Prenatal Glucocorticoid Administration Accelerates Maturation in the Hepatocytes of Fetal Rats

Prenatal glucocorticoid (GC) is clinically administered to pregnant women at risk of preterm birth for maturation of the 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 regarding maturity of the liver in preterm rats. Dexamethasone (DEX) was administered to pregnant Wistar rats on gestational day 17 and 19 before cesarean section. Real time polymerase chain reaction (RT-PCR) was then used to analyze the mRNA levels (albumin, HNF4α, HGF, Thy-1, cyclin B, and CDK1) in the liver samples. Immunohistochemical staining and enzyme-linked immunosorbent assay (ELISA) were used to analyze protein production. Hepatocyte size enlarged because of growth and administration of prenatal DEX. Albumin, HNF4α, and hepatocyte growth factor (HGF) increased secondary to growth and administration of prenatal DEX. Cell cycle markers, cyclin B, and CDK1 gradually decreased during growth and by administration of DEX. These results suggest that prenatal GC administration achieves hepatocyte maturation via expression of HNF4α and HGF in premature fetuses.


Introduction
The prevalence rates of low birth weight and preterm delivery infants are high among developed countries, especially, in Japan. Therefore, the importance of research focusing on the fetal and childhood periods is advocated (Sata 2016  Prenatal GC administration in mothers with babies at risk of being preterm delivery between 26 and 32 weeks gestation in commonly is used to accelerate the pulmonary circulation and reduce mortalities (Hayes et al. 2008). We previously demonstrated that prenatal GC administration growth with development of cardiac functions in premature infant model rats (Sakurai et  The neonatal liver is generally immature and develops the structure and function during the early postnatal period. However, preterm infants with immature liver function are subject to risk of hypoglycemia, hyperbilirubinemia, cholestasis, bleeding, and impaired drug metabolism (Grijalva and Vakili 2013). Furthermore, low birth weight and premature birth infants often develop physiological jaundice with immature fatal liver function ( (Snykers et al. 2009).
In contrast, repeated administration of prenatal GC has been reported to decrease the liver weight of mice (Ozdemir et al. 2003). 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 maturation of the liver during fetal development. The fetal liver cells are structural immature and have many stem cells. Various factors can be linked to maturation in the fetal period (Shiojiri 1997).
In this study, we aimed to elucidate the process of differentiation and maturation of hepatocytes that accompanies growth in the fatal liver. from CLEA (Tokyo, Japan). Treatment of 2 doses of DEX given every 24 hours has been performed in clinical practice. Samatani et al. have been described the optimal regimen of DEX for fetal lung maturation during late gestation by pharmacokinetic/pharamcodynamics (PK/PD) simulation, when they injected the dose of total amount 6µM/kg in pregnant rats (Samtani et al. 2006a, b). We determined the dose of DEX by referring to these investigations. After mating, the virginal smear was taken every morning, the day of nding sperm was designated as day 1 of pregnancy. The pregnant rats give birth to 10-14 neonates on day 22 of gestation. On day 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-dayold neonates, 5-day-old neonates, and 8-week-old-adult rat. The 8-week-old adult rats were male.

1) Animal experiments
Rats were kept at a constant temperature (23 ± 1°C), constant humidity (55 ± 5%), arti cial lighting (6-18 o'clock), and darkness in the Laboratory Animal Care and Management Facility of St. Marianna University School of Medicine. They were kept under free drinking water under a cycle (12 hours light period, 12 hours dark period). This study has complied with the "Guiding principles for the care and use of laboratory animals" by The Japanese Pharmacological Society and was approved by the Experimental After mating, the virginal smear was taken every morning, the day of nding sperm was designated as day 1 of pregnancy. We selected equally or randomly the respective fetuses born out of three pregnant rats. The liver samples were used from different pregnant rats.

2) Histological evaluation
Rat liver sections were preserved in 10% formalin, and embedded in para n, 5 µm sections. The sections were stained with hematoxylin and eosin (H&E) solution.
We evaluated hepatocyte size as the total number of hepatocytes by area of view (2.8 × 10 4 µm 2 ) under microscope with high magni cation (×400).
3) The mRNA extraction and real time reverse transcription -polymerase chain reaction (RT-PCR) Total RNA in the liver tissue was isolated using cell lysates, a 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 total RNA concentration at an optical density of 260 nm. The 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). Table 1 shows the used primers. The PCR conditions were 95°C, 20 s (denaturation), 60°C, 30 s (annealing), and 72°C, 40 s (extension) 45 cycles.
Gene expression was determined using the relative values of standard curve values. In each sample the relative value was normalized to the housekeeping gene for hypoxanthine phosphoribosyltransferase (HPRT). Table 1 and Supplement 1 show the used primers.
The OLYMPUS CX41 upright microscope (Olympus, Tokyo, Japan) was used to acquire immunochemistry digital images. ImageJ 1.49v. processing software was used to calculate the density of the positive area. 5) HNF4α enzyme-linked immunosorbent assay (ELISA) HNF4α protein levels were measured using Rat HNF4A/HNF4 ELISA Kit (LifeSpan Biosciences, WA, USA) according to the manufacturer's instructions. The tissues samples were prepared using CelLytic™ MT cell lysis reagent for mammalian tissues (Sigma-Aldrich, MO, US), and were quanti ed 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
Results are shown as mean ± standard error of mean (SEM). JMP pro 13 (SAS Institute Inc., Cary, NC, USA) was used to perform the statistical analyses. All mRNA expression levels were analyzed statistically using non-parametric test, Steel test. Evaluation of protein levels was analyzed using Dunnett's test from the reason that the sample size is small. To compare the characteristics in the respective group, two-way ANOVA was used for the continuous variables, such as DEX treatment × age × measure. Thy-1 mRNA levels tended to decrease in 21F with administration of DEX 1.0 mg/kg (0.43-fold) and 2.0 mg/kg (0.42-fold) compared with that of the untreated group; however, Dlk1 mRNA levels in the liver were unchanged after administration of prenatal DEX (Fig. 3B, F).

4) Alpha-fetoprotein (AFP) and albumin mRNA and protein levels
In untreated groups, AFP mRNA levels gradually decreased from 19F to adult rats (Fig. 4A). Although AFP levels mRNA were increased in 19F with DEX 2.0 mg/kg administration, those were unchanged in the 21F with administration of DEX (Fig. 4B).
In contrast, albumin mRNA levels in the liver of 19F were low, and the levels gradually increased during growth (Fig. 4C, E, F).
Albumin mRNA (5.79-fold) and protein levels increased in the 19F with administration of DEX 2.0 mg/kg compared with the untreated group (Fig. 4D, E, G).
G6Pase and TAT mRNA levels in livers of 8W rats were higher than those of 19F rats; however, prenatal DEX administration did not affect 19F and 21F (Fig.S2).

5) HGF mRNA levels and protein levels
HGF levels increased two-fold in the 1N group compared with 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 of respective untreated groups (Fig. 5B).
HNF4α is involved in maturation of hepatocytes. HNF4α mRNA and protein levels in adult rats were higher than those in 19F rats (Fig. 6A, C).

7) Inter-treatment and age interacted
The obtained all data was compared using a two-way ANOVA variance test to detect any differences between groups, considering in the age, DEX treatment, and both factors.
The effect of age or DEX treatment was signi cant difference in the fetal hepatocyte size by two-way ANOVA. However, there was not signi cant difference between the hepatocyte size and both of the age and DEX (Table 2).
Signi cant difference between mRNA levels of Ki-67, Dlk1, TAT, and G6Pase and age was observed.
However, there was no signi cant difference between those mRNA levels both of age and DEX treatment. The mRNA levels of cyclin B, CDK1, AFP, albumin, HGF, and HNF4α showed signi cant differences between age or DEX treatment. There were not a signi cant differences between the respective gene expressions and both factors of the age and DEX treatment. In, Thy1, mTOR and Largen mRNA levels, there was no signi cant difference in the effects of age and DEX respectively. (Table 3).
The protein levels of albumin, HGF, and HNF4α had differences between the age and DEX treatment. Under the in uence of both factors of age and DEX treatment, was greater than the one age or DEX treatment (Table 4).
These results indicate that effects of the age and DEX treatment on gene expressions and protein production are independent. However, the mechanisms of fetal liver growth by prenatal GC administration remains unclear. In this study, prenatal DEX administration increased the size of hepatocytes in fetal liver and maturation-related factors. This suggests that DEX induces fetal hepatocyte growth. First, we found that hepatocyte size gradually enlarged from 19-day fetuses (19F) to 5-day-old neonates (5N). Additionally, enlargement of hepatocytes was also observed in adult rats. Prenatal DEX administration enlarged hepatocyte size in 19F, but not 21-day fetuses (21F). The results of the two-way ANOVAs (DEX treatment x age) presented signi cant main effects for DEX treatment and age but no interaction. Therefore, We cannot conclude that the increase in cell size due to DEX administrate is different between 19F and 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 studied whether hepatocyte enlargement depends on cell proliferation or differentiation. High levels of cell proliferation marker Ki-67 mRNA were expressed in fetal hepatocytes, and their levels gradually decreased. This indicates that the capacity for cell proliferation declines with liver growth. However, prenatal DEX administration did not signi cantly change  2004) reported that Dlk-1 expression is strongest at murine fetal day 10.5 and is downregulated after fetal day 16.5. Because Dlk-1 expressions in the livers of 19F and 21F were scanty, DEX may not have had any effect. The early rat fetal liver also contains Thy-1-positive cells, which gradually decrease with growth. Thy-1 mRNA and protein levels were sustained until 21F, therefore indicating that DEX may have been in uential.

Discussion
Hepatic maturation is characterized according to the stage of liver growth with speci c genes (Derman et al. 1981;Panduro et al. 1987). Some studies have reported a high level of AFP in fetal rat hepatocytes, and the AFP gradually decreased with hepatic maturity. In contrast, albumin or G6Pase levels are increased in mature hepatocytes (Jochheim et al. 2004;Sell 2008). Levels of AFP mRNA decreased in 19F liver during growth. Our nding corresponded with that obtained by Zhang et al. The level of AFP 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 (Zhang et al. 2012). Prenatal DEX administration did not change AFP mRNA levels in fetal liver. Albumin, TAT, and G6Pase mRNA levels increased from the fetal liver to neonatal liver with growth. DEX administration only increased albumin production in 19F. Human hepatic progenitor cells (HPCs) morphologically and functionally differentiate into hepatocytes and cholangiocytes. Freshly isolated HPCs co-expressed G6Pase, glycogen, albumin, and gamma glutamyl transpeptidase, and could differentiate into functional liver cells (Nava et al. 2005). A combination of HGF, oncostatin M, and DEX induces hepatocyte maturation. HGF, in the presence of DEX, induces expression of G6Pase, TAT, and albumin in fetal hepatocytes (Kamiya et al. 2001). HGF appears in the liver in the rst few days after birth. We found that HGF mRNA and protein levels were enhanced in the 19F liver with prenatal DEX administration. This result suggests that DEX may have a direct effect on HGF increase. The mechanism is the subject for future study.
Hepatocyte nuclear factor 4-alpha (HNF4α) has essential roles in structure formation and function maturity of the hepatocyte (Li et al. 2000). HPCs are negative for HNF4α. They start expressing this transcription factor when they differentiate into hepatocytes (Parviz et al. 2003). 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 (Dean et al. 2010).
HNF4α mRNA and protein levels signi cantly increase in the liver with growth. Prenatal DEX administration also increased their levels in 19F and 21F livers. Nyirenda et al. (2006)  The obtained all data was compared using a two-way ANOVA variance test to detect any differences between groups, considering in the age, DEX treatment, and both factors. Almost the gene and protein levels, except for some gene and protein were signi cant differences between the age or DEX treatment. These results indicate that effects of fetal age and DEX treatment on gene expression and protein production are independent. There is little effect on interaction between the age and DEX treatment.
Prenatal GC is used on late birth at < 36 week's gestation. Pregnant rats are normally deliver on gestational day 22, ie., full term birth. We examined day 17 and 19 of gestation period because at this gestational age the rats are premature enough to satisfy the de nition of premature enough to satisfy the de nition of prematurity yet mature enough to that the majority of them are able to survive without the need for supportive care (ex, mechanical ventilation) (Chen Y et al. 1994). In animal model, the remarkable maturation in the heart and the lung were demonstrated in our studies by prenatal GC administration. Therefore, we think the importance whether antenatal GC acts other organs including the liver in this study. Although, this study has not showed whether prenatal GC acts through the glucocorticoid receptor and other factors including the metabolic enzymes in liver function, we demonstrated antenatal GC contributes the maturation of liver. This knowledge is new. In the future, it is necessary to investigate the liver function and adverse effects of prenatal glucocorticoid administration. We attempt to obtain a new interpretation about before 17-day fetuses in the further study.

Conclusion
These results suggest that prenatal GC administration induces hepatocyte differentiation, and liver maturation is achieved via the expressions 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 generate or analyzed during this study are included in this publish article and its supplementary information les.

Not applicable
Author' contributions TK and YT contributed to the study conception and design. Material preparation, experiment, and data collection were performed by TK. TK, YT, YO, MO, and KK contributed analysis and interpretation of data for the manuscript. The rst draft of the manuscript was written by TK. YT contributed to assist in the preparation of the manuscript. MW contributed Resources. NM and TI contributed supervision and funding acquisition. All authors commented on previous versions of the manuscript. All authors read and approved the nal manuscript. The authors declare that all data were generated in-house and that no paper mill was used.

Ethics approval
This study has complied with the "Guiding principles for the care and use of laboratory animals" by The Japanese   Figure 1 Histology of the hepatocyte in liver tissue with growth. (A) Histology of hepatocytes on hematoxylin and eosin staining in day-19 fetuses (19F), day-21 fetuses (21F), 1-day-old (1N), 3-day-old (3N), 5-day-old (5N) neonatal, and 8-weeks adult rats (8W) in liver tissue with growth and in day-19 and day-21 fetal rats after prenatal DEX administration. The photos show images at high magni cation (×400), scale bar = 20 µm. The area of 2.8 × 104 µm2 at eld of view was divided by total hepatocyte number to obtain a hepatocyte size in liver tissues with growth (B), and in the fetal liver rats after DEX. Fetal liver, neonatal liver, and adult rat liver tissues: n = 3. Two non-adjacent 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.

Figure 3
Thy1 and Dlk1 mRNA and protein levels. Thy-1 and Dlk1 mRNA levels were analyzed in liver tissues from 19F to 8W rats (A) and (E). Thy-1 and Dlk1 mRNA levels were evaluated in the 19F and 21F rats after prenatal DEX administration (B) and (F). Fetal, neonatal, and adult rat mRNA sample: n = 7. In immunohistochemistry, images of Thy-1 production in liver tissue after prenatal DEX (C). Thy-1 positive staining intensity was analyzed using NIH image analysis. 19F rat tissues specimen: n = 4. 21F, 1N, and 8W rat tissues specimen: n = 3. Two non-adjacent locations were photographed, for each tissue specimen (D). **P < 0.01 vs. the respective 19 fetus group. †P < 0.05 vs the respective untreated group. AFP mRNA and albumin mRNA and protein levels. AFP and albumin mRNA levels were analyzed in liver tissues from 19F to 8W (A) and (C). AFP and albumin mRNA levels were evaluated in 19F and 21F rats after prenatal DEX administration (B) and (D). Fetal, neonatal, and adult rat mRNA sample: n = 7. In immunohistochemistry, albumin production increased in liver tissue after prenatal DEX (E). Albumin positive staining intensity was analyzed using NIH image analysis. 19F rat tissues specimen: n = 4. 21F, 1N, and adult rat tissues specimen: n = 3. Two non-adjacent 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. HGF mRNA and protein levels. HGF mRNA levels were analyzed in liver tissues from 19F to 8W (A). HGF mRNA levels were evaluated in the 19F and 21F rats after prenatal DEX administration (B). Fetal, neonatal, and adult rat mRNA sample: n = 7. In immunohistochemistry, HGF production increased in liver tissue of 19F after prenatal DEX (C). HGF positive staining intensity was analyzed using NIH image analysis. 19F rat tissues specimen: n = 4. 21F, 1N, and 8W rat tissues specimen: n = 3. Two non-adjacent locations were photographed, for each tissue specimen (D). *P < 0.05 vs. the respective 19 fetus group. †P < 0.05, † †P < 0.01, and † † †P < 0.001 vs the respective untreated group.