DOI: 10.5061/dryad.280gb5mzf
title: Maternal progesterone and adipose mPRε in pregnancy regulate the embryonic nutritional state

description: Sex steroid hormones such as progesterone play a pivotal role in
 reproductive functions and maintaining pregnancy; however, the impact of
 progesterone on the interaction between mother and embryo is unclear.
 Here, we demonstrate that the relationship between maternal progesterone
 and membrane progesterone receptor epsilon (mPRε) in adipose tissue
 regulates embryonic nutritional environment and growth after birth in
 mice. The activation of adipose mPRε by increased progesterone during
 pregnancy enhanced maternal insulin resistance through the production of
 prostaglandins by enhancing PLA2 activity, thereby efficiently providing
 glucose to embryos. The offspring of mPRε-deficient mothers exhibited
 metabolic dysfunction, whereas mPRε-deficient mothers with
 high-fat-diet-induced obesity exhibited improved insulin sensitivity.
 These findings establish the importance of progesterone as a nutritional
 regulator between mother and embryo and suggest that mPRε modulators could
 be developed to treat pregnant glycemic control disorders such as
 gestational diabetes mellitus, as well as metabolic syndrome in offspring.
description: # Maternal progesterone and adipose mPRε in pregnancy regulate the
 embryonic nutritional state
 [https://doi.org/10.5061/dryad.280gb5mzf](https://doi.org/10.5061/dryad.280gb5mzf\\) We have submitted a description of the data and file structure, sharing/access information, and computational scripts. ## Description of the data and file structure **Figure 1: mPRε is sufficiently expressed in white adipose tissues** (A) mPRε mRNA expression in mouse tissues (postnatal day 49: P49) measured by quantitative RT-PCR (n = 3–4). WATs: white adipose tissues (epididymal adipose tissue); BAT: brown adipose tissue. Control 18S rRNA expression. NC, normal chow; HFD, high-fat diet. Statistical analysis was performed using Student’s t-test. (B) Expression of mPR family subtypes in mouse WAT (n = 4; P49). (C) mPRε expression in mature adipocytes (MA) and a stromal vascular fraction (SVF) of mice fed NC or HFD (n = 4). (D) mPRε mRNA expression in MEF-derived adipocytes during adipogenesis (n = 6–10). (E) Expression of the progesterone nuclear receptor PGR in mouse WATs (left, n = 4). PGR expression in the MA and SVF of mice fed NC or HFD (right, n = 3–8). (P49). PGR, progesterone receptor. *p &lt; 0.05, **p &lt; 0.01 (Student’s t-test). Results are presented as the mean ± standard error of the mean (SE). **Figure 2: Progesterone enhances insulin resistance via adipose mPRε** (A) Changes in body weight of WT and mPRε-/- mice under high-fat diet (HFD) feeding (n = 4–6).  (B) Plasma steroid levels at 16 weeks of age (n = 4–6). (C, E) An oral glucose tolerance test (OGTT) in male (C) or female (E) WT and mPRε-/- mice was performed at 15 min post-progesterone subcutaneous injection. (n = 7–10, *p &lt; 0.05, vs. WT: progesterone).  (D, F) Insulin tolerance test (ITT) in male (D) or female (F) WT and mPRε-/- mice was performed at 15 min post-progesterone subcutaneous injection (n = 7–10, *p &lt; 0.05 vs WT: progesterone). *p &lt; 0.05, **p &lt; 0.01 (Student’s t-test).  (H) Inhibitory effects of progesterone on insulin signaling. After pretreatment with progesterone for 30 min, a bolus of insulin (0.5 U/kg, i.p.) with or without progesterone (5 mg/kg, s.c.) was administered. Akt phosphorylation of Ser473 in WAT of wild-type mice after a 5 h of fast (n = 7–9).  (I) Effect of progesterone on glucose uptake in mouse embryonic fibroblast (MEF)-derived adipocytes from WT or mPRε-/- mice. Glucose uptake was determined by measuring 2-deoxyglucose uptake using an enzymatic photometric assay (n = 4–10); P4: progesterone. *p &lt; 0.05, **p &lt; 0.01 (Dunn’s post hoc test). The results are presented as the mean ± standard error of the mean (SE). N.S.: not significant. See also Figures S1, S2, and S3. **Figure 3: Maternal progesterone-adipose mPRε control blood glucose in embryos** (A) Plasma progesterone levels in mice (male, non-pregnant, GD13.5, and GD16.5 pregnant female mice) (n = 4–6).  (B) Oral glucose tolerance test (OGTT) in WT and mPRε-/- GD16.5 female mice (n = 4–8, *p &lt; 0.05, **p &lt; 0.01 vs WT).  (C) Blood glucose levels in WT and mPRε-/- GD16.5 female mice (left) and E16.5 embryos (right) (n = 9–10, **p &lt; 0.01 vs WT).  (D) OGTT in non-pregnant female mice n = 5–8, *p &lt; 0.05, **p &lt; 0.01 vs WT)  (E) OGTT in HFD-induced gestational diabetes mellitus model WT and mPRε-/- GD16.5 female mice (n = 4–8). *p &lt; 0.05, **p &lt; 0.01 (Mann–Whitney U test). Results are presented as the mean ± standard error of the mean (SE). See also Figures S4 and S5. **Figure 4: Offspring from mPRε-deficient female mice exhibits metabolic dysfunctions** (B) Body weight change during the growth of offspring from homozygous crosses or heterozygous crosses (n = 7–8). *p &lt; 0.05, **p &lt; 0.01 (Dunn’s post hoc test).  (C) Changes in body weight of WT and mPRε-/- offspring mice under high-fat diet (HFD) feeding in males (left) or females (right) (n = 4–10).  (D) Tissue weights of 16-week-old mPRε-/- offspring mice under high-fat diet feeding in males (left) or females (right) (n = 8–10). *p &lt; 0.05, **p &lt; 0.01 (Mann–Whitney U test). Results are presented as the mean ± standard error of the mean (SE). N.S.: not significant. See also Figures S6 and S7. **Figure 5: Prostaglandin production is increased during pregnancy via mPRε in WATs** (A) Beta diversity via the principal component analysis (PCA) based on genes from KEGG (ID mmu00590; mmu04064; mmu04910) in the WATs of WT and mPRε-/- mice with non-pregnant or GD16.5 (n = 5). The compositional similarity was compared using the permutational multivariate analysis of variance.  (B) KEGG enrichment analysis related to molecular function in WATs of GD16.5 mPRε-/- females. p-values adjusted based on the false discovery rate (FDR).  (C) Heat map of prostaglandin synthesis pathway, chronic inflammation, insulin resistance-related gene profiles of the WATs from WT and mPRε-/- non-pregnant or GD16.5 female mice (n = 5). Among non-pregnant vs GD16.5 females, 66 identified genes are differentially expressed (absolute log2 fold change &gt; 0.5, p &lt; 0.05: red open square).  (D) KEGG pathway analysis related to arachidonic acid metabolism in WATs of mPRε-/- GD16.5 females. P-values were adjusted based on the FDR.  (E) Comprehensive analysis of lipid mediators in WATs of mPRε-/- GD16.5 females; heat map of the top 40% relative lipid metabolite profiles of the WATs of WT and mPRε-/- non-pregnant or GD16.5 female mice (n = 5). Among non-pregnant vs GD16.5 females, 75 genes were differentially expressed (absolute log2 fold change &gt; 0.5, p &lt; 0.05: red open square).  (F) Arachidonic acid (left) and PGE2 (right) in the WATs of non-pregnant or GD16.5 females. (n = 6–7 per group for arachidonic acid; n = 6–7 per group for PGE2).  (G) Arachidonic acid levels in response to progesterone (100 nM) in Flp-In mPRε T-REx HEK293 cells (n = 6–8). Cells were induced mPRε expression by treatment with doxycycline (10 μg/mL) for 24 hr. Dox; doxycycline. *p &lt; 0.05, **p &lt; 0.01 (Mann–Whitney U test). The results are presented as the mean ± standard error of the mean (SE). N.S.: not significant. See also Figures S8, S9, S10, and S11. **Figure 6: Progesterone-induced activation of mPRε increases insulin resistance via adipose PGE2 production** (A) Arachidonic acid (left) and PGE2 (right) levels in the white adipose tissues (WATs) of WT and mPRε-/- female mice; 15 min after subcutaneous progesterone injection mice are treated with or without ibuprofen (100 mg/kg) (n = 4–5 per group for arachidonic acid; n = 3–5 per group for PGE2) *p &lt; 0.05, **p &lt; 0.01 (Dunn’s post-hoc). #p &lt; 0.05 (Mann–Whitney U test).  (B) Oral glucose tolerance test (OGTT) in WT and mPRε-/- female mice was performed at 15 min post-progesterone subcutaneous injection with or without ibuprofen (100 mg/kg). (n = 4–5). P4, progesterone. #p &lt; 0.05, ##p &lt; 0.01 between WT_P4 and WT_P4+ibuprofen (Dunn’s post-hoc test). NS: not significant. **Figure S1: Schematic representation of the mPRε gene structure and expression, related to Figure 2** (C) Expression of mPRε mRNA in mouse adipose tissues of wild-type and mPRε-/- mice (n = 3–4). The expression of 18S rRNA was used as an internal control. **Figure S2: Metabolic parameters in mPRε-/- offspring from mPRε+/- male and mPRε+/- female mice, related to Figure 2** (A) Tissue weight after 12 weeks in mPRε-/- male mice under high-fat diet (HFD) feeding (n = 4–6 from four litters; independent experiments). Epi, epididymal; peri, perirenal; sub, subcutaneous; BAT, brown adipose tissue; WATs, white adipose tissues. (B) Blood glucose, plasma triglyceride, non-esterified fatty acid (NEFA), and total cholesterol levels in WT and mPRε-/- male mice after HFD intervention for 12 weeks (n = 4–6). (C) Plasma insulin levels in male mice (n = 4–6 from four litters; independent experiments). (D) Tissue weight after 12 weeks in mPRε-/- female mice under HFD feeding (n = 4–5 from four litters; independent experiments). Gona, gonadal; par, parametrial. (E) Blood glucose, plasma triglyceride, NEFAs, and total cholesterol levels in WT and mPRε-/- female mice after HFD intervention for 12 weeks (n = 4–5). (F) Plasma insulin levels in female mice (n = 4–5 from four litters; independent experiments). All data are presented as the mean ± standard error of the mean (SE). **Figure S3: Functional analysis of adipose mPRε on glucose homeostasis, related to Figure 2** (A) Plasma progesterone levels 30 min after progesterone administration (s.c.) in male (left) and female (right) mice (n = 6–8). (B) Plasma insulin levels 30 min after progesterone administration (s.c.) in male (left) and female (right) mice (n = 6–9). (C, D) Expression of mPR family members in mouse liver (C, n = 4) and muscle (D, n = 4) tissues (postnatal day 49, P49). (E) Oil Red O staining of differentiated adipocytes derived from mPRε-/- mouse embryonic fibroblasts (MEFs) treated with 10 μM pioglitazone and inducer during adipogenesis (n = 3–4). (F) Oil Red O staining of MEF-derived adipocytes of mPRε-/- mice with progesterone addition (n = 3–10). *p &lt; 0.05, **p &lt; 0.01 (Dunn’s test). Results are presented as the mean ± standard error of the mean (SE). N.S.: not significant. **Figure S4: Functional analysis of maternal adipose mPRε in pregnancy, related to Figure 3** (A) Plasma progesterone levels in GD16.5 females (n = 9–11). (B) Plasma insulin levels in GD16.5 females (n = 9–11). (C) Comparison of adipose mPRε expression between non-pregnant and GD16.5 female mice (n = 6). Results are presented as the mean ± standard error of the mean (SE). **Figure S5: mPRs expression and RNA sequencing in the placenta of mPRε-/- mice, related to Figure 3** (A) Expression of mPR subtypes in GD16.5 placenta (n = 4). (B) Placenta progesterone levels in GD16.5 females (n =4). (C) Beta diversity determined via principal component analysis (PCA) based on genes from KEGG (ID mmu00590; mmu04064; mmu04910) in the placenta of WT and mPRε-/- GD16.5 female mice (n = 4). Compositional similarity is compared using the permutational multivariate analysis of variance. (D) KEGG enrichment analysis related to molecular function in the placenta of mPRε-/- GD16.5 female mice. p-values adjusted based on the false discovery rate (FDR). N.S.: not significant. **Figure S6: Body and tissue weights in offspring from mPRε-/- mothers, related to Figure 4** (A, B) Changes in body weight (left), tissue weights (middle), and lean mass (right) of 4-week-old male (A) or female (B) offspring from mPRε-/- mothers (n = 5–8). (C, D) Normalization of tissue weight by body weight of 16-week-old males (left) and females (right) offspring from mPRε-/- mothers under high-fat diet feeding (n = 8–10). *p &lt; 0.05, **p &lt; 0.01 (Dunn’s test: A and B; Student’s t-test: C and D). All data are presented as the mean ± standard error of the mean (SE). N.S.: not significant. **Figure S7: Metabolic parameters in mPRε-/- offspring from mPRε-/- male and mPRε-/- female mice, related to Figure 4** (A) Blood glucose, plasma triglyceride, non-esterified fatty acid (NEFA), and total cholesterol levels in WT and mPRε-/- male offspring mice after HFD intervention for 12 weeks (n = 8–10). (B) Plasma insulin levels in male offspring mice (n = 8–10 from three litters; independent experiments). (C) Plasma steroid levels in 16-week-old male offspring mice (n = 8–9). (D) Blood glucose, plasma triglyceride, NEFA, and total cholesterol levels in WT and mPRε-/- female offspring mice after HFD intervention for 12 weeks (n = 9–10). (E) Plasma insulin levels in female offspring mice (n = 10 from three litters; independent experiments). (F) Plasma steroid levels in 16-week-old female offspring mice (n = 11–12). *p &lt; 0.05, **p &lt; 0.01 (Mann–Whitney U test). All data are presented as the mean ± standard error of the mean (SE). **Figure S8: RNA sequencing in the liver of pregnant mPRε-/- mice, related to Figure 5** (A) Beta diversity via principal component analysis (PCA) based on genes from KEGG (ID mmu00590; mmu04064; mmu04910) in the liver of WT and mPRε-/- GD16.5 female mice (n = 5). Compositional similarity is compared using the permutational multivariate analysis of variance. (B) KEGG enrichment analysis related to molecular function in the liver of mPRε-/- GD16.5 females. p-values adjusted based on the false discovery rate (FDR). **Figure S9: Progesterone-stimulated mPRε activation promotes PLA2 activity, related to Figure 5** (A) Membrane phospholipids of phosphatidylserine in the WATs of non-pregnant and GD16.5 female mice (n = 4 per group). (B) PLA2 activity in WATs of mPRε-/- GD16.5 female mice (n = 9–10). (C) PLA2 activity in response to progesterone (100 nM) in Flp-In mPRε T-REx HEK293 cells treated with or without doxycycline (Dox) (n = 4). Dox; doxycycline. *p &lt; 0.05, **p &lt; 0.01 (Mann–Whitney U test). The results are presented as the mean ± standard error of the mean (SE). **Figure S10: Phospholipid analysis in WATs from pregnant mPRε-/- mice, related to Figure 5** Quantitation of membrane phospholipids in WATs of mPRε-/- GD16.5 female mice (n = 4). All data are presented as the mean ± standard error of the mean (SE). **Figure S11: mPRε overexpressing HEK293 cells, related to Figure 5** (A) mRNA expression of mPRε in Flp-In mPRε T-REx HEK293 cells (n = 4). (B) cAMP levels (n = 3–6) and (C) ERK1/2 phosphorylation (n = 4) in response to progesterone in a dose-dependent manner in Flp-In mPRε T-REx HEK293 cells treated with or without doxycycline (Dox). Intracellular cAMP levels were determined using a cAMP assay kit, and each data point is presented relative to the forskolin-induced cAMP levels. Cells were induced mPRε expression by treatment with Dox (10 μg/mL) for 24 hr. Dox; doxycycline. All data are presented as means ± standard error of the mean (SE). ## Sharing/Access information All data supporting the findings of this study are available within the article and its supplemental information. Source data, including images, have been deposited in DRYAD: [https://doi.org/10.5061/dryad.280gb5mzf](https://doi.org/10.5061/dryad.280gb5mzf) and the DNA Data Bank of Japan (DDBJ) under the accession nos. E-GEAD-852, E-GEAD-853, E-GEAD-854, and E-GEAD-894.