Porcine Oviductal Epithelial Cells Research Articles

Genes involved in angiogenesis and circulatory system development are differentially expressed in porcine epithelial oviductal cells during long-term primary in vitro culture – a transcriptomic study

Abstract An oviduct is an essential organ for gamete transport, oocyte maturation, fertilization, spermatozoon capacitation and early embryo development. The epithelium plays an important role in oviduct functioning. The products of secretory cells provide an optimal environment and influence gamete activities and embryonic development. The oviduct physiology changes during the female cycle, thus, the ratio of the secreted molecules in the oviduct fluid differs between phases. In this study, a differential gene expression in porcine oviduct epithelial cells was examined during the long-term primary in vitro culture. The microarray expression analysis revealed 2552 genes, 1537 of which were upregulated and 995 were downregulated after 7 days of culture, with subsequent changes in expression during 30 day-long culture. The obtained genes were classified into 8 GO BP terms, connected with angiogenesis and circulatory system development, extracted by DAVID software. Among all genes, 10 most up-regulated and 10 most down-regulated genes were selected for further investigation. Interactions between genes were indicated by STRING software and REACTOME FIViz application to the Cytoscape 3.6.0 software. Most of the genes belonged to more than one ontology group. Although studied genes are mostly responsible for angiogenesis and circulatory system development, they can also be found to be expressed in processes connected with fertilization and early embryo development. The latter function is focused on more, considering the fact that these genes were expressed in epithelial cells of the fallopian tube which is largely responsible for reproductive processes.

Ontology groups representing angiogenesis and blood vessels development are highly up-regulated during porcine oviductal epithelial cells long-term real-time proliferation – a primary cell culture approach

Abstract The morphological and biochemical modification of oviductal epithelial cells (OECs) belongs to the group of compound processes responsible for proper oocyte transport and successful fertilization. The cellular interactions between cumulus-oocyte complexes (COCs) and oviductal epithelial cells (OECs) are crucial for this unique mechanism. In the present study we have analyzed angiogenesis and blood vessel development processes at transcript levels. By employing microarrays, four ontological groups associated with these mechanisms have been described. Differentially expressed genes belonging to the “angiogenesis”, “blood circulation”, “blood vessel development” and “blood vessel morphogenesis” GO BP terms were investigated as a potential markers for the creation of new blood vessels in cells under in vitro primary culture conditions.

Fatty Acids Related Genes Expression Undergo Substantial Changes in Porcine Oviductal Epithelial Cells During Long-Term Primary Culture

Abstract The process of reproduction requires several factors, leading to successful fertilization of an oocyte by a single spermatozoon. One of them is the complete maturity of an oocyte, which is acquired during long stages of folliculogenesis and oogenesis. Additionally, the oviduct, composed of oviductal epithelial cells (OECs), has a prominent influence on this event through sperm modification and supporting oocyte’s movement towards uterus. OECs were isolated from porcine oviducts. Cells were kept in primary in vitro culture for 30 days. After 24h and on days 7, 15 and 30 cells were harvested, and RNA was isolated. Transcript changes were analyzed using microarrays. Fatty acids biosynthetic process and fatty acids transport ontology groups were selected for analysis and described. Results of this study indicated that majority of genes in both ontology groups were up-regulated on day 7, 15 and 30 of primary in vitro culture. We analyzed genes involved in fatty acids biosynthetic process, including: GGT1, PTGES, INSIG1, SCD, ACSL3, FADS2, FADS1, ACSS2, ALOX5AP, ACADL, SYK, ACACA, HSD17B8, FADS3, OXSM, and transport, including: ABCC2, ACSL4, FABP3, PLA2G3, PPARA, SYK, PPARD, ACACA and P2RX7. Elevated levels of fatty acids in bovine and human oviducts are known to reduce proliferation capacity of OECs and promote inflammatory responses in their microenvironment. Most of measured genes could not be connected to reproductive events. However, the alterations in cellular proliferation, differentiation and genes expression during in vitro long-term culture were significant. Thus, we can treat them as putative markers of changes in OECs physiology.

In Vitro Mimicking of Estrous Cycle Stages: Dissecting the Impact of Estradiol and Progesterone on Oviduct Epithelium.

We have previously mimicked the morphological and functional changes occurring in the oviduct epithelium during the estrous cycle in vitro by using an air-liquid interface (ALI) culture system and basolateral application of 17β-estradiol (E2) and progesterone (P4). In the current study we aimed to explore the transcriptomic changes elicited by E2 and P4 together during estrous cycle simulation and to dissect the individual effects of E2 and P4 on oviduct epithelium physiology. Primary porcine oviduct epithelial cells (POECs) (N = 6 animals) were cultured at the ALI. After differentiation for 11 days, we sequentially simulated diestrus (10 days) and estrus (2.5 days) by adding serum levels of E2 and P4 to the basolateral compartment either in combination (mix trial) or separately (P4 trial and E2 trial, respectively). Cell response was evaluated by microarray analysis (mix and P4 trials), quantitative RT-PCR, and histomorphometry (all trials). When we compared simulated diestrus with estrus stage in the mix trial, there were 169 (142 upregulated and 27 downregulated) differentially expressed genes (DEGs; fold change ≥1.5). In the P4 trial, 108 DEGs (83 upregulated and 25 downregulated) were detected. Gene enrichment analysis revealed that immune-related pathways were exclusively affected in the mix trial. In both mix and P4 trials, POECs exhibited in vivo-like morphological changes regarding epithelium height and portion of ciliated cells. However, E2 alone did not trigger morphological changes. We deduce that P4 mainly drives structural variations, and E2 is imperative for regulating immune function of the oviduct epithelium during estrous cycle.

Amino acids metabolism and degradation is regulated during porcine oviductal epithelial cells (OECs) primary culture in vitro – a signaling pathways activation approach

Abstract The ovary is part of the reproductive system, possessing very important functions in the reproduction process (ovum and embryo transfer, providing a suitable environment for sperm capacitation, etc.). There are two types of cells in the fallopian tubes: alveolar and secretive cells. These study shows the metabolic processes in pig oviductal epithelial cells associated with the activation of signaling pathways of amino acids metabolism and degradation during long-term in vitro culture. Oviductal epithelial cells from 45 colonies in the anestrous phase of the estrous cycle have been utilized in this study. RNA extract from the OEC primary cultures was pooled after 24h, 7days, 15 days and 30 days from the beginning of culture and the transcriptome investigated by Affymetrix® Porcine Gene 1.1 ST. From the whole transcript that consisted of 2009 different genes, 1537 were upregulated and 995 were downregulated after 7 days of culture, 1471 were upregulated and 1061 were downregulated after 15 days of culture and 1329 were upregulated and 1203 were downregulated after 30 days of culture. The results of these studies provide, for the first time, information on the activation of metabolic pathways of amino acids such as valine, leucine, isoleucine, cysteine, and methionine in the investigated tissue. They also indicate genes that may be OECs-specific genetic markers that are expressed or upregulated during long-term in vitro culture.

Periovulatory follicular fluid levels of estradiol trigger inflammatory and DNA damage responses in oviduct epithelial cells.

ObjectiveOvarian steroid hormones (mainly E2 and P4) regulate oviduct physiology. Serum-E2 acts on the oviduct epithelium from the basolateral cell compartment. Upon ovulation, the apical compartment of the oviduct epithelium is temporarily exposed to follicular fluid, which contains much higher levels of E2 than serum. The aim of this study was to evaluate the effects of human periovulatory follicular fluid levels of E2 on oviduct epithelial cells using two porcine in vitro models.MethodsA cell line derived from the porcine oviductal epithelium (CCLV-RIE270) was characterized (lineage markers, proliferation characteristics and transformation status). Primary porcine oviduct epithelial cells (POEC) were cultured in air-liquid interface and differentiation was assessed histologically. Both cultures were exposed to E2 (10 ng/ml and 200 ng/ml). Proliferation of CCLV-RIE270 and POEC was determined by real-time impedance monitoring and immunohistochemical detection of Ki67. Furthermore, marker gene expression for DNA damage response (DDR) and inflammation was quantified.ResultsCCLV-RIE270 was not transformed and exhibited properties of secretory oviduct epithelial cells. Periovulatory follicular fluid levels of E2 (200 ng/ml) upregulated the expression of inflammatory genes in CCLV-RIE270 but not in POEC (except for IL8). Expression of DDR genes was elevated in both models. A significant increase in cell proliferation could not be detected in response to E2.ConclusionsCCLV-RIE270 and POEC are complementary models to evaluate the consequences of oviduct exposure to follicular fluid components. Single administration of periovulatory follicular fluid E2 levels trigger inflammatory and DNA damage responses, but not proliferation in oviduct epithelial cells.

Characterisation of extracellular vesicles produced by the Porcine oviductal epithelial cells using size exclusion chromatography

Characterisation of extracellular vesicles produced by the Porcine oviductal epithelial cells using size exclusion chromatography

Transepithelial electrical resistance (TEER): a functional parameter to monitor the quality of oviduct epithelial cells cultured on filter supports.

Cultivation of oviduct epithelial cells on porous filters fosters in vivo-like morphology and functionality. However, due to the optical properties of the filter materials and the cells’ columnar shape, cell quality is hard to assess via light microscopy. In this study, we aim to evaluate transepithelial electrical resistance (TEER) measurement as a prognostic quality indicator for the cultivation of porcine oviduct epithelial cells (POEC). POEC were maintained in four different types of media for 3 and 6 w to achieve diverse culture qualities, and TEER was measured before processing samples for histology. Culture quality was scored using morphological criteria (presence of cilia, confluence and cell polarity). We furthermore analyzed the correlation between cellular height (as a measure of apical–basal polarization) and TEER in fully differentiated routine cultures (biological variation) and in cultures with altered cellular height due to hormonal stimulation. Fully differentiated cultures possessed a moderate TEER between 500 and 1100 Ω*cm2. Only 5 % of cultures which exhibited TEER values in this defined range had poor quality. Sub-differentiated cultures showed either very low or excessively high TEER. We unveiled a highly significant (P < 0.0001) negative linear correlation between TEER and epithelial height in well-differentiated cultures (both routine and hormone stimulated group). This may point toward the interaction between tight junction assembly and epithelial apical–basal polarization. In conclusion, TEER is a straightforward quality indicator which could be routinely used to monitor the differentiation status of oviduct epithelial cells in vitro.Electronic supplementary materialThe online version of this article (doi:10.1007/s00418-015-1351-1) contains supplementary material, which is available to authorized users.

Long-term culture of primary porcine oviduct epithelial cells: Validation of a comprehensive in vitro model for reproductive science

Recently, we established a protocol for the cultivation of primary porcine oviduct epithelial cells (POEC), which promoted tissue-like morphology for a prolonged culture period. The present study focuses on developing this model into a comprehensive, standardized culture system, as a candidate tool for reproductive toxicity testing and basic research. We cultivated POEC isolated from 25 animals in our culture system for both 3 and 6 weeks and systematically analyzed effects of medium conditioning, supplementation with standardized sera, and culture duration in both freshly isolated and cryopreserved cells. The differentiation status was evaluated via histomorphometry, transepithelial electrical resistance (TEER) measurement, and expression analyses. The culture system possessed high reproducibility, more than 95% of cultures achieved a fully differentiated phenotype. Cells recapitulated in vivo–like morphology and ultrastructure from 3 to 6 weeks. Cryopreservation of the cells prior to cultivation did not affect culture quality of POEC. Employment of conditioned medium ensured optimal promotion of POEC differentiation, and different standardized sera induced fully differentiated phenotypes. Consistent TEER establishment indicated the presence and maintenance of cell type–specific intercellular junctions. The functionality of POEC was proven by consistent mucin secretion and stable expression of selected markers over the whole culture duration. We conclude that POEC are suitable for experiments from 3 weeks up to at least 6 weeks of culture. Therefore, this culture system could be used for in vitro estrous cycle simulation and long-term investigation of toxic effects on oviduct epithelium.

Simulation of the estrous cycle in a long-term culture system for porcine oviductal epithelial cells (POECs)

Simulation of the estrous cycle in a long-term culture system for porcine oviductal epithelial cells (POECs)

Effect of a pre-freezing treatment with cholesterol-loaded cyclodextrins on boar sperm longevity, capacitation dynamics, ability to adhere to porcine oviductal epithelial cells in vitro and DNA fragmentation dynamics

The aim of this work was to examine how a pre-freezing treatment with cholesterol-loaded cyclodextrins (CLC) affects boar sperm longevity, capacitation dynamics, ability to bind to a porcine telomerase-immortalised oviductal epithelial cell line (TERT-OPEC) in vitro and DNA integrity dynamics after freeze-thawing. Although the samples treated with CLC exhibited lower sperm quality than the control samples (P<0.05) immediately after thawing, these differences disappeared (P>0.05) after long-term incubation (26h at 37 or 16°C). Additionally, the CLC-treated spermatozoa underwent similar capacitation and DNA fragmentation dynamics as the control spermatozoa (P>0.05). However, CLC-treated spermatozoa were better able to bind to TERT-OPEC in vitro (P<0.0001). In conclusion, the pre-freezing treatment of boar spermatozoa with CLC enhanced the ability of the spermatozoa to bind to TERT-OPEC in vitro, which could have an effect on the establishment of the sperm reservoir in the ampullary--isthmic junction in vivo. Additionally, frozen-thawed spermatozoa can be stored at 16°C for at least 6h without a significant observable decline in sperm quality, which could be beneficial for the transport of thawed diluted doses of spermatozoa from the laboratory to the farm.

Modelling the porcine oviduct epithelium: A polarized in vitro system suitable for long-term cultivation

For exploring the processes leading to successful reproduction, differentiated long-term in vitro systems modelling the mammalian oviduct are needed. Therefore, in the present study culture conditions for primary porcine oviductal epithelial cells were optimized with regard to morphological differentiation and usability for extended cultivation periods. To evaluate different growth media for the primary cells, we used morphological criteria as well as real-time impedance measurement. After an initial media testing, the cells were grown on hanging membranes and the culture settings (conventionally cultured, serum gradient over the membrane and air-liquid interface) were assessed by histology and electron microscopy. We proved long-term expression of an oviduct specific marker (oviductal glycoprotein 1) and showed a hormone responsiveness of the culture system by means of quantitative reverse transcription-PCR. Differentiated epithelial cells could reproducibly be cultured up to 6 weeks in an air-liquid interface. After 3 weeks of culturing, the cells were clearly polarized and exhibited cilia. The model maintains physiological properties such as morphological features (mixed cell population of ciliated and secretory cells, apical cell-cell contacts typical for columnar epithelial cells) and oviduct-specific markers showing hormone responsiveness. We established a polarized long-term in vitro-system of the porcine oviductal epithelium preserving detailed features of the porcine oviduct. Therefore, we provide a useful tool to elucidate unsolved scientific questions concerning reproductive physiology.

The Effects of Fulvic Acid on Copper Bioavailability to Porcine Oviductal Epithelial Cells

This study assessed the effect of dissolved organic matter on the copper (Cu) bioavailability to mammalian cells, porcine oviductal epithelial cells (POEC), in order to imply its effect onto humans. Cu toxicity was investigated in the presence of with and without fulvic acid (FA). Dissociation and exchange rate constants were calculated by using competing ligand Chelex-100, and optical parameters were employed to help explain the complexation of their aromatic and aliphatic structures. Their morphological change was observed using transmission electron microscope (TEM), and Cu species were calculated using MINTEQA2 program. The results showed that the dissociation rate constant of Cu(2+)-FA was equal to 9.08 x 10(-4) s(-1), which was slower than the exchange rate at 1.95 x 10(-3) s(-1). Although Cu-FA was significantly absorbed into the cells higher than Cu(2+), it showed less damage than tested with Cu(2+). TEM and optical studies showed many aggregations around nucleus suggesting the amphipathic character of FA helped binging to the nuclear surfaces of both Cu-FA and FA treatments. Even though the MINTEQA2 calculations showed that there was free Cu(2+) in the mixed solutions around 39.2%, it could not bind with the cell surface. This suggested that the effect of FA was strong and had a lot of influence on the living surface of POEC, modifying the effect of Cu toxicity.

Effects of Donor Cell Passage, Size and Type on Development of Porcine Embryos Derived from Somatic Cell Nuclear Transfer

The aim of this study was to investigate the effects of donor cell passage, size and type on the development of nuclear transfer embryos. Porcine cumulus cells, fetal fibroblasts and oviductal epithelial cells from 1-2, 3-6 and 7-10 passages were used for the nuclear transfer. In the oocytes with the cumulus donor cells, fusion and cleavage rates of oocytes and cell numbers per blastocyst among the three different passage groups did not show any differences, but the rates of blastocyst formation from 1-2 and 3-6 passage groups were higher than those from 7-10 passage group. The rates of fusion, cleavage and blastocyst formation, and the cell numbers per blastocyst were higher in the embryos with the sizes of cumulus donor cells compared to the >20 cumulus donor cell. In the oocytes with the fetal fibroblast donor cells, the rate of blastocyst formation from the 3-6 passage group was higher than from 1-2 and 7-10 passage groups. The embryos with the size of 20 fetal fibroblast donor cell showed higher rate of blastocyst formation compared to those with 20 donor cells. In the oocytes with the oviductal epithelial cells, the rates of blastocyst formation from 1-2 and 3-6 passage groups were higher compared to those from 7-10 passage group. The embryos with the sizes of oviductal epithelial donor cells had a higher rate of blastocyst formation compared to those with >20 donor cell. Fusion and cleavage rates of oocytes, and cell numbers per blastocyst among the three different donor cell types from the 3-6 passage did not show any differences. However, the rate of blastocyst formation of somatic cell nuclear transfer (SCNT) embryos with the fetal fibroblast donor cell was higher than that of blastocyst formation of SCNT embryos with the cumulus and oviductal epithelial donor cells.

In vitro toxicology assessment of cadmium bioavailability on primary porcine oviductal epithelial cells

This study assessed the in vitro effects of cadmium ion (Cd 2+) and cadmium–fulvic acid complexes (Cd 2+–FA) on porcine oviductal epithelial cells (POEC) using transmission electron microscopy (TEM). Fresh POEC were cultured for 24 h and then exposed for 3 h in the tested solutions. Absorbed cadmium was analyzed by graphite furnace atomic absorption spectrophotometer (AAS). Dissociation and exchange rate constants were determined using the competing ligand, Chelex-100 and predicted cadmium species were calculated using the MINTEQA2 program. The results showed that the dissociation rate constant of Cd 2+–FA was equal to 1.023 × 10 −3 s −1 which was slower than the exchange rate at 2.062 × 10 −3 s −1. Nevertheless, the absorbed concentrations of Cd 2+ and Cd 2+–FA of POEC were similar and were 47 ± 6.25 μg L −1 and 54 ± 3.61 μg L −1, respectively. Although both levels of absorptions were not significantly different ( t-test p = 0.168 at α 0.01), their morphological effects as examined by TEM were substantially different with the effects being most marked with Cd 2+ > Cd 2+–FA > FA. Aggregations around nuclei and nuclear membranes were observed with FA treatment whilst Cd 2+–FA treatment produced more cytoplasmic damage. Cd 2+ treatment caused nuclear deformities. In conclusion, FA appears to penetrate the cells but was less likely to enter the nucleus. It also reduced the toxicity of Cd 2+ as the nuclei from the Cd 2+–FA treatment appeared normal. Nevertheless, some Cd 2+ could still enter the nucleus. This might be because there was still 67.8% Cd 2+ left unbound from the Cd 2+–FA treatment as calculated from the MINTEQA2 program compared to 99.1% Cd 2+ with Cd 2+ treatment, thus underlining the inherent toxicity of soluble cadmium ion.

Characterization of porcine oviductal epithelial cells, cumulus cells and granulosa cells-conditioned media and their ability to induce acrosome reaction on frozen-thawed bovine spermatozoa

The mammalian female reproductive tract cells have been wildely used in in vitro fertilization. We studied the secretory proteins of porcine oviductal epithelial cells (POEC), and cumulus cells (CC) co-cultured with granulosa cells (GC) in conditioned media (CM), and their ability in inducing acrosome reaction (AR) on frozen-thawed bovine spermatozoa. POEC and CC + GC were cultured in M 199 medium for 48, 96 and 144 h prior to investigation of protein secretion. The results from SDS-PAGE showed secretory proteins sizes of about 17, 22 and >220 kDa in both CM from POEC and CC + GC. To test the ability of CM from POEC and CC + GC in inducing AR, the CM was frozen for 1–3 months before incubating with frozen-thawed bovine spermatozoa. Percentages of the spermatozoa with AR were determined under an inverted microscope and it was found that the tested group 1 (incubated with fresh and frozen CM from POEC for 1–3 months) were 78.44 ± 7.25, 75.78 ± 4.41, 65.22 ± 5.59 and 50.56 ± 6.25, respectively. The tested group 2 (incubated with fresh and frozen CM from CC + GC for 1–3 months) had the percentages of the spermatozoa with AR at 88.67 ± 4.03, 82.22 ± 3.46, 71.00 ± 3.16, and 58.56 ± 4.69, respectively. Statistical analysis of all group percentages indicated that they were significantly different ( P < 0.05). Transmission electron microscope studies demonstrated that there were morphological changes on the sperm heads which resulted in the leakage of acrosin and subsequent AR. We concluded that the CM from both POEC and CC + GC efficiently increased in vitro AR on frozen-thawed bovine spermatozoa. Identification and functions of these secretory proteins are currently under investigation which may lead us to a better understanding of other beneficial effects in the utilization of both CM conditions.

In vivo and in vitro study of porcine oviductal epithelial cells, cumulus oocyte complexes and granulosa cells: A scanning electron microscopy and inverted microscopy study

The morphology and structure of porcine oviductal epithelial cells (POEC), cumulus-oocyte complexes (COCs) and granulosa cells (GC) were investigated in vivo and in vitro conditions using scanning electron microscopy (SEM) and inverted microscopy. The POEC contained columnar ciliated cells and spherical shaped non-ciliated cells. Both non- and ciliated cells appeared either in groups or distributing among each other. However, the isolation of cells was observed after culture for 48 h. A total of 921 oocytes from 20 ovaries was isolated resulting in an average of 46 oocytes per ovary. They were round in shape, surrounded by zona pellucida with layers of cumulus cells ranging between 89.16 and 144.68 μm in size. As for COCs, they were classified into 4 types; intact-, multi-, partial-cumulus cell layers and completely denuded oocyte. Interestingly, changes in morphology of COCs with intact and multi-cumulus cell layers were observed in the in vitro study. The GCs in the follicular fluid were also round in shape and found as clusters. After culturing in in vitro for 48 h, no change in morphology was observed. The GC appeared in smaller clusters or were present as single cells and their sizes ranged from 6 to 8 μm. The results obtained from this study allow us to have a better understanding of the morphology and nature of cells under both in vivo and in vitro conditions. This information is also important for the study of their secretions and biochemical compositions, which is of great importance to the use of cells as feeder cells in in vitro fertilization in current studies.

396 CHANGES OF PLASMINOGEN ACTIVATOR ACTIVITY IN OVIDUCTAL EPITHELIAL CELLS DURING THE ESTROUS CYCLE IN PIGS

Plasminogen activator (PA) activity in rat, porcine, and bovine embryos and in oocytes have been demonstrated, but reports of PA activity in the uterus and oviduct are limited. The present study was undertaken to identify changes of PAs in porcine oviductal epithelial cells (pOECs) during the estrous cycle in pigs. pOECs obtained from pigs in post-ovulatory (Post-Ov), early-to-mid luteal (Early-mid L), and pre-ovulatory (Pre-Ov) stages were washed twice with Hank&amp;apos;s balanced salt solution (HBSS); the oviductal lumen was flushed with 10 mL HBSS, and centrifuged at 300g for 10 min at 4�C. The collected pOECs were washed twice with DMEM containing 0.1% BSA and centrifuged at 300g for 10 min at 4�C. To examine the changes in activity of PAs in pOEC, 2.0 � 105 fresh cells were cultured in a 4-well dish with DMEM/Ham&amp;apos;s F-12 medium containing inactivated 10% FBS and amphotericin under a humidified atmosphere of 5% CO2 in air at 38�C for 1, 3, 5, and 7 days. The culture medium was replaced every 48 h with fresh medium during culture for 7 days, after which cells and conditioned medium were stored separately at -20�C until they were used for zymographic analysis. PA activities of pOECs were measured using SDS-PAGE, casein-agar zymography, and densitometry. The urokinase-type PA (uPA) was observed at 5–7 days of culture in pOECs of different stages of the estrous cycle in pigs. On the other hand, uPA, tissue-type (tPA), and tPA-PA inhibitor (tPA-PAI) activities were observed at days 1, 3, 5, and 7 of culture in medium conditioned with pOEC from different stages of the estrous cycle. uPA activity was decreased during prolonged culture of Post-Ov pOECs. At this time, uPA activity was significantly (P &amp;lt; 0.05) higher at 1 day than at 3–7 days of culture in Post-Ov pOEC-conditioned medium. tPA activity was significantly (P &amp;lt; 0.05) higher at 5 days than at 1–3 days or 7 days of culture in Post-Ov pOEC-conditioned medium. tPA-PAI activity was significantly (P &amp;lt; 0.05) higher at 7 days than at 5 days of culture in Pre-Ov pOEC-conditioned medium, and tPA-PAI activity was significantly (P &amp;lt; 0.05) higher at Post-Ov than Pre-Ov or Early-mid L in 7 days of culture in pOEC-conditioned medium of different stages of the estrous cycle. All tPA activities were increased until Day 5 and were decreased at Day 7 of culture in pOEC-conditioned medium. uPA activity was observed in pOEC-conditioned medium throughout the estrous cycle. uPA activity was significantly (P &amp;lt; 0.05) different during culture of Post-Ov in pOECs-conditioned medium. tPA-PAI activity was observed at all stages, and tPA-PAI activity was significantly (P &amp;lt; 0.05) different among different stages of the estrous cycle. These results suggest that uPA, tPA, and tPA-PAI are produced by pOEC, and that variations of PA activity are found at different stages of the estrous cycle in porcine oviductal epithelial cells. This work was supported by grant No. R01-2003-000-10500-0 from the Basic Research Program of Korea Science &amp;amp; Engineering Foundation.

Effects of type and state of co-culture cells on in-vitro development of porcine oocytes matured and fertilized in vitro

The present study was to investigate the impact of type and state of co-culture cells on developmental competence of porcine oocytes matured and fertilized in vitro. Porcine zygotes were co-cultured with granulosa cells (GCs) (Group 1) or porcine oviductal epithelial cells (pOECs) at follicular stage (Group 2), ovulation stage (Group 3) or corpus luteum (CL) stage (Group 4) or cultured in a medium without co-culture cells (control group). The proportion of oocytes developed to 2-cell stage embryos in Group 2 was similar to that in control group, but significantly (p < 0.05) lower than that in Groups 1, 3 and 4. The proportions of oocytes developed to > or = 4-cell stage embryos in Groups 3 and 4 were significantly (p < 0.05) higher than that in Groups 1 and 2. At 144 h after insemination, 12.0, 14.8 and 20.0% of oocytes developed to blastocysts in Groups 1, 3 and 4, respectively. However, no embryos in control group developed beyond 4-cell stage and no embryos in Group 2 developed to blastocyst stage. As compared with GCs and pOECs at follicular stage, the pOECs at ovulation and CL stages had a better competence to support porcine embryo development under in vitro conditions.

Immunophenotype of porcine oviduct epithelial cells during the oestrous cycle: a double-labelling immunohistochemical study

The luminal epithelium of the porcine oviduct is composed of ciliated cells and secretory cells, but it is assumed for several species that under the control of steroid hormones secretory cells are able to be transformed into ciliated cells. In order to better understand such physiological changes during the different stages of the oestrous cycle, we evaluated epithelial cell proliferation together with oestrogen receptor alpha (ERalpha) expression of porcine ampullary oviducts. To identify the immunophenotype of proliferating cells, double immunohistochemistry was performed using anti-chromogranin A antibody (anti-CgA) as the second primary antibody. Anti-CgA, recently shown to be an immunocytochemical marker of ciliated cells of the cow, also labelled specifically the luminal surface of ciliated cells of the pig. Double labelling of sections with the monoclonal antibody MIB-1 against the proliferation-associated nuclear epitope Ki-67 and anti-CgA clearly demonstrates that MIB-1 was selectively localised in the nuclei of secretory cells. Proliferative activity was not observed in CgA-positive ciliated cells in all examined stages of the oestrous cycle. The percentage of Ki-67-positive epithelial cells was higher at pro-oestrus, compared with the other stages of the oestrous cycle. Furthermore, ERalpha immunoreactivity was exclusively detected in the nuclei of the epithelial cells, which were negative for CgA. We conclude, therefore, that oestrogen may induce the initial proliferation of secretory cells and promote the differentiation into ciliated cells.