Michael C. Henson

Endocrine disruption by cadmium, a common environmental toxicant with paradoxical effects on reproduction.

Cadmium (Cd2+) is a common environmental pollutant and a major constituent of tobacco smoke. Exposure to this heavy metal, which has no known beneficial physiological role, has been linked to a wide range of detrimental effects on mammalian reproduction. Intriguingly, depending on the identity of the steroidogenic tissue involved and the dosage used, it has been reported to either enhance or inhibit the biosynthesis of progesterone, a hormone that is inexorably linked to both normal ovarian cyclicity and the maintenance of pregnancy. Thus, Cd2+ has been shown to exert significant effects on ovarian and reproductive tract morphology, with extremely low dosages reported to stimulate ovarian luteal progesterone biosynthesis and high dosages inhibiting it. In addition, Cd2+ exposure during human pregnancy has been linked to decreased birth weights and premature birth, with the enhanced levels of placental Cd2+ resulting from maternal exposure to industrial wastes or tobacco smoke being associated with decreased progesterone biosynthesis by the placental trophoblast. The stimulatory effects of Cd2+ on ovarian progesterone synthesis, as revealed by the results of studies using stable porcine granulosa cells, appear centered on the enhanced conversion of cholesterol to pregnenolone by the cytochrome P450 side chain cleavage (P450scc). However, in the placenta, the Cd2+-induced decline in progesterone synthesis is commensurate with a decrease in P450scc. Additionally, placental low-density lipoprotein receptor (LDL-R) mRNA declines in response to Cd2+ exposure, suggesting an inhibition in the pathway that provides cholesterol precursor from the maternal peripheral circulation. Potential mechanisms by which Cd2+ may affect steroidogenesis include interference with the DNA binding zinc (Zn2+)-finger motif through the substitution of Cd2+ for Zn2+ or by taking on the role of an endocrine disrupting chemical (EDC) that could mimic or inhibit the actions of endogenous estrogens. Divergent, tissue-specific (ovary vs. placenta) effects of Cd2+ also cannot be ruled out. Therefore, in consideration of the data currently available and in light of the potentially serious consequences of environmental Cd2+ exposure to human reproduction, we propose that priority should be given to studies dedicated to further elucidating the mechanisms involved.

Leptin in Pregnancy

Abstract Leptin is a polypeptide hormone that aids in the regulation of body weight and energy homeostasis and is linked to a variety of reproductive processes in both animals and humans. Thus, leptin may help regulate ovarian development and steroidogenesis and serve as either a primary signal initiating puberty or as a permissive regulator of sexual maturation. Perhaps significantly, peripheral leptin concentrations, adjusted for adiposity, are dramatically higher in females than in males throughout life. During primate pregnancy, maternal levels that arise from adipose stores and perhaps the placenta increase with advancing gestational age. Proposed physiological roles for leptin in pregnancy include the regulation of conceptus growth and development, fetal/placental angiogenesis, embryonic hematopoiesis, and hormone biosynthesis within the maternal-fetoplacental unit. The specific localization of both leptin and its receptor in the syncytiotrophoblast implies autocrine and/or paracrine relationships in this endocrinologically active tissue. Interactions of leptin with mechanisms regulating pre-eclampsia and maternal diabetes have also been suggested. Collectively, therefore, reports suggest that a better understanding of the regulation of leptin and its role(s) throughout gestation may eventually impact those causes of human perinatal morbidity and mortality that are exacerbated by intrauterine growth retardation, macrosomia, placental insufficiency, or prematurity.

Leptin in Pregnancy: An Update

Abstract Leptin influences satiety, adiposity, and metabolism and is associated with mechanisms regulating puberty onset, fertility, and pregnancy in various species. Maternal hyperleptinemia is a hallmark of mammalian pregnancy, although both the roles of leptin and the mechanisms regulating its synthesis appear to be taxa specific. In pregnant humans and nonhuman primates, leptin is produced by both maternal and fetal adipose tissues, as well as by the placental trophoblast. Specific receptors in the uterine endometrium, trophoblast, and fetus facilitate direct effects of the polypeptide on implantation, placental endocrine function, and conceptus development. A soluble isoform of the receptor may be responsible for inducing maternal leptin resistance during pregnancy and/or may facilitate the transplacental passage of leptin for the purpose of directly regulating fetal development. The steroid hormones are linked to the regulation of leptin and the leptin receptor and probably interact with other pregnancy-specific, serum-borne factors to regulate leptin dynamics during pregnancy. In addition to its effects on normal conceptus development, leptin is linked to mechanisms affecting a diverse array of pregnancy-specific pathologies that include preeclampsia, gestational diabetes, and intrauterine growth restriction. Association with these anomalies and with mechanisms pointing to a fetal origin for a range of conditions affecting the individuals health in adult life, such as obesity, diabetes mellitus, and cardiovascular disease, reiterate the need for continued research dedicated to elucidating leptins roles and regulation throughout gestation.

Expression of placental leptin and leptin receptor transcripts in early pregnancy and at term.

Objective To demonstrate the expression of messenger RNA (mRNA) transcripts for placental leptin and leptin receptor in early gestation and at term, to quantitate transcriptional changes relative to stage of gestation, to localize transcripts within specific placental cell types, and to determine if transcripts also are expressed in cultured cells. Methods Expression of leptin and leptin receptor was assessed by reverse transcriptase-polymerase chain reaction, and leptin quantitated against a leptin mRNA competitor (MIMIC), in human placental villous tissue collected at term cesarean deliveries and earlier during gestation (7–14 weeks) upon elective terminations. In situ hybridization was used to identify cell types exhibiting transcripts for genes of interest. Additionally, tissue was dispersed enzymatically, cytotrophoblast cells progressed to syncytiotrophoblastic maturity in culture, and transcripts were assessed. Results Placental leptin and leptin receptor transcripts were identified in early (n = 6) and late (n = 5) gestation. Although no changes (P > .05) were apparent for receptor, leptin mRNA declined (P < .005) from (mean ± standard error) 1.815 ± .491 attomoles/μg total RNA early in gestation to .013 ± .003 attomoles/μg total RNA at term. Leptin and leptin receptor transcripts were localized in trophoblast by in situ hybridization and were expressed in culture. Conclusion Results suggest an ontogenetic decline in leptin mRNA with advancing gestation. Localization of leptin and leptin receptor transcripts in syncytiotrophoblasts, cells also responsible for the production of hormones vital to pregnancy maintenance, suggest a potential for autocrine or paracrine interactions within this tissue. Finally, transcript expression in cultured cells suggests the suitability of in vitro paradigms for future studies of leptin in pregnancy.

Effects of estrogen on leptin gene promoter activation in MCF-7 breast cancer and JEG-3 choriocarcinoma cells: selective regulation via estrogen receptors α and β

Leptin is a potential regulator of conceptus development. We have previously suggested that in primate pregnancy, leptin biosynthesis is regulated by estrogen in a tissue-specific manner. Therefore, the objective of the current study was to determine the mechanism of estrogen action on LEP promoter activation in divergent cell types. The effects of estrogen were investigated in estrogen receptor (ER)-positive MCF-7 breast cancer cells and in ER-negative JEG-3 choriocarcinoma cells. Cells were transfected with a leptin-luciferase or an estrogen responsive element (ERE)-luciferase reporter construct, in conjunction with ERalpha, ERbeta, or empty vector expression plasmids. Cells were treated with estradiol and/or the specific estrogen antagonists, ICI-182,780 or 4-hydroxytamoxifen. In MCF-7 cells, estradiol stimulated (P<0.05) ERE-luciferase activity and was inhibited by ICI-182,780, but did not stimulate leptin-luciferase activity. However, leptin-luciferase was stimulated by estradiol (P<0.05) and inhibited by antiestrogens in JEG-3 cells that were co-transfected with ERalpha. Both antiestrogens stimulated leptin-luciferase activity (P<0.05) in JEG-3 cells co-transfected with ERbeta. Results suggested that LEP promoter activation may depend upon co-activators present in leptin-producing cells and may be inhibited by repressors present in non-leptin producing cells. Divergent effects of estrogen may be owed to differences in the type of ER (alpha or beta) expressed in target tissues.

Metals in human placenta: focus on the effects of cadmium on steroid hormones and leptin.

Cadmium and other metallic ions can act as metalloestrogens and endocrine disruptors of reproductive tissues and fetal development in mammals, including humans. The detrimental effects occur with respect to the synthesis of both steroid and polypeptide hormones in the placenta. Leptin is produced by the trophoblast and may regulate fetal organogenesis and development. In human term placentas, concentrations of toxic metals and their effects on steroidogenesis were assessed in healthy parturients (109 non‐smokers and 99 smokers) in relation to tobacco smoking. Trace elements (cadmium, lead, iron, zinc and copper) were analyzed in placentas using atomic absorption spectroscopy, and steroid hormones (progesterone and estradiol) were assayed in placental samples by an enzyme‐immunometric method. Cadmium concentrations were doubled in placentas of smokers as compared with non‐smokers, and placental lead and zinc concentrations increased significantly. Placental concentrations of iron, copper, progesterone and estradiol did not differ. In addition, human trophoblast cells were co‐cultured with 0, 5, 10 or 20  µm CdCl2 for 96  h and leptin mRNA assessed by quantitative polymerase chain reaction. Leptin mRNA declined dose‐responsively as a result of CdCl2 exposure. Collectively, the results confirm that human placental tissue offers a unique opportunity to biomonitor cadmium exposure in both the maternal and the internal fetal environments. In addition, the results strongly suggest that cadmium may cause a decline in placental leptin synthesis, as we have previously shown for placental progesterone production. This may constitute further evidence of the endocrine‐disrupting effects of cadmium, as a constituent of tobacco smoke, on reproduction in women. Copyright

Placental Endocrine Disruption Induced by Cadmium: Effects on P450 Cholesterol Side-Chain Cleavage and 3β-Hydroxysteroid Dehydrogenase Enzymes in Cultured Human Trophoblasts

Abstract We previously suggested that cadmium (Cd), an environmental toxicant and constituent of tobacco smoke, inhibits progesterone secretion in cultured human placental trophoblasts by inhibiting low-density lipoprotein receptor mRNA expression. In the current study, we investigated whether Cd also disrupts progesterone synthesis via P450 cholesterol side-chain cleavage (P450scc) and 3β-hydroxysteroid dehydrogenase (3β-HSD), enzymes that play important roles in placental steroidogenesis. Human cytotrophoblasts were purified by density gradient centrifugation and incubated in Dulbecco modified Eagle medium + 10% fetal bovine serum with 0, 5, 10, or 20 μM CdCl2 for 96 h. Cells progressed to syncytiotrophoblastic maturity regardless of treatment. No differences (P > 0.05) in cell protein and lactate dehydrogenase activity were observed between untreated trophoblasts and those treated with CdCl2. However, P450scc and 3β-HSD mRNA transcript levels declined in a dose-dependent manner (P <0.05) in trophoblasts cocultured with 5, 10, or 20 μM CdCl2. P450scc activity was similarly inhibited (P < 0.05) by CdCl2 treatment, although 3β-HSD activity was not significantly affected. Coculture with 8-bromo-cAMP enhanced progesterone secretion in untreated cultures but did not reverse the decline in progesterone secretion induced by CdCl2 treatment. CdCl2 failed to influence cAMP content in cultured cells. Collectively, results suggest that P450scc enzyme is another site at which Cd interferes with placental progesterone production. However, it is unlikely that an inhibition of cAMP is involved with the inhibition of progesterone biosynthesis by Cd in human trophoblasts.

Effects of cadmium on cell viability, trophoblastic development, and expression of low density lipoprotein receptor transcripts in cultured human placental cells

Previously, we have demonstrated that cadmium inhibits progesterone release in cultured human trophoblast cells. In the present study, we investigated potential mechanism(s) by which cadmium may elicit this effect. Cytotrophoblasts were obtained via enzymatic dispersion, purified by density gradient centrifugation, and cultured with increasing concentrations of cadmium. Cadmium-induced suppression of progesterone release seemed to be independent of cell death, as no significant decline in viability was observed with cadmium treatment. Further, immunocytochemical localization of cellular boundaries and nuclei indicated approximately 94% syncytial maturity was attained by both untreated and cadmium-treated cells, demonstrating that cadmium did not inhibit syncytial development. However, the abundance of LDL receptor (LDL-R) mRNA transcripts, as determined by competitive RT-PCR, was reduced (P < 0.05) by cadmium exposure in an apparent dose-dependent manner. Thus, the LDL-R, by which cholesterol substrate is supplied to the syncytiotrophoblast, is one site at which cadmium may interfere with placental progesterone production.

Leptin receptor transcripts are constitutively expressed in placenta and adipose tissue with advancing baboon pregnancy.

The baboon (Papio sp.) is an accepted nonhuman primate model for the study of the endocrinology of human pregnancy. To further characterize this model with regard to leptin function, messenger RNA transcripts for both long (Ob-RL) and short (Ob-RS) leptin receptor isoforms were identified in maternal tissues at various stages of gestation. Thus, placental villous, subcutaneous and omental adipose tissues were collected upon cesarean delivery at early (Days 60-62), mid (Days 98-102) and late (Days 159-164) pregnancy (term approximately 184 days). Additionally, amniochorion, decidua, and corpus luteum were collected in late gestation. Expression of Ob-RL and Ob-RS transcripts was determined in relation to constitutively expressed glyceraldehyde-3-phosphate dehydrogenase via reverse transcriptase-polymerase chain reaction, and transcripts were localized within specific placental cell types by in situ hybridization. Ob-RL and Ob-RS transcripts were present in amniochorion, decidua, and corpus luteum at term and appeared constitutively expressed throughout gestation in placenta and adipose tissues. Ob-RS was expressed in greater (P < 0.02) abundance than Ob-RL in all tissues. Within the placenta, receptor isoforms were localized predominantly to the syncytiotrophoblast. The expression of leptin receptor transcripts in maternal adipose tissues, as well as in the syncytiotrophoblast, amniochorion, decidua, and corpus luteum, suggests the potential for autocrine/paracrine roles for the polypeptide in the endocrinology of primate pregnancy. These are the first such observations in a nonhuman primate and support the use of the baboon as a model for the study of leptin in human pregnancy.

Cadmium accumulation and effects on progesterone release by cultured human trophoblast cells.

This study was designed to examine the characteristics of cadmium bioaccumulation by human trophoblast cells in culture and the subsequent effect of cadmium exposure on progesterone production and syncytial formation. The accumulation of cadmium suggested a time- and dose-dependent relationship, although it was not significant. The rate of metal accumulation was similar in all cadmium-treated groups. After 72 h of continuous exposure to cadmium concentrations of 5, 10, and 20 microM, progesterone release was diminished to 69, 51, and 38% of control values (P < 0.05), respectively. When cells were exposed to cadmium from 72 to 96 h (after syncytial development), progesterone release exhibited the same pattern of decline in response to increasing cadmium concentrations. Histologic evaluation of whole mounts of trophoblast cells exposed to 20 microM CdCl2 for 96 h revealed that syncytial formation seemed to be uninhibited. The pattern of cadmium-accumulation by normal cultured human trophoblast cells suggests a time- and dose-relationship with a concomitant decrease in progesterone release that occurs without apparent inhibition of syncytial development.