases after treatment. Similarly, we found no differences in serum leptin concentrations in women after oral contraceptive administration (37). Collectively, these results suggest that relatively modest changes in serum estrogen concentrations may be insufficient to bring about discernable changes in peripheral leptin levels. In this capacity, Yamada et al. (38) reported that leptin levels were not affected by the small increases associated with normal menstrual cyclicity, but were enhanced by the large increases resulting from ovulation induction. Therefore, considering the inconsistencies in recent findings and the possibility of estrogen serving as a permissive, rather than a dose-dependent, regulator, we postulated that estrogen administration may not constitute the best model for examining effects on leptin biosynthesis in vivo. Ovariectomy, however, has been extensively used to document the negative effects of estrogen withdrawal (19, 20). Therefore, we employed fetectomy in a similar manner to explore the effects of reduced estrogen availability on leptin dynamics. Fetectomy is a well characterized approach to investigate the regulation of placental endocrinology in the absence of the fetus (13, 14, 39). After fetectomy, placental morphology remains relatively unchanged for at least 60 days. Syncytiotrophoblasts in both pregnancy-intact and fetectomized baboons predominate and form a continuous surface covering of placental villi, as evidenced by their reaction with antisera against placental lactogen and pregnancy-specific ß1-glycoprotein (23). The placenta remains viable, and syncytiotrophoblasts retain their functional capacity, as evidenced by the aromatization of exogenous C19 steroids (40) and the formation of placental lactogen (41) and pregnancy-associated placental peptide A (42). Perhaps significantly, prior studies have determined the specific action of exogenous estrogen supplied to fetectomized baboons in regulating both placental low density lipoprotein receptor (23) and P450 side-chain cleavage enzyme (43). Interestingly, in the current study maternal androgen concentrations were noted to decline along with estrogen levels in fetectomized animals. In this respect, intermediates in the biosynthetic pathway leading to estrone and estradiol include testosterone and androstenedione. Our results suggest that a decrease in these steroid intermediates, which would have been derived from fetal precursors, may account for the decline in maternal androgens after fetectomy. The reduction of androgen levels after fetectomy indicates that approximately half of the production represents fetal adrenal-placental interaction, but the remainder is derived from maternal adrenal-placental interaction.

Because discrepancies in leptin production have been identified in divergent adipose depots (44), it was necessary to evaluate leptin production in both sc and omental repositories in the current study. To this end, although maternal estrogen concentrations were enhanced in pregnancy and increased with advancing gestation, we found no differences between cycling and pregnant baboons in LEP mRNA transcript abundance in sc adipose tissue or with advancing gestation in either adipose depot. However, compared with nonpregnant baboons, we did determine a significant increase in leptin protein in sc adipose tissue in early pregnancy as well as an increase in leptin protein with advancing gestation, results that may demonstrate the importance of posttranscriptional mechanisms to the regulation of leptin biosynthesis. In addition, both LEP mRNA and leptin protein were reduced in sc adipose tissue and enhanced in the placenta of fetectomized animals, inferring estrogen’s ability to divergently regulate leptin production in various tissues. Thus, results suggest that placental estrogen may act to enhance leptin production in maternal adipocytes, tissues that would bear primary responsibility for the manifestation of pregnancy-associated hyperleptinemia, while inhibiting leptin production in the placenta. The physiological relevance of these contrasting effects remains unknown. Certainly, the potential for tissue-specific regulatory mechanisms exists, as a functional enhancer for the LEP gene has been identified in choriocarcinoma cells, but is not present in adipocytes (21, 22). Furthermore, factors that induce transcription differ in adipocytes and trophoblasts (29, 45), and the capacity for leptin biosynthesis by adipose tissues (44) appears considerably greater than that for the placenta, although increases in syncytiotrophoblast mass with advancing gestation may be at least partly responsible for increases in maternal leptin concentrations. Collectively, our findings strongly suggest the presence of tissue-specific regulatory mechanisms that may be of functional significance to leptin biosynthesis. These results also support the proposal that estrogen serves as a permissive, rather than as a dose-dependent, stimulator of LEP transcription, as mRNA transcript abundance was unchanged in maternal adipose tissues as a result of pregnancy or with advancing gestation despite a concurrent increase in serum estrogen. However, LEP transcripts were significantly reduced in the sc fat of fetectomized baboons, suggesting that, as in humans (38), dramatic changes in serum estrogen concentrations are necessary to modulate transcription.

Although we detected tissue-specific modifications in leptin production after fetectomy, maternal serum leptin levels were unchanged from those in pregnancy-intact baboons. Therefore, although leptin expression declined in sc adipocytes, increased placental expression suggested a compensatory mechanism. In light of the accepted stimulatory effects of estrogens (11, 17, 29, 30) and the suppressive effects of androgens (46, 47), the effects of fetectomy on leptin transcript abundance may be 2-fold, including both a down-regulation of leptin in response to reduced estrogens as well as an up-regulation due to the removal of leptin-inhibiting androgens. Although testing these possibilities was beyond the scope of the present study, future experiments will attempt to distinguish between the specific effects of estrogen and the potentially suppressive effects of androgens inherent to primate pregnancy. Indeed, limitations of the fetectomy model in directly determining effects due to an intact conceptus make future experiments involving the inhibition of estrogen synthesis in pregnancy-intact baboons necessary to fully elucidate the mechanisms involved. Finally, we previously reported placental receptors to be constitutively expressed with advancing baboon pregnancy (8). However, with respect to the current study, LEP-RS transcripts declined in placental villous tissue after fetectomy, although no differences were detected in maternal adipose tissue or in LEP-RL transcript levels. This suggests that leptin receptor transcription is not generally regulated by estrogen in adipose tissues, but that permissive, isoform-specific regulatory mechanisms may function within the placenta.

In summary, elevated estrogen levels commonly associated with baboon pregnancy were associated with increased leptin protein in maternal adipose tissue. Intriguingly, maternal serum leptin levels were unchanged by fetectomy, and changes in LEP mRNA abundance after fetectomy suggested a permissive down-regulation of the gene in adipose tissue and a commensurate up-regulation in placenta. Collectively, therefore, these results imply a tissue-specific regulatory role for estrogen as well as the existence of nonestrogen-dependent regulatory mechanisms potentially dependent on an intact fetus and inherent to primate pregnancy.

Footnotes

1 This work was supported by NIH Research Grants R29-HD-32502 (to M.C.H.) and P51-RR-00164-35 (to the Tulane Regional Primate Research Center). Presented in part at the 32nd Annual Meeting of the Society for the Study of Reproduction, Pullman, Washington (1999), and the 47th Annual Meeting of the Society for Gynecologic Investigation, Chicago, Illinois (2000). Back

Received August 8, 2000.

Revised October 27, 2000.

Accepted December 8, 2000.

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