Porcine Luteal Cells Research Articles

Visfatin impact on the proteome of porcine luteal cells during implantation

Visfatin (VIS) is a hormone belonging to the adipokines’ group secreted mainly by the adipose tissue. VIS plays a crucial role in the control of energy homeostasis, inflammation, cell differentiation, and angiogenesis. VIS expression was confirmed in the hypothalamic–pituitary–gonadal (HPG) axis structures, as well as in the uterus, placenta, and conceptuses. We hypothesised that VIS may affect the abundance of proteins involved in the regulation of key processes occurring in the corpus luteum (CL) during the implantation process in pigs. In the present study, we performed the high-throughput proteomic analysis (liquid chromatography with tandem mass spectrometry, LC–MS/MS) to examine the in vitro influence of VIS (100 ng/mL) on differentially regulated proteins (DRPs) in the porcine luteal cells (LCs) on days 15–16 of pregnancy (implantation period). We have identified 511 DRPs, 276 of them were up-regulated, and 235 down-regulated in the presence of VIS. Revealed DRPs were assigned to 162 gene ontology terms. Western blot analysis of five chosen DRPs, ADAM metallopeptidase with thrombospondin type 1 motif 1 (ADAMTS1), lanosterol 14-α demethylase (CYP51A1), inhibin subunit beta A (INHBA), notch receptor 3 (NOTCH3), and prostaglandin E synthase 2 (mPGES2) confirmed the veracity and accuracy of LC–MS/MS method. We indicated that VIS modulates the expression of proteins connected with the regulation of lipogenesis and cholesterologenesis, and, in consequence, may be involved in the synthesis of steroid hormones, as well as prostaglandins’ metabolism. Moreover, we revealed that VIS affects the abundance of protein associated with ovarian cell proliferation, differentiation, and apoptosis, as well as CL new vessel formation and tissue remodelling. Our results suggest important roles for VIS in the regulation of ovarian functions during the peri-implantation period.

Visfatin Affects the Transcriptome of Porcine Luteal Cells during Early Pregnancy.

Visfatin/NAMPT (VIS), the hormone exerting a pleiotropic effect, is also perceived as an important factor in the regulation of reproductive processes and pregnancy maintenance. Previous studies confirmed its involvement in the control of porcine pituitary and ovary function. In this study, we hypothesized that VIS may affect the global transcriptome of luteal cells and thus regulate the functioning of the ovaries. Illumina's NovaSeq 6000 RNA sequencing was performed to investigate the differentially expressed genes (DEGs) and long non-coding RNAs (DELs) as well as the occurrence of differential alternative splicing events (DASs) in the porcine luteal cells exposed to VIS (100 ng/mL) during the implantation period. The obtained results revealed 170 DEGs (99 up- and 71 downregulated) assigned to 45 functional annotations. Moreover, we revealed 40 DELs, of which 3 were known and 37 were described for the first time. We identified 169 DASs events. The obtained results confirmed a significant effect of VIS on the transcriptome and spliceosome of luteal cells, including the genes involved in the processes crucial for successful implantation and pregnancy maintenance as angiogenesis, steroidogenesis, inflammation, cell development, migration, and proliferation.

CRISPR/Cas genome editing revealed non-angiogenic role of VEGFA gene in porcine luteal cells: a preliminary report.

The angiogenic cytokine vascular endothelial growth factor A (VEGFA) also exerts non-angiogenic effects on endocrine functionality of porcine luteal cells critical for progesterone (P4) production. The expression dynamics of VEGFA-FLT/KDR system were investigated using RT-qPCR during luteal stages and VEGFA gene knock out (KO) porcine luteal cells were generated using CRISPR/Cas9 technology. The downstream effects of VEGFA ablation were studied using RT-qPCR, Annexin V, MTT, ELISA for P4 estimation and scratch wound assay. Bioinformatics analysis of RNA-Seq data of porcine mid-luteal stage was conducted for exploring protein-protein interaction network, KEGG pathways, transcription factors and kinase mapping for VEGFA-FLT/KDR interactomes. The VEGFA-FLT/KDR system expressed throughout the luteal stages with highest expression during mid- luteal stage. Cellular morphology, structure and oil-red-o staining for lipid droplets did not differ significantly between VEGFA KO and wild type cells, however, VEGFA KO significantly decreased (p < 0.05) viability and proliferation efficiency of edited cells on subsequent passages. Expression of apoptotic gene, CASP3 and hypoxia related gene, HIF1A were significantly (p < 0.05) upregulated in KO cells. The relative mRNA expression of VEGFA and steroidogenic genes STAR, CYP11A1 and HSD3B1 decreased significantly (p < 0.05) upon KO, which was further validated by the significant (p < 0.05) decrease in P4 output from KO cells. Bioinformatics analysis mapped VEGFA-FLT/KDR system to signalling pathways associated with steroidogenic cell functionality and survival, which complemented the findings of the study. The ablation of VEGFA gene resulted in decreased steroidogenic capability of luteal cells, which suggests that VEGFA exerts additional non-angiogenic regulatory effects in luteal cell functionality.

Phoenixin-14 as a novel direct regulator of porcine luteal cell functions†.

Phoenixin is a neuropeptide with a well-established role in the central regulation of reproductive processes; however, knowledge regarding its role in the ovary is limited. One of the main active phoenixin isoforms is phoenixin-14, which acts through G protein-coupled receptor 173. Our research hypothesis was that phoenixin-14 is expressed in porcine corpus luteum and exerts luteotropic action by affecting the endocrine function of luteal cells through G protein-coupled receptor 173 and protein kinase signaling. Luteal cells were cultured to investigate the effect of phoenixin-14 (1-1000nM) on endocrine function. We showed that phoenixin-14 and G protein-coupled receptor 173 are produced locally in porcine corpus luteum and their levels change during the estrous cycle. We detected phoenixin-14 immunostaining in the cytoplasm and G protein-coupled receptor 173 in the cell membrane. Plasma phoenixin levels were highest during the early luteal phase. Interestingly, insulin, luteinizing hormone, progesterone, and prostaglandins decreased phoenixin-14 levels in luteal cells. Phoenixin-14 increased progesterone, estradiol, and prostaglandin E2 secretion, but decreased prostaglandin F2α, upregulated the expression of steroidogenic enzymes, and downregulated receptors for luteinizing hormone and prostaglandin. Also, phoenixin-14 increased the expression of G protein-coupled receptor 173 and the phosphorylation of extracellular signal-regulated kinase 1/2, protein kinase B, inhibited the phosphorylation of protein kinase A, and had mixed effect on AMP-activated protein kinase alpha and protein kinase C. G protein-coupled receptor 173 and extracellular signal-regulated kinase 1/2 mediated the effect of phoenixin-14 on endocrine function of luteal cells. Our results suggest that phoenixin is produced by porcine luteal cells and can be a new regulator of their function.

Resistin as A New Regulator of Luteolysis: in Vitro Effect of Resistin on Proliferation, Apoptosis, and Autophagy in Porcine Luteal Cells

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Resistin as a new player in the regulation of porcine corpus luteum luteolysis: in vitro effect on proliferation/viability, apoptosis and autophagy.

The formation and luteolysis of the corpus luteum (CL) is strictly controlled by many factors. Imbalance between proliferation and apoptosis processes leads to deficiency of the luteal phase and infertility. Our previous study showed resistin expression in porcine luteal cells and an inhibitory effect on progesterone synthesis. Thus, the aim of the present study was to examine the in vitro effect of resistin on the proliferation/viability, apoptosis and autophagy of porcine luteal cells as well as the involvement of mitogen-activated kinase (MAP3/1), protein kinase B (AKT) and signal transducer and activator of transcription 3 (STAT3) in these processes. Porcine luteal cells were incubated with resistin (0.1-10 ng/mL) for 24-72 h and viability was assessed using the alamarBlue or 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Then, the time-dependent effect of resistin on mRNA and protein expression of proliferating cell nuclear antigen (PCNA), caspase 3, BCL2-like protein 4 (BAX), B-cell lymphoma 2 (BCL2), beclin1, microtubule-associated protein 1A/1B-light chain 3 (LC3) and lysosomal-associated membrane protein 1 (LAMP1) was measured by real-time polymerase chain reaction (PCR) and immunoblotting, respectively. We found that resistin enhanced luteal cell viability with no effect on caspase 3 mRNA and protein, increased the BAX/BCL2 mRNA and protein ratio and significantly stimulated the initiation of autophagy, which promotes the maintenance of CL function rather than its regression. Additionally, using pharmacological inhibitors of MAP3/1 (PD98059), AKT (LY294002) and STAT3 (AG490), we observed that the effect of resistin was reversed to the control level in viability and, by influence, MAP3/1 and STAT3 in autophagy. Taken together, our results suggest that resistin, in addition to its well-known effect on granulosa cell function has direct influence on CL luteolysis and the formation and maintenance of luteal cell function.

Resistin as a new player in the regulation of porcine corpus luteum luteolysis: in vitro effect on proliferation/viability, apoptosis and autophagy.

The formation and luteolysis of the corpus luteum (CL) is strictly controlled by many factors. Imbalance between proliferation and apoptosis processes leads to deficiency of the luteal phase and infertility. Our previous study showed resistin expression in porcine luteal cells and an inhibitory effect on progesterone synthesis. Thus, the aim of the present study was to examine the in vitro effect of resistin on the proliferation/viability, apoptosis and autophagy of porcine luteal cells as well as the involvement of mitogen-activated kinase (MAP3/1), protein kinase B (AKT) and signal transducer and activator of transcription 3 (STAT3) in these processes. Porcine luteal cells were incubated with resistin (0.1-10 ng/mL) for 24-72 h and viability was assessed using the alamarBlue or 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Then, the time-dependent effect of resistin on mRNA and protein expression of proliferating cell nuclear antigen (PCNA), caspase 3, BCL2-like protein 4 (BAX), B-cell lymphoma 2 (BCL2), beclin1, microtubule-associated protein 1A/1B-light chain 3 (LC3) and lysosomal-associated membrane protein 1 (LAMP1) was measured by real-time polymerase chain reaction (PCR) and immunoblotting, respectively. We found that resistin enhanced luteal cell viability with no effect on caspase 3 mRNA and protein, increased the BAX/BCL2 mRNA and protein ratio and significantly stimulated the initiation of autophagy, which promotes the maintenance of CL function rather than its regression. Additionally, using pharmacological inhibitors of MAP3/1 (PD98059), AKT (LY294002) and STAT3 (AG490), we observed that the effect of resistin was reversed to the control level in viability and, by influence, MAP3/1 and STAT3 in autophagy. Taken together, our results suggest that resistin, in addition to its well-known effect on granulosa cell function has direct influence on CL luteolysis and the formation and maintenance of luteal cell function.

Coculture with porcine luteal cells during in vitro porcine oocyte maturation affects lipid content, cortical reaction and zona pellucida ultrastructure.

In pigs, in vitro fertilisation (IVF) is associated with high polyspermy rates, and for this reason, in vitro embryo production (IVP) is still an inefficient biotechnology. Coculture with somatic cells is an alternative to improve suboptimal in vitro maturation (IVM) conditions. This study was conducted to test a coculture system of porcine luteal cells (PLC) and cumulus-oocyte complexes (COC) to improve oocyte metabolism. COC were matured in vitro with PLC. Oocyte lipid content, mitochondrial activity, zona pellucida (ZP) digestibility and pore size, cortical reaction and in vitro embryo development were assessed. Coculture reduced cytoplasmic lipid content in the oocyte cytoplasm without increasing mitochondrial activity. Although ZP digestibility and ZP pore number were not different between culture systems, ZP pores were smaller in the coculture. Coculture impacted the distribution of cortical granules as they were found immediately under the oolemma, and more of them had released their content in the ZP. Coculture with porcine luteal cells during IVM increased monospermic penetration and embryo development after IVF. The coculture of COC with PLC affects the metabolism of the oocyte and benefits monospermic penetration and embryo development. The coculture system with PLC could be an alternative for the conventional maturation medium in pigs.

Androgen receptor gene deficiency results in the reduction of steroidogenic potential in porcine luteal cells

Luteal steroidogenesis is critical to implantation and pregnancy maintenance in mammals. The role of androgen receptors (AR) in the progesterone (P4) producing luteal cells of porcine corpus luteum (CL) remains unexplored. The aim of the present study was to establish AR gene knock out (KO) porcine luteal cell culture system model by CRISPR/Cas9 genome editing technology and to study the downstream effects of AR gene deficiency on steroidogenic potential and viability of luteal cells. For this purpose, genomic cleavage detection assay, microscopy, RT-qPCR, ELISA, annexin, MTT, and viability assay complemented by bioinformatics analysis were employed. There was significant downregulation (p < 0.05) in the relative mRNA expression of steroidogenic marker genes STAR, CYP11A1, HSD3B1 in AR KO luteal cells as compared to the control group, which was further validated by the significant (p < 0.05) decrease in the P4 production. Significant decrease (p < 0.05) in relative viability on third passage were also observed. The relative mRNA expression of hypoxia related gene HIF1A was significantly (p < 0.05) downregulated in AR KO luteal cells. Protein-protein interaction analysis mapped AR to signaling pathways associated with luteal cell functionality. These findings suggests that AR gene functionality is critical to luteal cell steroidogenesis in porcine.

Chemerin Impact on Alternative mRNA Transcription in the Porcine Luteal Cells.

Chemerin participates in the regulation of processes related to physiological and disorder mechanisms in mammals, including metabolism, obesity, inflammation, and reproduction. In this study, we have investigated chemerin influence on alternative mRNA transcription within the porcine luteal cell transcriptome, such as differential expression of long non-coding RNAs (DELs) and their interactions with differentially expressed genes (DEGs), differences in alternative splicing of transcripts (DASs), and allele-specific expression (ASEs) related to the single nucleotide variants (SNVs) frequency. Luteal cells were collected from gilts during the mid-luteal phase of the oestrous cycle. After in vitro culture of cells un-/treated with chemerin, the total RNA was isolated and sequenced using the high-throughput method. The in silico analyses revealed 24 DELs cis interacting with 6 DEGs and trans-correlated with 300 DEGs, 137 DASs events, and 18 ASEs. The results enabled us to analyse metabolic and signalling pathways in detail, providing new insights into the effects of chemerin on the corpus luteum functions related to inflammatory response, leukocyte infiltration, the occurrence of luteotropic and luteolytic signals (leading to apoptosis and/or necroptosis). Validation of the results using qPCR confirmed the predicted expression changes. Chemerin at physiological concentrations significantly modifies the transcription processes in the porcine luteal cells.

Chemerin as a modulator of angiogenesis and apoptosis processes in the corpus luteum of pigs: an in vitro study†.

The corpus luteum (CL) undergoes rapid changes, and its functional capabilities are influenced by processes such as angiogenesis and apoptosis. According to the literature, chemerin-a protein that participates in the regulation of energy homeostasis and the immune response, may also affect angiogenesis and apoptosis. Therefore, the aim of this study was to investigate the in vitro effect of chemerin on angiogenesis and apoptosis in porcine luteal cells (Lc) during specific phases related to CL physiology. Luteal cells were harvested from gilts during the early-, mid-, and late-luteal phases of the estrous cycle. The cells were preincubated for 48h and incubated for 24h with chemerin or a serum-free medium (controls). The abundance of angiogenesis- and apoptosis-related proteins was determined by enzyme-linked immunosorbent assay (ELISA) in spent culture media, or by ELISA and Western blot in protein extracts. The current study demonstrated that chemerin stimulates the production of vascular endothelial growth factor A (VEGF-A) and basic fibroblast growth factor (bFGF) by porcine Lc and increases the protein abundance of angiogenic factors' receptors (VEGFR1, VEGFR2, VEGFR3, FGFR1, FGFR2) in these cells. The study also revealed that chemerin exerts a modulatory effect (stimulatory/inhibitory, depending on the phase of the cycle) on the protein abundance of first apoptosis signal (Fas), Fas ligand, B-cell lymphoma 2, and caspase-3 in porcine Lc. These results imply that chemerin may affect angiogenesis and apoptosis processes in the porcine CL, as evidenced by its modulatory effect of chemerin on the protein abundance of crucial angiogenesis- and apoptosis-related factors, observed in an in vitro study of porcine Lc.

Coculture of porcine luteal cells during in vitro porcine oocyte maturation affects blastocyst gene expression and developmental potential

Oocyte maturation in culture is still the weakest part of in vitro fertilization (IVF) and coculture with somatic cells may be an alternative to improve suboptimal culture conditions, especially in the pig in which maturation takes more than 44 h. In the present study, we investigated the effect of a coculture system of porcine luteal cells (PLC) during in vitro maturation (IVM) on embryo development and gene expression. Cumulus-oocyte complexes were matured in vitro in TCM-199 with human menopausal gonadotrophin (control) and in coculture with PLC. IVF was performed with frozen-thawed boar semen in Tris-buffered medium. Presumptive zygotes were cultured in PZM for 7 days. The coculture with PLC significantly increased blastocysts rates. Gene expression changes were measured with a porcine embryo-specific microarray and confirmed by RT-qPCR. The global transcription pattern of embryos developing after PLC coculture exhibited overall downregulation of gene expression. Following global gene expression pattern analysis, genes associated with lipid metabolism, mitochondrial function, endoplasmic reticulum stress, and apoptosis were found downregulated, and genes associated with cell cycle and proliferation were found upregulated in the PLC coculture. Canonical pathway analysis by Ingenuity Pathway revealed that differential expression transcripts were associated with the sirtuin signaling pathway, oxidative phosphorylation pathway, cytokines and ephrin receptor signaling. To conclude, the coculture system of PLC during IVM has a lasting effect on the embryo until the blastocyst stage, modifying gene expression, with a positive effect on embryo development. Our model could be an alternative to replace the conventional maturation medium with gonadotrophins with higher rates of embryo development, a key issue in porcine in vitro embryo production.

Novel Insights on the Corpus Luteum Function: Role of Vaspin on Porcine Luteal Cell Angiogenesis, Proliferation and Apoptosis by Activation of GRP78 Receptor and MAP3/1 Kinase Pathways.

Formation and limited lifespan of corpus luteum (CL) are important for proper ovarian periodicity and fertility. Failed vascularization, imbalance between proliferation and apoptosis leads to luteal phase deficiency and infertility. The aim of this study was to examine the effect of vaspin on angiogenesis, apoptosis and proliferation as well as the involvement of 78-kDa glucose-regulated protein receptor (GRP78) and mitogen-activated kinase (MAP3/1) in these processes. Porcine luteal cells were incubated with vaspin (0.1–10 ng/mL) for 24 h to 72 h and then mRNA and protein expression of angiogenesis: vascular endothelial growth factor (VEGFA), fibroblast growth factor 2 (FGF2), angiopoietin 1 (ANGPT1), VEGFA receptors (VEGFR1, VEGFR2), apoptosis: caspase 3, bcl-2-like protein 4 (BAX), B-cell lymphoma (BCL2), and proliferation: proliferating cells nuclear antigen (PCNA), cyclin A factors as well as secretion of VEGFA, FGF2, ANGT1 were measured by real-time polymerase chain reaction (PCR), immunoblotting and enzyme-linked immunosorbent assay (ELISA), respectively. Moreover, apoptosis was assessed by caspase activity using the Caspase-Glo 3/7 assay, while proliferation was by alamarBlue. We found that vaspin enhanced luteal cell angiogenesis, proliferation, and significantly decreased apoptosis. Additionally, using GRP78 siRNA and the pharmacological inhibitor of MAP3/1 (PD98059), we observed that the effect of vaspin was reversed to the control level in all investigated processes. Taken together, our results suggest that vaspin is a new regulator of female fertility by direct regulation of CL formation and maintenance of luteal cell function.

Transcription Analysis of the Chemerin Impact on Gene Expression Profile in the Luteal Cells of Gilts.

Chemerin is a recently discovered adipokine that participates in the regulation of many physiological and disorder-related processes in mammals, including metabolism, inflammatory reactions, obesity, and reproduction. We investigated how chemerin affects the transcriptome profile of porcine luteal cells. The luteal cells were acquired from mature gilts. After the in vitro culturing with and without chemerin, the total RNAs were isolated and high-throughput sequencing was performed. Obtained datasets were processed using bioinformatic tools. The study revealed 509 differentially expressed genes under the chemerin influence. Their products take part in many processes, important for the functions of the corpus luteum, such as steroids and prostaglandins synthesis, NF-κB and JAK/STAT signal transducing pathways, and apoptosis. The expression of the CASP3, HSD3B7, IL1B, and PTGS2 genes, due to their important role in the physiology of the corpus luteum, was validated using the quantitative real-time polymerase chain reaction (qPCR) method. The qPCR confirmed the changes of gene expression. Chemerin in physiological concentrations significantly affects the expression of many genes in luteal cells of pigs, which is likely to result in modification of physiological processes related to reproduction.

Coculture of porcine cumulus-oocyte complexes with porcine luteal cells during IVM: effect on oocyte maturation and embryo development.

Coculture with somatic cells is an alternative to improve suboptimal invitro culture conditions. In pigs, IVF is related to poor male pronuclear formation and high rates of polyspermy. The aim of this study was to assess the effect of a coculture system with porcine luteal cells (PLCs) on the IVM of porcine cumulus-oocyte complexes (COCs). Abattoir-derived ovaries were used to obtain PLCs and COCs. COCs were matured invitro in TCM-199 with or without the addition of human menopausal gonadotrophin (hMG; C+hMG and C-hMG respectively), in coculture with PLCs from passage 1 (PLC-1) and in PLC-1 conditioned medium (CM). In the coculture system, nuclear maturation rates were significantly higher than in the C-hMG and CM groups, but similar to rates in the C+hMG group. In cumulus cells, PLC-1 coculture decreased viability, early apoptosis and necrosis, and increased late apoptosis compared with C+hMG. PLC-1 coculture also decreased reactive oxygen species levels in cumulus cells. After IVF, monospermic penetration and IVF efficiency increased in the PLC-1 group compared with the C+hMG group. After invitro culture, higher blastocysts rates were observed in the PLC-1 group. This is the first report of a coculture system of COCs with PLCs. Our model could be an alternative for the conventional maturation medium plus gonadotrophins because of its lower rates of polyspermic penetration and higher blastocysts rates, key issues in porcine invitro embryo production.

Progesterone inhibits cytokine/TNF-α production by porcine CL macrophages via the genomic progesterone receptor

In pigs, luteolytic sensitivity to PGF-2α (=LS) is delayed until d 13 of the estrous cycle. While the control of LS is unknown, it is temporally associated with macrophage (MAC; which secretes tumor necrosis factor [TNF]-α) infiltration into the corpora lutea (CL), and previous studies have shown that TNF-α induces LS in porcine luteal cells (LCs) in culture. This study was designed to explore the control of LS by CL macrophage (CL MAC)/TNF-α by progesterone (P4), and to examine the hypothesis that P4 acting via the genomic P4 receptor (PGR) inhibits CL MAC TNF-α and thus plays a key role in regulating LS during the pig estrous cycle. In experiment 1, the effects of LCs on CL MAC cytokine/TNF-α mRNA expression in co-culture were examined (MID cycle; ~d 7–12; no LS); results showed that LC was inhibitory to cytokine/TNF-α. In experiment 2, the effects of P4 or R5020 (PGR-agonist) on CL MAC cytokine/TNF-α mRNA expression were examined (MID cycle; ~d 7–12; no LS); results showed that both P4 and R5020 dose-dependently inhibited TNF-α. In experiment 3, CL MACs were isolated from CL at MID (~d 7–12; no LS) and LATE (~d 13–18; + LS) cycle, and TNF-α/PGR mRNA measured. Results indicated that while TNF-α mRNA was 4.2-fold greater in CL MACs from LATE vs MID cycle, PGR mRNA was 4.5-fold greater in CL MACs from MID vs LATE cycle. These data support our hypothesis and suggest that progesterone, acting via PGR, plays a critical physiological role in the control of TNF-α production by CL MACs and LS during the pig estrous cycle.

Mitogen-activated protein kinase kinase kinase 8 (MAP3K8) mediates the LH-induced stimulation of progesterone synthesis in the porcine corpus luteum.

Progesterone (P4) synthesized by the corpus luteum (CL) plays a key role in the establishment and maintenance of pregnancy. The LH signal is important for luteinisation and P4 synthesis in pigs. In a previous study, we demonstrated that mitogen-activated protein kinase kinase kinase 8 (MAP3K8) regulates P4 synthesis in mouse CL, but whether the function and mechanism of MAP3K8 in the pig is similar to that in the mouse is not known. Thus, in the present study we investigated the effects of MAP3K8 on porcine CL. Abundant expression of MAP3K8 was detected in porcine CL, and, in pigs, MAP3K8 expression was higher in mature CLs (or those of the mid-luteal phase) than in regressing CLs (late luteal phase). Further functional studies in cultured porcine luteal cells showed that P4 synthesis and the expression of genes encoding the key enzymes in P4 synthesis are significantly reduced when MAP3K8 is inhibited with the MAP3K8 inhibitor Tpl2 kinase inhibitor (MAP3K8i, 10μM). After 12-24h treatment of luteal cells with 100ngmL-1 LH, MAP3K8 expression and P4 secretion were significantly upregulated. In addition, the 10μM MAP3K8 inhibitor blocked the stimulatory effect of LH on P4 synthesis and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in porcine luteal cells. The LH-induced increases in MAP3K8 phosphorylation and expression, ERK1/2 phosphorylation and P4 synthesis were all blocked when protein kinase A was inhibited by its inhibitor H89 (20 μM) in porcine luteal cells. In conclusion, MAP3K8 mediates the LH-induced stimulation of P4 synthesis through the PKA/mitogen-activated protein kinase signalling pathway in porcine CL.

Prostaglandin (PTG) E and F receptors in the porcine corpus luteum; effect of tumor necrosis factor-α

The porcine corpus luteum (CL) is NOT sensitive to the luteolytic effects of PGF-2α until days 12–13 of cycle. The control of “luteolytic sensitivity” (LS) of the pig CL to PGF-2α is unknown, but it is temporally associated with macrophage infiltration into the CL. Since macrophages are the predominant source of TNF-α in the porcine CL, in other studies we examined the effects of TNF-α on porcine luteal cells in culture and showed that TNF-α induces LS in vitro. In Experiment 1 of this study possible mechanisms involved in the control of LS were examined, and involved measurement of the protein levels of PTGER2/EP-2, and PTGER3/EP-3 in porcine CL collected before (days 7–10), versus after (day 13), the onset of the LS. In Experiment 2, an examination of potential mechanisms involved in the control of LS by TNF-α, was carried out in which the effects of TNF-α on mRNA and protein expression of EP-2, EP-3 and FP in cultured luteal cells, were examined. The results of Experiment 1 showed that PTGER-3/EP-3 (but not PTGER-2/EP-2) levels decreased in porcine CLs after (day 13) compared to before (day 7–10) LS. In Experiment 2, the data obtained showed that TNF-α decreased PTGER-3/EP-3 and increased PTGFR/FP protein (in EARLY stage CL). In conclusion, these studies suggest a role for PTGER-3/EP-3 in the acquisition of LS, and support the hypothesis that TNF-α from CL macrophages plays a critical role in the control of LS in the porcine CL, by increasing PTGFR/FP, and decreasing PTGER-3/EP-3 protein.

Establishment and characterization of a telomerase immortalized porcine luteal cells

Luteal cells play a crucial role in pregnancy through secreting progesterone to maintain pregnancy and support of fetus. However, low cellular yields and inability to passage primary porcine luteal cells (PLCs) in vitro limit the luteal cell study. Therefore, developing an immortalized porcine luteal cell line is necessary for studying luteal cells activity and function in different diseases. In this study, primary PLCs were obtained from gilts at day 30 to day 50 of gestation and immortalized by human telomerase reverse transcriptase (hTERT). The porcine corpus luteal cell line (hTERT-PLCs) expressed hTERT gene steady, maintained high hTERT activity and normal karyotype. The phase contrast microscope and transmission electron microscope observation showed primary PLCs and hTERT-PLCs were polygonal and exhibited abundant mitochondria, smooth endoplasmic reticulum and lipid droplets. 3β hydroxysteroid dehydrogenase (3βHSD) and Oil-Red-O staining showed that hTERT-PLCs at passage 30 and 50 were similar to primary PLCs. The hTERT-PLCs expressed steroidogenesis-related proteins, enzymes and receptors, such as steroidogenic acute regulatory protein, P450 cholesterol side-chain cleavage, 3βHSD, 20αHSD, luteinizing hormone receptor, progesterone receptor, prolactin receptor, estrogen receptorα/β, as well as primary PLCs. Consequently, hTERT-PLCs could secret progesterone and exhibited similar responses to luteinizing hormone and prostaglandin F2α as primary PLCs. In addition, the hTERT-PLCs did not show neoplastic transformation or anchorage independent growth. In summary, we developed an immortalized porcine luteal cell line which maintained its originally morphological, biological and functional characteristics.

Culture of porcine luteal cells as a substrate for in vitro maturation of porcine cumulus oocyte complexes. Establishment and characterization

Fil: Teplitz, Gabriela Maia. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigacion y Tecnologia en Reproduccion Animal; Argentina. Consejo Nacional de Investigaciones Cientificas y Tecnicas; Argentina