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.

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.

Nucleotide-binding oligomerization domain receptors (NOD-like receptors, NLRs) have critical effects on interfaces of the immune and reproductive systems, and the spleen plays a key role in both innate and adaptive immune functions. It is hypothesized that NLR family participates in maternal splenic immune regulation during early pregnancy in sheep. In this study, maternal spleens were collected on day 16 of the estrous cycle, and days 13, 16 and 25 of gestation (n = 6 for each group) in ewes. Expression of NLR family, including NOD1, NOD2, class II transactivator (CIITA), NLR family apoptosis inhibitory protein (NAIP), nucleotide-binding oligomerization domain, Leucine rich repeat and Pyrin domain containing 1 (NLRP1), NLRP3 and NLRP7, was analyzed using quantitative real-time PCR, Western blot and immunohistochemistry analysis. The results revealed that expression levels of NOD1, NOD2, CIITA and NLRP3 were downregulated at days 13 and 16 of pregnancy, but expression of NLRP3 was increased at day 25 of pregnancy. In addition, expression values of NAIP and NLRP7 mRNA and proteins were improved at days 16 and 25 of pregnancy, and NLRP1 was peaked at days 13 and 16 of pregnancy in the maternal spleen. Furthermore, NOD2 and NLRP7 proteins were limited to the capsule, trabeculae and splenic cords. In summary, early pregnancy changes expression of NLR family in the maternal spleen, which may be related with the maternal splenic immunomodulation during early pregnancy in sheep.