• Journal of Embryo Transfer
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• v.28 no.3
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• pp.273-280
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• 2013

The aim of this study was to establish a three dimensional (3D) culture system of endometrial cells and to examine the plasminogen activators (PAs) activity in porcine uterine. The 3D culture system in porcine endometrial cells was composed to mixture 3D gel, stromal cells and epithelial cells. The 3D culture system was used to identify normal structure search as uterine tissue and PAs expression in this study. In results, porcine endometrium epithelial cells forming a top monolayer and endometrium stromal cells developed as fibroblast-like within 3D matrix scaffold. Expression of urokinase-type PA (uPA) and tissue-type PA (tPA) were observed during the 3D culture using immunofluorescence. PA activity in 3D-cultured endometrial cells was no significant difference between the tissue type, but 2D culture system were significantly lower than in 3D-cultured endometrial cells (P<0.05). Therefore, basic system and functional aspect of 3D culture could be established with similar system of endometrium tissue. We suggest that this study was assumed applicable as baseline data to investigate mechanism between porcine uterus cells in vitro.

• Animal Bioscience
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• v.36 no.3
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• pp.441-450
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• 2023

Objective: Serum amyloid A3 (SAA3), an acute phase response protein, plays important roles in opsonization, antimicrobial activity, chemotactic activity, and immunomodulation, but its expression, regulation, and function at the maternal-conceptus interface in pigs are not fully understood. Therefore, we determined the expression of SAA3 in the endometrium throughout the estrous cycle and at the maternal-conceptus interface during pregnancy. Methods: Endometrial tissues from pigs at various stages of the estrous cycle and pregnancy and with conceptuses derived from somatic cell nuclear transfer (SCNT), conceptus tissues during early pregnancy, and chorioallantoic tissues during mid- to late pregnancy were obtained and the expression of SAA3 was analyzed. The effects of the steroid hormones, interleukin-1β (IL1B), and interferon-γ (IFNG) on the expression of SAA3 were determined in endometrial explant cultures. Results: SAA3 was expressed in the endometrium during the estrous cycle and pregnancy, with the highest level on day 12 of pregnancy. The expression of SAA3 in the endometrium was significantly higher on day 12 of pregnancy than during the estrous cycle. Early-stage conceptuses and chorioallantoic tissues during mid to late pregnancy also expressed SAA3. The expression of SAA3 was primarily localized to luminal epithelial cells in the endometrium. In endometrial explant cultures, the expression of SAA3 was induced by increasing doses of IL1B and IFNG. Furthermore, the expression of SAA3 decreased significantly in the endometria of pigs carrying conceptuses derived from SCNT on day 12 of pregnancy. Conclusion: These results suggest that the expression of SAA3 in the endometrium during the implantation period increases in response to conceptus-derived IL1B and IFNG. The failure of those appropriate interactions between the implanting conceptus and the endometrium leads to dysregulation of endometrial SAA3 expression, which could result in pregnancy failure. In addition, SAA3 could be a specific endometrial epithelial marker for conceptus implantation in pigs.

• Asian-Australasian Journal of Animal Sciences
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• v.24 no.7
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• pp.919-928
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• 2011

The pig exhibits true epitheliochorial placentation, where the fetal membrane maintains attachment throughout pregnancy but does not invade into the maternal uterine endometrium. Accordingly, the expression and function of cell adhesion molecules are very important for embryo implantation and the establishment of pregnancy. In our recent microarray analysis, we found that activated leukocyte cell adhesion molecule (ALCAM) was expressed in the uterine endometrium during pregnancy in pigs. To better understand the roles of ALCAM in the establishment and maintenance of pregnancy, we examined ALCAM expression in the uterine endometrium during the estrous cycle and pregnancy in pigs. Real-time RT-PCR analysis showed that ALCAM was differentially expressed in the uterine endometrium during the estrous cycle and pregnancy, with the highest levels on D12 of pregnancy. ALCAM mRNA was localized to the luminal and glandular epithelial cells and to the trophectoderm of conceptuses during early pregnancy. The steroid hormones estrogen and progesterone had no effect on ALCAM expression in an endometrial explant culture study. Further, we found that ALCAM expression in the uterine endometrium from gilts with somatic cell nuclear transfer-derived embryos was not different from that in gilts with embryos from natural mating. ALCAM was expressed in a pregnancy stage- and cell type-specific manner in the uterine endometrium and conceptuses during pregnancy. These findings suggest that ALCAM may play a role in the establishment of pregnancy. Further analysis of ALCAM will provide insight into the implantation process and establishment of pregnancy in pigs.

• Journal of Embryo Transfer
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• v.28 no.3
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• pp.257-263
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• 2013

The endometrium undergoes a cyclic growth and tissue remodeling as changes of epithelial cells, and plasminogen activators (PAs) are related to endometrium tissue remodeling. This study was to evulate expression of urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) in porcine uterine epithelial cells. In results, the uPA and tPA were expressed in uterine tissue, epithelium and secretory glands in porcine endometrial cell. In addition, the uPA and tPA were expressed in cultured epithelial cells, and it were mainly expressed in cytoplasm. In porcine uterine tissue and epithelial cells, uPA activity was higher than activity in tPA. In PAs mRNA expression levels, uPA mRNA level was significantly higher than tPA mRNA level (P<0.05). The fluorescence intensity of uPA protein was also higher than fluorescence intensity of tPA protein, and uPA protein expression was significantly higher than in tPA protein expression (P<0.05). Therefore, we suggest that a physiological function in porcine uterine epithelial cells should be more influenced by uPA than in tPA during pre-ovulatory phase.

• Journal of Animal Reproduction and Biotechnology
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• v.37 no.4
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• pp.255-265
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• 2022

The cellular communication network factor (CCN) family proteins regulate many biological events such as angiogenesis, tumor growth, placentation, implantation, and embryogenesis. The expression and function of CCN1, CCN2, and CCN3 at the maternal-conceptus interface are established in humans and rodents, but little is known about the role of CCN4 to CCN6 in the reproductive organs in any other species. Several studies in transcriptome analysis in pigs have shown that the expression of CCN4 and CCN6 increases in the endometrium during early pregnancy. However, their expression, regulation, and function in the endometrium throughout the estrous cycle and pregnancy have not been fully understood in pigs. Thus, we determined the expression, localization, and regulation of CCN4 and CCN6 during the estrous cycle and at the maternal-conceptus interface in pigs. We found that the levels of CCN4, but not CCN6, changed during the estrous cycle. The levels of CCN4 were greater during mid- to late pregnancy than in the early stage, and the levels of CCN6 were greatest on Day 15 of pregnancy. CCN4 and CCN6 were detected in conceptus tissues during early pregnancy and in chorioallantoic tissues during the later stage of pregnancy. CCN4 mRNA was mainly localized to epithelial cells, CCN6 mRNAs to epithelial and stromal cells in the endometrium. In endometrial explant cultures, CCN4 expression was increased by progesterone, and CCN6 expression by interferon-𝛾. These results suggest that CCN4 and CCN6 may play roles in the establishment and maintenance of pregnancy by regulating the endometrial epithelial cell functions in pigs.

• Asian-Australasian Journal of Animal Sciences
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• v.18 no.12
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• pp.1708-1714
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• 2005

Keratinocyte growth factor (KGF) functions in epithelial growth and differentiation in many tissues and organs. KGF is expressed in the uterine endometrial epithelial cells during the estrous cycle and pregnancy in pigs, and receptors for KGF (KGFR) are expressed by conceptus trophectoderm and endometrial epithelia. KGF has been shown to stimulate the proliferation and differentiation of conceptus trophectoderm. However, the role of KGF on the endometrial epithelial cells has not been determined. Therefore, this study determined the effect of KGF on proliferation and differentiation of endometrial epithelial cells in vitro and in vivo using an immortalized porcine luminal epithelial (pLE) cell line and KGF infusion into the uterine lumen of pigs between Days 9 and 12 of estrous cycle. Results showed that KGF did not stimulate proliferation of uterine endometrial epithelial cells in vitro and in vivo determined by the $^3$H]thymidine incorporation assay and the proliferating cell nuclear antigen staining, respectively. Effects of KGF on expression of several markers for epithelial cell differentiation, including integrin receptor subunits $\alpha$4, $\alpha$5 and $\beta$1, plasmin/trypsin inhibitor, uteroferrin and retinol-binding protein were determined by RT-PCR, Northern and slot blot analyses, and immunohistochemisty, and KGF did not affect epithelial cell differentiation in vitro and in vivo. These results show that KGF does not induce epithelial cell proliferation and differentiation, suggesting that KGF produced by endometrial epithelial cells acts on conceptus trophectoderm in a paracrine manner rather than on endometrial epithelial cells in an autocrine manner.

• Applied Microscopy
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• v.14 no.2
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• pp.29-37
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• 1984

Morphological changes of the epithelium of the endometrium by prolonged treatment of $17{\beta}$-estradiol or progesterone in ovariectomized rats was studied at the ultrastructural level. The epithelium of the endometrium in ovariectomized rats was characterized by the appearance of a number of vacuoles which was contained with the membraneous structures, lipid droplets and the others. The epithelium was low cuboidal, and a few short microvilli were present at the cell surface. Secretory granules are rarely found. After estradiol treatment, the epithelium was high columnar in shape. The mitochondria was appeared throughout the cytoplasm, however, long or swelling mitochondria was often found. Golgi apparatus and rER were relatively well-developed. Relatively long and sparse microvilli were present at the cell surface. After progesterone treatment, the epithelium was characterized by the appearance of numerous vesicles at the apical region and numerous lipid droplets at the subnuclear region. At the cell surface a number of short and blunt microvilli were found. These data indicated that the endometrium was dependent on estrogen and progesterone for changes in both its morphological and functional state and suggested that each hormone exerted a unique effect on the epithelial cells.

• Korean Journal of Veterinary Research
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• v.35 no.2
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• pp.217-228
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• 1995

The study was designed to investigate the effects of progesterone on the reproductive system. This investigation was performed by immunohistochemical methods using anti-bromodeoxyuridine-antibody following bromodeoxyuridine(Brdur) injection for labeling proliferating cells in the uterus and ovary of rats. Sixteen female rats(Wistar), weighing initially 300g, were randomly allotted into ovariectomized and unovariectomized large groups. These two large groups were subdivided into three subgroups of control, 3-day and 6-day groups, respectively. 3-days and 6-days group were injected with 1mg of progesterone/rat/day for 3 or 6 days, respectively. In gross findings, the uterus of ovariectomized groups markedly atrophied, and were not hypertrophied by progesterone injection for 3 days or 6 days and the uterus of unovariectomized groups also were not hypertrophied. Labeling index(LI, %) was measured by counting the number of Brdur-positive cells from 300 to 3,000 cells per layer in the uterus tissue. The average LI of the uterus in unovariectomized groups was higher than that of ovariectomized groups. The subgroups with higher LI in unovariectomized groups were ordered as 6-day group, 3-day group. So progesterone considerably effected to the proliferating of the cells in the uterus of unovariectomized groups. The layers with higher LI in the uterus wall were ordered as the functional zone of endometrium, epithelial layer of endometrium, basal zone of endometrium, myometrium and perimetrium. The cell types with higher LI in the uterus of unovariectomized groups were ordered as the surface epithelial cells, stromal cells, glandular epithelial cells and muscle cells. Growing follicles with proliferating cells from secondary and tertiary follicles in the ovary of unovariectomized groups appeared to be 37.66% in control group, 39.23% in 3-day groups, 39.47% in 6-day groups. Mature follicles in the ovary were more number in control group than those in 3-day groups but not appeared in 6-day groups. So progesterone not nearly effects to the number of the growing follicles but appeared to be related to suppression of the development and protrusion of the mid-tertiary and mature follicles on the ovary surface. The cell types with higher LI in the ovary of unovariectomized groups were respectively ordered as granulosa cells, theca interns cells in secondary follicles; theca interna cells, granulosa cells, theca externa cells in tertiary follicles; fibroblasts, theca in terns cells in atretic follicles; fibroblasts, luteal cells in corpus luteum.

• Clinical and Experimental Reproductive Medicine
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• v.21 no.3
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• pp.315-323
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• 1994

Mammalian oviductal epithelial cells have been known to improve in vitro fertilization and embryonic development. Recently, co-cultured human embryos with the epithelial cells in human genital tract has been reported to improve the pregnancy rate. The purpose of the study was to investigate the effects of the epithelial cells of human genital tract on the development of mouse early embryos and human fertilized oocytes. The epithelial cells of human genital tract were collected from the fallopian tubes which were obtained during hysterectomy in fertile women and from the endometrium during endometrium biopsy. Collected human ampullary cells(HACs) and endometrial cells(HECs) were cultured for 10 days to establish primary monolayer. Second passaged HACs and HECs were obtained by trypsinization were cryopreserved in PBS with 1.5 M DMSO for later use. To investigate the effect when co-cultured with HACs and HECs, we tried to apply strict quality control on mouse embryo, from two cell to blastocyst prior to human trial. The results of quality control were as follows; In Group I (Ham's F10 with 10% FCS), Group IT (co-cultured with HACs) and Group ill (co-cultured with HECs), developmental rates to blastocyst were 63.3%(253/400), 76.0%(304/ 400),74.0%(296/400), respectively. Hatching rates were 36.8%(147/400), 41.80/0(167/400), 38.0%(152/400), respectively(p<0.05). To perform the human IVF, cryopreserved HACs were thawed at 37$^{\circ}C$ waterbath, seeded on the well dish and cultured for 48 hI'S. The pronuclear stage embryos were transferred to the seeded well dish. After 24 hRS, co-cultured embryos were examined and transferred to patient's uterus. The results of human IVF when co-cultured with HACs were that fertilization and developmental rates were 61.8% (256/414), 95.3% (244/256) as compared with 57.2% (279/488) and 94.6%(264/279) in Ham's F10 supplemented with 10% FCS(control). However, 62.9% (161/256) of co-cultured human embryos showed good embryos(no or slight fragmentation) as compared with 53.8 % (150/279) in control(p < 0.05). Pregnancy rate was 40.0% (12/30) when co-cultured with HACs whereas 30.6%(11/36) in control. In conclusions, co-culture system using HACs and HECs improved the developmental and hatching rates of mouse embryo. Also, in human IVF system when co-cultured with HACs, it improved both the quality of human embryos and the pregnancy rate.

• The Korean Journal of Zoology
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• v.25 no.1
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• pp.9-28
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• 1982

Cyclical changes in the fine structures of the surface epithelial, stroma and glandular cells of guinea pig endometrium during the estrous cycle were studied by transmission and scanning electron microscopy. Cytochemical studies were made in order to investigate the ultrastructural localization of the acid phosphatase, alkaline phosphatase and ATPase in these cells. The results obtained are as follows: 1. The endometrial surface epithelium was pseudostratified columnar during estrus and meterstrus, and simple columnar during proestrus and diestrus. The characteristic features observed in these cells include increased nucleocytoplasmic ratio at proestrus, elongated shapes of both the nucleus and the entire cell, increased volume of the cytoplasm and cytoplasmic bulding into the lumen during estrus, and smaller surface epithelial cells during metestrus. 2. In the cytoplasm of surface epithelial cells, the numbers of mitochondria and free ribosomes were increased, and rough endoplasmic reticulum and Golgi complex appeared during estrus, and the degenerated cells, lipid droplets, multilamellated bodies and lysosomes appeared during diestrus. 3. During estrus, scanning electron microscopic observations of endometrial surface showed a regular arrangement with polygonal outlines of epithelial cells, distinct intercellular border, and bulged surface into the lumen, whereas flat surface and indistinct cell border were characteristic during meterstrus and diestrus. 4. Microvilli which aligned on the surface were longer and most abundant during estrus while short and aparse during other phases. 5. Cytochemical studies indicated that during metestrus acid phosphatase activities were localized in the microvilli and vacuoles, and alkaline phosphatase activities were significant around luminal surface and lateral cell membrane in the surface epithelial cells. ATPase activities were present on the microvilli and cell membrane during proestrus and estrus.