The effects of the activation of TLR2/TLR1 on in vitro angiogenesis in an immortalized ovine luteal endothelial cell line.

In brief: Activation of TLR2/TLR1 alters in vitro formation of capillary-like structures and induces inflammatory processes in ovine luteal endothelial (OLENDO) cells. Abstract: Postpartum bacterial infections of the uterus affect uterine physiology and ovarian activity, causing fertility problems. The outer membrane component of Gram-negative bacteria, lipopolysaccharide, is involved in the initiation of the local inflammatory processes, and other bacterial toxins, particularly lipopeptides, have also been shown to be potent cytokine inducers, acting via Toll-like receptor-2 (TLR2). However, the possible adverse effects of TLR2 on ovarian and luteal activities have not yet been investigated in depth. The strong expression of TLR2 in the blood vessels of the corpus luteum led us to hypothesize that TLR2 activation might participate in the disruption of luteal vascular functionality. Therefore, we analyzed the effects of Pam3CSK4 (Pam3CysSerLys4), a synthetic triacylated lipopeptide and TLR2/TLR1 ligand, on the functionality of gap junctional intercellular communication (GJIC), endothelial cell invasion, and in vitro capillary-like network formation in an immortalized ovine luteal endothelial (OLENDO) cell line. Pam3CSK4 treatment of OLENDO cells disrupted in vitro tube formation but had no effect on GJIC or migration of OLENDO cells. Furthermore, Pam3CSK4 induced the expression of NFKB, IL6, and IL8 in OLENDO cells. Additionally, the basal availability of TLRs (TLR1-10) and TLR co-receptors (MYD88, LY96/MD2, and CD14) in OLENDO cells was confirmed by conventional PCR. Finally, the activation of TLR2/TLR1 appears to alter in vitro formation of capillary-like structures and induce inflammatory processes in OLENDO cells.

The p38 signaling pathway mediates the TGF-ß1-induced increase in type I collagen deposition in human granulosa cells.

Type I collagen, which is mainly composed of collagen type I alpha 1 chain (COL1A1), is the most abundant extracellular matrix (ECM) protein in the mammalian ovary; and the cyclical remodeling of the ECM plays an essential role in the regulation of corpus luteum formation. Our previous studies have demonstrated that TGF-ß1 is a potent inhibitor of luteinization in human granulosa-lutein (hGL) cells. Whether TGF-ß1 can regulate the expression of COL1A1 during the luteal phase remains to be elucidated. The aim of this study was to investigate the effect of TGF-ß1 on the regulation of COL1A1 expression and the underlying molecular mechanisms using an immortalized hGL cell line (SVOG cells) and primary hGL cells (obtained from 20 consenting patients undergoing IVF treatment). The results showed that TGF-ß1 significantly upregulated the expression of COL1A1. Using inhibition approaches, including pharmacological inhibition (a specific p38 inhibitor, SB203580, and a specific ERK1/2 inhibitor, U0126) and specific siRNA-mediated knockdown inhibition, we demonstrated that TGF-ß1 promoted the expression and production of COL1A1 in hGL cells, most likely via the ALK5-mediated p38 signaling pathway. Our findings provide insights into the molecular mechanisms by which TGF-ß1 promotes the deposition of type I collagen during the late follicular phase in humans.

The effect of cellular and physiological indicators on gender determination.

Gender determination, in addition to having special value to parents, has particular importance in sex-linked diseases. This study aimed to investigate the cellular indicators (i.e. BMP-6 protein and PPAR? protein expression levels in granulosa cells) and the physiological indicators on gender determination. For this purpose, on 68 infertile patients referred to the clinic, ovarian stimulation was performed by different protocols and then ruptured by different HCG. Follow-up of patients was performed after they became pregnant after five months. U/S was done for knowing the gender of the baby then after labor rechecked another time. Also, granulosa-luteal cells (GLCs) were isolated from the follicular fluid of 68 women participating in the study. BMP-6 protein and PPAR? protein were measured using Western blotting. Results showed that the total number of delivered babies was 68, 41 males (60.3%) and 27 females (39.7%). About physiological indicators results, there was no significant association between the age of the mother and sex of the baby (P=0.934). No significant association was detected between the month during which the conception occurred and the sex of the baby (P=0.734). The same result was obtained for the follicle side (P=0.236), and follicle size (P=0.659), there was no significant association between the sex of the baby with the following factors: protocol of treatment (P=0.417), IVF after HCG (P=0.237), HCG type (P=0.572), parity (P=0.282), and type of infertility (P=0.376). The cellular indicators results showed that the BMP-6 protein level in granulosa cells of mothers with daughters was almost twice as high as mothers with sons (P=0.043). But there was no significant difference between mothers with daughters and mothers with sons in PPAR? protein level (P=0.12). It can be concluded that except for BMP-6 protein level, none of the cellular and physiological indicators affects gender determination. Therefore, this cell indicator can probably be evaluated as an effective indicator in determining gender.

Luteinizing Hormone Regulation of Inter-Organelle Communication and Fate of the Corpus Luteum.

The corpus luteum is an endocrine gland that synthesizes the steroid hormone progesterone. luteinizing hormone (LH) is a key luteotropic hormone that stimulates ovulation, luteal development, progesterone biosynthesis, and maintenance of the corpus luteum. Luteotropic and luteolytic factors precisely regulate luteal structure and function; yet, despite recent scientific progress within the past few years, the exact mechanisms remain largely unknown. In the present review, we summarize the recent progress towards understanding cellular changes induced by LH in steroidogenic luteal cells. Herein, we will focus on the effects of LH on inter-organelle communication and steroid biosynthesis, and how LH regulates key protein kinases (i.e., AMPK and MTOR) responsible for controlling steroidogenesis and autophagy in luteal cells.

Pentraxin-3 mediates prosurvival actions of interferon tau in bovine luteinized granulosa cells.

Pentraxin 3 (PTX3), a multimeric glycoprotein, is implicated in various biological functions. PTX3 was shown to be elevated in the corpus luteum (CL) of early pregnant ewes; however, its role in sheep or other ruminants' CL during this reproductive stage or how it is regulated remain unknown. Here we explored the role of PTX3 and its relationship with interferon-tau (IFNT; the pregnancy recognition signaling molecule during early pregnancy in domestic ruminants) in bovine luteinized granulosa cells (LGCs). IFNT robustly elevated PTX3 expression in bovine LGCs, and significantly stimulated its expression in luteal endothelial cells, along with CL slices; yet, LGCs were the most responsive and sensitive among these luteal models. ALK2/ALK3/ALK6 kinase inhibitor, dorsomorphin, dose-dependently inhibited basal and IFNT-elevated PTX3 expression in LGCs. In contrast, ALK4/5/7 inhibitor, SB431542, did not alter basal and TGFB1-induced PTX3. We found that recombinant human PTX3 itself moderately but significantly increases LGC numbers. Because PTX3 is highly expressed in bovine LGCs, we next examined the impact of lowering endogenous PTX3 levels with siRNA. PTX3 silencing decreased the viable cell numbers and reversed IFNT actions on cell viability, percentage of proliferating cells, and on two key survival/death genes: BIRC5 encoding surviving protein, and FASL - a death-inducing signal. Interestingly, thrombospondin-1, a known luteal proapoptotic factor, was inversely related to PTX3 in LGCs. Together, these findings suggest a novel role for PTX3 during early pregnancy, as mediator of IFNT prosurvival actions supporting CL maintenance during this reproductive stage.

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.

Prolyl oligopeptidase regulates progesterone secretion via the ERK signaling pathway in murine luteal cells.

Prolyl oligopeptidase (POP), one of the most widely distributed serine endopeptidases, is highly expressed in the ovaries. However, the physiological role of POP in the ovaries is not clear. In this study, we investigated the significance of POP in the corpus luteum. Murine luteal cells were cultured in vitro and treated with a POP selective inhibitor, (2S)-1[[(2 S)-1-(1-oxo-4-phenylbutyl)-2-pyrrolidinyl carbonyl]-2-pyrrolidinecarbonitrile (KYP-2047). We found that KYP-2047 treatment decreased progesterone secretion. In contrast, POP overexpression increased progesterone secretion. Three essential steroidogenic enzymes, including p450 cholesterol side-chain cleavage enzyme (CYP11A), 3ß-hydroxysteroid dehydrogenase (3ß-HSD), and the steroidogenic acute regulatory protein (StAR), were regulated by POP. Further studies showed that POP overexpression increased ERK1/2 phosphorylation and increased the expression of steroidogenic factor 1 (SF1), while KYP-2047 treatment decreased ERK1/2 phosphorylation and SF1 expression. To clarify the role of ERK1/2 signaling in POP-regulated progesterone synthesis, U0126-EtOH, an inhibitor of the ERK signaling pathway, was used to treat luteal cells. We found that U0126-EtOH decreased progesterone production and the expression of steroidogenic enzymes and SF1. POP overexpression did not reverse the effects of U0126-EtOH. Overall, POP regulates progesterone secretion by stimulating the expression of CYP11A, 3ß-HSD, and StAR in luteal cells. ERK signaling and downstream SF1 expression contribute to this process.

[Slit/Robo pathway participates in luteal cells apoptosis].

This study was aimed to examine the expression and function of Slit/Robo family members in mouse ovary. Real-time PCR was used to assess the mRNA expression levels of Slit/Robo family members, and immunohistochemistry was used to examine the location of Slit2 and Robo1 in the ovary. The mRNA and protein expression levels of Slit2 and Robo1 in early-, middle- and late-phase corpus luteum (CL) were examined by real-time PCR and immunohistochemistry, respectively. Blocking agent ROBO1/Fc chimera was used in the luteal cells in vitro to examine the function of Slit/Robo signaling pathway in mouse CL. The results showed that, among the Slit/Robo family members, the expression levels of ligand Slit2 and receptor Robo1 were the highest in mouse ovarian tissue. Moreover, both of them were specifically expressed in mouse luteal cells. Compared with proestrus ovaries, the expression levels of Slit2 and Robo1 mRNA in the ovaries during diestrus were significantly up-regulated (P < 0.01, P < 0.001). The mRNA expression levels of Slit2 and Robo1 in late-phase CL were significantly increased when compared with pregnant CL. Furthermore, blocking Slit/Robo signaling pathway with ROBO1/Fc chimera in the luteal cells in vitro significantly decreased the apoptotic rate of late luteal cells. These results suggest that Slit/Robo family members are mainly expressed in the late-phase CL of ovary and participate in luteal cells apoptosis.

Fibroblast growth factor-2 and transforming growth factor-beta1 oppositely regulate miR-221 that targets thrombospondin-1 in bovine luteal endothelial cells.

Thrombospondin-1 (THBS1) affects corpus luteum (CL) regression. Highly induced during luteolysis, it acts as a natural anti-angiogenic, proapoptotic compound. THBS1 expression is regulated in bovine luteal endothelial cells (LECs) by fibroblast growth factor-2 (FGF2) and transforming growth factor-beta1 (TGFB1) acting in an opposite manner. Here we sought to identify specific microRNAs (miRNAs) targeting THBS1 and investigate their possible involvement in FGF2 and TGFB1-mediated THBS1 expression. Several miRNAs predicted to target THBS1 mRNA (miR-1, miR-18a, miR-144, miR-194, and miR-221) were experimentally tested. Of these, miR-221 was shown to efficiently target THBS1 expression and function in LECs. We found that this miRNA is highly expressed in luteal cells and in mid-cycle CL. Consistent with the inhibition of THBS1 function, miR-221 also reduced Serpin Family E Member 1 [SERPINE1] in LECs and promoted angiogenic characteristics of LECs. Plasminogen activator inhibitor-1 (PAI-1), the gene product of SERPINE1, inhibited cell adhesion, suggesting that PAI-1, like THBS1, has anti-angiogenic properties. Importantly, FGF2, which negatively regulates THBS1, elevates miR-221. Conversely, TGFB1 that stimulates THBS1, significantly reduces miR-221. Furthermore, FGF2 enhances the suppression of THBS1 caused by miR-221 mimic, and prevents the increase in THBS1 induced by miR-221 inhibitor. In contrast, TGFB1 reverses the inhibitory effect of miR-221 mimic on THBS1, and enhances the upregulation of THBS1 induced by miR-221 inhibitor. These data support the contention that FGF2 and TGFB1 modulate THBS1 via miR-221. These in vitro data propose that dynamic regulation of miR-221 throughout the cycle, affecting THBS1 and SERPINE1, can modulate vascular function in the CL.

miR-210 and GPD1L regulate EDN2 in primary and immortalized human granulosa-lutein cells.

Endothelin-2 (EDN2), expressed at a narrow window during the periovulatory period, critically affects ovulation and corpus luteum (CL) formation. LH (acting mainly via cAMP) and hypoxia are implicated in CL formation; therefore, we aimed to elucidate how these signals regulate EDN2 using human primary (hGLCs) and immortalized (SVOG) granulosa-lutein cells. The hypoxiamiR, microRNA-210 (miR-210) was identified as a new essential player in EDN2 expression. Hypoxia (either mimetic compound-CoCl2, or low O2) elevated hypoxia-inducible factor 1A (HIF1A), miR-210 and EDN2 Hypoxia-induced miR-210 was suppressed in HIF1A-silenced SVOG cells, suggesting that miR-210 is HIF1A dependent. Elevated miR-210 levels in hypoxia or by miR-210 overexpression, increased EDN2 Conversely, miR-210 inhibition reduced EDN2 levels, even in the presence of CoCl2, indicating the importance of miR-210 in the hypoxic induction of EDN2 A molecule that destabilizes HIF1A protein, glycerol-3-phosphate dehydrogenase 1-like gene-GPD1L, was established as a miR-210 target in both cell types. It was decreased by miR-210-mimic and was increased by miR-inhibitor. Furthermore, reducing GPD1L by endogenously elevated miR-210 (in hypoxia), miR-210-mimic or by GPD1L siRNA resulted in elevated HIF1A protein and EDN2 levels, implying a vital role for GPD1L in the hypoxic induction of EDN2 Under normoxic conditions, forskolin (adenylyl cyclase activator) triggered changes typical of hypoxia. It elevated HIF1A, EDN2 and miR-210 while inhibiting GPD1L Furthermore, HIF1A silencing greatly reduced forskolin's ability to elevate EDN2 and miR-210. This study highlights the novel regulatory roles of miR-210 and its gene target, GPD1L, in hypoxia and cAMP-induced EDN2 by human granulosa-lutein cells.

Dysregulated genes and their functional pathways in luteinized granulosa cells from PCOS patients after cabergoline treatment.

Polycystic ovarian syndrome (PCOS) is a common reproductive disorder frequently associated with a substantial risk factor for ovarian hyperstimulation syndrome (OHSS). Dopamine receptor 2 (D2) agonists, like cabergoline (Cb2), have been used to reduce the OHSS risk. However, lutein granulosa cells (LGCs) from PCOS patients treated with Cb2 still show a deregulated dopaminergic tone (decreased D2 expression and low dopamine production) and increased vascularization compared to non-PCOS LGCs. Therefore, to understand the PCOS ovarian physiology, it is important to explore the mechanisms that underlie syndrome based on the therapeutic effects of Cb2. Here, LGCs from non-PCOS and PCOS patients were cultured with hCG in the absence/presence of Cb2 (n = 12). Subsequently, a transcriptomic-paired design that compared untreated vs treated LGCs within each patient was performed. After transcriptomic analysis, functions and genes were prioritized by systems biology approaches and validated by RT-qPCR. We identified that similar functions were altered in both PCOS and non-PCOS LGCs treated with Cb2; however, PCOS-treated LGCs exhibited more significant changes than non-PCOS. Among the prioritized functions, dopaminergic synapse, vascular endothelial growth factor (VEGF) signaling, apoptosis and ovarian steroidogenesis were highlighted. Finally, network modeling showed CASP9, VEGFA, AKT1, CREB, AIF, MAOA, MAPK14 and BMAL1 as key genes implicated in these pathways in Cb2 response, which might be potential biomarkers for further studies in PCOS.

Influence of omega-3 fatty acids on bovine luteal cell plasma membrane dynamics.

Fish oil is a rich source of omega-3 fatty acids which disrupt lipid microdomain structure and affect mobility of the prostaglandin F2α (FP) receptor in bovine luteal cells. The objectives of this study were to determine the effects of individual omega-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on 1) membrane fatty acid composition, 2) lipid microdomain structure, and 3) lateral mobility of the FP receptor in bovine luteal cells. Ovaries were collected from a local abattoir (n=5/experiment). The corpus luteum was resected and enzymatically digested using collagenase to generate a mixed luteal cell population. In all experiments, luteal cells were treated with 0, 1, 10 or 100µM EPA or DHA for 72h to allow incorporation of fatty acids into membrane lipids. Results from experiment 1 show that culturing luteal cells in the presence of EPA or DHA increased these luteal fatty acids. In experiment 2, both EPA and DHA increased spatial distribution of lipid microdomains in a dose-dependent manner. Single particle tracking results from experiment 3 show that increasing both EPA and DHA concentrations increased micro- and macro-diffusion coefficients, increased domain size, and decreased residence time of FP receptors. Collectively, results from this study demonstrate similar effects of EPA and DHA on lipid microdomain structure and lateral mobility of FP receptors in cultured bovine luteal cells. Moreover, only 10µM of either fatty acid was needed to mimic the effects of fish oil.

Bone morphogenetic protein 2 regulates cell-cell communication by down-regulating connexin43 expression in luteinized human granulosa cells.

STUDY QUESTION: Does bone morphogenetic protein 2 (BMP2) regulate connexin43 (Cx43) and modulate cell-cell communication in luteinized human granulosa cells? SUMMARY ANSWER: BMP2 decreases gap junction intercellular communication (GJIC) of luteinized human granulosa cells by down-regulating Cx43 expression through an activin receptor-like kinase (ALK)2/ALK3-mediated Sma- and Mad-related protein (SMAD)-dependent signaling pathway. WHAT IS KNOWN ALREADY: BMP2 and its putative receptors are highly expressed in the human corpus luteum and are involved in the process of luteolysis. Cx43-coupled gap junctions play a critical role in the development and maintenance of corpus luteum. STUDY DESIGN DURATION: This is a laboratory study conducted over a 1-year period. At least three independent experiments with three replicates were conducted and the experimental samples were compared with the appropriate vehicle controls for all of the inhibition-approach, concentration-dependent or time-course studies. PARTICIPANTS/MATERIALS, SETTING, METHODS: SVOG cell line (immortalized human granulosa-lutein cells derived from in vitro fertilization patients in an academic research center) was used as the study model. The changes of Cx43 expression and levels of phosphorylated SMAD1/5/8 protein were evaluated after exposure to recombinant human BMP2. Real-time quantitative PCR and Western blot analysis were used to examine the specific mRNA and protein levels, respectively. The BMP/TGF-ß type I receptor inhibitors (Dorsomorphin, DMH-1 and SB431542) and target depletion small interfering RNAs (ALK2, ALK3, ALK6 and SMAD4) were used to investigate the underlying molecular mechanisms. A scrape loading and dye transfer assay was used to evaluate the GJIC between the SVOG cells. MAIN RESULTS AND THE ROLE OF CHANCE: Treatment with BMP2 down-regulated the expression of Cx43 and decreased the GJIC activity, whereas it increased the phosphorylated SMAD1/5/8 protein in SVOG cells (P < 0.05). These biological effects were abolished by pre-treatment with the BMP type I receptor inhibitors, Dorsomorphin and DMH-1 (P < 0.05), but not SB431542. Additionally, the individual or concomitant small interfering RNA-mediated knockdown of ALK2 and ALK3, but not ALK6 attenuated the BMP2-induced increases in phosphorylated SMAD1/5/8 and down-regulation of Cx43 expression (P < 0.05). The knockdown of SMAD4 completely abolished the BMP2-induced down-regulation of Cx43 expression (P < 0.05). LIMITATIONS REASONS FOR CAUTION: This experimental study was conducted in an in vitro cell culture system, and may not reflect a realistic intra-ovarian environment. WIDER IMPLICATIONS OF THE FINDINGS: Our results suggested that BMP2 may be involved in the local modulation of cell-cell communication in the luteal phase. This study also represents the first comprehensive research of molecular mechanisms of BMP2 in the down-regulation Cx43 in luteinized human granulosa cells. Such data may provide valuable insights into ovarian physiology and benefit the development of potential therapeutic methods for patients suffering from luteal insufficiency. LARGE SCALE DATA: N/A. STUDY FUNDING AND COMPETING INTEREST(s): This research was supported by an operating grant from the China-Canadian Joint Health Research Initiative Grants Program to P.C.K. Leung and J.Z. Sheng. The authors declare no competing interest with the contents of this article.

Isolation of luteal endothelial cells and functional interactions with T lymphocytes.

The objectives of this study were to optimize the isolation of luteal endothelial cells (LEC) and examine their functional interactions with autologous T lymphocytes. Analysis by flow cytometry showed that the purity of LEC isolated by filtration was nearly 90% as indicated by Bandeiraea simplicifolia (BS)-1 lectin binding. LEC expressed mRNA for progesterone receptor (PGR), prostaglandin receptors (PTGFR, PTGER2 and 4, and PTGIR), tumor necrosis factor receptors (TNFRSF1A&B) and interleukin (IL) 1B receptors (IL1R1&2). LEC were pretreated with either vehicle, progesterone (P4; 0-20 µM), prostaglandin (PG) E2 or PGF2α (0-0.2 µM), and further treated with or without TNF and IL1B (50 ng/mL each). LEC were then incubated with autologous T lymphocytes in an adhesion assay. Fewer lymphocytes adhered to LEC after exposure to high compared to low P4 concentrations (cubic response; P < 0.05). In contrast, 0.2 µM PGE2 and PGF2α each increased T lymphocyte adhesion in the absence of cytokines (P < 0.05). LEC induced IL2 receptor alpha (CD25) expression and proliferation of T lymphocytes. In conclusion, filtration is an effective way of isolating large numbers of viable LEC. It is proposed that PGs and P4 modulate the ability of endothelial cells to bind T lymphocytes, potentially regulating extravasation, and that LEC activate T lymphocytes migrating into or resident in the CL.

The influence of adiponectin on the transcriptomic profile of porcine luteal cells.

Reproductive functions are closely related to nutritional status. Recent studies suggest that adiponectin may be a hormonal link between them. Adiponectin is an adipocytokine, abundantly expressed in adipose tissues. It plays a dominant role in lipid and carbohydrate metabolism by stimulating fatty acid oxidation, decreasing plasma triglycerides, and increasing cells' sensitivity to insulin and has direct antiatherosclerotic effects. The hormone is also postulated to play a modulatory role in the regulation of the reproductive system. The aim of this study was to identify differentially expressed genes (DE-genes) in response to adiponectin treatment of porcine luteal ovarian cells. The global expression of genes in the porcine ovary was investigated using the Porcine (V2) Two-color gene expression microarray, 4 × 44 (Agilent, USA). Analysis of the microarray data showed that 701 genes were differentially expressed and 389 genes showed a fold change greater than 1.2 (p < 0.05). Among this number, 186 genes were up-regulated and 203 were down-regulated. The list of DE-genes was used for gene ontology analyses. The biological process list was generated from up-regulated and down-regulated DE-genes. We found that up-regulated products of DE-genes take part in 30 biological processes and down-regulated products in 9. Analysis of the interaction network among DE-genes showed that adiponectin interacts with genes involved in important processes in luteal cells. These results provide a basis for future work describing the detailed interactions and relationships explaining local regulation of adiponectin actions in the ovary of pigs.

Human granulosa-luteal cells initiate an innate immune response to pathogen-associated molecules.

The microenvironment of the ovarian follicle is key to the developmental success of the oocyte. Minor changes within the follicular microenvironment can significantly disrupt oocyte development, compromising the formation of competent embryos and reducing fertility. Previously described as a sterile environment, the ovarian follicle of women has been shown to contain colonizing bacterial strains, whereas in domestic species, pathogen-associated molecules are concentrated in the follicular fluid of animals with uterine infection. The aim of this study is to determine whether human granulosa-luteal cells mount an innate immune response to pathogen-associated molecules, potentially disrupting the microenvironment of the ovarian follicle. Human granulosa-luteal cells were collected from patients undergoing assisted reproduction. Cells were cultured in the presence of pathogen-associated molecules (LPS, FSL-1 and Pam3CSK4) for 24h. Supernatants and total RNA were collected for assessment by PCR and ELISA. Granulosa-luteal cells were shown to express the molecular machinery required to respond to a range of pathogen-associated molecules. Expression of TLR4 varied up to 15-fold between individual patients. Granulosa-luteal cells increased the expression of the inflammatory mediators IL1B, IL6 and CXCL8 in the presence of the TLR4 agonist E. coli LPS. Similarly, the TLR2/6 ligand, FSL-1, increased the expression of IL6 and CXCL8. Although no detectable changes in CYP19A1 or STAR expression were observed in granulosa-luteal cells following challenge, a significant reduction in progesterone secretion was measured after treatment with FSL-1. These findings demonstrate the ability of human granulosa-luteal cells to respond to pathogen-associated molecules and generate an innate immune response.

Activin A upregulates PTGS2 expression and increases PGE2 production in human granulosa-lutein cells.

Activin A is one of the members of transforming growth factor-ß superfamily that is expressed in human large luteal cells, and may act in an autocrine/paracrine manner to regulate luteal function. Prostaglandin-endoperoxide synthase 2 (PTGS2) enzyme and its derivative, prostaglandin E2 (PGE2), play significant roles in the regulation of corpus luteum formation and maintenance. To date, whether activin A can induce the expression of PTGS2 and the production of PGE2 in human granulosa-lutein cells is largely unknown. The aim of this study was to examine the effects of activin A on the regulation of PTGS2 expression and PGE2 production in human granulosa-lutein cells, and to investigate the underlying signal transduction mechanisms. In this study, the immortalized (SVOG cells) and primary human granulosa-lutein cells were used as the cell models. A TGF-ß/activin type I receptor inhibitor, SB431542 and small interfering RNAs were used to investigate the activin A-induced downstream signaling pathway. We have demonstrated that activin A upregulated the expression of PTGS2 and increased the production of PGE2 via an ACVR1B-mediated SMAD2/3-SMAD4 signaling pathway. Our results suggest that activin A may be involved in the modulation of human corpus luteum formation via the induction of PTGS2 expression and PGE2 production.

Regulated C-C motif ligand 2 (CCL2) in luteal cells contributes to macrophage infiltration into the human corpus luteum during luteolysis.

Intense macrophage infiltration is observed during luteolysis in various animals including women; however, we still do not know how macrophage infiltration into the human corpus luteum (CL) during luteolysis is regulated. In this study, we examined the expression, localization and regulation of an important chemokine for the recruitment of monocyte/macrophage lineages, C-C motif ligand 2 (CCL2), in the human CL across the luteal phase and in cultured human luteinized granulosa cells (LGCs), with special reference to the number of infiltrating macrophages and luteal cell function. CCL2 mRNA increased in the non-functional regressing CL during menstruation (P < 0.01), corresponding to an elevated mRNA expression of a macrophage-derived cytokine, tumor necrosis factor (TNF), and an increased number of infiltrating macrophages positively stained with a macrophage marker, CD68. CCL2 protein was immunohistochemically localized to the cytoplasm of granulosa-lutein and theca-lutein cells, and CCL2 mRNA was significantly reduced by hCG both in vivo (P < 0.05) and in vitro (P < 0.01). CCL2 was also down-regulated by luteotrophic prostaglandin (PG) E (P < 0.0001), but up-regulated by luteolytic PGF (P < 0.05) in vitro. Administration of TNF significantly enhanced the CCL2 mRNA expression in cultured LGCs (P < 0.01). A greater abundance of infiltrating macrophages were found around granulosa-lutein cells lacking 3ß-HSD or PGE synthase (PGES) immunostaining. CCL2 mRNA expression was negatively correlated with both HSD3B1 and PGES, suggesting that locally produced progesterone and PGE suppress macrophage infiltration into the CL. Taken together, the infiltration of macrophages in the human CL is regulated by endocrine and paracrine molecules via regulation of the CCL2 expression in luteal cells.

Expression and functional implications of luteal endothelins in pregnant and non-pregnant dogs.

Luteal development is regulated by many locally produced mediators, e.g., prostaglandins and angiogenic factors. However, the role and function of vasoactive factors in the canine corpus luteum (CL) remain largely unknown. Consequently, expression of the endothelin (ET) receptors-A and -B (ETA and ETB, revealing vasoconstriction and vasodilator properties respectively), the ET-converting enzyme (ECE1) and ET1, -2 and -3 were investigated in CL from non-pregnant dogs (days 5, 15, 25, 35, 45 and 65 post-ovulation), and at selected stages of pregnancy (pre-implantation, post-implantation, mid-gestation), and during normal and antigestagen-induced prepartum luteolysis/abortion. The interrelationship between PGE2 and the ET system was investigated in PGE2-treated canine primary lutein cells from early CL. ET1 did not change significantly over time; ET2, ECE1 and ETB were elevated in early CL and were downregulated towards the mid/late-luteal phase. The prepartum increase of ET2 was significant. ET3 increased gradually, and was highest in late CL and/or at prepartum luteolysis. ETA remained constant until the late CL phase and increased only during prepartum luteolysis. ET1 was localized to the luteal cells, and ET2, ET3 and ETA to vascular endothelium. ECE1 and ETB were detected at both locations. Except for upregulated ET1 and lack of effect on ET2, antigestagen applied to mid-pregnant dogs evoked similar changes to those observed during normal luteolysis. PGE2 upregulated ETB in treated cells; ETA and ET1 remained unaffected, and ET2 decreased. A modulatory role of the ETs in canine CL, possibly in association with other factors (e.g., PGE2 and progesterone receptor), is strongly indicated.

Lysophosphatidic acid modulates prostaglandin signalling in bovine steroidogenic luteal cells.

We examined whether lysophosphatidic acid affects prostaglandin biosynthesis, transport, and signalling in bovine steroidogenic luteal cells. The aim of the present study was to determine the influence of LPA on PGE2 and PGF2α synthesis and on the expression of enzymes involved in PG biosynthesis (PTGS2, mPGES-1, cPGES, mPGES-2, PGFS and 9-KPR), prostaglandin transporter (PGT), and prostaglandin receptors (EP1, EP2, EP3, EP4 and FP) in bovine steroidogenic luteal cells. We found that LPA inhibited PGF2α synthesis in steroidogenic luteal cells. Moreover, LPA increased mPGES1 and cPGES and decreased PGFS expression in cultured bovine steroidogenic luteal cells. Additionally, LPA stimulated EP2 and EP4 receptor and PGT expression. This study suggests that LPA activity in the bovine CL directs the physiological intraluteal balance between the two main prostanoids towards luteotropic PGE2.