Conselho Nacional de Desenvolvimento Científico e Tecnológico / The objective of the present study is to evaluate interferon-tau (IFNT) endocrine action in extra uterine tissues. The first approach consists of collecting samples from ewes on days 12, 13, 14 and 15 of the estrous cycle or early pregnancy. The second, consists of installing osmotic pumps to deliver a continuous concentration of recombinant ovine (ro) IFNT into the uterine vein for 24 or 72 hours. Our hypothesis is that endocrine release of IFNT into the uterine vein occurs as early as Day 13 of pregnancy. Also, that 24 and 72 hours infusion of roIFNT induces ISGs in the CL, liver and endometrium and IFNT has endocrine action on the CL to modulate major genes involved in luteolysis. Endometrium, liver, corpus luteum, uterine vein, jugular and uterine vein blood were collected from cyclic (NP) and pregnant (P) ewes on Days 12, 13, 14 and 15. Instalation of 24 and 72 hour pumps was done on Day 10 of the estrous cycle. Half were infused with BSA and the other half with roIFNT. Only in the 24 hour infusion, half of each group was challanged with a PGF injection at 12 hours following pump installation. Concentrations of progesterone in serum were similar on Days 12 and 13 and then declined (P<0.05) to less than 1 ng/ml in NP ewes between Days 14 and 15. Endometrial ISG15 mRNA increased (P<0.05) in P versus NP ewes by Day 13 and remained greater through Day 15. Endometrial ESR1 and OXTR mRNAs were up-regulated (P<0.05) in NP compared to P ewes by Day 14, and remained up-regulated on Day 15. Uterine vein ISG15 mRNA was not affected by pregnancy status. IFNAR1 and IFNAR2 mRNA were present in the CL, but did not change due to pregnancy status. ISG15 mRNA in CL and liver increased (P<0.05) by Day 14 and remained up-regulated on Day 15 in P compared to NP ewes. The expression of ESR1, OXTR, PTGFR, PTGER2, PTGER3 and PTGER4 did not change in the corpus luteum during Days 12, 13, 14 and 15 related to pregnancy status. Following the 24 hour infusion, ESR1, OXTR, PTGER2 and PTGER3 mRNA were not affected, but SLCO2A1 mRNA decreased (P<0.001) in BSA+PGF, roIFNT and roIFNT+PGF compared to BSA-infused ewes. PTGFR mRNA was down regulated (P<0.001) in roIFNT and roIFNT+PGF compared to BSA and BSA+PGF-infused ewes. PTGER4 mRNA was greater (P<0.05) in roIFNT and roIFNT+PGF compared to BSA, but not compared to BSA+PGF-infused ewes. After 72 hours, concentrations of progesterone did not differ in roIFNT- compared to BSA-infused ewes. ISG15 mRNA was induced in the CL, liver and endometrium in roIFNT- compared with BSA-infused ewes. Endocrine action of IFNT occurred through up-regulation of ISG15 mRNA in CL and liver by Day 14 of pregnancy. It is concluded that IFNT has endocrine effects on the CL between Days 13 and 14 of pregnancy and may protect the CL through mechanisms that are complementary, yet independent to its paracrine effects on the OXTR endometrial pathway. / O objetivo deste trabalho foi avaliar a resposta do interferon-tau (IFNT) em tecidos extra uterinos utilizando dois modelos experimentais. O primeiro, com a utilização de amostras coletadas nos dias 12, 13, 14 e 15 do ciclo estral ou da gestação e o segundo com a instalação de bombas osmóticas de infusão contínua por 24 ou 72 horas. Formulou-se a hipótese que o IFNT é liberado na veia uterina em torno do dia 13 da gestação. Também, que após 24 ou 72 horas de infusão na veia uterina, o interferon-tau é capaz de induzir a expressão de genes estimulados pelo interferon (ISGs) no corpo lúteo, fígado e endométrio. A resposta no corpo lúteo é capaz de modular genes envolvidos na luteólise. Para isso, foram coletados endométrio, corpo lúteo, fígado, veia uterina, sangue das veias jugular e uterina, nos dias 12, 13, 14 e 15 do ciclo estral ou gestação. A instalação das bombas osmóticas de infusão por 24 e 72 horas foi realizada no dia 10 do ciclo estral, onde metade dos animais receberam infusão de albumina bovina (grupo BSA) e outra metade de IFNT recombinante ovino (ro; grupo roIFNT). Nos animais infundidos por 24 horas, metade de cada grupo BSA ou roIFNT, recebeu um desafio com prostaglandina F2 alfa (PGF; grupos BSA+PGF e roIFNT + PGF) 12 horas após a instalação das bombas. Concentrações sericas de progeterona foram iguais nos dias 12 e 13, e diminuiram (P<0,05) a menos de 1 ng/ml entre os dias 14 e 15 nos animais cíclicos. A expressão de ISG15 endometrial aumentou (P<0,05) no dia 13 em ovelhas prenhes, e permaneceu elevada até o dia 15. A expressão dos receptores de estrógeno alfa (ESR1) e de ocitocina (OXTR) aumentaram (P<0,05) em ovelhas cíclicas comparadas com prenhes no dia 14, permanecendo elevadas no dia 15. A expressão de ISG15 na veia uterina não apresentou diferença em função da gestação. A expressão dos receptores de Interferon tipo I não apresentou diferença em função da prenhez no corpo lúteo. No corpo lúteo e fígado, a expressão de ISG15 aumentou no dia 14 da gestação, permanecendo no dia 15. A expressão luteal de ESR1, OXTR, receptores de PGF (PTGFR), e E2 subtipos 2 (PTGER2), 3 (PTGER3) e 4 (PTGER4) não apresentaram diferença em função da gestação nos dias 12, 13 14, e 15. Após 24 horas de infusão, a expressão luteal de ESR1, OXTR, PTGER2 e PTGER3, não apresentaram diferenças. O transportador de prostaglandinas diminuiu nos grupos BSA+PGF, roIFNT e roIFNT+PGF (P<0,001). O receptor PTGFR diminuiu a expressão (P<0,001) nos grupos roIFNT e roIFNT+PGF, e o receptor PTGER4 aumentou (P<0,05) nos grupos roIFNT e roIFNT+PGF. Em 72 horas de infusão, não foi observada diferença na secreção de progesterona. A expressão de ISG15 nos animais do grupo roIFNT aumentou (P<0,01) no corpo lúteo, fígado e endométrio. Assim, a função endócrina do IFNT ocorre com a expressão de ISG15 no dia 14 da gestação no corpo lúteo e no fígado. Portanto, a ação endócrina do IFNT acontece entre os dias 13 e 14 da gestação e pode proteger o corpo lúteo por mecanismos complementares ao mecanismo anti luteolítico parácrino endometrial.

In ruminants, prostaglandin F2 alpha (PGF2α) is released from the endometrium in a pulsatile pattern at the time of luteolysis. The luteolytic PGF2α pulses are transported from the uterus to the corpus luteum (CL) through the utero-ovarian plexus (UOP) to cause luteolysis. At the time of establishment of pregnancy, interferon tau (IFNT) secreted by the conceptus suppresses the pulsatile release of PGF2α and thereby rescues the CL and maintains its secretion of progesterone. However, basal concentrations of PGF2α are higher in pregnant ewes than in cyclic ewes. The pulsatile release of PGF2α likely requires selective carrier-mediated transport and cannot be supported by a simple diffusion mechanism. The molecular and functional aspects of carrier mediated transport of PGF2α from the uterus to the ovary through the utero- ovarian plexus (UOP) at the time of luteolysis and recognition/establishment of pregnancy are largely unknown ruminants. Results indicate that intrauterine inhibition of (PGT) prevents the pulsatile release of PGF2α independently of spatial expressions of estrogen receptor (ESR-1) and oxytocin receptor (OXTR) proteins by the endometrium at the time of luteolysis in sheep. PGT protein is expressed in the UOP during the estrous cycle and pharmacological inhibition of PGT prevents transport of luteolytic PGF2α pulse through the UOP in sheep. IFNT activates novel JAK-SRC-EGFR-RAS-RAF-ERK1/2-EGR-1 signaling modules in endometrial luminal epithelial (LE) cells and regulates PGT- mediated release of PGF2α through these novel cell-signaling pathways. IFNT stimulates ERK1/2 pathways in endometrial LE cells and inhibition of ERK1/2 inhibits IFNT action and restores spatial expression of OXTR and ESR-1 proteins in endometrial LE cells and restores endometrial luteolytic pulses of PGF2α in sheep. Collectively, the results of the present study provide the first evidence to indicate that transport of endometrial luteolytic PGF2α pulses from the uterus to the ovary through the UOP is controlled by a PGT-mediated mechanism in sheep, new mechanistic insight into molecular mechanisms regulating cellular and compartmental transport of PGF2α at the time of luteolysis, and new mechanistic understanding of IFNT action and release of PGF2α from the endometrial LE cells and thus opens a new arena of research in IFNT signaling and PGT function.