Hyocheol Bae

Brain‐derived neurotrophic factor improves proliferation of endometrial epithelial cells by inhibition of endoplasmic reticulum stress during early pregnancy

Brain‐derived neurotrophic factor (BDNF) is a member of the neurotrophin family binds to two transmembrane receptors; neurotrophic receptor tyrosine kinase 2 (NTRK2) with high affinity and p75 with low affinity. Although BDNF‐NTRK2 signaling in the central nervous system is known, signaling in the female reproductive system is unknown. Therefore, we determined effects of BDNF on porcine endometrial luminal epithelial (pLE) cells isolated from Day 12 of pregnancy, as well as expression of BDNF and NTRK2 in endometria of cyclic and pregnant pigs. BDNF‐NTRK2 genes were expressed in uterine glandular (GE) and luminal (LE) epithelia during early pregnancy. In addition, their expression in uterine GE and LE decreased with increasing parity of sows. Recombinant BDNF increased proliferation in pLE cells in a dose‐dependent, as well as expression of PCNA and Cyclin D1 in nuclei of pLE cells. BDNF also activated phosphorylation of AKT, P70S6K, S6, ERK1/2, JNK, P38 proteins in pLE cells. In addition, cell death resulting from tunicamycin‐induced ER stress was prevented when pLE cells were treated with the combination of tunicamycin and BDNF which also decreased cells in the Sub‐G1 phase of the cell cycle. Furthermore, tunicamycin‐induced unfolded protein response genes were mostly down‐regulated to the basal levels as compared to non‐treated pLE cells. Our finding suggests that BDNF acts via NTRK2 to induce development of pLE cells for maintenance of implantation and pregnancy by activating cell signaling via the PI3K and MAPK pathways and by inhibiting ER stress.

Stimulatory effects of fibroblast growth factor 2 on proliferation and migration of uterine luminal epithelial cells during early pregnancy

Fibroblast growth factor 2 (FGF2) is a mitogen that induces proliferation, differentiation, and migration of cells, as well as angiogenesis and carcinogenesis via autocrine or paracrine actions. Fibroblast growth factor 2 expression is abundant in porcine conceptuses and endometrium during the estrous cycle and peri-implantation period of pregnancy. However, its intracellular actions in uterine epithelial cells have not been reported. The results of this study indicated abundant expression of FGFR1 and FGFR2 predominantly in uterine luminal and glandular epithelia during early pregnancy and that their expression decreased with increasing parity of the sows. Treatment of porcine uterine luminal epithelial (pLE) cells with FGF2 increased proliferation and DNA replication based on increases in proliferating cell nuclear antigen (PCNA) and initiation of G1/S phase progression. In addition, FGF2 increases phosphorylation of AKT, P70S6K, S6, ERK1/2, JNK, P38, and P90RSK in a time-dependent manner, and increases in their expression was suppressed by Wortmannin (a phosphatidylinositol 3-kinase [PI3K] inhibitor), U0126 (an ERK1/2 inhibitor), SP600125 (a JNK inhibitor), and SB203580 (a P38 inhibitor) based on western blot analyses. Also, the abundance of cytoplasmic p-AKT protein was decreased by Wortmannin and U0126, and p-ERK1/2 protein was reduced only by U0126. Furthermore, inhibition of each signal transduction protein reduced the ability of FGF2 to stimulate proliferation and migration of pLE cells. Collectively, these results indicate that activation of FGFR1 and FGFR2 by uterine- and endometrial-derived FGF2 stimulates PI3K/AKT and mitogen-activated protein kinase pathways for development of the porcine uterus and improvement of litter size.Abstract Fibroblast growth factor 2 (FGF2) is amitogen that induces proliferation, differentiation, and migration of cells, as well as angiogenesis and carcinogenesis via autocrine or paracrine actions. Fibroblast growth factor 2 expression is abundant in porcine conceptuses and endometrium during the estrous cycle and peri-implantation period of pregnancy. However, its intracellular actions in uterine epithelial cells have not been reported. The results of this study indicated abundant expression of FGFR1 and FGFR2 predominantly in uterine luminal and glandular epithelia during early pregnancy and that their expression decreased with increasing parity of the sows. Treatment of porcine uterine luminal epithelial (pLE) cells with FGF2 increased proliferation and DNA replication based on increases in proliferating cell nuclear antigen (PCNA) and initiation of G1/S phase progression. In addition, FGF2 increases phosphorylation of AKT, P70S6K, S6, ERK1/2, JNK, P38, and P90RSK in a time-dependent manner, and increases in their expression was suppressed by Wortmannin (a phosphatidylinositol 3-kinase [PI3K] inhibitor), U0126 (an ERK1/2 inhibitor), SP600125 (a JNK inhibitor), and SB203580 (a P38 inhibitor) based on western blot analyses. Also, the abundance of cytoplasmic p-AKT protein was decreased by Wortmannin and U0126, and p-ERK1/2 protein was reduced only by U0126. Furthermore, inhibition of each signal transduction protein reduced the ability of FGF2 to stimulate proliferation and migration of pLE cells. Collectively, these results indicate that activation of FGFR1 and FGFR2 by uterine- and endometrial-derived FGF2 stimulates PI3K/AKT and mitogen-activated protein kinase pathways for development of the porcine uterus and improvement of litter size. Summary Sentence Activation of FGFR1 and FGFR2 by uterine- and endometrial-derived FGF2 enhances uterine development for implantation and placentation and improvement of litter size.

A Critical Role for Adiponectin-Mediated Development of Endometrial Luminal Epithelial Cells During the Peri-implantation Period of Pregnancy.

Adiponectin is one of the adipokines in the collagen superfamily. It is secreted primarily by white adipocytes and influences reproductive processes including ovarian and uterine functions. Adiponectin regulates energy homeostasis, insulin sensitivity, and is anti‐inflammatory in various tissues. Its receptors (ADIPOR1 and ADIPOR2) are widely expressed in mammalian tissues, including porcine conceptuses and endometrial during the estrous cycle and peri‐implantation period of pregnancy. However, regulatory effects of adiponectin on endometrial epithelial cells are unknown. Therefore, we investigated the effects of parity on expression of ADIPOR1 and ADIPOR2 and the effects of adiponectin in the porcine endometrium during early pregnancy. Results of this study revealed robust expression of ADIPOR1 and ADIPOR2 in uterine luminal (LE) and glandular (GE) epithelia during early pregnancy and expression decreased as with increasing parity. For porcine luminal epithelial (pLE) cells, adiponectin enhanced proliferation, and increased phosphorylation of AKT, P70S6K, S6, ERK1/2, JNK, P38, and P90RSK in a time‐dependent manner. Moreover, the abundance of adiponectin‐activated signaling molecules were suppressed by pharmacological inhibitors including wortmannin, U0126, SP600125, and SB203580, respectively, in pLE cells. Furthermore, inhibition of each targeted signal transduction molecule influenced proliferation of adiponectin‐stimulated pLE cells. In addition, adiponectin inhibited tunicamycin‐induced endoplasmic reticulum (ER)‐stress through effects on ER stress regulated proteins in pLE cells. Collectively, these results suggest that adiponectin affects development of porcine uterine epithelia and reproductive performance through modulation of PI3K/AKT and MAPK cell signaling pathways.

Cell-specific expression and signal transduction of C-C motif chemokine ligand 2 and atypical chemokine receptors in the porcine endometrium during early pregnancy

&NA; Chemokines and atypical chemokine receptors (ACKRs; also known as chemokine decoy receptors) play an important role in reproductive immunology by recruiting leukocytes during early pregnancy. The aim of this study was to determine the expression of C‐C motif chemokine ligand 2 (CCL2) and ACKRs in the endometrium during estrous cycle and early pregnancy, and to investigate the functional effects of CCL2 on porcine uterine luminal epithelial (pLE) cells. Our results indicated that CCL2, ACKR1, ACKR3, and ACKR4 were strongly detected in the glandular and luminal epithelium of the endometrium during early pregnancy compared to that in non‐pregnant pigs. Recombinant CCL2 improved pLE cell proliferation via activation of the PI3K and MAPK pathways and suppression of endoplasmic reticulum (ER) stress by reducing the expression of ER stress regulatory genes. Collectively, these results provide novel insights into CCL2‐mediated signaling mechanisms in the porcine endometrium at the maternal‐fetal interface during early pregnancy. HighlightsCCL2‐ACKR system is regulated in the porcine uterine endometrium.CCL2 induces the proliferation and cell cycle progression of porcine uterine cells.CCL2 activates PI3K/AKT and MAPK pathways for cellular proliferation in the uterus.CCL2 reduces ER stress regulatory gene expression in porcine luminal epithelia cells.CCL2 may play an important role in maternal‐fetal interaction during early pregnancy.

Functional Roles of Eph A‐Ephrin A1 System in Endometrial Luminal Epithelial Cells During Early Pregnancy

Eph and ephrin regulate diverse biological events such as proliferation, adhesion, migration, and angiogenesis through cell‐to‐cell interactions. However, little is known of their functional role and mechanisms of action in uterine endometrial cells. In the present study, we demonstrated the effects of the Eph and ephrin on interactions between blastocysts and endometrial luminal epithelial (pLE) cells in the pig that is regarded as an excellent biomedical animal model for research on the peri‐implantation period of pregnancy. Results of this study indicated that among eight members of the Eph A family, expression of Eph A1, A2, A4, and A7 was strongly detected in endometrial epithelial cells during early pregnancy. Of these, for identification of signal transduction pathways induced by ephrin A1, a major ligand for Eph A, cell proliferation assays, and immunofluorescence and cell cycle regulation were analyzed following treatment of pLE cells with ephrin A1. Ephrin A1 stimulated proliferation of pLE cells as evidenced by abundant PCNA expression and an increase in the G2/M phase. Western blot analysis showed that ephrin A1 activated PI3K and MAPK signaling proteins in a time‐dependent manner. Moreover, phosphorylation of AKT, ERK1/2, P38, and JNK proteins were suppressed by their inhibitors wortmannin, U0126, SB203580, and SP600125, respectively. Also, phosphor‐AKT was reduced by ERK1/2 and P38 inhibitors. Ephrin A1‐induced proliferation and migration of pLE cells was also blocked by those inhibitors. Collectively, these results suggest that ephrin A1 enhances interactions between porcine blastocysts and endometrial luminal epithelial cells by activating PI3K and MAPK signal transduction pathways. J. Cell. Physiol. 232: 1527–1538, 2017.

Bifunctional role of ephrin A1-Eph system in stimulating cell proliferation and protecting cells from cell death through the attenuation of ER stress and inflammatory responses in bovine mammary epithelial cells

Structural and functional development of the mammary gland is constant in the mammary gland life cycle. Eph receptors and their ligands, ephrins, control events through cell‐to‐cell interactions during embryonic development, and adult tissue homeostasis; however, little information on participation of ephrin A1, a representative ligand of the Eph receptor, in the development and function of normal mammary glands is known. In this study, we demonstrated functional effects of the ephrin A1‐Eph system and mechanisms of its action on bovine mammary epithelial (MAC‐T) cells. The in vitro cultured MAC‐T cells expressed the ephrin A1 ligand and EphA1, A2, A4, A7, and A8 among the eight members of the Eph A family. Our results revealed that ephrin A1 induced MAC‐T cell cycle progression and stimulated cell proliferation with abundant expression of nucleic PCNA and cyclin D1 proteins. Additionally, ephrin A1 induced activation of intracellular signaling molecules involved in PI3 K/AKT and MAPK signaling, and the proliferation‐stimulating effect of ephrin A1 was mediated by activation of these pathways. Furthermore, ephrin A1 influenced expression and activation of various ER stress‐related proteins and protected MAC‐T cells from stress‐induced cell death. Finally, ephrin A1 alleviated LPS‐induced cell death through down‐regulation of inflammatory cytokines. In conclusion, the results of this study suggest that the Eph A‐ephrin A1 system is a positive factor in the increase and maintenance of epithelial cells in mammary glands of cows; the signaling system contributes to development, remodeling, and functionality of normal mammary glands and could overcome mastitis in cows and other mammals.

Differential expression and functional roles of chemokine (C-C motif) ligand 23 and its receptor chemokine (C-C motif) receptor type 1 in the uterine endometrium during early pregnancy in pigs

Abstract Many chemokines expressed by cells of the uterine endometrium of mammals are involved in cell‐cell interactions. However, little is known about expression and functional roles of chemokine (C‐C motif) ligand 23 (CCL23) in the uterine endometrium. Results of this study demonstrated that CCL23 and its receptor, chemokine (C‐C motif) receptor type 1 (CCR1), are up‐regulated in porcine endometria during pregnancy. CCL23 and CCR1 mRNAs were strongly expressed in endometrial glandular (GE) and luminal (LE) epithelial cells. Treatment of porcine uterine luminal epithelial (pLE) cells with recombinant CCL23 increased the abundances of PCNA and cyclin D1, and enhanced proliferation and cell cycle progression in pLE cells. CCL23 also stimulated phosphorylation of cell signaling molecules including AKT and MAPKs in pLE cells. Furthermore, ER stress‐related molecules were reduced by CCL23. These results suggest that CCL23‐CCR1 signaling is important for endometrial development and establishment of pregnancy in pigs. HighlightsCCL23‐CCR1 system is up‐regulated in the uterine endometrium during early pregnancy.CCL23 induces the proliferation and cell cycle progression of pLE cells.CCL23 activates intracellular signaling pathways including PI3K/AKT and MAPK.CCL23 reduces tunicamycin‐induced ER stress in pLE cells.CCL23‐CCR1 is important for development of the endometrium during early pregnancy.

Differential expression of apolipoprotein D in male reproductive system of rats by high-fat diet

Apolipoprotein D, a 29‐kDa secreted glycoprotein that belongs to the lipocalin superfamily, is widely expressed in various tissues and associated with lipid metabolism as a component of high‐density lipoproteins. Although Apolipoprotein D binds to small hydrophobic ligands including cholesterol, little is known about effects of high‐fat diet with cholesterol on expression of Apolipoprotein D in the male reproductive tract. Therefore, we investigated Apod expression in penises, prostate glands, and testes from rats fed a high‐fat diet including a high amount of cholesterol. Our previous research indicated that a high‐fat diet induces dyslipidemia leading to histological changes and dysfunction of male reproduction in rats. Consistent with these results, Apod mRNA expression was significantly (p < 0.001) decreased in penises and prostate glands (p < 0.01) and testes (p < 0.01) from rats fed a high‐fat diet as compared with normal diet. In addition, Apod mRNA and protein were detected predominantly in urethral epithelium and penile follicle from rats. Moreover, changes in expression of specific microRNAs (miR‐229b‐3p, miR‐423‐3p, and miR‐490‐3p) regulating Apod in the penises and prostate glands were negatively associated with Apod expression. Collectively, results of this study suggest that Apod is a novel regulatory gene in the male reproductive system, especially in penises of rats fed a high‐cholesterol diet, and that expression of Apod is regulated at the posttranscriptional level by target microRNAs.

Aquaporin 3 is regulated by estrogen in the chicken oviduct and is involved in progression of epithelial cell-derived ovarian carcinomas

Aquaporins (AQPs) are membrane proteins that passively deliver water across the plasma membrane to play an important role in maintaining cell shape. Members of the AQP family are distributed in most of the tissues in the human body and perform a variety of functions based on the water homeostasis suitable for each organ. However, there is little known about the expression and regulation of AQP family members in chickens. Therefore, we determined the expression of AQPs in various tissues of chickens. Among 13 isotypes, AQP3 was highly expressed in the chicken oviduct. Expression of AQP3 messenger RNA (mRNA) increased in the magnum (P < 0.001) and isthmus (P < 0.001) of chick oviducts treated with diethylstilbestrol. Consistent with these results, the localization of AQP3 was detected in the glandular and luminal epithelia of the magnum and isthmus of oviducts of diethylstilbestrol-treated chicks. In addition, the pattern of expression of AQP3 changed in an estrogen-dependent manner during the molting period. During the regenerative period of the oviduct after molting, expression of AQP3 mRNA increased coordinately with increasing concentrations of estradiol (P < 0.001), whereas expression of AQP3 mRNA decreased as concentrations of estradiol in plasma decreased in response to induced molting (P < 0.001). Also, expression of the AQP3 increased (P < 0.001) in cancerous ovaries of laying hens. In conclusion, AQP3 does not simply function to transport water into and out of cells but also appears to be closely involved in development of the chicken oviduct, which is regulated by estrogens. Furthermore, our results suggest AQP3 as a new diagnostic for early detection and treatment of epithelial cell-derived ovarian carcinomas.

Diethylstilbestrol regulates expression of avian apolipoprotein D during regression and recrudescence of the oviduct and epithelial-derived ovarian carcinogenesis

Apolipoprotein D (APOD) is a glycoprotein which is widely expressed in mammalian tissues. It is structurally and functionally similar to the lipocalins which are multiple lipid-binding proteins that transport hydrophobic ligands and other small hydrophobic molecules, including cholesterol and several steroid hormones. Although multiple functions for APOD in various tissues have been reported, its expression, biological function, and hormonal regulation in the female reproductive system are not known. Thus, in this study, we focused on correlations between APOD and estrogen during development, differentiation, regression, and regeneration of the oviduct in chickens and in the development of ovarian carcinogenesis in laying hens. Results of the present study indicated that APOD messenger RNA (mRNA) expression increased (P < 0.001) in the luminal and glandular (GE) epithelia of the chicken oviduct in response to diethylstilbestrol (a nonsteroidal synthetic estrogen). In addition, the expression of APOD mRNA and protein decreased (P < 0.001) as the oviduct regressed during induced molting, and gradually increased (P < 0.001) with abundant expression in GE of the oviduct during recrudescence after molting. Furthermore, APOD mRNA and protein were predominantly localized in GE of cancerous, but not normal ovaries from laying hens. Collectively, results of the present study suggest that APOD is a novel estrogen-stimulated gene in the chicken oviduct which likely regulates growth, differentiation, and remodeling of the oviduct during oviposition cycles. Moreover, up-regulated expression of APOD in epithelial cell-derived ovarian cancerous tissue suggests that it could be a candidate biomarker for early detection and treatment of ovarian cancer in laying hens and in women.