Skip to main content
NCBI home page
Biology of Reproduction logoLink to Biology of Reproduction
. 2018 Jul 31;100(1):208–216. doi: 10.1093/biolre/ioy167

Sexual dimorphism of mitochondrial function in the hypoxic guinea pig placenta†

Hong Song 1, Bhanu P Telugu 2,3, Loren P Thompson 1,✉
PMCID: PMC6335207  PMID: 30085007

Abstract

Placental hypoxia can stimulate oxidative stress and mitochondrial dysfunction reducing placental efficiency and inducing fetal growth restriction (FGR). We hypothesized that chronic hypoxia inhibits mitochondrial function in the placenta as an underlying cause of cellular mechanisms contributing to FGR. Pregnant guinea pigs were exposed to either normoxia (NMX) or hypoxia (HPX; 10.5% O2) at 25 day gestation until term (65 day). Guinea pigs were anesthetized, and fetuses and placentas were excised at either mid (40 day) or late gestation (64 day), weighed, and placental tissue stored at −80°C until assayed. Mitochondrial DNA content, protein expression of respiratory Complexes I-V, and nitration and activity rates of Complexes I and IV were measured in NMX and HPX male (N = 6 in each treatment) and female (N = 6 in each treatment) placentas. Mitochondrial density was not altered by HPX in either mid- or late-term placentas. In mid gestation, HPX slightly increased expression of Complexes I-III and V in male placentas only, but had no effect on either Complex I or IV activity rates or nitrotyrosine expression. In late gestation, HPX significantly decreased CI/CIV activity rates and increased CI/CIV nitration in male but not female placentas exhibiting a sexual dimorphism. Complex I-V expression was reduced from mid to late gestation in both male and female placentas regardless of treatment. We conclude that chronic HPX decreases mitochondrial function by inhibiting Complex I/IV activity via increased peroxynitrite in a sex-related manner. Further, there may be a progressive decrease in energy metabolism of placental cell types with gestation that increases the vulnerability of placental function to intrauterine stress.

Keywords: pregnancy, hypoxia, mitochondria, respiratory complex

Chronic maternal hypoxia impairs mitochondrial function as an underlying cause of placental dysfunction, which may contribute to altered placental and fetal growth.

Introduction

Maternal chronic hypoxia is one of the most significant clinical challenges during pregnancy and impacts maternal, placental, and fetal compartments. Maternal hypoxia can result from several adverse conditions such as high-altitude hypoxia [1, 2], maternal cardiorespiratory disease, preeclampsia, and placental insufficiency [3, 4]. Both nutrient/O2 transfer across and hormone synthesis by the placenta may be disrupted by hypoxia [5, 6], mediated by altered metabolism of syncytiotrophoblasts, vascular smooth muscle, and endothelial cells. Mitochondria play an important role in energy metabolism and cellular processes required for successful placentation such as trophoblast proliferation, differentiation, spiral artery remodeling [7–9], and nutrient transport across the maternal-fetal circulations during placental growth [6].

The mitochondria generate cellular ATP to the cell via oxidative phosphorylation. During placental development, cells increase their reliance on mitochondrial oxidative phosphorylation as tissue oxygenation increases with blood vessel development [10]. Chronic hypoxia impacts mitochondrial respiration by reducing oxygen availability, inhibiting electron flux along the respiratory chain, increasing reactive oxygen species (ROS) generation at respiratory complexes, and disrupting normal energy balance within the cell [11, 12]. Placental hypoxia can generate oxidative stress [1] as a result of ROS generation from mitochondria, NADPH oxidases, xanthine oxidases, uncoupled nitric oxide synthases, and cytosolic metabolic enzymes [1, 13]. Mitochondria respond to hypoxic stress by altering their rate of respiration and ATP synthesis, biogenesis, and protein expression [11, 13]. These dynamic responses can disrupt mitochondrial function in ways not clearly understood but may be an underlying cause of altered metabolism during placental development.

Previous studies have identified an important effect of gestational hypoxia on mitochondrial function in placental development. During early gestation, the embryo relies on anaerobic glycolysis for its ATP supply due to low oxygen conditions [14]. As spiral arteries undergo remodeling, there is an increase in tissue oxygenation that contributes to a metabolic shift toward oxidative phosphorylation [8]. Maternal chronic hypoxia can disrupt this process of normal placental development by disrupting trophoblast differentiation, endocrine function (e.g., syncytiotrophoblast), and nutrient exchange (e.g., endothelial/vascular smooth muscle cell) in different cell types, thereby increasing the risk of fetal growth restriction [1, 15, 16]. Further, evidence exists to support sexual dimorphism in placental dysfunction with adverse intrauterine conditions such as gestational hypoxia, obesity, and diabetes [17–21].

We postulate that chronic hypoxia disrupts mitochondrial function as an underlying cause of placental dysfunction during the course of gestation. Mitochondrial function will be assessed by measuring mitochondrial density, protein expression of Complexes I-V, catalytic enzyme activity, and nitrotyrosine expression of Complexes I and IV at both mid and late gestation in male and female guinea pig placentas.

Materials and methods

Animal model and sample collection (n = 6 fetuses for each group)

All animal procedures using guinea pigs were approved by the University of Maryland Institution Animal Care and Use Committee in accordance with Association for Assessment and Accreditation of Laboratory Animal Care—accredited procedures (Animal Welfare Assurance No. A3200-01). Time-mated pregnant Dunkin-Hartley guinea pigs (term = 65 days) (Elm Hill Laboratories, Chelmsford, MA) were housed in either room air (normoxia; 21% O2, NMX) or in a Plexiglas chamber (hypoxia; 10.5% O2, HPX). Pregnant sows were exposed to 10.5% O2 at 25-day gestation for either 15- or 39-day duration. Animals were anesthetized (ketamine, 80 mg/kg i.p. and xylazine, 1 mg/kg i.p.) and placentas and fetuses extracted at either 40-day (mid-term) or 64-day gestation (late term, term = 65 days) following a subcutaneous lidocaine injection (1%), an abdominal skin incision, and hysterectomy. A total of 28 pregnant guinea pigs were used to obtain 24 fetuses. Two fetuses of different sexes were selected from a single sow when possible or a single fetus was selected if there was not a matching sibling of the opposite sex. Fetuses were obtained from a position closest to the cervix as representative of the entire litter. Fetuses and placentas were collected, weighed and placentas immediately frozen in liquid N2 and stored at −80°C until analysis. Fetal weight was normalized to its respective placental weight as relative fetal weight (fetal body weight/placental weight ratio).

Mitochondrial DNA content

Mitochondrial DNA content was measured as an index of mitochondrial mass. Total genomic DNA was isolated from guinea pig placenta from each treatment group (i.e., mid-term NMX/HPX and late-term NMX/HPX) using QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Genomic DNA concentration was determined by Nanodrop (ThermoFisher Scientific, Walthem, MA). Relative quantification of mitochondrial DNA content for each group was determined by real-time quantitative PCR (qPCR) using primers for mitochondrial gene (mt-ND1, forward 5′-CTAAAAACCCTTGCGCTCAC-3′; reverse 5′-TGGGAAGGGAAATGTGTCAT-3′) and nuclear gene (β-actin, forward 5′-ACTCTCCACCTTCCAGCAGA -3′; reverse 5′-AAAGCCATGCCAATCTCATC-3′). qPCR was performed with a two-step cycling program by using SYBR Green ROX qPCR Mastermix (Qiagen) and read on QuantStudio (Thermo Fisher, Rockford, IL) and expressed as a ratio of mtDNA/nucDNA. The relative copy number between each group was analyzed by using the 2−ΔΔCt method [22].

Mitochondrial protein isolation

Mitochondrial proteins were isolated from the labyrinth of guinea pig placentas from each of the treatment groups (mid-term—NMX/HPX and late-term—NMX/HPX) using a standard differential centrifugation protocol [23, 24] for subsequent assays of western immunoblotting and complex enzyme activity assays. Briefly, frozen placentas (30–40 mg) were ground to a fine powder in liquid N2, and frozen tissues were each transferred to a 1.5-ml microtube where they were suspended in 1 ml of ice-cold Homogenization Buffer (0.25 M sucrose, 5 mM HEPES, 1mM EDTA, pH 7.2). Tissues were kept on ice at all times during the preparation. Zirconium oxide homogenization beads were placed into tubes and tissues homogenized in a VWR Multi tube Vortexer at a power setting of 9 for 10 min at 4°C in the cold room. Samples were centrifuged twice at 600 × g for 10 min at 4°C to remove cellular debris. The supernatant was carefully transferred to a clean 1.5-ml tube and recentrifuged at 12 500× g for 10 min to generate an enriched mitochondrial fraction. The pellet containing the mitochondrial fraction was resuspended in 100 μl 1× RIPA Lysis buffer supplemented with a protease inhibitor (Bio-Rad, Hercules, CA) for western blot or was solubilized with 0.1 mM N-Dodecyl β-D-maltoside (Sigma, St. Louis, MO) for measurement of Complex I and IV enzyme activity. The mitochondrial protein concentration of each sample was determined by the Bio-Rad Protein Assay (Bio-Rad).

Purity of enriched mitochondrial fraction

To quantify the degree of contamination of other proteins in our enriched mitochondrial fraction, we generated three fractions from a single sample from two separate male NMX placentas and probed for cytoplasmic (i.e., α-tubulin), nuclear (i.e., Lamin), and mitochondrial (i.e., VDAC, volume-dependent anion selective channel) proteins. To obtain total protein, samples were extracted, sonicated, and heated to 95°C before loading. To obtain cytosolic proteins, samples were homogenized followed by centrifugation at 12 500 rpm for 10 min and the supernatant was removed for analysis. The mitochondrial protein fraction was generated as described above. An equal amount of protein (10 μg) was loaded onto the same gel and probed separately using primary antibodies of anti-α-tubulin (1:1000, CST, Danver, MA), anti-Lamin (1:2000, CST, Danver, MA), and anti-VDAC (1:2000, Abam, Inc. Cambridge, MA) followed by a peroxidase-conjugated secondary detection antibody.

Expression and nitration of mitochondrial respiratory complex proteins

Mitochondrial complex subunit expression of mid- and late-term NMX/HPX guinea pig placentas was measured by western blot analysis. Isolated mitochondrial proteins (10 μg) were separated on 4%–15% precast gradient gels (Bio-Rad) and then transferred to PVDF membranes. The membranes were blocked with 5% nonfat milk in TBS-T (TBS Tween-20 Buffer) for 2 h and incubated overnight at 4°C with primary antibody diluted in 5% nonfat milk in TBST, and then detected using an appropriate peroxidase-conjugated secondary antibody. Protein bands were targeted with a primary antibody cocktail containing antibodies for complex subunits (I-V; 1:500, Abcam, Cambridge, MA) and polyclonal anti-VDAC antibody (1:2000, Boster Biological Technology Co., Pleasanton, CA) and visualized by ChemiDoc Touch Imaging System (Bio-Rad). Nitration of mitochondrial proteins of the same samples was identified by the anti-nitrotyrosine antibody (1:1000, Millipore, Billerica, MA) in separate gels. Density of the bands was quantified by the Bio-Rad Image Lab System software. To determine the effect of HPX on protein expression, gels were generated to compare NMX and HPX placentas at mid- and late-term gestation.

To compare the effect of gestational age on protein expression independent of treatment, gels were generated to compare either NMX or HPX placentas at mid- and late-term gestation for each sex. Surprisingly, we found that the loading control, VDAC, as well as TOM, another mitochondrial membrane protein (not shown) was reduced by gestation, thereby disallowing us to use VDAC for normalization. Instead, total density of all mitochondrial proteins was measured to confirm equal loading on the gel and for normalization of band density of Complexes I-V.

Complex I and IV enzyme activity

Mitochondrial Complex I enzyme activity of guinea pig placenta was measured as the oxidation of NADH to NAD+ using a colorimetric assay kit (Abcam, Inc., Cambridge, MA). Briefly, mitochondrial proteins were isolated as previously described and 15 μg added to each of the microplate wells precoated with antibody specific for Complex I. After 3 h of incubation at 25°C, OD values at 450 nm were recorded using a 96-well plate reader (BioTek, Winooski, VT) and enzyme activity expressed as the change in OD values per minute per mg sample mitochondrial protein. The Complex IV activity is measured colorimetrically by monitoring the oxidation of cytochrome c. Briefly, mitochondrial proteins were isolated as before and 15 μg added to each of a 96-well plate containing the assay buffer (10 mM Tri-HCl, pH 7.0 and 120 mM KCl plus reduced cytochrome c) [25]. The OD value was measured at 550 nm in a 96-well plate reader (BioTek, Winooski, VT) for 30 min at 20 s intervals. The rate of the reaction was calculated in the linear range according to the following formula: cytochrome c oxidase activity (units/mg mitochondrial protein) = ΔOD/time (Δt)/ϵ*protein (mg), (ϵ (extinction coefficient) = 7.04 mM−1cm−1).

Statistics

All values are presented as mean ± SEM. Comparisons of weight, ΔCt values, and Complex I/IV activities and protein expression of Complexes I-V between NMX and HPX for mid- and late-term placentas were made using a two-way ANOVA followed by post hoc comparisons test of Bonferroni correction method or Student's t-test. Significant differences were identified as a P value < 0.05.

RESULTS

Effects of prenatal hypoxia on fetal and placenta weights

Fetal body weight, independent of sex, was 15.8 ± 1.3 g at mid-term gestation (N = 12; N = 6 males, N = 6 females) and increased to 91.3 ± 3.2g at late-term gestation (N = 12) in NMX animals (Figure 1). Prenatal HPX had no effect on fetal body weight at mid-term (14.6 ± 1.0) but significantly decreased (P < 0.05) weight at late-term gestation (64.5 ± 3.2). Placental weight was 2.65 ±0.13 g at mid-term gestation in NMX animals (N = 12) and increased by 71% to 4.54 ± 0.20 g by late gestation (N = 12). Chronic HPX had no effect on absolute placental weights at either mid- or late-term gestation. Relative fetal weight (calculated as fetal/placental weight ratio) at mid-term gestation was 5.94 ± 0.36 compared to 20.37 ± 0.82 at late gestation in NMX animals. Prenatal HPX significantly decreased (P < 0.05) the relative fetal weight in both mid- and late-term gestation. With regard to sex differences, in mid-term gestation, HPX had no effect on fetal body weight (males: 15.9 ± 1.9 vs 14.6 ± 1.4 g, females: 15.6 ± 1.8 vs 14.6 ± 0.3 g, NMX vs HPX, respectively), placental weight (males: 2.59 ± 0.14 vs 3.19 ± 0.32 g, females: 2.72 ± 0.24 vs 2.98 ± 0.21 g) or relative fetal weights (males: 6.14 ± 0.64 vs 4.71 ± 0.50, females: 5.74 ± 0.36 vs 4.91 ± 0.50) for both males (N = 6) and females (N = 6). In late gestation, HPX significantly (P < 0.05) decreased fetal body weight in both sexes (males: 94.2 ± 4.3 vs 64.1 ± 3.3 g, females: 88.5 ± 4.7 vs 65.0 ± 4.4 g), had no effect on placental weight in either sex (males: 4.80 ± 0.33 vs 4.55 ± 0.39 g, females: 4.28 ± 0.23 vs 3.79 ± 0.18 g), and significantly (P < 0.05) decreased relative fetal weight in both males (19.8 ± 0.8 vs 14.7 ± 1.5; N = 6) and females (20.9 ± 1.49 vs 17.07 ± 1.12; N = 6, NMX vs HPX, respectively).

Figure 1.

Figure 1.

Open in a new tab

Effects of hypoxia on fetal body and placenta weights (wts) of combined male and female guinea pigs. Fetal body and placenta wts were obtained from normoxic (NMX, open bar, N = 12) and hypoxic guinea pigs (HPX, hashed bar, N = 12, 10.5% O2) at mid- (40-day gestation) and late-term gestation (64 days; term = 65-day gestation). Relative placenta wts were normalized to their respective fetal body weights as placenta wt/fetal body wt ratios. Values are mean ± SEM. Significant differences from NMX controls are indicated as an “*” for P < 0.05.

Mitochondrial density

Neither prenatal HPX nor gestational age had any effect on mitochondrial DNA content at either mid- or late-term gestation in either sexes (Figure 2).

Figure 2.

Figure 2.

Open in a new tab

Effects of hypoxia on mitochondrial density of male and female guinea pig placentas. Placentas were obtained from normoxic (NMX, N = 6 male, N = 6 female) and hypoxic (HPX, 10.5% O2, N = 6 male, N = 6 female) at mid- (40-day gestation) and late-term gestation (64 days, term = 65-day gestation). Mitochondrial density is expressed as delta Ct values from qPCR as mean ± SEM. There were no significant differences among treatment groups.

Protein expression of mitochondrial Complexes I-V

The relative purity of the enriched mitochondrial fraction is illustrated in Figure 3. Proteins of Lamin (nuclear), α-tubulin (cytoplasmic), and VDAC (mitochondrial) were all found in the total cellular fraction as expected. The cytosolic fraction contained predominantly cytoplasmic and mitochondrial but no nuclear proteins. In both samples, VDAC was predominantly identified with no other significant contamination by other cytosolic and/or nuclear proteins. This illustrates the relative purity of the enriched mitochondrial fraction compared to other proteins and suggests that the total amount of mitochondrial protein loaded onto gels and/or used for enzyme activity assays is not compromised by significant contamination.

Figure 3.

Figure 3.

Open in a new tab

Protein expression of cellular fractions. Two separate samples of male normoxic (NMX) placentas were fractionated into three fractions of total (T) (i.e., mitochondrial, nuclear, and cytoplasmic proteins), cytosolic (C), and mitochondrial (M) proteins. Ten microgram of each sample was loaded onto a gel and probed separately for lamin A/C (i.e., nuclear protein), α-tubulin (i.e., cytosolic protein), and VDAC (i.e., mitochondrial protein) to determine the relative purity of the enriched mitochondrial fraction. Relative to the total and cytosolic fractions, the enriched mitochondrial fraction exhibited no significant contamination of lamin A/C or α-tubulin.

Images of western blots and graphic analysis of protein expression of Complexes I-V in both male and female placentas are shown in Figure 4. In male placentas, HPX increased expression of complexes I-III and V at mid- but not late-term gestation. In female placentas, HPX had no effect on any of the Complexes at either mid- or late-term gestation. Complex V is associated with ATP synthase and its expression was highly abundant in all placentas compared to Complexes I-IV.

Figure 4.

Figure 4.

Open in a new tab

Effects of hypoxia on Complex I-V protein expression of male and female guinea pig placentas. Mitochondria were isolated from placentas obtained from normoxic (NMX, N = 6 male, N = 6 female) and hypoxic (HPX, 10.5% O2, N = 6 male, N = 6 female) at mid- (40-day gestation, top graphs) and late-term gestation (64-day gestation, bottom graphs). (A) Immunoblot images illustrate band density of protein subunits targeted by a mitochondrial antibody complex cocktail for Complexes I-V. Band density was normalized to VDAC protein illustrated as the loading control. (B) Graphic analysis of normalized band densities for Complexes I-V of male and female NMX/HPX placentas at mid and late gestation. Values are mean ± SEM. Significant differences from NMX controls are indicated as an “*” for P < 0.05.

Complex I and IV activity rates

Prenatal HPX significantly (P < 0.05) decreased both CI and CIV activity in male placentas in late gestation but had no effect at mid-term gestation (Figure 5). There was no effect of hypoxia on either CI or CIV activity in female placentas regardless of gestational age.

Figure 5.

Figure 5.

Open in a new tab

Effects of hypoxia on enzyme activities of mitochondrial Complex I and IV proteins. Mitochondria were isolated from placentas obtained from normoxic (NMX, N = 6 male, N = 6 female) and hypoxic (HPX, 10.5% O2, N = 6 male, N = 6 female) at mid- (40-day gestation) and late-term gestation (64-day gestation). Complex I activity is measured as mOD/min/mg protein and Complex IV as activity units/mg/protein. Values are mean ± SEM. Significant differences from NMX controls are indicated as an “*” for P < 0.05.

Nitration of Complex I and IV proteins

Expression of nitrotyrosine is an indicator of nitration by peroxynitrite. Prenatal HPX had no significant effect of nitrotyrosine expression at mid-term in either male or female placentas (Figure 6). However, prenatal HPX significantly (P < 0.05) increased nitrotyrosine expression of Complexes I and IV in late-term gestation of only male placentas. No bands were identified at any other MW indicating a selective nitration of Complexes I and IV. In the western blot, nitrotyrosine targets only two bands corresponding to ∼20 and ∼35 kDa, which are associated with bands corresponding to Complex I and Complex IV subunits, respectively. Since only mitochondrial proteins are loaded onto the gel and only two bands are identified, then it is reasonable to assume that nitrotyrosine binding is associated with Complexes I and IV.

Figure 6.

Figure 6.

Open in a new tab

Effects of hypoxia on protein expression of nitrotyrosine of mitochondrial Complex I and IV proteins. Mitochondria were isolated from placentas obtained from normoxic (NMX, N = 6 male, N = 6 female) and hypoxic (HPX, 10.5% O2, N = 6 male, N = 6 female) at mid- (40-day gestation) and late-term gestation (64-day gestation). Immunoblot images from late gestation only illustrate band density of protein subunits targeted by a nitrotyrosine antibody along with VDAC as a loading control. Graphic analysis of normalized band densities for nitrated proteins correspond to MW associated with Complexes I (top) and IV (bottom) only. Values are mean ± SEM. Significant differences from NMX controls are indicated as an “*” for P < 0.05.

Effect of gestation on mitochondrial protein expression

To compare the effect of gestational age on mitochondrial complex expression, total mitochondrial protein from samples of NMX/HPX male and female placentas (Figure 7) were loaded onto separate gels and targeted with the mitochondrial protein antibody cocktail for Complexes I-V and VDAC antibody. Mitochondrial Complexes I, III, and V and VDAC were significantly (P < 0.05) decreased at late- compared to mid-term gestation in both males and females. In addition, Complexes II and IV were variably decreased in late vs mid gestation. This indicates a gestational age-dependent decrease in mitochondrial protein expression of not only the respiratory complexes but also proteins of outer mitochondrial membranes (i.e., VDAC) as well. Equal amounts of total mitochondrial protein were confirmed by measuring total band density for the entire column (not shown) and used to normalize the band densities of Complexes I-V.

Figure 7.

Figure 7.

Open in a new tab

Effects of gestational age on Complex I-V protein expression of male and female guinea pig placentas. Mitochondria were isolated from placentas obtained from normoxic (NMX, N = 6 male, N = 6 female) and hypoxic (HPX, 10.5% O2, N = 6 male, N = 6 female) at mid- (40-day gestation) and late-term gestation (64-day gestation). (A) Immunoblot images illustrate band density of protein subunits targeted by a mitochondrial antibody complex cocktail for Complexes I-V. Equal amounts of total mitochondrial protein confirmed equal loading. (B) Graphic analysis of absolute band densities for Complexes I-V of mid vs late gestation of normoxic (NMX) and hypoxic (HPX) male and female placentas. Values are mean ± SEM. Significant differences from NMX controls are indicated as an “*” for P < 0.05.

Discussion

This study indicates that maternal hypoxia alters mitochondrial function in the guinea pig placenta in a sex- and gestational age-related manner. Mitochondrial function was assessed by measurement of mitochondrial DNA content, protein expression of respiratory Complexes, Complexes I and IV activities and nitration of Complexes I and IV protein subunits. In general, hypoxia had a greater inhibitory effect on mitochondrial function in males compared to females in late gestation as indicated by reduced activities of Complexes I and IV and increased nitration of the same complexes with little or no change in respiratory complex protein expression. Lastly, independent of hypoxia and sex, advancing gestation decreased expression of specific respiratory Complexes and VDAC in all groups indicating the term placenta may be exhibiting signs of respiratory impairment.

The placenta is the site of nutrient/O2 transfer from the maternal to fetal circulation and a major determinant of fetal growth. As gestation advances, placental growth is expected to increase providing a sufficient nutrient/O2 supply to match the energy demand of the rapidly growing fetus [5, 17]. Under chronic hypoxia, an imbalance between the nutrient supply/demand ratio contributes to fetal growth restriction [26]. In the current study, at mid gestation, both placenta and fetal growth were unaffected by hypoxia, although the relative fetal weight was decreased as a result of a slight increase in placental weight. With prolonged hypoxia, the decrease in relative fetal weight occurred as a result of a reduction in fetal body weight. Previous studies reported that hypoxia alters placental weight in a variable manner exhibiting increased [17, 27], decreased [28], and no change [29, 30]. This suggests different strategies by which the placenta compensates to hypoxic stress such as increasing its surface area for exchange and diffusing capacity [17, 29, 31, 32] in the labyrinthine zone [32–34] or altering its endocrine function in the junctional zone [5]. The disproportionate decrease in fetal weight relative to placental weight reflects altered efficiency of placental function, particularly in late gestation. Despite sex-related differences in indices of mitochondrial function, there was no sexual dimorphism in the response of relative fetal weight to hypoxia.

We evaluated the effect of placental hypoxia on mitochondrial function at mid and late gestation to understand whether this may contribute as an underlying cause in placental dysfunction. Mitochondrial DNA content was unaltered by hypoxia and there were small but significant changes in Complex I-V protein expression. In mid gestation, there was a paradoxical or compensatory increase in expression of CI, II, III, and CV in hypoxic male placentas. This was not sustained in late gestation nor present in female placentas at either gestational age. This may indicate that mitochondrial protein expression in the females is either insensitive to hypoxic stress or recovers following prolonged hypoxia.

Both Complexes I (NADH dehydrogenase) and IV (cytochrome c oxidase) are important for electron transfer and oxygen consumption in the respiratory chain. The effect of hypoxia on respiratory function can be measured as enzymatic activity. A reduction in enzyme activity of any of these complexes in vivo could inhibit the flux of electrons and the rate of oxidative phosphorylation [35]. Enzyme activities of both Complexes I and IV were reduced by hypoxia in late but not mid gestation of male placentas whereas hypoxia had no effect in female placentas, identifying a sex difference to hypoxic stress favoring the female. An impaired enzyme activity of these complexes may reduce ATP generation, as well as, increase ROS generation [35], both of which impair oxidative phosphorylation [6, 15]. We attribute the decrease in Complex I and IV activity to an increased hypoxia-induced nitration by peroxynitrite. Peroxynitrite generation is a molecular footprint of nitrative stress as it is generated from the interaction of superoxide anion and nitric oxide [36, 37]. Upon its formation, peroxynitrite can nitrate tyrosine moieties of protein subunits forming nitrotyrosine through covalent modification and thereby inhibit enzyme activity [37, 38]. Only in late gestation was there a significant increase in nitrotyrosine expression associated with both Complexes I and IV in hypoxic male placentas, which may indicate a redox imbalance favoring nitrative stress [36]. This differs in the hypoxic female placentas where enzyme activities and nitration of Complexes I and IV were unaffected by hypoxia. An increased antioxidant capacity of female placentas [19] may account for the difference in response to hypoxia in the current study. Sex dimorphism in the placenta has been measured in response to stressful intrauterine environments and dependent on gestational age of exposure [21]. For example, recent studies have identified an increased risk of male human placentas to impaired mitochondrial biogenesis in maternal diabetes [39], reduced antioxidant capacity of male placentas from obese women [19], and a greater inhibition of mitochondrial respiration by microRNA 210 in female placentas [20, 40]. In the current study, the differential effect of hypoxia on reducing mitochondrial function may be mediated by a greater protective effect in female compared to male placentas, consistent with that measured in other animal studies [41–44].

This study also identified a gestational age effect on mitochondrial protein expression in both males and females, independent of hypoxia. By comparing protein expression between mid and late gestation on the same gel, we identified a significant decrease in complex and VDAC expression in each sex and regardless of treatment. This may contribute to a reduced capacity of mitochondrial respiration and/or nutrient transfer efficiency in the placenta at term. Further, the effect of gestational age on mitochondrial protein expression may increase the vulnerability of the term placenta to hypoxic stress as pregnancy advances. It should be noted that mitochondrial DNA content or mass of the placenta does not change with gestational age. This likely reflects differing mechanisms regulating mitochondrial dynamics and transcriptional regulation of respiratory Complex proteins with gestational age. Further, a lack of change in mitochondrial DNA content may represent an integrated response of a heterogeneous cell population within the placenta.

In summary, the current study suggests a mitochondrial dysfunction by hypoxia attributed to the decrease in CI/CIV activity concomitant with nitration of each of these complexes. This phenotype was predominant in late but not mid gestation, suggesting a progressive decline in indices of respiratory function to prolonged hypoxia. The effect of gestational age on reducing mitochondrial protein expression may identify an additional effect that may contribute to a progressive decrease in placental function. The labyrinth placenta is made up of multiple cell types including cytotrophoblasts, syncytiotrophoblasts, endothelial cells, and vascular smooth muscle cells. Labyrinthine trophoblasts, in particular, rely on energy metabolism for terminal differentiation from proliferating cytotrophoblasts to invasive extravillous trophoblasts [45], nutrient transport across syncytiotrophoblasts [46], and steroidogenesis of syncytiotrophoblasts [47, 48]. While this study did not distinguish between different placental cell types, it identified specific changes in indices of oxidative phosphorylation associated with prolonged hypoxia, sex, and gestation, identifying the mitochondrion as an important target organelle in oxidative metabolism during placental development.

Supplementary Material

Supplemental File
Click here for additional data file. (11.3KB, xlsx)

Acknowledgments

The authors acknowledge technical assistance by Gerard Pinkas in generating treatment groups and maintaining the animal colony.

Notes

Conference Presentation: Presented in part at the Annual Meeting of the Society of Reproductive Investigation on March 16–18, 2017, Orlando, Florida, and March 7–10, 2018, San Diego, California.

Edited by Dr. Haibin Wang, PhD, Xiamen University

Footnotes

†

Grant Support: The project described is supported by UMB-UMCP Seed Grant to LT and BT and National Institute of Health (NIH HL126859, LPT). The content is solely the responsibility of the authors and does not necessarily represent the official view of the NIH.

References

  • 1. Herrera EA, Krause B, Ebensperger G, Reyes RV, Casanello P, Parra-Cordero M, Llanos AJ. The placental pursuit for an adequate oxidant balance between the mother and the fetus. Front Pharmacol 2014; 5:149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2. Niermeyer S, Andrade Mollinedo P, Huicho L. Child health and living at high altitude. Arch Dis Child 2009; 94(10):806–811. [DOI] [PubMed] [Google Scholar]
  • 3. Hutter D, Kingdom J, Jaeggi E. Causes and mechanisms of intrauterine hypoxia and its impact on the fetal cardiovascular system: a review. Int J Pediat 2010; 2010:1–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4. Herrera EA, Riquelme RA, Ebensperger G, Reyes RV, Ulloa CE, Cabello G, Krause BJ, Parer JT, Giussani DA, Llanos AJ. Long-term exposure to high-altitude chronic hypoxia during gestation induces neonatal pulmonary hypertension at sea level. Am J Physiol Regul Int Comp Physiol 2010; 299:R1676–R1684. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5. Sferruzzi-Perri AN, Camm EJ. The Programming power of the placenta. Front Physiol 2016; 7:33. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6. Dimasuay KG, Boeuf P, Powell TL, Jansson T. Placental responses to changes in the maternal environment determine fetal growth. Front Physiol 2016; 7:12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7. Genbacev O, Joslin R, Damsky CH, Polliotti BM, Fisher SJ. Hypoxia alters early gestation human cytotrophoblast differentiation/invasion in vitro and models the placental defects that occur in preeclampsia. J Clin Invest 1996; 97(2):540–550. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8. Genbacev O, Zhou Y, Ludlow JW, Fisher SJ. Regulation of human placental development by oxygen tension. Science 1997; 277(5332):1669–1672. [DOI] [PubMed] [Google Scholar]
  • 9. Caniggia I, Mostachfi H, Winter J, Gassmann M, Lye SJ, Kuliszewski M, Post M. Hypoxia-inducible factor-1 mediates the biological effects of oxygen on human trophoblast differentiation through TGFbeta(3). J Clin Invest 2000; 105(5):577–587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10. Xie Y, Zhou S, Jiang Z, Dai J, Puscheck EE, Lee I, Parker G, Huttemann M, Rappolee DA. Hypoxic stress induces, but cannot sustain trophoblast stem cell differentiation to labyrinthine placenta due to mitochondrial insufficiency. Stem Cell Res 2014; 13(3):478–491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11. Angelova PR, Abramov AY. Functional role of mitochondrial reactive oxygen species in physiology. Free Radic Biol Med 2016; 100:81–85. [DOI] [PubMed] [Google Scholar]
  • 12. Lushchak VI. Free radicals, reactive oxygen species, oxidative stress and its classification. Chem Biol Interact 2014; 224:164–175. [DOI] [PubMed] [Google Scholar]
  • 13. Aljunaidy MM, Morton JS, Cooke CM, Davidge ST. Prenatal hypoxia and placental oxidative stress: linkages to developmental origins of cardiovascular disease. Am J Physiol Regul Int Comp Physiol 2017; 313(4):R395–R399. [DOI] [PubMed] [Google Scholar]
  • 14. De Marco CS, Caniggia I. Mechanisms of oxygen sensing in human trophoblast cells. Placenta 2002; 23(Suppl A):S58–S68. [DOI] [PubMed] [Google Scholar]
  • 15. Murray AJ. Oxygen delivery and fetal-placental growth: beyond a question of supply and demand? Placenta 2012; 33(Suppl 2):e16–e22. [DOI] [PubMed] [Google Scholar]
  • 16. Myatt L. Placental adaptive responses and fetal programming. J Physiol 2006; 572(1):25–30. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17. Matheson H, Veerbeek JH, Charnock-Jones DS, Burton GJ, Yung HW. Morphological and molecular changes in the murine placenta exposed to normobaric hypoxia throughout pregnancy. J Physiol 2016; 594(5):1371–1388. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18. Adibi J, Burton GJ, Clifton V, Collins S, Frias AE, Gierman L, Grigsby P, Jones H, Lee C, Maloyan A, Markert UR, Morales-Prieto DM et al. IFPA meeting 2016 workshop report II: Placental imaging, placenta and development of other organs, sexual dimorphism in placental function and trophoblast cell lines. Placenta 2017; 60(Suppl 1):S10–S14. [DOI] [PubMed] [Google Scholar]
  • 19. Evans L, Myatt L. Sexual dimorphism in the effect of maternal obesity on antioxidant defense mechanisms in the human placenta. Placenta 2017; 51:64–69. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20. Muralimanoharan S, Guo C, Myatt L, Maloyan A. Sexual dimorphism in miR-210 expression and mitochondrial dysfunction in the placenta with maternal obesity. Int J Obes 2015; 39(8):1274–1281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21. Kalisch-Smith JI, Simmons DG, Dickinson H, Moritz KM. Review: sexual dimorphism in the formation, function and adaptation of the placenta. Placenta 2017; 54:10–16. [DOI] [PubMed] [Google Scholar]
  • 22. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCt Method. Methods 2001; 25:402–408. [DOI] [PubMed] [Google Scholar]
  • 23. Gostimeskaya I, Galkin A. Preparation of highly coupled rat heart mitochondrial. J Vis Exp 2010; 43:3–5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24. Al-Hasan YM, Evans LC, Pinkas GA, Dabkowski ER, Stanley WC, Thompson LP. Chronic hypoxia impairs cytochrome oxidase activity via oxidative stress in selected fetal guinea pig organs. Reprod Sci 2013; 20(3):299–307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25. Spinazzi M, Casarin A, Pertegato V, Salviati L, Angelini C. Assessment of mitochondrial respiratory chain enzymatic activities on tissues and cultured cells. Nat Protoc 2012; 7(6):1235–1246. [DOI] [PubMed] [Google Scholar]
  • 26. Zamudio S, Torricos T, Fik E, Oyala M, Echalar L, Pullockaran J, Tutino E, Martin B, Belliappa S, Balanza E, Illsley NP. Hypoglycemia and the origin of hypoxia-induced reduction in human fetal growth. PLoS One 2010; 5(1):e8551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27. Thompson LP, Pence L, Pinkas G, Song H, Telugu BP. Placental hypoxia during early pregnancy causes maternal hypertension and placental insufficiency in the hypoxic guinea pig model. Biol Reprod 2016; 95(6):128–128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28. Richter HG, Camm EJ, Modi BN, Naeem F, Cross CM, Cindrova-Davies T, Spasic-Boskovic O, Dunster C, Mudway IS, Kelly FJ, Burton GJ, Poston L et al. Ascorbate prevents placental oxidative stress and enhances birth weight in hypoxic pregnancy in rats. J Physiol 2012; 590(6):1377–1387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29. Bacon BJ, Gilbert RD, Kaufmann P, Smith AD, Trevino FT, Longo LD. Placental anatomy and diffusing capacity in guinea pigs following long-term maternal hypoxia. Placenta 1984; 5(6):475–487. [DOI] [PubMed] [Google Scholar]
  • 30. Phillips TJ, Scott H, Menassa DA, Bignell AL, Sood A, Morton JS, Akagi T, Azuma K, Rogers MF, Gilmore CE, Inman GJ, Grant S et al. Treating the placenta to prevent adverse effects of gestational hypoxia on fetal brain development. Sci Rep 2017; 7(1):9079. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31. Gilbert RD, Cummings LA, Juchau MR, Longo LD. Placental diffusing capacity and fetal development in exercising or hypoxic guinea pigs. J Appl Physiol Respir Environ Exerc Physiol 1979; 46(4):828–834. [DOI] [PubMed] [Google Scholar]
  • 32. Higgins JS, Vaughan OR, Fernandez de Liger E, Fowden AL, Sferruzzi-Perri AN. Placental phenotype and resource allocation to fetal growth are modified by the timing and degree of hypoxia during mouse pregnancy. J Physiol 2016; 594(5):1341–1356. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33. Lewis RM, Doherty CB, James LA, Burton GJ, Hales CN. Effects of maternal iron restriction on placental vascularization in the rat. Placenta 2001; 22(6):534–539. [DOI] [PubMed] [Google Scholar]
  • 34. Cuffe JS, Walton SL, Singh RR, Spiers JG, Bielefeldt-Ohmann H, Wilkinson L, Little MH, Moritz KM. Mid- to late term hypoxia in the mouse alters placental morphology, glucocorticoid regulatory pathways and nutrient transporters in a sex-specific manner. J Physiol 2014; 592(14):3127–3141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35. Waypa GB, Smith KA, Schumacker PT. O2 sensing, mitochondria and ROS signaling: the fog is lifting. Mol Aspects Med 2016; 47–48:76–89. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36. Myatt L, Rosenfield RB, Eis AL, Brockman DE Greer I, Lyall F. Nitrotyrosine residues in placenta: evidence of peroxynitrite formation and action. Hypertension 1996; 28:488–493. [DOI] [PubMed] [Google Scholar]
  • 37. Murray J, Taylor SW, Zhang B, Ghosh SS, Capaldi RA. Oxidative damage to mitochondrial complex I due to peroxynitrite. J Biol Chem 2003; 278(39):37223–37230. [DOI] [PubMed] [Google Scholar]
  • 38. Behn C, Araneda OF, Llanos AJ, Celedón G, González G. Hypoxia-related lipid peroxidation: evidences, implications and approaches. Respir Physiol Neurobiol 2007; 158(2–3):143–150. [DOI] [PubMed] [Google Scholar]
  • 39. Jiang S, Teague AM, Tryggestad JB, Aston CE, Lyons T, Chernausek SD. Effects of maternal diabetes and fetal sex on human placenta mitochondrial biogenesis. Placenta 2017; 57:26–32. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40. Myatt L, Muralimanoharan S, Maloyan A. Effect of preeclampsia on placental function: influence of sexual dimorphism, microRNA’s and mitochondria. Adv Exp Med Biol 2014; 814:133–146. [DOI] [PubMed] [Google Scholar]
  • 41. Borras C, Sastre J, Garcia-Sala D, Lloret A, Pallardo FV, Vina J. Mitochondria from females exhibit higher antioxidant gene expression and lower oxidative damage than males. Free Radic Biol Med 2003; 34:546–552. [DOI] [PubMed] [Google Scholar]
  • 42. Stark MJ, Hodyl NA, Wright IM, Clifton VL. Influence of sex and glucocorticoid exposure on preterm placental pro-oxidant-antioxidant balance. Placenta 2011; 32(11):865–870. [DOI] [PubMed] [Google Scholar]
  • 43. Al-Gubory KH, Garrel C. Sex-specific divergence of antioxidant pathways in fetal brain, liver, and skeletal muscles. Free Radic Res 2016; 50(3):366–373. [DOI] [PubMed] [Google Scholar]
  • 44. Pajovic SB, Saicic ZS. Modulation of antioxidant enzyme activities by sexual steroid hormones. Physiol Res 2008; 57(6):801–811. [DOI] [PubMed] [Google Scholar]
  • 45. James JL, Stone PR, Chamley LW. The regulation of trophoblast differentiation by oxygen in the first trimester of pregnancy. Hum Reprod Update 2006; 12(2):137–144. [DOI] [PubMed] [Google Scholar]
  • 46. Jones HN, Powell TL, Jansson T. Regulation of placental nutrient transport – a review. Placenta 2007; 28(8–9):763–774. [DOI] [PubMed] [Google Scholar]
  • 47. Meigs RA, Sheean LA. Mitochondria from human term placenta. Biochim Biophys Acta 1977; 489(2):225–235. [DOI] [PubMed] [Google Scholar]
  • 48. Martínez F, Meaney A, Espinosa-García MT, Pardo JP, Uribe A, Flores-Herrera O. Characterization of the F1F0-ATPase and the tightly-bound ATPase activities in submitochondrial particles from human term placenta. Placenta 1996;17(5–6):345–350. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental File
Click here for additional data file. (11.3KB, xlsx)

Articles from Biology of Reproduction are provided here courtesy of Oxford University Press

RESOURCES