Evolutionary traits and functional roles of chemokines and their receptors in the male pregnancy of the Syngnathidae.

Vertebrates have developed various modes of reproduction, some of which are found in Teleosts. Over 300 species of the Syngnathidae (seahorses, pipefishes and seadragons) exhibit male pregnancies; the males have specialized brood pouches that provide immune protection, nourishment, and oxygen regulation. Chemokines play a vital role at the mammalian maternal-fetal interface; however, their functions in fish reproduction are unclear. This study revealed the evolutionary traits and potential functions of chemokine genes in 22 oviparous, ovoviviparous, and viviparous fish species through comparative genomic analyses. Our results showed that chemokine gene copy numbers and evolutionary rates vary among species with different modes of reproduction. Syngnathidae lost cxcl13 and cxcr5, which are involved in key receptor-ligand pairs for lymphoid organ development. Notably, Syngnathidae have site-specific mutations in cxcl12b and ccl44, suggesting immune function during gestation. Moreover, transcriptome analysis revealed that chemokine gene expression varies among Syngnathidae species with different types of brood pouches, suggesting adaptive variations in chemokine functions among seahorses and their relatives. Furthermore, challenge experiments on seahorse brood pouches revealed a joint immune function of chemokine genes during male pregnancy. This study provides insights into the evolutionary diversity of chemokine genes associated with different reproductive modes in fish. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-023-00205-x.

Seahorse Male Pregnancy as a Model System to Study Pregnancy, Immune Adaptations, and Environmental Effects.

Seahorses, together with sea dragons and pipefishes, belong to the Syngnathidae family of teleost fishes. Seahorses and other Syngnathidae species have a very peculiar feature: male pregnancy. Among different species, there is a gradation of paternal involvement in carrying for the offspring, from a simple attachment of the eggs to the skin surface, through various degrees of egg coverage by skin flaps, to the internal pregnancy within a brood pouch, which resembles mammalian uterus with the placenta. Because of the gradation of parental involvement and similarities to mammalian pregnancy, seahorses are a great model to study the evolution of pregnancy and the immunologic, metabolic, cellular, and molecular processes of pregnancy and embryo development. Seahorses are also very useful for studying the effects of pollutants and environmental changes on pregnancy, embryo development, and offspring fitness. We describe here the characteristics of seahorse male pregnancy, its regulatory mechanisms, the development of immune tolerance of the parent toward the allogeneic embryos, and the effects of environmental pollutants on pregnancy and embryo development.

Orphan gene expressed in flame cone cells uniquely found in seahorse epithelium.

The seahorse is one of the most unique teleost fishes in its morphology. The body is surrounded by bony plates and spines, and the male fish possess a brooding organ, called the brood pouch, on their tail. The surfaces of the brood pouch and the spines are surrounded by characteristic so-called flame cone cells. Based on our histological observations, flame cone cells are present in the seahorse Hippocampus abdominalis, but not in the barbed pipefish Urocampus nanus or the seaweed pipefish Syngnathus schlegeli, both of which belong to the same family as the seahorse. In the flame cone cells, we observed expression of an "orphan gene" lacking homologs in other lineages. This gene, which we named the proline-glycine rich (pgrich) gene, codes for an amino acid sequence composed of repetitive units. In situ hybridization and immunohistochemical analyses detected pgrich-positive signals from the flame cone cells. Based on a survey of the genome sequences of 15 teleost species, the pgrich gene is only found from some species of Syngnathiformes (namely, the genera Syngnathus and Hippocampus). The amino acid sequence of the seahorse PGrich is somewhat similar to the sequence deduced from the antisense strand of elastin. Furthermore, there are many transposable elements around the pgrich gene. These results suggest that the pgrich gene may have originated from the elastin gene with the involvement of transposable elements and obtained its novel function in the flame cone cells during the evolution of the seahorse.

Characterization and expression patterns of lysozymes reveal potential immune functions during male pregnancy of seahorse.

Seahorses are one of the most amazing ovoviviparous fishes in the ocean because males, and not females, have evolved a brood pouch for incubating embryos. During male pregnancy, paternal seahorses need to develop effective immune protection for embryos in the brood pouch from potential infection by pathogens. Lysozymes (Lyz) are a group of antibacterial enzymes of the innate immune system that play an important role in resisting pathogen invasion. However, the immune function of lysozymes in the brood pouch of the pregnancy-lined seahorse (Hippocampus erectus) remains unknown. In this study, we found three different lysozymes in the lined seahorse: HeLyzC, HeLyzG1, and HeLyzG2. Synteny analysis revealed that HeLyzG1 and HeLyzG2 were generated by species-specific expansion rather than tandem duplication. Tissue expression patterns showed that the highest mRNA expression levels of the three lysozymes occurred in the brood pouches. Immunostimulation-induced expression analysis showed that all three HeLyzs in the brood pouches up-regulated their mRNA expression levels after Vibrio parahaemolyticus infection, but only the HeLyzG2 was upregulated after Poly(I:C) injection. Similarly, except for HeLyzC, upregulated expressions of HeLyzG1 and HeLyzG2 were found quickly in brood pouches injected with LPS. The upregulated levels of HeLyzC and HeLyzG2 in brood pouches during pregnancy were significantly higher than those in non-pregnancy, implying that seahorse lysozymes might function in the immune defense in brood pouches during pregnancy. In addition, the expression levels of HeLyzs were low in embryos in the brood pouch but significantly increased in neonates. This implies that embryos in the brood pouch might not necessarily express more lysozymes by themselves due to paternal immune protection. In conclusion, our study demonstrated that HeLyzs play an important role in immune protection during male seahorse gestation, and the synergistic effect of multiple HeLyzs may contribute to improved neonatal survival.

Seahorse brood pouch morphology and control of male parturition in Hippocampus abdominalis.

INTRODUCTION: Syngnathids (seahorses, pipefishes and seadragons) are among the few vertebrates that display male pregnancy. During seahorse pregnancy, males incubate developing embryos embedded in a placenta within a fleshy brood pouch, before expelling fully developed neonates at parturition. The mechanisms underpinning seahorse parturition are poorly understood. METHODS: We examined the morphology of the brood pouch using microcomputed tomography and histological techniques, in combination with physiological assays, to examine how male pot-bellied seahorses (Hippocampus abdominalis) control labour. In female-pregnant vertebrates, nonapeptide hormones (such as vasopressin- and oxytocin-like hormones) produce contractions of gestational smooth muscle to produce labour. RESULTS: Histological analysis of the seahorse brood pouch reveals only scattered small smooth muscle bundles in the brood pouch, and in-vitro application of isotocin (a teleost nonapeptide hormone) to the brood pouch do not produce measurable muscle contractions. Micro-computed tomography shows differences in size and orientation of the anal fin assembly between male and female pot-bellied seahorses, and histological analysis reveals large skeletal muscle bundles attached to the anal fin bones at the male brood pouch opening. DISCUSSION: We conclude that seahorse parturition may be facilitated by contraction of these muscles, which, in combination with body movements, serves to gape open the pouch and expel the neonates. Future biomechanical studies are needed to test this hypothesis.

Genome and gene evolution of seahorse species revealed by the chromosome-level genome of Hippocampus abdominalis.

Seahorses belong to the teleost family Syngnathidae that evolved a distinct body plan and unique male pregnancy compared to other teleosts. As a classic model for studying evolution of viviparity and sexual selection of teleosts, seahorse species still lack a publicly available high-quality reference genome. Here, we generated the genome assembly of the big-belly seahorse, Hippocampus abdominalis with long-read and Hi-C technologies. We managed to place over 99% of the total length of 444.7 Mb of assembled genome into 21 linkage groups with almost no gaps. We reconstructed a phylogenomic tree with the big-belly seahorse genome and other representative Syngnathidae and teleost species. We also reconstructed the historical population dynamics of four representative Syngnathidae species. We found the gene families that underwent expansion or contraction in the Syngnathidae ancestor were enriched for immune-related or ion transporter gene ontology terms. Many of these genes were also reported to show a dynamic expression pattern during the pregnancy stages of H. abdominalis. We also identified putative positively selected genes in the Syngnathidae ancestor or in H. abdominalis, whose mouse mutants are enriched for abnormal craniofacial and limb morphological phenotypes. Overall, our study provides an important genome resource for evolutionary and developmental studies of seahorse species, and candidate genes for future experimental works.

Effects of tributyltin on gonad and brood pouch development of male pregnant lined seahorse (Hippocampus erectus) at environmentally relevant concentrations.

The male pregnancy of seahorses is unique, but their reproductive response to environmental disturbances has not yet been clarified. Tributyltin (TBT) is known to have an endocrine disrupting effect on the reproductive system of coastal marine organisms. This study evaluated the potential effects of exposure to environmentally relevant concentrations of TBT on the development of gonads and brood pouch of the lined seahorse (Hippocampus erectus). Physiological, histological, and transcriptional analyses were conducted, and results showed that high levels of TBT bioaccumulation occurred in male and female seahorses. TBT led to ovarian follicular atresia and apoptosis with the elevation of androgen levels, accompanied by the induction of genes associated with lysosomes and autophagosomes. Comparative transcriptional analyses revealed the likely inhibition of spermatogenesis via the suppression of cyclic AMP and androgen synthesis. Notably, the transcriptional profiles showed that TBT potentially affects the immune system, angiogenesis, and embryo nourishment of the brood pouch, which indicates that it has negative effects on the male reproductive system of seahorses. In summary, this study reveals that environmental levels of TBT potentially affect the reproductive efficiency of seahorses, and may ultimately lead to a reduction in their populations in coastal environments.

Paternal nutrient provisioning during male pregnancy in the seahorse Hippocampus abdominalis.

Vertebrates that incubate embryos on or within the body cavity exhibit diverse strategies to provide nutrients to developing embryos, ranging from lecithotrophy (solely yolk-provided nutrition) to substantial matrotrophy (supplemental nutrients from the mother before birth). Syngnathid fishes (seahorses, pipefishes and sea dragons) are the only vertebrates to exhibit male pregnancy. Therefore, they provide a unique opportunity for comparative evolutionary research, in examining pregnancy independent of the female reproductive tract. Here, we tested the hypothesis that the most complex form of syngnathid pregnancy involves nutrient transport from father to offspring. We compared the dry masses of newly fertilised Hippocampus abdominalis eggs with those of fully developed neonates to derive a patrotrophy index. The patrotrophy index of H. abdominalis was 1, indicating paternal nutrient supplementation to embryos during gestation. We also measured the lipid content of newly fertilised eggs and neonates and found that there was no significant decrease in lipid mass during embryonic development. Since lipids are likely to be the main source of energy during embryonic development, our results suggest that lipid yolk reserves being depleted by embryonic metabolism are replaced by the brooding father. The results of our study support the hypothesis that nutrient transport occurs in the most advanced form of male pregnancy in vertebrates.

The evolution and physiology of male pregnancy in syngnathid fishes.

The seahorses, pipefishes and seadragons (Syngnathidae) are among the few vertebrates in which pregnant males incubate developing embryos. Syngnathids are popular in studies of sexual selection, sex-role reversal, and reproductive trade-offs, and are now emerging as valuable comparative models for the study of the biology and evolution of reproductive complexity. These fish offer the opportunity to examine the physiology, behavioural implications, and evolutionary origins of embryo incubation, independent of the female reproductive tract and female hormonal milieu. Such studies allow us to examine flexibility in regulatory systems, by determining whether the pathways underpinning female pregnancy are also co-opted in incubating males, or whether novel pathways have evolved in response to the common challenges imposed by incubating developing embryos and releasing live young. The Syngnathidae are also ideal for studies of the evolution of reproductive complexity, because they exhibit multiple parallel origins of complex reproductive phenotypes. Here we assay the taxonomic distribution of syngnathid parity mode, examine the selective pressures that may have led to the emergence of male pregnancy, describe the biology of syngnathid reproduction, and highlight pressing areas for future research. Experimental tests of a range of hypotheses, including many generated with genomic tools, are required to inform overarching theories about the fitness implications of pregnancy and the evolution of male pregnancy. Such information will be widely applicable to our understanding of fundamental reproductive and evolutionary processes in animals.

Regulatory Role of Retinoic Acid in Male Pregnancy of the Seahorse.

Seahorses epitomize the exuberance of evolution. They have the unique characteristic of male pregnancy, which includes the carrying of many embryos in a brood pouch that incubates and nourishes the embryos, similar to the mammalian placenta. However, the regulatory networks underlying brood pouch formation and pregnancy remain largely unknown. In this study, comparative transcriptomic and metabolomic profiling on the lined seahorse Hippocampus erectus, with unformed, newly formed, and pregnant brood pouches identified a total of 141 and 2,533 differentially expressed genes together with 73 and 121 significantly differential metabolites related to brood pouch formation and pregnancy, respectively. Specifically, integrative omics analysis revealed that retinoic acid (RA) synthesis and signaling pathway played essential roles in the formation of the brood pouch and pregnancy. RA might function upstream of testosterone and progesterone, thereby directly influencing brood pouch formation by regulating the expression of fshr and cyp7a1. Our results also revealed that RA regulates antioxidant defenses, particularly during male pregnancy. Alternatively, pregnancy caused a consistent decrease in RA, canthaxanthin, astaxanthin, and glutathione synthetase, and an increase in susceptibility to oxidative stress, which may balance brood pouch development and reproduction in seahorses and pave the way to successful gestation.

Morphology of brood pouch formation in the pot-bellied seahorse Hippocampus abdominalis.

BACKGROUND: The reproductive strategies of vertebrates are diverse. Seahorses (Pisces: Syngnathidae) possess the unique characteristic of male pregnancy; i.e., males, not females, incubate embryos in a specialized structure called a 'brood pouch'. The brood pouch is formed along the ventral midline of the tail. The lumen of the brood pouch is surrounded by loose connective tissue, called pseudoplacenta, and dermis. RESULTS: We visualized and evaluated the morphology of brood pouch formation in Hippocampus abdominalis to gain generalizable insights into this process in seahorses. First, we employed several staining methods to characterize the pseudoplacenta and dermis of the brood pouch of mature male seahorses. The pseudoplacenta is composed mainly of reticular fibers, while the dermis is composed mainly of collagenous fibers. Further observations showed that pouch formation is initiated by linear projections of epithelia on both ventrolateral sides of the body. These projections elongated toward the ventral midline, eventually fused together, and then formed a baggy structure composed of a single dermis layer with neither smooth muscle nor pseudoplacenta. Finally, the pseudoplacenta was formed, together with two layers of dermis and smooth muscle. Thus, a fully developed brood pouch was established. The morphology of the luminal epithelium also changed during pouch formation. We analyzed the localization of C-type lectins as markers; haCTL II was localized in both the outer and luminal epithelia of the brood pouch throughout development in the male seahorse, whereas haCTL IV, which was not detected in the early stage of seahorse development, became localized only in the luminal epithelium as development proceeded. CONCLUSIONS: We categorized the processes of brood pouch formation during male seahorse development into three stages: (1) the early stage, characterized by formation of a baggy structure from the primordium; (2) the middle stage, characterized by the differentiation and establishment of brood pouch-specific tissues; and (3) the late stage, characterized by a fully formed pouch with developing blood vessels and a pouch fold ultimately capable of carrying and incubating embryos.

Plasma levels of immune factors and sex steroids in the male seahorse Hippocampus erectus during a breeding cycle.

To better understand the endocrine- and immune-response pattern during reproduction in a fish species having parental care behaviors and also to accumulate the endocrine- and immune-related data for future explanations of the low reproductive efficiency in seahorse species, the variations of immune factors and sex steroids in the plasma of the male lined seahorse Hippocampus erectus at different breeding stages, i.e., pre-pregnancy, pregnancy (early, middle, and late periods), and post-pregnancy, were investigated in the present study. The immune factors included monocytes/leucocytes (M/L), leucocyte phagocytic rate (LPR), immunoglobulin M (Ig M), interleukin-2 (IL-2), interferon-α (IFN-α), and lysozyme (LZM). The sex steroids included testosterone (T), 11-ketotestosterone (11-KT), 11ß-hydroxytestosterone (11ß-OHT), 17α-methyltestosterone (17α-MT), 17ß-estradiol (E2), and 17α-hydroxy-20ß-dihydroprogesterone (17α-20ß-P). Moreover, the immune metabolic activity of epithelium cells in the brood pouch at different breeding stages was also analyzed through ultrastructural observations of the abundance of cytoplasmic granules, mitochondria, endoplasmic reticulum, lysosomes, and exocytosis. The results show that a higher immune level was observed during pregnancy, particularly in the early and middle periods, and a lower immune level was noted during pre-pregnancy. Correspondingly, the epithelium cells in the brood pouch also showed a stronger immune metabolic activity during pregnancy and weaker activity during pre-pregnancy. Four sex steroids of T, 11ß-OHT, 17α-MT, and E2 were higher during pre-pregnancy and lower during post-pregnancy, whereas 11-KT and 17α-20ß-P, which were positively correlated with part immune factors, were higher during pregnancy. No negative correlations between sex steroids and immune factors were observed. In conclusion, the higher immune competence during pregnancy may indicate that parental care could improve immunity, which may be the major factor for no immunosuppressive effect of sex steroids during reproduction in the seahorse H. erectus, unlike noncaregiving fishes in which inhibitions of sex steroids on immunity are frequently observed. Moreover, higher 11-KT and 17α-20ß-P during pregnancy than during pre-pregnancy and post-pregnancy may suggest that these two steroids are also involved in parental care regulation.

Seahorse Brood Pouch Transcriptome Reveals Common Genes Associated with Vertebrate Pregnancy.

Viviparity (live birth) has evolved more than 150 times in vertebrates, and represents an excellent model system for studying the evolution of complex traits. There are at least 23 independent origins of viviparity in fishes, with syngnathid fishes (seahorses and pipefish) unique in exhibiting male pregnancy. Male seahorses and pipefish have evolved specialized brooding pouches that provide protection, gas exchange, osmoregulation, and limited nutrient provisioning to developing embryos. Pouch structures differ widely across the Syngnathidae, offering an ideal opportunity to study the evolution of reproductive complexity. However, the physiological and genetic changes facilitating male pregnancy are largely unknown. We used transcriptome profiling to examine pouch gene expression at successive gestational stages in a syngnathid with the most complex brood pouch morphology, the seahorse Hippocampus abdominalis. Using a unique time-calibrated RNA-seq data set including brood pouch at key stages of embryonic development, we identified transcriptional changes associated with brood pouch remodeling, nutrient and waste transport, gas exchange, osmoregulation, and immunological protection of developing embryos at conception, development and parturition. Key seahorse transcripts share homology with genes of reproductive function in pregnant mammals, reptiles, and other live-bearing fish, suggesting a common toolkit of genes regulating pregnancy in divergent evolutionary lineages.