Testosterone Decreases Placental Mitochondrial Content and Cellular Bioenergetics

Abstract

Placental mitochondrial dysfunction plays a central role in the pathogenesis of preeclampsia. Since preeclampsia is a hyperandrogenic state, we hypothesized that elevated maternal testosterone levels induce damage to placental mitochondria and decrease bioenergetic profiles. To test this hypothesis, pregnant Sprague-Dawley rats were injected with vehicle or testosterone propionate (0.5 mg/kg/day) from gestation day (GD) 15 to 19. On GD20, the placentas were isolated to assess mitochondrial structure, copy number, ATP/ADP ratio, and biogenesis (Pgc-1α and Nrf1). In addition, in vitro cultures of human trophoblasts (HTR-8/SVneo) were treated with dihydrotestosterone (0.3, 1.0, and 3.0 nM), and bioenergetic profiles using seahorse analyzer were assessed. Testosterone exposure in pregnant rats led to a 2-fold increase in plasma testosterone levels with an associated decrease in placental and fetal weights compared with controls. Elevated maternal testosterone levels induced structural damage to the placental mitochondria and decreased mitochondrial copy number. The ATP/ADP ratio was reduced with a parallel decrease in the mRNA and protein expression of Pgc-1α and Nrf1 in the placenta of testosterone-treated rats compared with controls. In cultured trophoblasts, dihydrotestosterone decreased the mitochondrial copy number and reduced PGC-1α, NRF1 mRNA, and protein levels without altering the expression of mitochondrial fission/fusion genes. Dihydrotestosterone exposure induced significant mitochondrial energy deficits with a dose-dependent decrease in basal respiration, ATP-linked respiration, maximal respiration, and spare respiratory capacity. In summary, our study suggests that the placental mitochondrial dysfunction induced by elevated maternal testosterone might be a potential mechanism linking preeclampsia to feto-placental growth restriction.

Keywords: NRF1; PGC-1α; mitochondria; oxygen consumption; placenta; preeclampsia; respiration; testosterone; trophoblast.

Figure 1

Plasma T levels and placental and fetal weights in control and T-treated pregnant rats. Pregnant rats were treated with vehicle (sesame oil) or T propionate from gestation day 15 to 19 and euthanized on day 20. (A) Plasma T levels were quantified using ELISA. (B) Placental and (C) Fetal weights were measured. Data presented as mean ± SEM of 6 rats in each group. * p < 0.05 vs. control.

Figure 2

Characterization of mitochondrial structure in the placenta. (A) Representative electron micrographs of placental mitochondria from control (left) and T-treated pregnant rats (right). Images show less abundant mitochondria and abnormal mitochondrial structure with condensed matrix and cristae in the placenta of T-treated rats. (B) Quantification of the percentage of morphologically abnormal mitochondrial showing condensed matrix and cristae in the placenta of control and T-treated rats. n = 4 in each group. * p < 0.05 vs. control.

Figure 3

Mitochondrial copy number and ATP levels in the placenta of control and T-treated pregnant rats. (A) Mitochondrial DNA copy number was quantified using qRT-PCR based analysis. Placental (B) ATP and (C) ADP content was quantified using ApoSENSOR ADP/ATP kit. (D) Measurement of the ATP/ADP ratio. Data presented as mean ± SEM of 6 rats in each group. * p < 0.05 vs. control.

Figure 4

Expression of mitochondrial biogenesis indicators in the placenta of control and T-treated pregnant rats. Real-time PCR was used to assess (A) Pgc-1a and (B) Nrf1 mRNA expression in the placenta. Quantitation of placental Pgc-1a and Nrf1 mRNA expression was normalized relative to β-actin. (C) Representative Western blots for Pgc-1α, Nrf1, and β-actin are shown at top; blot density obtained from densitometric scanning of Pgc-1α and Nrf1 normalized to β-actin is shown at the bottom. Data presented as means ± SEM of 6 rats in each group. * p < 0.05 vs. control.

Figure 5

Mitochondrial copy number and expression of fission/fusion and biogenesis indicators. Trophoblasts cells were treated with vehicle (ethanol) or dihydrotestosterone (DHT) for 24 h. (A) Mitochondrial DNA copy number was quantified using qRT-PCR based analysis. (B) Cell viability after exposure to DHT was assessed using lactate dehydrogenase (LDH) cytotoxicity assay. The LDH levels were measured and expressed as the fold change compared to vehicle control. Real-time PCR was used to assess the relative mRNA expression levels of (C) fission/fusion genes (FIS-1, DRP-1, MFN-1, MFN-2, and OPA-1), and biogenesis indicators (D) PGC-1α and (E) NRF1, normalized to β-actin. (F) Representative Western blots for PGC-1α, NRF-1, and β-actin are shown at the left; blot density obtained from densitometric scanning of PGC-1α and NRF-1 normalized to β-actin is shown at the right data presented as means ± SEM of 4 biologically independent replicates. * p < 0.05 vs. control.

Figure 6

Bioenergetics profile of trophoblasts. Trophoblasts cells were treated with vehicle (ethanol) or dihydrotestosterone (DHT) for 24 h; mitochondrial respiratory parameters were measured using Seahorse. (A) Representative traces of mitochondrial respiration, (B) basal oxygen consumption rates (OCR), (C) proton leak, (D) ATP production-linked respiration (OCR after oligomycin administration), (E) maximal respiration (OCR after carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) administration) and (F) spare respiratory capacity (Difference between basal and maximal OCR). Data are presented as means ± SEM. The studies were done in duplicate from samples obtained from 4 biologically independent replicates. * p < 0.05 vs. vehicle control.