Prenatal Glucocorticoid Administration Accelerates the Maturation of Fetal Rat Hepatocyte

Prenatal glucocorticoid (GC) is clinically administered to pregnant women who are at risk of preterm birth for maturation of cardiopulmonary function. Preterm and low-birth-weight infants often experience liver dysfunction after birth because the liver is immature. However, the effects of prenatal GC administration on the liver remain unclear. We aimed to investigate the effects of prenatal GC administration on the maturation of liver hepatocytes in preterm rats. Dexamethasone (DEX) was administered to pregnant Wistar rats on gestational days 17 and 19 before cesarean section. Real time-polymerase chain reaction (RT-PCR) was performed to determine the mRNA levels of albumin, HNF4α, HGF, Thy-1, cyclin B, and CDK1 in the liver samples. Immunohistochemical staining and enzyme-linked immunosorbent assay were performed to examine protein production. The hepatocytes enlarged because of growth and prenatal DEX administration. Albumin, HNF4α, and HGF levels increased secondary to growth and prenatal DEX administration. The levels of the cell cycle markers cyclin B and CDK1 gradually decreased during growth and with DEX administration. The results suggest that prenatal GC administration leads to hepatocyte maturation via expression of HNF4α and HGF in premature fetuses.


Introduction
The prevalence rates of low-birth-weight and preterm infants are high among developed countries, especially in Japan. Therefore, research focusing on the fetal and childhood periods is advocated [1].
Preterm birth occurs before the physiological increase in endogenous glucocorticoid (GC) levels. GC action is important in the structural and functional maturation of various organs [2,3].
Prenatal GC administration in mothers with infants at risk of delivery between 26 and 32 weeks of gestation is commonly used to accelerate pulmonary circulation and reduce the incidence of mortality [4].
We previously demonstrated that prenatal GC administration promotes growth and development of cardiac functions in premature infant rats [5][6][7]. The neonatal liver is generally immature and develops structure and function during the early postnatal period. However, preterm infants with immature liver function are at risk of hypoglycemia, hyperbilirubinemia, cholestasis, bleeding, and impaired drug metabolism [8]. Furthermore, low-birth-weight and preterm infants often develop physiological jaundice with immature fetal liver function [8][9].
The role of GC has been demonstrated in association with the differentiation of hepatocytes and the bile duct [10][11]. Dexamethasone (DEX) is administered for the differentiation of hepatic stem progenitor cells established from induced pluripotent stem cells into hepatocytes and their subsequent maturation [12].
In contrast, repeated administration of prenatal GC has been reported to decrease the liver weight of mice [13]. However, there is no explanation regarding the development of the fetal liver subsequent to prenatal GC administration.
Therefore, we predicted that prenatal GC administration would promote liver maturation during fetal development. Fetal hepatocytes are structurally immature and have many stem cells. Various factors have been associated with maturation in the fetal period [14].
In this study, we aimed to determine the process of differentiation and maturation of hepatocytes that accompanies growth in the fetal liver.

1) Animal experiments
We determined the dose of GC based on previous reports [5][6][7]. Wistar rats were purchased from CLEA (Tokyo, Japan). DEX (Fuji lm Wako Pure Chemical, Osaka, Japan) dissolved in sesame oil (Kanto Chemical, Tokyo)-at doses of 1.0 and 2.0 mg/kg-was administered subcutaneously on days 17 and 19 of the gestation period to 8-week-old pregnant Wistar rats. In clinical practice, two doses of DEX have been administered every 24 h. Samatani et al. administered a total dose of 6 µM/kg in pregnant rats and described the optimal regimen of DEX for fetal lung maturation during the late gestation period using pharmacokinetic/pharamcodynamics simulation [18,19]. We determined the dose of DEX by referring to these previous literatures. After mating, vaginal smears were taken every morning; the day of nding sperm was designated as day 1 of pregnancy. The pregnant rats gave birth to 10-14 neonates on day 22 of gestation. On days 19 and 21, fetal rats were delivered by cesarean section, and liver samples were extracted under inhaled iso urane anesthesia. To evaluate the change the factor with growth, liver samples were also extracted from spontaneously delivered 1-day-old neonates, 3-day-old neonates, 5-dayold neonates, and 8-week-old adult rat. The 8-week-old adult rats were male.
The rats were kept at a constant temperature (23 ± 1°C) and constant humidity (55 ± 5%) at the Laboratory Animal Care and Management Facility of St. Marianna University School of Medicine. They were provided unlimited drinking water and kept at a 12-h light/12-h dark cycle. This study complied with the "Guiding principles for the care and use of laboratory animals" by The Japanese Pharmacological Society and was approved by the Experimental Animal Research Committee of St. Marianna University Graduate School of Medicine (approval number: 2002008).

2) Histological evaluation
Rat liver sections were preserved in 10% formalin, embedded in para n, and cut into 5-µm sections. The sections were stained with hematoxylin and eosin. The hepatocyte size was determined as the total number of hepatocytes by area of view (2.8 × 10 4 µm 2 ) under a microscope with high magni cation (×400).

3) mRNA extraction and real time reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was extracted from the liver tissues using an RNeasy® sepasol-RNA I super G kit (Nakarai, Kyoto, Japan). A NanoDrop One/OneC Spectrophotometer (Thermo Fisher Scienti c, Waltham, MA, USA) was used to measure the concentration of total RNA at an optical density of 260 nm. cDNA was synthesized using the Transcriptor First Strand cDNA Synthesis Kit (Roche Diadnostic GmbH, Mannheim, Germany). RT-PCR was performed using Step One Plus and Power SYBR® Green PCR Master Mix (Thermo Fisher Scienti c). The PCR conditions were as follows: 45 cycles of denaturation at 95°C for 20 s, annealing at 60°C for 30 s, and extension at 72°C for 40 s.
Gene expression was determined using the relative values of standard curve values. In each sample, the relative value was normalized to the housekeeping gene, hypoxanthine phosphoribosyltransferase (HPRT). The primers used are shown in Table 1 and Table Supplement 1.
The OLYMPUS CX41 upright microscope (Olympus, Tokyo, Japan) was used to acquire immunochemistry digital images. ImageJ version 1.49 software was used to calculate the density of the positive area.

5) Determination of HNF4α levels
HNF4α protein levels were measured using Rat HNF4A/HNF4 enzyme-linked immunosorbent assay (ELISA) Kit (LifeSpan Biosciences, WA, USA) according to the manufacturer's instructions. The tissues samples were lysed using CelLytic™ MT cell lysis reagent for mammalian tissues (Sigma-Aldrich, MO, US) and quanti ed total protein using Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad, CA, USA). Absorbance was measured at a wavelength of 450 nm using Viento nano (DS Pharma Biomedical, Osaka, Japan)

6) Statistical analysis
The results are presented as mean ± standard error of the mean. JMP pro 13 (SAS Institute Inc., Cary, NC, USA) software was used to perform statistical analyses. All mRNA expression levels were analyzed statistically using the nonparametric Steel test. The protein levels were analyzed using Dunnett's test because the sample size was small.

1) Hepatocyte size
With growth, the size of the hepatocytes gradually increased from the fetuses (19- (Fig. 1C). Largen mRNA and mTOR levels were not affected in the 19F and 21F groups, the process of growth, or by prenatal DEX administration (Fig. S1).

3) Thy-1 and Dlk1 expression levels
High mRNA expression levels of Thy-1 (Fig. 3A) and delta-like 1 homolog (Dlk1) (Fig. 3E) were observed in 19F rats, and the levels gradually decreased with growth. The mRNA levels of Thy-1 tended to decrease in 21F rats following DEX 1.0 mg/kg (0.43-fold) and 2.0 mg/kg (0.42-fold) administration compared with that in the untreated group; however, the mRNA levels of Dlk1 in the liver were unchanged after prenatal DEX administration (Fig. 3B, F). The number of Thy-1-protein-positive cells also decreased in 21F rats following DEX 2.0 mg/kg administration (28.7 ± 2.6%) compared with that in the untreated group (42.3 ± 1.4%) (Fig. 3C, 3D). 4) Alpha-fetoprotein (AFP) and albumin mRNA and protein levels In the untreated group, the mRNA levels of AFP gradually decreased from 19F rats to adult rats (Fig.  4A). Although the mRNA levels of AFP were increased in 19F rats following DEX 2.0 mg/kg administration, they were unchanged in 21F rats following DEX administration (Fig. 4B). In contrast, the liver mRNA levels of albumin in 19F rats were low and gradually increased with growth (Fig. 4C, E, F). The albumin mRNA (5.79-fold) and protein levels increased in 19F rats following DEX 2.0 mg/kg administration compared with that in the untreated group (Fig. 4D, E, G).
The G6Pase and TAT mRNA levels in the livers of 8W rats were higher than those in the livers of 19F rats; however, prenatal DEX administration did not affect 19F and 21F rats (Fig.S2).

5) HGF mRNA levels and protein levels
The HGF levels increased two-fold in the 1N group compared with that in the 19F group (Fig.  5A). Administration of 1.0 mg/kg DEX in the 19F group increased 1.6-fold, and administration of 2.0 mg/kg DEX in the 21F group increased 2.9-fold compared with that in the respective untreated groups (Fig. 5B).

6) HNF4α mRNA and protein levels
HNF4α is involved in hepatocyte maturation. The HNF4α mRNA and protein levels in adult rats were higher than those in 19F rats (Fig. 6A, C).

Discussion
Preterm infants are at a risk of hepatic insu ciency because their immaturity results in a delay in achieving detoxi cation and synthetic functions [20].
Prenatal GC administration accelerates the development of cardiopulmonary functions for reducing respiratory distress syndrome and mortality in premature infants [21][22]. GC induces organ maturation and is necessary for sustaining extrauterine life [23]. We previously con rmed the expression of MRP2 and albumin following prenatal GC administration [24]. However, the mechanisms of fetal liver growth following prenatal GC administration remain unclear. In this study, prenatal DEX administration increased the size of fetal rat hepatocytes as well as increased the levels of maturation-related factors. This suggests that DEX induces the growth of fetal hepatocyte. We found that from 19F to 5N, the hepatocyte size gradually increased. Additionally, enlargement of hepatocytes was observed in the adult rats. Prenatal DEX administration increased the size of hepatocytes in 19F but not in 21F. Processes essential to attaining adequate liver mass and function during the fetal period include activation of speci c cell lineage early in development and cell proliferation or differentiation, accomplished by enzymes and transcriptional factors. We therefore investigated whether hepatocyte enlargement depends on cell proliferation or differentiation. High mRNA levels of the cell proliferation marker Ki-67 were found in fetal hepatocytes, which gradually decreased with growth. This indicates that the capacity for cell proliferation declines with liver growth. However, prenatal DEX administration did not signi cantly change Ki-67 mRNA expression i.e., DEX administration may have a negligible effect on cell proliferation in fetal hepatocytes. However, other cell proliferation markers (e.g., PCNA, MCM-2, and BrdU) should be evaluated in the future.
Hepatocyte proliferation and differentiation for functional growth occur simultaneously during late mammalian gestation. Gruppuso et al. (1999) reported the presence of independent signaling pathways for controlling the proliferation and differentiation in developing hepatocytes [25]. The cell cycle of hepatocytes is controlled from G(0) to mitosis by the regulation of cyclins and CDKs. Cyclin B1 forms a complex with CDK1, and the activation of this complex leads to the initiation of mitosis [26]. The mRNA expressions of cyclin B and CDK1 signi cantly decreased from 19F to 1N to adult rats. Additionally, prenatal DEX administration decreased the mRNA expression levels of cyclin B and CDK1 in 19F and 21F rats. The transcriptional inhibition of the cyclin B-CDK1 complex by prenatal DEX administration limited proliferation. Premature hepatocytes include hepatic progenitors during hepatocyte development [27]. Thy-1 and Dlk-1 are stem cell markers that can identify stem cell character [28,29]. With growth, the mRNA expression levels of Thy-1 and Dlk-1 signi cantly decreased in the fetal liver. Prenatal DEX administration tended to decrease Thy-1 mRNA levels in 21F rats. Tanimizu et al. (2004) reported that Dlk-1 expression was strongest at murine fetal day 10.5 and was downregulated after fetal day 16.5. Because Dlk-1 expressions in the livers of 19F and 21F rats were negligible, DEX may not have had any effect [30]. The early rat fetal liver also contains Thy-1-positive cells, which gradually decrease in number with growth. The mRNA and protein levels of Thy-1 were sustained until 21F, indicating that DEX may have been in uential.
Hepatic maturation is characterized according to the stage of liver growth with speci c genes [31][32]. Some studies reported a high level of AFP in fetal rat hepatocytes, and the AFP level gradually decreased with hepatic maturity. In contrast, albumin and G6Pase levels were reported to be increased in mature hepatocytes [33][34]. The mRNA levels of AFP decreased in the liver of 19F rats during growth. Our ndings are in agreement with those obtained by Zhang et al. The AFP level decreased as differentiation progressed in human hepatic progenitor cells. Cells exposed to a mixture of oncostatin M, DEX, and HGF gradually featured differentiated hepatic functions, including albumin production in vitro [35].Prenatal DEX administration did not change AFP mRNA levels in the fetal liver. The mRNA levels of albumin, TAT, and G6Pase increased in the liver from fetal rats to neonatal rats. DEX administration only increased albumin production in 19F rats. Human hepatic progenitor cells (HPCs) morphologically and functionally differentiate into hepatocytes and cholangiocytes. Freshly isolated HPCs coexpressed G6Pase, glycogen, albumin, and gamma glutamyl transpeptidase and could differentiate into functional liver cells [36]. A combination of HGF, oncostatin M, and DEX induced hepatocyte maturation. HGF in the presence of DEX induced the expression of G6Pase, TAT, and albumin in fetal hepatocytes [37]. HGF appears in the liver in the rst few days after birth. We found that the mRNA and protein levels of HGF were increased in the liver of 19F rats following prenatal DEX administration. This result suggests that DEX may have a direct effect on HGF level elevation. The underlying mechanism is the subject of future research.
HNF4α plays essential roles in structure formation and functional maturity of hepatocytes [38]. HPCs are negative for HNF4α. They start expressing HNF4α following their differentiation into hepatocytes [39]. The HNF4α gene has two promoters of P1 and P2 isoforms in mouse liver. Transcription starts via P2 promoter during fetal life but switches to P1 at birth. Exposure to DEX in the fetal rat liver suppresses P2 but enhances the expression of transcripts from P1 [40].
The mRNA and protein levels of HNF4α were signi cantly increased in the liver with growth. Prenatal DEX administration also increased their levels in the liver of 19F and 21F rats. Nyirenda et al. reported that continuous administration of prenatal DEX increased HNF4α mRNA expression in rat livers [41]. Our results are close to clinical therapeutics and are consistent with those obtained by Nyirenda et al. Thus, an increase in hepatic HNF4α expression is related to liver maturation.
This study did not show whether prenatal GC acts through the glucocorticoid receptor or indirectly for liver maturation. Further studies are necessary to elucidate these mechanisms using isolated fetal hepatocytes.

Conclusion
These results suggest that prenatal GC administration induces hepatocyte differentiation, and liver maturation is achieved via the expression of HNF4α and HGF in premature fetuses.

Competing interests
The authors declare that they have no competing interests.

Availability of data and materials
All data generated or analyzed during this study are included in this article and its supplementary information.  Figure 1 Histology of hepatocytes in liver tissue with growth (A) Histology of hepatocytes with hematoxylin and eosin staining in day-19 fetuses (19F), day-21 fetuses (21F), 1-day-old (1N) rats, 3-day-old (3N) rats, 5day-old (5N) rats, and 8-weeks adult rats (8W) in liver tissue with growth and in 19F and 21F rats after prenatal DEX administration. The photos show images at high magni cation (×400), scale bar = 20 µm.
The eld of view of 2.8 × 104 µm2 was divided by total hepatocyte number to obtain the hepatocyte size in liver tissues with growth (B) and in the liver of fetal rats after DEX administration. Fetal liver, neonatal liver, and adult rat liver tissues: n = 3. Two nonadjacent locations were photographed, for each tissue specimen (C). *P < 0.05, **P < 0.01, and ***P < 0.001 vs. 19F group. † † †P < 0.001 vs the respective untreated group.  Thy1 and Dlk1 mRNA and protein levels Thy-1 and Dlk1 mRNA levels were determined in liver tissues in 19F and 8W rats (A) and (E). Thy-1 and Dlk1 mRNA levels were determined in 19F and 21F rats after prenatal DEX administration (B) and (F). Fetal, neonatal, and adult rat mRNA samples: n = 7. In immunohistochemistry, images of Thy-1 production in liver tissue after prenatal DEX administration (C). Thy-1 positive staining intensity was analyzed using NIH image analysis. 19F rat tissues specimens: n = 4. 21F, 1N, and 8W rat tissues specimens: n = 3. Two nonadjacent locations were photographed for each tissue specimen (D). **P < 0.01 vs. the respective 19-day fetus group. †P < 0.05 vs. the respective untreated group.

Figure 4
AFP mRNA and albumin mRNA and protein levels AFP and albumin mRNA levels were determined in liver tissues from 19F to 8W (A) and (C). AFP and albumin mRNA levels were determined in 19F and 21F rats after prenatal DEX administration (B) and (D). Fetal, neonatal, and adult rat mRNA samples: n = 7. In immunohistochemistry, albumin production increased in liver tissue after prenatal DEX administration (E). Albumin positive staining intensity was analyzed using NIH image analysis. 19F rat tissues specimens: n = 4. 21F, 1N, and adult rat tissues specimens: n = 3. Two nonadjacent locations were photographed, for each tissue specimen (F) and (G). *P < 0.05, **P < 0.01, and ***P < 0.001 vs. the respective 19 fetus group. †P < 0.05 and † †P < 0.01 vs the respective untreated group. Figure 5