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Lymph nodes are distributed all over the body and are part of the lymphatic system. The interferon-stimulated gene 15 kDa protein (ISG15) and prostaglandins (PGs) are involved in the establishment of pregnancy and are expressed in the uterus during early pregnancy in sheep. In this study, the ovine lymph nodes were obtained on Day 16 of the estrous cycle, and Days 13, 16, and 25 of pregnancy, and the expression of ISG15 and PG synthases, including cyclooxygenase 1 (COX-1), COX-2, prostaglandin E (PGE) synthase (PTGES), and a PGF synthase (aldo-keto reductase family 1, member B1, AKR1B1) were detected by quantitative real-time polymerase chain reaction, western blot analysis, and immunohistochemistry analysis. Our results showed that there were peaks in the expression of mRNAs and the proteins of ISG15, COX-1, COX-2, PTGES, and AKR1B1 in the lymph nodes during early pregnancy and that the COX-2 and AKR1B1 proteins were limited to the subcapsular sinus and lymph sinuses. In conclusion, the ISG15, COX-1, COX-2, PTGES, and AKR1B1 were upregulated in the maternal lymph nodes, which may be beneficial for the development of conceptus, maternal systemic immunoregulation, and anti-luteolysis during early pregnancy in sheep., (© 2018 Wiley Periodicals, Inc.)

The conceptus-derived signals that initiate maternal recognition of pregnancy act primarily on the endometrium to inhibit the development of luteolysis, thus modifying the expression of genes in the corpus luteum. The involvement of peripheral blood mononuclear cells (PBMCs) in the formation of this anti-luteolytic mechanism during early pregnancy is uncertain. In this study, PBMCs from non-pregnant and early-pregnant cows were sampled to explore the expression of genes associated with luteolysis, including AKR1B1 (aldo-keto reductase family 1, member B1; a bovine prostaglandin F synthase), PTGFR (PGF2α receptor), OXT (oxytocin), PTGES (PGE synthase), PTGER1 (PGE2 receptor 1), and PGR (progesterone receptor). OXT and PTGFR transcript abundance was low in PBMCs at Day 18 in pregnant individuals. PGR and PTGER1 mRNA abundance was significantly higher at Day 30 in pregnant individuals. AKR1B1 and PTGES transcript abundance was significantly higher at Day 18 in PBMCs from non-pregnant individuals, yet AKR1B1 and PTGES protein abundance was elevated at Day 30 in pregnant individuals-although AKR1B1 dimer may be significantly higher at Day 18 in non-pregnant PBMCs. In conclusion, changes in bovine PBMC gene expression are associated with luteolysis during early pregnancy, which implicate the influence of circulating blood components in controlling luteolysis. Mol. Reprod. Dev. 83: 509-515, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Simple Summary: The thymus is implicated in central immune tolerance and plays essential roles during normal pregnancy. The inhibitor of the NF-κB (IκB) family is a key component of the NF-κB pathway; however, the modulation of the κB family in the maternal thymus by early gestation has not been fully understood. It has been found that there changes in the expression of the IκB family in the maternal thymus take place during early pregnancy, suggesting that IκB proteins are involved in modulating immunologic tolerance and pregnancy establishment. The thymus is an essential component of maternal immune systems that play key roles in recognizing the placenta as immunologically foreign. The inhibitor of the NF-κB (IκB) family has essential effects on the NF-κB pathway; however, it is unclear whether early pregnancy modulates the expression of the IκB family in the thymus. In this study, maternal thymuses were sampled on day 16 of nonpregnancy and different gestation stages in the ovine, and the expression of IκB proteins was analyzed. The data showed that B cell leukemia-3 and IκBβ increased; however, IκBα, IκBε, and IKKγ deceased during gestation. Furthermore, there was an increase in IκBNS and IκBζ expression values on day 13 of pregnancy; however, this decreased on day 25 of gestation. In summary, the expression of the IκB family was modulated in the thymus during early gestation, suggesting that the maternal thymus can be associated with maternal immunologic tolerance and pregnancy establishment in ewes. [ABSTRACT FROM AUTHOR]

Early pregnancy modulates the maternal immune system, including the spleen and lymph nodes, which participate in maternal innate and adaptive immune responses. Methods: Ovine spleens and lymph nodes were sampled at day 16 of the estrous cycle, and at days 13, 16 and 25 of gestation, and qRT-PCR, Western blot and immunohistochemistry analysis were used to analyze the expression of the IκB family, including BCL-3, IκBα, IκBβ, IκBε, IKKγ, IκBNS and IκBζ. Early pregnancy induced expression of BCL-3, IκBα, IκBε, IKKγ and IκBζ, and expression of BCL-3, IκBβ and IκBNS peaked at day 16 of pregnancy in the spleen. However, early pregnancy suppressed the expression of BCL-3 and IκBNS, but stimulated the expression of IκBβ and IκBζ, and expression levels of IκBα, IκBβ, IκBε and IKKγ peaked in lymph nodes at days 13 and/or 16 of pregnancy. Early pregnancy changed the expression of the IκB family in the maternal spleen and lymph node in a tissue-specific manner, suggesting that the modulation of the IκB family may be involved in regulation of maternal functions of the spleen and lymph nodes, which are necessary for the establishment of maternal immune tolerance during early pregnancy in sheep. [ABSTRACT FROM AUTHOR]

Simple Summary: An inhibitor of the NF-κB (IκB) family is involved in regulating innate immune responses. In this study, early pregnancy induces upregulation of IκBα, but changes and downregulates the expression of BCL-3, IκBε, IKKγ and IκBNS, and changes expression of IκBβ and IκBζ in the maternal liver, which are related to maternal peripheral tolerance and pregnancy establishment. During normal pregnancy, there is a dynamic regulation of the maternal immune system, including the liver, to accommodate the presence of the allogeneic foetus in the uterus. However, it was unclear that the expression of the IkappaB (IκB) family was regulated in the ovine maternal liver during early pregnancy. In this study, sheep livers were collected at day 16 of the oestrous cycle (NP16), and days 13, 16 and 25 of gestation (DP13, DP16 and DP25), and RT-qPCR, Western blot and immunohistochemistry analysis were used to analyse the expression of the IκB family, including B cell leukemia-3 (BCL-3), IκBα, IκBβ, IκBε, IKKγ, IκBNS and IκBζ. The results revealed that expression of BCL-3, IκBβ, IκBε and IKKγ peaked at DP16, and the expression of IκBα was increased during early pregnancy. In addition, the expression of IκBζ peaked at DP13 and DP16, and IκBNS peaked at DP13. IκBβ and IKKγ proteins were located in the endothelial cells of the proper hepatic arteries and portal veins, and hepatocytes. In conclusion, early pregnancy changed the expression of the IκB family, suggesting that the modulation of the IκB family may be related to the regulation of maternal hepatic functions, which may be favourable for pregnancy establishment in sheep. [ABSTRACT FROM AUTHOR]

Nucleotide-binding oligomerization domain receptors (NOD-like receptors, NLRs) are involved in modulating the innate immune responses of the trophoblast and the placenta in normal pregnancy. The thymus participates in regulation of innate and adaptive immune responses. However, it is unclear whether expression of NLR is modulated in the maternal thymus during early pregnancy. In this study, thymuses were sampled at day 16 of the estrous cycle, and at days 13, 16 and 25 of gestation (n = 6 for each group) from ewes after slaughter. Different stages were chosen because the maternal thymus was under the different effects of interferon-tau and/or progesterone or not. RT-qPCR, Western blot and immunohistochemistry analysis were used to analyze the expression of the NLR family, including NOD1; NOD2; major histocompatibility complex class II transactivator (CIITA); NLR family apoptosis inhibitory protein (NAIP); nucleotide-binding oligomerization domain and Leucine-rich repeat and Pyrin domain containing protein 1 (NLRP1), NLRP3 and NLRP7. The results showed that expression level of NOD1 was changed with the pregnancy stages, and expression levels of NOD2, CIITA, NAIP, NLRP1, NLRP3 and NLRP7 mRNA and proteins were peaked at day 13 of pregnancy. The levels of NOD2 and CIITA were increased during early pregnancy. The stainings for NOD2 and NLRP7 proteins were located in epithelial reticular cells, capillaries and thymic corpuscles. In summary, pregnancy stages changed expression of NLR family in the maternal thymus, which may be related to the modulation of maternal thymic immune responses, and beneficial for normal pregnancy in sheep. [ABSTRACT FROM AUTHOR]

Simple Summary: NOD receptors mediate adaptive immune responses and immune tolerance. In this study, the results demonstrated that early pregnancy increased expression of NOD1, CIITA, NLRP1, NLRP3 and NLRP7. However, early pregnancy inhibited NAIP expression in maternal lymph nodes. There is immune regulation of the lymph nodes, which is necessary during ovine gestation. NOD receptors (NLRs) mediate adaptive immune responses and immune tolerance. Nevertheless, it is not clear if gestation modulates the NLR signaling pathway in lymph nodes of ewes. In this study, lymph nodes of ewes were collected at day 16 of the estrous cycle, and at days 13, 16 and 25 of gestation (n = 6 for each group). RT-qPCR, Western blot and immunohistochemistry analysis were used to analyze the expression of the NLR family, including NOD1, NOD2, CIITA, NAIP, NLRP1, NLRP3 and NLRP7. The data showed that early gestation enhanced expression of NOD1, CIITA, NLRP1, NLRP3 and NLRP7 mRNA, as well as proteins at day 16 of gestation, and the expression levels of NOD2, CIITA, NLRP1 and NLRP7 were higher at days 13 and 25 of gestation than day 16 of the estrous cycle. However, NOD1 expression was lower on days 13 and 25 of gestation compared to day 16 of the estrous cycle, and early gestation suppressed NAIP expression. In summary, early pregnancy modulated expression of the NLR family in ovine lymph nodes, which participates in immune regulation, and this modulation may be necessary for pregnancy establishment in ewes. [ABSTRACT FROM AUTHOR]

There is a pregnant maternal immunological tolerance that protects the fetus and promotes its growth, and nuclear factor kappa B (NF-κB) family participates in the regulation of innate immune and adaptive immune responses. The thymus is related to establishing central tolerance, and early pregnancy has effects on expression of a good number of genes and proteins in the maternal thymus in sheep. However, it is unclear whether early pregnancy changes expression of NF-κB subunits in the ovine thymus. In this study, the thymic samples were collected from day 16 of non-pregnant ewes, and days 13, 16 and 25 of pregnant ewes, and the expression of NF-κB members (NF-κB1, NF-κB2, RelA, RelB and c-Rel) was analyzed through real-time quantitative PCR, Western blot and immunohistochemical analysis. The results showed that c-Rel mRNA and protein upregulated at day 25 of pregnancy, and NF-κB1 mRNA and proteins increased at days 16 and 25 of pregnancy, and RelB mRNA and proteins enhanced during early pregnancy. However, expression levels of NF-κB2 and RelA were decreased during early pregnancy, but upregulated from day 13 to 25 of pregnancy. In addition, the RelA protein was located in the epithelial reticular cells, capillaries and thymic corpuscles. This paper reported for the first time that early pregnancy induced expression of NF-κB1, RelB and c-Rel, but inhibited expression of NF-κB2 and RelA in the maternal thymus during early pregnancy, which is involved in the central immune tolerance, and helpful for successful pregnancy in sheep. [ABSTRACT FROM AUTHOR]

Toll-like receptors (TLRs) participate in maternal immune regulation at multiple maternal-fetal interfaces. The liver is crucial for innate and adaptive immunity during pregnancy. The objective of this study was to measure the expression of genes and proteins related to TLR signaling. In this study, livers were obtained from ewes on day 16 of the estrous cycle, and on days 13, 16 and 25 of gestation, and RT-qPCR, western blot and immunohistochemistry were employed to measure the expression of genes and proteins related to TLR signaling, including TLR2, TLR3, TLR4, TLR5, TLR7, TLR9, myeloid differentiation primary-response protein 88 (MyD88), tumor necrosis factor receptor associated factor 6 (TRAF6) and interleukin-1-receptor-associated kinase 1 (IRAK1). Our data showed that the expression levels of TLR2, TLR3, TLR5, TLR9 and TRAF6 were increased, but those of TLR4 and TLR7 were decreased in the liver during early pregnancy. In addition, the expression levels of MyD88 and IRAK1 were the highest on days 13 and 16 of pregnancy, respectively, and the MyD88 protein was located in endothelial cells of proper hepatic arteries and portal veins and in hepatocytes. In conclusion, early pregnancy modulates TLR signaling in the maternal liver in sheep, which may participate in maternal immune regulation. [ABSTRACT FROM AUTHOR]

Complement pathways participate in the regulation of innate immune system, and complement activation is inhibited in normal pregnancy. The liver plays key roles in the modulation of immunity and tolerance, but it is unclear that early pregnancy induces the changes in expression of complement components in the ovine maternal liver. The aim of the present study was to explore the expression of complement components in the liver using quantitative real‐time polymerase chain reaction (PCR), Western blot, and immunohistochemistry. Maternal livers were collected on Day 16 of the estrous cycle and Days 13, 16, and 25 of gestation. The results indicated that early pregnancy suppressed the expression of C1q, C1r, C1s, C2, C4a, C5b, and C9 in the maternal liver, but C3 expression was increased. In addition, C3 protein was located in the endothelial cells of the proper hepatic arteries and portal veins and hepatocytes. In summary, the downregulaltion of C1q, C1r, C1s, C2, C4a, C5b, and C9 may be involved in the suppression of complement activation, and upregulation of C3 is related to the modulation of maternal immune tolerance in ovine liver. [ABSTRACT FROM AUTHOR]

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The spleen combines the innate and adaptive immune systems, and the early conceptus must regulate the maternal intrauterine immune and systemic immune response during early pregnancy in sheep. However, it is unclear whether early pregnancy exerts its effects on the characterization of the T helper (Th) cytokines profile in the spleen during early pregnancy in ewes. In this study, spleens were obtained at day 16 of the oestrous cycle and at days 13, 16 and 25 of pregnancy (n = 6 for each group) from ewes, and qRT-PCR, western blot and immunohistochemistry analysis were used to analyze the Th cytokines profile of the spleens. Our results showed that there was down-regulation of IFN-γ at days 13 and 16 of pregnancy, but there was upregulation of IL-2, IL-5 and IL-6 at day 25 of pregnancy, TNF-β at days 16 and 25 of pregnancy, and IL-4 and IL-10 in all pregnant groups. Immunohistochemistry results showed that the IL-6 protein was limited to the capsule, trabeculae and splenic cords. This paper reported, for the first time, that characterization of the Th cytokines profile varied in the maternal spleen during early pregnancy, which may be essential for successful pregnancy in sheep. [ABSTRACT FROM AUTHOR]

The spleen is a unique lymphoid organ that plays a key role in immune regulation. Interferon‐tau induces upregulation of interferon‐stimulated gene 15‐kDa protein (ISG15) in the uterus during early pregnancy in sheep. Prostaglandins (PGs) have important effects in both the activation and the inhibition of immune response through an autocrine and paracrine manner. In this study, splenic samples were obtained at Day 16 of the estrous cycle, and Days 13, 16, and 25 of pregnancy from ewes, and the expression of ISG15 and PG synthases, including cyclooxygenase 1 (COX‐1), COX‐2, PGE synthase (PTGES), and PGF synthase (aldo‐keto reductase family 1, member B1, AKR1B1), was detected through quantitative real‐time PCR, western blot, and immunohistochemistry analysis. Our results showed that there was upregulation of COX‐2 mRNA and protein at Day 25 of pregnancy, and ISG15 mRNA and conjugated proteins, and AKR1B1 mRNA and dimer at Days 16 and 25 of pregnancy. COX‐2 and AKR1B1 proteins were limited to the capsule, trabeculae, and splenic cords. However, the expression of COX‐1 and PTGES was not affected by early pregnancy. In conclusion, ISG15‐conjugated proteins, COX‐2 and AKR1B1 upregulated in maternal spleen during early pregnancy, which may be beneficial for the placentation in sheep. [ABSTRACT FROM AUTHOR]

A fetus changes immune responses in the uterus and the maternal immune system, and lymph nodes are associated with regulating maternal adaptive immunity. Complement activation is associated with abnormal pregnancy in mice and humans. The aim of the present study was to explore the expression levels of complement components in maternal lymph nodes during early pregnancy in sheep. Maternal inguinal lymph nodes were sampled on day 16 of the estrous cycle, and days 13, 16 and 25 of gestation in ewes. Reverse transcription-quantitative PCR, western blotting and immunohistochemical analyses were used to detect the expression levels of complement components C1q, C1r, C1s, C2, C3, C4a, C5b and C9 in the lymph nodes. The results revealed that the protein and mRNA levels of C1q, C1s and C5b were enhanced during early pregnancy, and that C1r and C4a were upregulated at day 25 of pregnancy. The mRNA and protein levels of C2 and C9 peaked at day 16 of pregnancy, but C3 was decreased at day 25 of pregnancy. C3 protein was located in the subcapsular sinuses and lymph sinuses of the maternal lymph node. In summary, the present study detected changes in the expression levels of complement components in maternal lymph nodes, which may be associated with maternal immune regulation during early pregnancy in sheep. [ABSTRACT FROM AUTHOR]

Simple Summary: Complement regulation is related to fetal growth during early pregnancy in humans. The results show that C1q increased during early pregnancy, and C1r, C1s, C2, C3 and C5b enhanced at day 25 of gestation, and C4a and C9 increased on days 13 and 16 of pregnancy in the ovine maternal spleen. In summary, a complement pathway participates in maternal immune regulation during early gestation. During early gestation in humans, complement regulation is essential for normal fetal growth. It is supposed that a complement pathway participates in maternal splenic immune regulation at the early stage of gestation in ewes. The aim of this study was to analyze the effects of early pregnancy on the expression of complement components in the maternal spleen of ewes. In this study, ovine spleens were sampled on day 16 of nonpregnancy, and days 13, 16 and 25 of gestation. RT-qPCR, Western blot and immunohistochemical analysis were used to detect the changes in expression of complement components in the ovine maternal spleens. Our results reveal that C1q was upregulated during early gestation, C1r, C1s, C2, C3 and C5b increased at day 25 of gestation and C4a and C9 peaked at days 13 and 16 of gestation. In addition, C3 protein was located in the capsule, trabeculae and splenic cords. In conclusion, our results show for the first time that there was modification in the expression of complement components in the ovine spleen at the early stage of gestation, and complement pathways may participate in modulating splenic immune responses at the early stage of gestation. [ABSTRACT FROM AUTHOR]

Lymph nodes have functions in the adaptive immune response, and interferon-tau (IFNT), a primary pregnancy recognition signal in domestic ruminants has effects on immune regulation. It, however, is unclear whether early pregnancy induces an increase in the abundance of interferon-stimulated gene (ISG) mRNA transcripts and proteins in lymph nodes of sheep. In this study, lymph nodes were obtained on day 16 of the estrous cycle from non-pregnant ewes and days 13, 16 and 25 of gestation from pregnant ewes, and the abundance of ISG mRNA transcripts, including signal transducer and activator of transcription 1 (STAT1), phosphorylated STAT1 (p-STAT1), 2′,5′-oligoadenylate synthetase (OAS1), myxovirus resistance protein 1 (MX1) and C-X-C motif chemokine 10 (CXCL10), was analyzed using real-time quantitative PCR. Furthermore, Western blot and immunohistochemistry analysis was conducted to assess relative abundance of proteins encoded by these genes. The results indicated that there was a larger abundance of STAT1 mRNA transcript and protein, and p-STAT1 protein in the maternal lymph node at days 16 and 25 of gestation, and that abundances of OAS1, MX1 and CXCL10 mRNA transcripts and protein were greatest on day 16 of gestations. In addition, STAT1 protein was located in the subcapsular sinus, lymph sinuses, B cells and T cells. The larger relative abundances of STAT1, p-STAT1, OAS1, MX1 and CXCL10 mRNA transcripts and/or protein in the lymph nodes of ewes may be associated with maternal immunoregulation through blood circulation and lymph circulation during early pregnancy. [ABSTRACT FROM AUTHOR]

Simple Summary: Interferon-tau is a maternal recognition factor in ruminants, and spleen plays an essential role in regulating innate and adaptive immune responses. We found that interferon-tau derived from conceptus induces expression of STAT1, OAS1, and CXCL10 in ovine maternal spleen, which may be helpful for maternal immune regulation. Interferon-tau is a maternal recognition factor in ruminant species, and spleen plays an essential role in regulating innate and adaptive immune responses. However, it is not fully understood that early pregnancy induces expression of interferon stimulated genes (ISGs) in the spleen during early pregnancy in ewes. In this study, spleens were collected from ewes at day 16 of the estrous cycle, and on days 13, 16, and 25 of gestation (n = 6 for each group), and RT-qPCR, western blot and immunohistochemistry analysis were used to detect the expression of signal transducer and activator of transcription 1 (STAT1), 2′,5′-oligoadenylate synthetase 1 (OAS1), myxovirusresistance protein 1 (Mx1) and C-X-C motif chemokine 10 (CXCL10). The results revealed that STAT1, OAS1 and CXCL10 mRNA and proteins were upregulated in the spleens during early pregnancy, and STAT1 protein was located in connective tissue cells in the capsule and trabeculae, and blood cells and lymphocytes in the red pulp. However, early pregnancy had no significant effects on expression of MX1 mRNA and protein. In conclusion, early pregnancy induces expression of STAT1, OAS1 and CXCL10 in maternal spleen, suggesting that maternal spleen is involved in immune regulation of pregnancy in sheep. [ABSTRACT FROM AUTHOR]

Simple Summary: The thymus is a main organ of the immune system. Immune tolerance exists in maternal immune system during pregnancy. Helper T (Th)1 and Th2 cytokines regulate the functions of immune system. We found that early pregnancy affected the production of Th1 and Th2 cytokines in maternal thymus in sheep, which may be beneficial for normal pregnancy. There is an immune tolerance in maternal immune system during pregnancy, and thymus is a main organ of the immune system. Helper T (Th)1 and Th2 cytokines are involved in the regulation of immune system, but the modulation of Th cytokines in the thymus during early pregnancy is unclear in ewes. Thymuses were collected on day 16 of the estrous cycle, and on days 13, 16, and 25 of pregnancy in ewes. qRT-PCR, Western blot, and immunohistochemistry were used to analyze the expression of Th1 and Th2 cytokines in the thymuses. There was a peak in the expression of interferon-gamma (IFN-γ) on day 16 of pregnancy, an upregulation of tumor necrosis factor beta (TNF-β), and a sustained expression of interleukin-2 (IL-2) and IL-4. Furthermore, there was a peak in the expression of IL-6 on day 13 of pregnancy, no expression of IL-6 on day 16 of the estrous cycle and day 25 of pregnancy, and an upregulation of IL-5 and IL-10 in the thymuses during early pregnancy. The immunohistochemistry results revealed that the IFN-γ and IL-6 proteins were limited to the stromal cells, capillaries, and thymic corpuscles. In conclusion, early pregnancy influenced the production of Th1 and Th2 cytokines of maternal thymus in sheep. [ABSTRACT FROM AUTHOR]

Thymus is a primary lymphoid organ, must adapt to the presence of fetal alloantigens. Prostaglandins (PGs) have diverse effects to activate or inhibit the immune response, but effects of early pregnancy on the expression of PG synthases in ovine maternal thymus are unclear. In this study, ovine thymic samples were obtained at day 16 of the estrous cycle, and days 13, 16 and 25 of pregnancy. The expression of PG synthases, including cyclooxygenase 1 (COX-1), COX-2, PGE2 synthase (PTGES), and a prostaglandin F2α synthase (Aldo-keto reductase family 1, member B1, AKR1B1), was evaluated using quantitative real-time PCR, Western blot and immunohistochemistry analysis. In addition, the thymus/body ratio was also calculated. Our results showed that the expression of COX-2 mRNA and protein, AKR1B1 mRNA and dimer were up-regulated on day 25 of pregnancy (P < 0.05), and expression of COX-1, PTGES mRNA and protein, AKR1B1 monomer and thymus/body ratio were similar at different stages of pregnancy and the estrous cycle. The immunohistochemistry results showed that the COX-2 and AKR1B1 proteins were located in the stromal cells, capillaries and thymic corpuscles. This is the first study to report that expression of COX-2 and AKR1B1 dimer is up-regulated in the maternal thymus during early pregnancy, suggesting that early pregnancy exerts its effects on maternal thymus, which is involved in immunomodulation during early pregnancy in sheep. • Up-regulation expression of COX-2 mRNA and protein in the thymus on day 25 of pregnancy. • Up-regulation expression of AKR1B1 mRNA and dimer in the thymus on day 25 of pregnancy. • COX-2 and AKR1B1 proteins were limited to the stromal cells, capillaries and thymic corpuscles in the thymus. [ABSTRACT FROM AUTHOR]

Abstract As a fetal allograft to the mother, early conceptus regulates the intrauterine immune and systemic immune responses during early pregnancy in sheep. However, expression of T helper 1 (Th1) and Th2 cytokines in maternal lymph nodes is unclear during early pregnancy in sheep. In this study, inguinal lymph nodes were obtained on day 16 of the estrous cycle and on days 13, 16 and 25 of pregnancy (n = 4 for each group) in ewes, and qRT-PCR, western blot and immunohistochemistry were used to compare the expression of Th1 and Th2 cytokines in the lymph nodes. Our results showed that there were the highest levels of Th1 cytokines (IFN-γ, TNF-β and IL-2) and Th2 cytokines (IL-4, IL-5, IL-6 and IL-10) in the lymph nodes on day 13 or 16 of pregnancy. Furthermore, there were a downregulation of TNF-β and IL-2 and an upregulation of IL-5 and IL-10 on day 25 of pregnancy compared with that in nonpregnancy, with no significant difference in the expression of IFN-γ, IL-4, and IL-6 between the ewes on day 25 of pregnancy and nonpregnancy. The immunohistochemistry results showed that the IL-2 and IL-10 proteins were limited to the subcapsular sinus and trabeculae in the cortex, lymph sinus. In conclusion, early pregnancy exerted its effects on the lymph node and induced a Th2-biased response, which was essential for a normal pregnancy in sheep. Highlights • Downregulation of tumor necrosis factor beta and IL-2 in the lymph node. • Upregulation of IL-5 and IL-10 in the lymph node. • IL-2 and IL-10 were localized in the subcapsular sinus and trabeculae in the cortex, medullary sinuses. [ABSTRACT FROM AUTHOR]

The spleen plays a key role in immune regulation. Progesterone (P4) exhibits different immunological effects through binding to different types of receptor. In the present study, splenic samples were obtained at day 16 of non-pregnancy and days 13, 16, and 25 of pregnancy to study the expression of P4 receptor (PGR) and P4-induced blocking factor (PIBF) in ewes through quantitative real-time PCR, Western blot and immunohistochemistry analysis. Our results showed that the relative expression levels of PGR and PIBF mRNA increased from day 13–16 in pregnant ewes, but decreased from day 16–25, and the 26 kDa isoform of PGR and the 22 kDa variant of PIBF were down-regulated in the spleens at day 25 of pregnancy compared with expression at day 16 of the oestrous cycle and days 13 and 16 of pregnancy. The immunohistochemistry test results confirmed that PGR and PIBF proteins were localized in the cytoplasm of cells in the capsule, trabeculae and splenic cords. This paper reports for the first time that the 26 kDa isoform of PGR and the 22 kDa variant of PIBF were down-regulated during early pregnancy, which may be essential for normal pregnancy in ewes. [ABSTRACT FROM AUTHOR]

Lymph nodes are the sites where the immune reaction or suppression takes place. Progesterone (P4) exerts an essential effect of the immunomodulation on the maternal uterus during early pregnancy in ruminants. At present study, the inguinal lymph nodes were obtained at day 16 of non-pregnancy, days 13, 16 and 25 of pregnancy (n = 3 for each group) in ewes, and RT-PCR assay, western blot and immunohistochemistry analysis were used to analyze to the effect of early pregnancy on the expression of P4 receptor (PGR) and progesterone-induced blocking factor (PIBF) in the lymph nodes. Our results showed that the PGR and PIBF mRNA were up-regulated in the lymph nodes in pregnant ewes, and the PGR isoform (60 kDa) and the PIBF variant (75 kDa) were expressed constantly in the lymph nodes. However, there was no expression of the PGR isoform (40 kDa) and the PIBF variant (48 kDa) at day 16 of the estrous cycle. The immunohistochemistry results confirmed that the PGR and PIBF proteins were limited to the subcapsular sinus and trabeculae in the cortex, medullary sinuses, and were localized in the cytoplasm of the specific cells. This paper reports for the first time that early pregnancy exerts its effect on the specific cells in the lymph nodes through P4, which results in the up-regulated expression of the PGR mRNA and 40 kDa isoform, the PIBF mRNA and 48 kDa variant, and is involved in the immunoregulation of the lymph nodes through a cytosolic pathway in ewes. [ABSTRACT FROM AUTHOR]