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Low density lipoprotein (LDL) is accepted as the primary source of cholesterol for progesterone biosynthesis in the primate placental syncytiotrophoblast. We hypothesized that the syncytiotrophoblast may, however, derive significant amounts of cholesterol from sources in addition to the LDL pathway, especially during early pregnancy or when faced with a paucity of lipoprotein-cholesterol. To test this, alternate cholesterol-providing pathways were assessed in placentae at early (Days 60-61), mid (Days 98-102), and late (Days 160-167) gestation in the baboon (Papio sp., term approximately 184 days). Expression of LDL receptor mRNA transcripts in an enriched fraction of syncytiotrophoblast cells was approximately 13-fold greater (P < 0.05) in mid and late gestation than in early pregnancy, although no differences were observed in whole villous tissue. The abundance of transcripts for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the enzyme responsible for de novo cholesterol synthesis, remained unchanged in syncytiotrophoblast cells; however, HMG-CoA reductase activity declined approximately 2-fold from early to late pregnancy (P < 0.01), with a commensurate decline in immunoreactive HMG-CoA reductase protein. Activities for acyl-coenzyme A:cholesterol acyl transferase (ACAT), a rate-limiting enzyme for cholesterol esterification, were greater (P < 0.05) at early and mid pregnancy in placental homogenates than in those from late pregnancy, while ACAT-1 mRNA concentrations and cholesterol ester hydrolase activity remained unchanged. These results, taken together, suggest that although de novo synthesis has the potential to provide a measure of the cholesterol used for placental progesterone production during early baboon pregnancy, its contribution declines with advancing gestational age as LDL receptor-derived cholesterol becomes the major source of substrate. Changes in LDL receptor mRNA abundance suggest differences in mechanisms regulating cholesterol homeostasis in steroidogenically active syncytiotrophoblasts vs. proliferative nonendocrine cell types in the placenta.

Leptin is a polypeptide hormone originally thought to be produced exclusively by adipocytes. Recently, however, both leptin messenger ribonucleic acid (mRNA) and leptin protein were identified in human placental trophoblast cells, suggesting a potential role in primate pregnancy. In the present study, venous blood samples were collected at 5-day intervals during gestation from baboons (Papio sp), an established model for the study of human pregnancy, as well as from nonpregnant baboons, and leptin concentrations were determined by RIA. Additionally, placental villous tissue was collected upon cesarean delivery at early (days 60–62; n = 5), mid (days 98–102; n= 5), and late (days 159–167; n = 5) gestation (term = ∼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.

Maternal low density lipoprotein (LDL) is the principal source of cholesterol substrate for progesterone biosynthesis in the primate placental syncytiotrophoblast. The relationship of LDL-cholesterol availability and other potential cholesterol-yielding pathways to placental progesterone production have not, however, been demonstrated in vivo in a nonhuman primate. Therefore, maternal peripheral lipoprotein-cholesterol and progesterone concentrations were determined in blood samples obtained by venipuncture, from day 72 until day 100, from pregnant baboons (Papio sp) that were either untreated (n = 4) or treated (n = 3) with the inhibitor of hepatic lipoprotein production, 4-aminopyrazolo [3-4-d]pyrimidine (4-APP, 10 mg/kg BW) on days 98-99 of pregnancy (term = 184 days). Although LDL-cholesterol and progesterone levels remained unchanged in untreated animals, LDL-cholesterol concentrations were 9-fold lower (P0.005) in baboons receiving 4-APP than in untreated baboons 2 days following initial administration. Commensurate progesterone levels were 3.5-fold lower (P0.03) in 4-APP-treated baboons than in untreated baboons. RT-PCR was used to approximate relative changes in transcription of messengers RNAs (mRNAs) for selected cholesterol-sensitive pathways in placental tissue collected on day 100. Thus, expression of mRNAs for LDL receptor and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase appeared enhanced, whereas acyl-coenzyme A:cholesterol acyl transferase (ACAT) mRNA was diminished in syncytiotrophoblast-enriched cell fractions as a result of 4-APP administration. No relative differences in mRNAs were apparent in whole placental villous tissue, however, as a result of 4-APP treatment. In summary, this experiment demonstrates a significant decline in progesterone production elicited by maternal LDL-cholesterol withdrawal, and attests to the efficacy of 4-APP administration during baboon pregnancy. These results also suggest a commensurate regulation of cholesterol-sensitive pathways in primate syncytiotrophoblast. However, no relative differences were apparent in mRNA levels for LDL receptor, HMG-CoA and ACAT in whole placental villous tissue as a result of LDL-cholesterol withdrawal, which may suggest potential disparities in the mechanisms regulating cholesterol homeostasis in steroidogenically active syncytiotrophoblasts vs. those in proliferative nonendocrine placental constituents.

Explant and cell culture methodologies are frequently employed in the investigation of the mechanisms that mediate placental hormone production. Recent reports suggest the presence of unknown regulatory factors in maternal serum that may impact significantly on the regulation of these biosynthetic pathways. The present study, therefore, determined the effects of sera obtained from pregnant women in the second to third trimester (PWS), nonpregnant women (NPWS), and men (MS) as well as commercially prepared FBS on hCG, estradiol, and progesterone release into medium by cultured human trophoblast cells. Placental villous tissue was enzymatically dispersed, and cytotrophoblast cells were purified via density gradient centrifugation and cultured (37 C; 90% air-10% CO2) in DMEM with 10% PWS, NPWS, MS, or FBS. All cytotrophoblast cultures aggregated and progressed to syncytial forms, although cells cultured with PWS exhibited notably larger multinucleated syncytial elements by 48 h in culture than cells cultured with FBS. Significant increases (P < 0.05) occurred in hCG, estradiol, and progesterone release due to the progression of cytotrophoblasts into the syncytiotrophoblast phase in all cultures. The quantity of hCG release was unaffected by serum origin. Cells cultured with human serum released greater (P < 0.05) amounts of estradiol than cells cultured with FBS. Cells cultured with MS released more (P < 0.01) estradiol than cells cultured with either PWS or NPWS, in a ratio to the concentration of endogenous androgen precursor available. Progesterone release was greater (P < 0.01) for PWS-cultured cells than for FBS-cultured cells. Progesterone release by NPWS- and MS-cultured cells was intermediate. Syncytiotrophoblasts cultured with PWS released approximately 3-fold more (P < 0.01) progesterone than syncytiotrophoblasts cultured with FBS and low density lipoprotein cholesterol, although the concentrations of available cholesterol substrate were similar. Culture of cells in steroid-depleted or lipoprotein-depleted PWS or FBS resulted in similar decreases (P < 0.01) in estradiol and progesterone release, respectively. In summary, increased estradiol release by placental cells cultured in intact human serum was attributed to aromatizable androgens, whereas enhanced progesterone release by cells cultured in human serum could be only partially attributed to higher concentrations of low density lipoprotein cholesterol substrate in human serum. Evidence of increased syncytial maturity and progesterone release by PWS-cultured cells may indicate the presence of undefined serum-borne regulators, which is enhanced during pregnancy.