The ERC Starting Grant MALEPREG explored how immunological tolerance coevolved with male pregnancy. We revealed a link between male pregnancy evolution and a contraction of immune system pathway-related gene families, supported by a complete loss of specific immune components (Roth et al., 2020; Liu et al., 2022). While most immune genes are lost in all syngnathids, the genomic modifications of the MHC II system correlate with progressive development of male pregnancy (Roth et al., 2020; Parker et al., 2023a). In Syngnathus pipefishes, key MHC II pathway genes are lost, making a successful MHC II immune response and efficient activation of CD4+ T-helper cells impossible. In contrast, seahorses (Hippocampus) feature high sequence divergence of the MHC II pathway, along with the partial loss of the invariant chain (CD74). A downregulation of the activity and specificity of CD4+ T-helper cells, along with other losses in the complement system and innate immune defense, can critically lower self-non-self discrimination, potentially facilitating the evolution of male pregnancy by reducing the risk of embryo rejection.
Genome-wide selection analyses illuminated further adaptations in syngnathids associated with male pregnancy evolution. We detected selection signatures on importantdevelopmental genes (e.g. sox4a, sox9a, hmx4, six3a). In the Syngnathus species we identified signatures of selection on key adaptive immune system genes – RAG1/RAG2, indicating continuation of adaptive immune system rearrangements. The immunological components that syngnathids have lost or are under selection are crucial for successful clearance of infectious diseases and autoimmune regulation in vertebrates.
To investigate how syngnathids can live without and compensate for vital components of the immune system, we characterized Syngnathus immune cells using single-cell transcriptomics (Parker et al., 2023). The putative T-cell cluster supposedly only consists of CD8+ T-cells, while some gene markers hint at the potential retention of Treg subsets (Parker et al., 2022). These adaptive rearrangements in the syngnathid gene repertoire (Roth et al., 2020; Liu et al., 2022) could have provided an opportunity for the unique male pregnancy evolution with internal gestation and placenta-like systems in Syngnathus and Hippocampus. The differential regulation of immune response pathways during male pregnancy support the regulation of immune responses as key for successful (male) pregnancy evolution (Parker et al., 2023; Hilgers et al., 2021; Roth et al., 2020). We have identified a surprising overlap in the specific immunological pathways that were differentially regulated in female vs. male pregnancy (Hilgers et al., 2021; Roth et al., 2020). Inflammation could be a key process explaining how evolutionary innovations such as pregnancy and placentation evolved convergently. An enrichment of mammalian placenta genes during brooding in both pelvic brooding ricefishes and male pregnant syngnathids indicates convergent gene co-option.
In a transplant experiment comparing immunological reactions against fin allografts in a pipefish species with MHC II (N. ophidion) versus one without MHC II (S. typhle) (Parker & Roth, 2022), immunological rejection of foreign tissue was reduced in the species without MHC II. The genomic modification/loss of the MHC II pathway initiated physiological adaptations required for the evolution of full male pregnancy with internal gestation. We have yet to develop a successful methodology for gene knockdown using CRISPR/Cas9 in syngnathids. Despite many trials, breeding syngnathid embryos detached from their father seems to be deadly, highlighting the importance of male pregnancy in protecting and supporting this critical life stage.
Initial microbial colonization during embryonic development can occur through various vertical transmission routes (eggs, pregnancy, birth, breastfeeding, parental care) and environmental horizontal transmission. The unique evolution of male pregnancy allows to disentangle maternal provisioning through the egg from paternal provisioning through pregnancy. Vaccinating both mothers and/ or fathers before reproduction against a disease agent shifted the offspring microbial community and protected the offspring from an infection with the same pathogen (Schneider et al., 2024). The internal gut microbiome of the developing embryo was shaped by bacteria assigned to a maternal origin, while the external microbiome was strongly associated with bacteria from the male placenta-like structure (Tanger et al., 2014). Both paternal antibiotic and relocation of paternal core microbes significantly influenced the microbial composition of their offspring's brood pouch tissue. Male pregnancy provides an important vertical route for transferring paternal microbes into the next generation, shaping offspring microbiome development.
