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Svetlana Farberov, Raghavendra Basavaraja and Rina Meidan

The multimodular matricellular protein thrombospondin-1 (THBS1) was among the first identified endogenous antiangiogenic molecules. Recent studies have shown THBS1-mediated suppression of angiogenesis and other critical activities for corpus luteum (CL) regression. THBS1 is specifically induced by prostaglandin F2alpha in mature CL undergoing regression, whereas luteinizing signals such as luteinizing hormone and insulin reduced its expression. THBS1 interacts both synergistically and antagonistically with other essential luteal factors, such as fibroblast growth factor 2, transforming growth factor beta1, and serpin family E member 1, to promote vascular instability, apoptosis, and matrix remodeling during luteal regression. Expression of THBS1 is also downregulated by pregnancy recognition signals to maintain the CL during early pregnancy. This dynamic pattern of luteal expression, the extensive interactivity with other luteal factors, and strong antiangiogenic and proapoptotic activities indicate that THBS1 is a major determinant of CL fate.

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Eyal Klipper, Tamar Gilboa, Nitzan Levy, Tatiana Kisliouk, Katharina Spanel-Borowski and Rina Meidan

Endothelium-derived endothelin-1 (ET-1) and nitric oxide (NO) are pivotal regulators of corpus luteum (CL) function. To have a better insight into their synthesis and action, members of the ET system (ET-1, ET converting enzyme (ECE-1) isoforms a–d, ETA and ETB receptors) along with NO synthase (NOS) isoforms – endothelial (e)NOS and inducible (i)NOS – were quantified in CL-derived endothelial cells (CLEC). The expression of these genes in microvascular CLEC, obtained by lectin-coated magnetic beads, was compared with cells removed from the luteal microenvironment and maintained in culture for different durations, and with endothelial cells (EC) derived from a large blood vessel (i.e. bovine aortic endothelial cells, BAEC). The profile of gene expression in the different EC types was determined by quantitative real-time PCR. Freshly isolated EC from mid-cycle CL exhibited high ET-1 receptor expression (both ETA and ETB), low ET-1 synthesizing ability (both prepro (pp) ET-1 and ECE-1), but elevated iNOS – the high throughput NOS isoform. The distinct phenotype of CLEC was lost soon after an overnight culture. ETA and ETB receptor levels declined, ppET-1 levels increased while iNOS was reduced. These changes were extenuated during long-term culture of CLEC. The general pattern of gene expression in BAEC and long-term cultured CLEC was similar yet some differences, reminiscent of freshly isolated CLEC, remained: ECE-1c, ETB receptor and NOS isoforms were expressed differently in BAEC as compared with lines of CLEC.

This study suggests that the luteal microenvironment is necessary to sustain the selective phenotype of its resident endothelial cells. The inverse relationship between ppET-1 and iNOS observed in freshly isolated CLEC and in cultured cells is physiologically significant and suggests that ET-1 and NO may modulate the production of each other.

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Raghavendra Basavaraja, Emilia Przygrodzka, Bartosz Pawlinski, Zdzislaw Gajewski, Monika M Kaczmarek and Rina Meidan

Interferon-tau (IFNT), a maternal recognition of pregnancy (MRP) signals in domestic ruminants, suppresses the release of luteolytic pulses of uterine prostaglandin F2a (PGF2a), thus extending the corpus luteum (CL) life span. We hypothesized that IFNT also exerts anti-luteolytic actions in bovine CL. To examine the direct effects of IFNT on bovine CL, luteal slices and enriched luteal endothelial cells (LECs) were utilized. We found that recombinant ovine IFNT (roIFNT) markedly elevates interferon-associated genes (STAT1, STAT2 and IRF9) and interferon-stimulated genes (ISGs: MX2, ISG15 and OAS1Y) in both models. Furthermore, IFNT time-dependently induced STAT1 phosphorylation in LECs without affecting total STAT1. roIFNT-stimulated viable LECs numbers and the knockdown of protein inhibitor of activated STAT1 (PIAS1) abolished this effect, suggesting that PIAS1 may mediate the proliferative effect of IFNT. IFNT significantly downregulated luteolytic genes such as TGFB1, thrombospondin-1 (THBS1), endothelin-1 (EDN1) and serpin family E member-1 (SERPINE1) in LECs. However, less robust effects were observed in luteal slices. Moreover, PGF2a alone induced THBS1, SERPINE1 and EDN1 mRNA in CL slices whereas in the presence of IFNT, THBS1 and SERPINE1 stimulation was abolished. Collectively, these results indicate that IFNT acts via STAT1- IRF9-dependent and independent pathways and affects diverse luteal functions. Most interestingly, this study suggests the existence of an anti-luteolytic effect of IFNT in bovine CL, namely, inhibiting key PGF2a-induced luteolytic genes. The proliferative effect of IFNT may constitute an additional mechanism that promotes luteal cell survival, thus, extending the luteal life span during early pregnancy in cows.

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Ronit Yalu, Adepeju Esther Oyesiji, Iris Eisenberg, Tal Imbar and Rina Meidan

Hypoxia-inducible factor 1 alpha (HIF1A) and endothelin 2 (EDN2) are transiently expressed during the same time window in the developing corpus luteum (CL). In this study, we sought to investigate the involvement of LH/cAMP, reactive oxygen species (ROS), and a hypoxia-mimetic compound (CoCl2) on HIF1A expression and how it affected EDN2 levels, using transformed human granulosa cells (thGCs) and primary bovine granulosa cells (GCs). CoCl2 elevated HIF1A protein levels in thGCs in a dose-dependent manner. Forskolin alone had no significant effect; however, forskolin and CoCl2 together further induced HIF1A protein and EDN2 mRNA expression in thGCs. Similarly, in primary GCs, LH with CoCl2 synergistically augmented HIF1A protein levels, which resulted in higher expression of EDN2 and another well-known hypoxia-inducible gene, VEGF (VEGFA). Importantly, LH alone elevated HIF1 A mRNA but not its protein. The successful knockdown of HIF1 A in thGCs using siRNA abolished hypoxia-induced EDN2 and also the additive effect of forskolin and CoCl2. We then examined the roles of ROS in thGCs: hydrogen peroxide (20 and 50 μM) elevated HIF1A protein as well as the expression of EDN2, implying that induction of HIF1A protein levels is sufficient to stimulate the expression of EDN2 (and VEGF) in normoxia. A broad-range ROS scavenger, butylated hydroxyanisole, inhibited CoCl2-induced HIF1A protein with a concomitant reduction in the mRNA expression of EDN2 and VEGF in thGCs. The results obtained in this study suggest that HIF1A, induced by various stimuli, is an essential mediator of EDN2 mRNA expression. The results may also explain the rise in the levels of HIF1A-dependent genes (EDN2 and VEGF) in the developing CL.

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Ketan Shrestha, Karolina Lukasik, Anja Baufeld, Jens Vanselow, Uzi Moallem and Rina Meidan

Prostaglandin endoperoxide synthase-2 (PTGS2), tumour necrosis factor-alpha-induced protein-6 (TNFAIP6), pentraxin-3 (PTX3), epidermal growth factor-like factors: amphiregulin (AREG) and epiregulin (EREG) are essential for successful ovulation. In this study, we compared the induction of these ovulatory genes in bovine granulosa cells (GCs) in vivo (after LH surge) and in vitro (forskolin (FRS) treatment). These genes were markedly stimulated in GCs isolated from cows 21 h after LH-surge. In isolated GCs, FRS induced a distinct temporal profile for each gene. Generally, there was a good agreement between the in vivo and in vitro inductions of these genes except for PTX3. Lack of PTX3 induction in isolated GCs culture suggests that other follicular compartments may mediate its induction by LH. Next, to study the role of PTGS2 and prostaglandins (PGs) in the cascade of ovulatory genes, PTGS2 was silenced with siRNA. PTGS2 siRNA caused a marked and specific knockdown of PTGS2 mRNA and PGE2 production (70% compared with scrambled siRNA) in bovine GCs. Importantly, PTGS2 silencing also reduced AREG, EREG and TNFAIP6 mRNA levels but not PTX3. Exogenous PGE2 increased AREG, EREG and TNFAIP6 mRNA levels, further confirming that these genes are prostanoid dependent. A successful and specific knockdown of PTGS2 was also achieved in endometrial cells (EndoCs) expressing PTGS2. Then, cholesterol-conjugated PTGS2 (chol-PTGS2) siRNA that facilitates cells' entry was investigated. In EndoCs, but not in GCs, chol-PTGS2 siRNA succeeded to reduce PTGS2 and PGE2 levels even without transfection reagent. PTGS2 knockdown is a promising tool to critically examine the functions of PTGS2 in the reproductive tract.

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Ketan Shrestha, Adepeju Esther Onasanya, Iris Eisenberg, Noa Wigoda, Simcha Yagel, Ronit Yalu, Rina Meidan and Tal Imbar

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.