Oxytocin modulates steroidogenesis-associated genes and estradiol levels in the placenta

Abstract Oxytocin (OXT) plays a significant role during pregnancy, especially toward the end of pregnancy. Some studies have reported that OXT is involved in the stimulation of steroidogenesis in several organs. However, the effects of OXT on placental steroidogenesis have not yet been established. In this study, we investigated the regulation of steroid hormones and steroidogenic enzymes by OXT-associated signaling in vitro and in vivo. OXT increased the gene expression of steroidogenic enzymes, which convert pregnenolone to progesterone and dehydroepiandrosterone (DHEA) in vitro. In OXT-administered pregnant rats, pregnenolone and DHEA levels were significantly enhanced in the plasma and the expression of the enzymes synthesizing DHEA, testosterone, and estradiol (E2) was increased in placental tissues. Furthermore, OXT was found to affect placental cell differentiation, which is closely related to steroid hormone synthesis. After treatment of the pregnant rats with atosiban, an antagonist of the OXT receptor, the concentration of E2 in the plasma and the expression of E2-synthesizing enzyme were reduced. This regulation may be due to OXT-mediated differentiation, because OXT increases the expression of corticotropin-releasing hormone, which is a biomarker of placental cell differentiation. Our findings suggest that OXT contributes to maintaining pregnancy by regulating the differentiation of placental cells and steroidogenesis during pregnancy.


Introduction
Oxytocin (OXT) is a neurohypophyseal hormone synthesized by the hypothalamus and several peripheral tissues such as the corpus luteum, gonad, uterus, and placenta (Soloff et al. 1977;Swann et al. 1984;Sugahara et al. 1985;Watson et al. 1999). OXT is secreted by magnocellular neurons and is stored in the posterior pituitary until it is released into the bloodstream (Thackare et al. 2006;Johnson 2018).
OXT is well known for playing many important roles in physiological processes such as maternal behavior, labor, lactation, and ejaculation (Bethlehem et al. 2013). Particularly in pregnant women, OXT contributes significantly to the milk injection reflex and uterine contractile activity for smooth childbirth (Gimpl and Fahrenholz 2001). In addition, OXT regulates the activity of steroidogenic enzymes and steroidogenesis in several organs, including the gonads (testes and ovaries) (Assinder et al. 2015). Although the physiological functions and signaling pathways of OXT in the brain, uterus, and reproductive organs have been well established, research on the placenta is limited.
The placenta is a pregnancy-specific and transient tissue that connects the developing fetus to the uterine wall for exchange of nutrients, excretion, antibody transport, gas exchange, and hormone secretion through the maternal-fetal interface (Benirschke et al. 1998;Burton and Fowden 2015). During pregnancy, placental development is an essential process for successful pregnancy and fetal growth (Kwak et al. 2019). For placental growth and development, the proliferation of mononucleated cytotrophoblast (CTB) cells and their morphological differentiation into multinucleated syncytiotrophoblast (STB) cells are required (Kliman et al. 1986). The differentiated STB plays various critical functions throughout pregnancy, including transport of ions, substrates, gases, and other factors between the maternal and fetal circulations (Hubert et al. 2010). In addition, during pregnancy, these cells synthesize and secrete a large amount of steroid hormones, which are essential for fetal growth, through steroidogenesis (Hubert et al. 2010). Among them, estradiol (E2) and progesterone (P4) are the major steroid hormones, and the serum levels of E2 and P4 increase throughout pregnancy (Kim et al. 2016;Young et al. 2016).
Steroidogenesis, which is an important process for successful pregnancy and occurs in STB cells during pregnancy, requires in-depth research because it occurs through a number of factors and complex processes comprising networks of intracellular signaling pathways (Tremblay 2015). It is a biological process that converts cholesterol into various steroid hormones by mediating steroidogenic enzymes (Miller 2013;Sato et al. 2014). The first step of steroidogenesis involves conversion of cholesterol to pregnenolone (P5) by the cholesterol side-chain cleavage enzyme (CYP11A1) (Arukwe 2008). Following this step, P5 is converted into dehydroepiandrosterone (DHEA) or P4 by steroid 17-a-hydroxylase/17,20 lyase (CYP17A1) or 3b-hydroxysteroid dehydrogenase/d5 4-isomerase type 1 (HSD3B1), respectively. Androgens, which contain testosterone (T), are synthesized from DHEA and P4 using 17b-dehydrogenase 3 (HSD17B3) and HSD3B1 (Arlt et al. 2001). In the final step, aromatase (CYP19A1) and HSD17B3 catalyze the conversion of androgens into E2.
According to our previous research, OXT has been reported to play an important role in the placenta during pregnancy (Kim et al. 2017). However, the effect of OXT on the differentiation of trophoblast cells and steroidogenesis in the placenta remains unclear. Therefore, in the present study, the effects of alterations in OXT-related signaling on steroidogenesis and placental development, which are essential for maintenance of pregnancy, were evaluated at the in vitro and in vivo levels.

OXT regulates steroidogenic enzymes in human placental JEG-3 cells
To evaluate the effect of OXT on steroidogenesis in human placental JEG-3 cells, we treated the cells with OXT at a dose of 1 mM for 24 h or 48 h. OXT did not affect the protein expression level of OXTR in JEG-3 cells ( Figure S1). Protein expression levels of steroidogenesis-associated enzymes in JEG-3 cells were analyzed through western blot analysis ( Figure 1A). The results revealed that the protein levels of HSD3B1 and CYP17A1 were increased by OXT (p < 0.05; Figure  1B,E), while CYP11A1 levels tended to increase ( Figure 1D). Additionally, the concentration of P4 from cell cultured media was increased after treatment with OXT compared the control group (p < 0.05) ( Figure 1G). The protein levels of CYP19A1 tended to increase in the OXT-treated group compared to the control group, but were not significant ( Figure 1F). However, the protein levels of HSD17B3 was not altered upon OXT treatment ( Figure 1C).

Expression of steroidogenic enzymes in placenta of pregnant rats treated with OXT
Pregnant female rats were injected with OXT subcutaneously at a dose of 0.25 mg/kg/day from gestational day (GD) 10 to GD 17 to evaluate the effect of OXT in the placenta, the major tissue where steroidogenesis occurs during pregnancy.
Protein expression levels of steroidogenesis-associated enzymes in the placenta of OXT-treated pregnant rats were evaluated through western blot analysis ( Figure 2A). As OXT was administered, the protein expression levels of steroidogenic enzymes were elevated in the placenta. Similar to our in vitro results, the levels of HSD3B1, CYP17A1, and CYP19A1 were significantly upregulated compared with those in the control group (p < 0.05; Figure 2(B,E,F), whereas the protein expression levels of HSD17B3 and CYP11A1 were not significantly changed ( Figure 2C,D).

Inhibition of OXT signaling by atosiban altered placental steroidogenesis
Atosiban, a specific OXTR antagonist, was administered to pregnant rats at 0.6 mg/kg/day or 1.2 mg/kg/day doses from GD 10 to GD 17. We analyzed the protein expression levels of enzymes related to steroidogenesis to evaluate whether inhibition of OXT signaling affects placental steroidogenesis during pregnancy ( Figure 3A). The protein expression levels of CYP19A1 were significantly downregulated following atosiban administration (p < 0.05; Figure  3F), while HSD3B1 levels tended to decrease in the atosiban 1.2 mg/kg/day dose group ( Figure 3B). The expression levels of other genes, including HSD17B3, CYP11A1, and CYP17A1, were not dramatically altered compared to the expression levels in the control group ( Figure 3C,D,E). These results suggest that the steroidogenesis process in the atosiban-administered and OXT-administered groups was the opposite.

Regulation of OXT-related signaling modulates concentration of steroid hormones in pregnant rats
Since treatment with OXT during pregnancy affected the expression of steroid hormone biosynthesis-related , and CYP19A1 (F) were quantified and normalized to those of ACTB. Additionally, to evaluate the effect of OXT on progesterone synthesis in JEG-3 cells, 1 lM of OXT for 48 h was treated for 48 h and the presence of P4 was analyzed using a specific ELISA (G). Experiments were performed three times with similar results, and a representative photograph was presented. Data are expressed as mean ± SD. Ã p < 0.05 compared to the control group. OXT: oxytocin; HSD3B1: 3b hydroxysteroid dehydrogenase/d5 4 isomerase type 1; HSD17B3: 17b dehydrogenase 3; CYP11A1: cholesterol side-chain cleavage enzyme; CYP17A1: 17a hydroxylase/17,20-lyase; CYP19A1: aromatase cytochrome P450; ELISA: enzyme-linked immunosorbent assay; SD: standard deviation.
enzymes, the effect of OXT on the concentration of steroid hormones including P5, P4, DHEA, T, and E2 in the blood produced through steroidogenesis in the placenta was measured via enzyme-linked immunosorbent assay (ELISA) ( Table 1). In plasma, the concentration of P5 and DHEA were higher in the OXT group than in the control group (p < 0.05). OXT did not affect the concentration of P4, T and E2.
Additionally, we examined the synthesis of steroid hormones in the groups treated with atosiban, which inhibits OXT signaling. We evaluated the concentration of steroid hormones, including P5, P4, DHEA, T, and E2 (Table 1). Following treatment with atosiban at a dose of 0.6 mg/kg/day, the concentration of P5 in plasma was significantly increased (p < 0.05), while the concentration of other steroid hormones was not altered. A high dose of atosiban (1.2 mg/kg/day) significantly decreased the concentration of E2 (p < 0.05). However, the plasma concentration of other hormones, including P4, DHEA, and T, did not show significant changes in the high-dose atosiban group. These results suggest that changes in OXT signaling during pregnancy affect the production of steroid hormones.
OXT and atosiban regulate trophoblast differentiation related gene expression Differentiated trophoblast cells are essential for placental development during pregnancy and are known to produce steroid hormones necessary for fetal growth (Hubert et al. 2010). To determine whether OXT and atosiban affect trophoblast differentiation, we analyzed the expression of corticotropin-releasing hormone (CRH), a major marker of placental cell differentiation (Riley et al. 1991;Warren and Silverman 1995), via immunocytochemistry ( Figure 4A). Interestingly, the CRH fluorescence intensity increased following administration of 1 mM OXT in human placental JEG-3 cells (p < 0.05).
In the next experiment, the effect of OXT on the protein expression level of CRH in vitro was evaluated through western blot analysis. In our study, OXT increased the protein expression of CRH in JEG-3 trophoblast cells (p < 0.05; Figure 4B). In addition, the protein expression of CRH was increased in the placenta of pregnant rats treated subcutaneously with OXT (p < 0.05; Figure 4C). However, in pregnant rats treated with atosiban, the protein expression level of , and CYP19A1 (F) were quantified and normalized to those of ACTB. Experiments were performed three times with similar results, and a representative photograph was presented. Data are expressed as mean ± SD. Ã p < 0.05 compared to the control group. OXT: oxytocin; HSD3B1: 3b hydroxysteroid dehydrogenase/d5 4 isomerase type 1; HSD17B3: 17b dehydrogenase 3; CYP11A1: cholesterol side-chain cleavage enzyme; CYP17A1: 17a hydroxylase/17,20-lyase; CYP19A1: aromatase cytochrome P450; SD: standard deviation.
CRH tended to decrease ( Figure 4D), while the result was not significant. These results suggest that alteration of OXT-related signaling may affect not only steroidogenesis, but also the differentiation of trophoblast cells.

Discussion
Steroid hormones are primarily produced by a biological process called steroidogenesis, which is the process by which cholesterol is converted into several other steroid hormones. Humans and various mammals use cholesterol to synthesize steroid hormones in the reproductive glands and placenta (Payne and Hales 2004). During pregnancy, multiple steroid hormones released by the placenta perform a variety of roles, including placental trophoblast differentiation, expansion and maturation of the placental vessels, and uterine endovascular invasion by the placental trophoblast (extravillous cytotrophoblast) (Pepe and Albrecht 2008). One study demonstrated that CTB isolated form human placentas are capable of differentiating and fusing spontaneously to form functional STB as well as producing steroid hormones (Kliman et al. 1986). In addition, various studies have conducted research on synthesis of steroid hormones using JEG-3 cells (Samson et al. 2009;Cao et al. 2017;Karahoda et al. 2021). Since the process of synthesizing steroid hormones in the placenta has a large impact on the mother and fetus during pregnancy, in-depth studies on placental steroidogenesis are inevitable.
Several previous studies have supported our hypothesis. One study reported that activation of Figure 3. Protein expression levels of steroidogenic enzymes in placenta of pregnant rats treated with atosiban. Pregnant female rats were treated with atosiban, a specific OXTR antagonist, at a dose of 0.6 mg/kg/day or 1.2 mg/kg/day from GD 10 to GD 17. The expression levels of placental steroidogenic enzymes were analyzed by Western blotting. Representative blot images of steroidogenic enzymes are shown in (A). The protein expression levels of HSD3B1 (B), HSD17B3 (C), CYP11A1 (D), CYP17A1 (E), and CYP19A1 (F) were quantified and normalized to those of ACTB. Experiments were performed three times with similar results, and a representative photograph was presented. Data are expressed as mean ± SD. Ã p < 0.05 compared to the control group. OXTR: oxytocin receptor; HSD3B1: 3b hydroxysteroid dehydrogenase/d5 4 isomerase type 1; HSD17B3: 17b dehydrogenase 3; CYP11A1: cholesterol side-chain cleavage enzyme; CYP17A1: 17a hydroxylase/17,20-lyase; CYP19A1: aromatase cytochrome P450; SD: standard deviation. OXT signaling in pre-pubertal mice promotes the expression of various steroidogenic factors including HSD3B1, thereby stimulates the generation of T. This study suggested that OXT plays a role in increasing the production of steroid hormone and spermatogenesis by activating testicular steroidogenesis (Anjum et al. 2018). OXT also induced the expression of steroidogenic enzymes such as CYP11A1 and HSD3B1 and involved in P4 synthesis in bovine granulosa cells, suggesting that OXT plays a role as an intragonadal regulator of follicular steroidogenesis (Aladin Chandrasekher and Fortune 1990; Berndtson et al. 1996). Taken together, these previous studies imply that OXT plays an important role in the reproductive system, where the synthesis of steroid hormones occurs.
In our previous study, the expression of OXT and OXTR in the placenta increased gradually in the late stage of pregnancy, suggesting that autocrine and paracrine regulation of OXT may play an important role in placental development and differentiation of trophoblast cells. However, studies on the effect of OXT on placental development and the production of steroid hormones, which are essential for successful The protein expression level of CRH in JEG-3 trophoblast cells treated with 1 lM of OXT was quantified (B). At the in vivo level, the CRH protein expression levels in pregnant rats treated with OXT (0.25 mg/kg/day) and atosiban (0.6 or 1.2 mg/kg/day) from GD 10 to GD 17 were quantified (C-D). All CRH protein expression levels were normalized to those of ACTB. Experiments were performed three times with similar results, and a representative photograph was presented. Data are expressed as mean ± SD. Ã p < 0.05 compared to the control group. OXT: oxytocin; CRH: corticotropin-releasing hormone; SD: standard deviation. pregnancy, are insufficient (Kim et al. 2017). Considering this, we examined the effect of the regulation of OXT-related signaling on steroidogenesis in the placenta and evaluated its effect on the differentiation of placental cells.
In our in vitro study, we evaluated the expression of placental steroidogenic enzymes in JEG-3 trophoblast cells after treatment with OXT. In the OXT treated group, the overall protein expression levels of steroidogenic enzymes tended to increase. In particular, the protein expression levels of HSD3B1 and CYP17A1, which play key roles in synthesizing P4 and DHEA, were significantly increased by OXT treatment. As a result of analyzing the level of P4 in OXTtreated JEG-3 cells, it significantly increased in the cell culture media. This result suggests that the level of P4 enhanced sequentially with the increase of protein expression level of HSD3B1.
In addition, CYP11A1, known to synthesize P5, a precursor of other steroid hormones such as P4 and DHEA, showed an increasing tendency in OXTtreated trophoblast cells. These results suggest that OXT may affect the synthesis of steroid hormones such as P5, P4, DHEA, T, and E2, which play important roles in maintaining pregnancy. However, since there is no feto-placental communication in in vitro models, there may be differences between the in vitro data and the results from animal and human studies.
To reliably establish the effect of regulation of OXT-related signaling on the steroidogenesis of the placenta, we conducted animal experiments. We treated pregnant female rats with OXT subcutaneously from GD 10 to GD 17. In the OXT treated group, similar to the results in vitro, the protein expression levels of HSD3B1, CYP17A1, and CYP19A1 were significantly increased compared with the untreated control group. For CYP11A1, there was a slight increase in the OXT treated group.
In this study, the effect of OXT on the steroidogenesis at the in vitro and in vivo levels was slightly different. During pregnancy, multi-hormonal interactions occur with activation of the OXT signaling system. It is possible that the treatment of OXT may have more influence to hypothalamus and pituitary that are higher regulatory endocrine organs in the hypothalamus-pituitary-gonadal axis (Henry 2015). In addition, the placental cells we used in this study were derived from human, and the physiological characteristics and structure of placenta are different in human and rat. Therefore, we assume that the results in vitro and in vivo studies may differ because the species-specific response of the placenta to OXT.
We also evaluated the levels of critical steroid hormones such as P5, P4 DHEA, T, and E2 in the plasma of pregnant animal models and found that plasma concentrations of P5 and DHEA were increased by OXT, which may be explained by the results of the analysis of CYP11A1 and CYP17A1 gene expression. The observed increases in HSD3B1, which converts DHEA to androstenedione, justifies the increases in DHEA observed in the plasma. One study demonstrated that DHEA increases vascular endothelial proliferation, migration, and vascular tube formation by activating the extracellular signal-regulated kinase 1/2 pathway (Liu et al. 2008). Although the protein levels of HSD3B1 were increased, the P4 concentration in the blood was not changed. It suggests that the produced P4 may be converted quickly to other steroid hormones. These results demonstrate that the synthesis of steroid hormones was increased by OXT, indicating that OXT could have a positive effect on the development of the placenta, which may affect both the fetus and the mother during pregnancy.
For the following experiment, we treated pregnant rats with atosiban from GD 10 to GD 17 to block the endogenous OXT signaling. It has been known that the endogenous OXT levels are increased following gestational age in animal and human (Douglas et al. 1993;Achie et al. 2016;Kim et al. 2017). The results showed that plasma E2 levels and its synthesizing enzyme, CYP19A1, were decreased in atosiban-treated animals. These results have a critical impact because E2 is the last hormone in the process of steroidogenesis and plays a critical role in maintaining pregnancy. Interestingly, atosiban increased the concentration of P5 in the plasma. This increase could be a compensatory regulation of the placenta to elevate the plasma levels of E2. The overall in vitro and in vivo results are shown in Figure 5. Our results suggest that disrupting OXT-related signaling may lead to abnormal steroidogenesis and possibly placental function.
Placental development and differentiation of trophoblast cells during pregnancy are among the most important events for a successful pregnancy. STB, a differentiated trophoblast cell, is important for maintaining pregnancy as it plays a variety of roles, including steroid hormone production (Hubert et al. 2010). In the present study, we investigated the effect of OXT on trophoblast cell differentiation. Various factors regulate the differentiation of placental cells. Among them, CRH is known to be a specific marker for terminally differentiated STB cells (Riley et al. 1991;Warren and Silverman 1995).
The results showed that the expression of CRH significantly increased after treatment with OXT in vitro. In addition, the expression levels of CRH protein were increased in the placenta of pregnant animals treated with OXT. However, CRH expression tended to decrease in the atosiban-treated group as opposed to the OXT-treated group. Based on these results, we suggest that OXT has a crucial effect not only on the production of steroid hormones during pregnancy but also on the differentiation of trophoblasts, which is important for maintaining pregnancy.
We analyzed the effects of OXT-related signaling on the placenta. OXT signaling influences placental cell differentiation. OXT-mediated differentiation may contribute to the synthesis of steroid hormones, specifically E2, since differentiation of trophoblasts to STB is a critical factor for the synthesis of steroid hormones from the placenta. These results suggest an important role for OXT in the function of the placenta and maintenance of pregnancy. However, future studies using in vivo models including OXT and atosiban co-treatment model, OXT-deficient model and null murine model are required for further validation. Additionally, further experiments such as more molecular and cellular biological experiments could be performed in future work to support our hypothesis.

Materials and methods
Cell culture and treatment JEG-3 human choriocarcinoma-derived cells (Korean Cell Line Bank, Seoul, Republic of Korea) were seeded on 6-well plates (5 Â 10 5 cells/well) containing Dulbecco's modified Eagle medium (Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (Hyclone), 100 IU/ml penicillin, and 100 lg/ml streptomycin and were incubated at 5% CO 2 and 37 C. The cells were treated with OXT (1 lM; BACHEM; H-2510.0025, Torrance, CA, USA) or EtOH as a vehicle control for 24 h in triplicate wells. The dose of OXT used in vitro was referenced to other previously performed studies (Zatkova et al. 2018;Gu et al. 2021). The experiments were repeated independently at least thrice.

Animals
Animal studies were approved (approval number: PNU-2022-0151) by the Ethics Committee of Pusan National University (Busan, Republic of Korea). All studies related to animals were performed in accordance with relevant guidelines and regulations, including the ARRIVE guideline (http://www.ARRIVEguide lines.org).
Twenty pregnant female Sprague-Dawley (GD 1, 200-250 g) rats were purchased from Central Lab. Animal, Inc. (Seoul, Republic of Korea). The rats were randomly divided into four groups as follows: control group (n ¼ 5), OXT group (n ¼ 5), atosiban (Sigma-Aldrich, St. Louis, MO, USA) low-dose group (n ¼ 5), and atosiban high-dose group (n ¼ 5). All rats were housed in individual cage under standard laboratory conditions with controlled temperature/humidity, a 12:12-hour light/dark cycle, and free access to food The OXT signaling pathway is significantly correlated with the steroidogenesis of the placenta during pregnancy. In our results, OXT increased the overall expression of placental steroidogenic enzymes, and inhibition of OXT signaling pathway tended to negatively regulate the placental steroidogenesis. Our results demonstrate that the OXT signaling pathway plays an important role in the function of the placenta and maintenance of pregnancy. OXT: oxytocin; HSD3B1: 3b hydroxysteroid dehydrogenase/d5 4 isomerase type 1; HSD17B3: 17b dehydrogenase 3; CYP11A1: cholesterol side-chain cleavage enzyme; CYP17A1: 17a hydroxylase/17,20-lyase; CYP19A1: aromatase cytochrome P450. and water at the Pusan National University Laboratory Animal Resources Center, which is accredited by the Korea Food and Drug Administration (KFDA) in accordance with the Laboratory Animal Act (Accredited Unit Number-000231) and AAALAC International according to the National Institutes of Health guidelines (Accredited Unit Number; 001525).

Animal treatment and sample collection
The OXT or atosiban (low dose and high dose) groups were subcutaneously injected daily with OXT (0.25 mg/kg/day/200 ll) or atosiban (low dose: 0.6 mg/kg/day/200 ll; high dose: 1.2 mg/kg/day/200 ll) from GD 10 to 17. The doses used for injection were referred to other studies previously conducted (Loyens et al. 2013;Babic et al. 2015;Kenkel et al. 2019). The control group was administered 0.9% saline (200 ll). Rats were injected every morning from 9 to 10 A.M. and had free access to food. The dosage was adjusted according to changes in body weight (BW). The condition of the pregnant rat was monitored daily during the experiment. On GD 18, the rats were euthanized between 10 A.M. and 12 P.M. in a gradually filled CO 2 gas chamber with a flow rate of 20-30% CO 2 of the chamber volume/min. In accordance with the American Veterinary Medical Association (AVMA) guidelines for animal euthanasia, humane euthanasia of animals was confirmed in response to lack of pulse, breathing, and toe pinching and all efforts were made to minimize suffering (Underwood and Anthony 2020). Following euthanasia, we confirmed death of the animal after breathing stops. Additionally, humane endpoints were set up as 20% BW loss, loss of mobility and activity after treatment.
Approximately 1 ml of blood was collected from the inferior vena cava in plastic tubes containing EDTA as an anticoagulant under aseptic conditions and was centrifuged at 10,483 g for 10 min at 4 C to separate the plasma. Plasma was collected and immediately frozen at À80 C until analysis. The placentas were weighed and stored at À80 C for western blot analysis.
For direct comparisons between the concentration levels of different signaling molecules, membranes were stripped and re-probed using Restore TM Stripping Buffer (Sigma-Aldrich) as detailed by the manufacturer and reprobed after verifying the absence of residual HRP activity of the membrane. Each blot was scanned using a Bio-Rad ChemiDoc XRS (Bio-Rad), and band intensities were normalized to ACTB levels.

ELISA
JEG-3 cells were seeded on 6-well plates (5 Â 10 5 cells/well) and treated with 1 lM of OXT for 48 h at 37 C. Cell cultured media were collected and stored at -80 C.

Immunocytochemical analysis
JEG-3 cells were seeded at 1 Â 10 4 cells/well in an 8well chamber slide (Thermo Scientific Nunc V R , Waltham, MA, USA). When the cells attained 70-80% confluence, they were treated with OXT for 24 h in a 37 C incubator. After washing three times with 1 Â PBS, the cells were fixed with 200 ll of 4% formaldehyde solution for 10 min at room temperature, permeabilized with 1% Triton X-100 (Biosesang Inc., Seonam, Korea) for 10 min at room temperature, and subsequently washed with 1 Â PBS three times. The cells were then blocked in 2% BSA in PBS for 1 h at room temperature, followed by the addition of a specific primary antibody (anti-CRH) at 1:100 dilution overnight at 4 C. After washing, the cells were incubated with a secondary antibody (goat anti-rabbit IgG-Texas red, Santa Cruz; cat. no. sc2780) for 1 h in the dark at room temperature. After washing once with 1 Â PBS, Fluorochrome DAPI (2 lg/mL, Santa Cruz; cat. no. sc3598) was applied to each well, and the samples were incubated for 10 min in the dark at room temperature. Finally, the cells were washed three times with 1Â PBS and mounted with Vectashield mounting medium (Vector Laboratories Inc., Burlington, Ontario, Canada). All cells were observed using a fluorescent microscope (Eclipse TX100, Nikon, Tokyo, Japan) at 200Â magnification.

Statistical analysis
Results are presented as means ± standard deviation (SD). Data were analyzed for statistical significance using one-way analysis of variance followed by Tukey's multiple comparison test using SPSS version 10.10 for Windows (SPSS, Inc., Chicago, IL, USA). Statistical significance was set at p < 0.05.

Ethics approval
The animal protocol used in this study was reviewed and approved based on ethical procedures and scientific care by the Pusan National University-Institutional Animal Care and Use Committee (PNU-IACUC) (Approval number; PNU-2022-0151). All in vivo experiments are reported in accordance to ARRIVE guidelines (http://www.ARRIVEguide lines.org).

Funding
The present study was supported by Biomedical Research Institute Grant from Pusan National University Hospital (grant no. 2019B006) and partially supported by the BK21 FOUR Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Korea.

Disclosure statement
No potential conflict of interest was reported by the author(s).