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Seahorses exhibit the unique characteristic of male pregnancy, which incubates numerous embryos in a brood pouch that plays an essential role in enhancing offspring survivability. The pot-belly seahorse (Hippocampus abdominalis) possesses the largest body size among seahorses and is a significant species in Chinese aquaculture. In this study, we revealed the cytological and morphological characteristics, as well as regulatory mechanisms, throughout the entire brood pouch development in H. abdominalis. The brood pouch originated from the abdominal dermis, extending towards the ventral midline. As the dermal layers thicken, the inner epithelium folds, the stroma loosens, and vascularization occurs, culminating in the formation of the brood pouch. Furthermore, through transcriptomic analysis of brood pouches at various developmental stages, 8 key genes (tgfb3, fgf2, wnt7a, pgf, mycn, tln2, jund, ccn4) closely related to the development of brood pouch were identified in the MAPK, Rap1, TGF-ß, and Wnt signaling pathways. These genes were highly expressed in the pseudoplacenta and dermal layers at the newly formed stage as examined by in situ hybridization (ISH). The angiogenesis, densification of collagen fibers, and proliferation of fibroblasts and endothelial cells in seahorse brood pouch formation may be regulated by these genes and pathways. Additionally, the expression of the androgen receptor gene (ar) was significantly upregulated during the formation of the brood pouch, and ISH confirmed the expression of the ar gene in the dermis and pseudoplacenta of the brood pouch, highlighting its role in the developmental process. Androgen and flutamide (androgen receptor antagonist) treatments significantly accelerated the formation of the brood pouch and completely inhibited its occurrence respectively, concomitant to the upregulated expression of differentially expressed genes involved above signaling pathways. These findings demonstrated that formation of the brood pouch is determined by androgen and the androgen receptor activates the above signaling pathways in the brood pouch through the regulation of fgf2, tgfb3, pgf, and wnt7a. Interestingly, androgen even induced the formation of the brood pouch in females. We firstly elucidated the formation of the seahorse brood pouch, demonstrating that androgens and their receptors directly induce the thickening, folding, and vascularization of the abdominal dermal layer into a placenta-like structure through multiple signaling pathways. These findings provide foundational insights to further exploring the evolution of male pregnancy and adaptive convergence in viviparity across vertebrates.

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Sexual dimorphism, the divergence in morphological traits between males and females of the same species, is often accompanied by sex-biased gene expression. However, the majority of research has focused on species with conventional sex roles, where females have the highest energy burden with both egg production and parental care, neglecting the diversity of reproductive roles found in nature. We investigated sex-biased gene expression in Syngnathus typhle, a sex-role reversed species with male pregnancy, allowing us to separate two female traits: egg production and parental care. Using RNA sequencing, we examined gene expression across organs (brain, head kidney and gonads) at various life stages, encompassing differences in age, sex and reproductive status. While some gene groups were more strongly associated with sex roles, such as stress resistance and immune defence, others were driven by biological sex, such as energy and lipid storage regulation in an organ- and age-specific manner. By investigating how genes regulate and are regulated by changing reproductive roles and resource allocation in a model system with an unconventional life-history strategy, we aim to better understand the importance of sex and sex role in regulating gene expression patterns, broadening the scope of this discussion to encompass a wide range of organisms.

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Early life microbial colonizers shape and support the immature vertebrate immune system. Microbial colonization relies on the vertical route via parental provisioning and the horizontal route via environmental contribution. Vertical transmission is mostly a maternal trait making it hard to determine the source of microbial colonization in order to gain insight into the establishment of the microbial community during crucial development stages. The evolution of unique male pregnancy in pipefishes and seahorses enables the disentanglement of both horizontal and vertical transmission, but also facilitates the differentiation of maternal versus paternal provisioning ranging from egg development, to male pregnancy and early juvenile development. Using 16S rRNA amplicon sequencing and source-tracker analyses, we revealed how the distinct origins of transmission (maternal, paternal and horizontal) shaped the juvenile internal and external microbiome establishment in the broad-nosed pipefish Syngnathus typhle. Our data suggest that transovarial maternal microbial contribution influences the establishment of the juvenile gut microbiome whereas paternal provisioning mainly shapes the juvenile external microbiome. The identification of juvenile key microbes reveals crucial temporal shifts in microbial development and enhances our understanding of microbial transmission routes, colonization dynamics and their impact on lifestyle evolution.

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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.

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Ocean antibiotics pose substantial risks to the adaptation and lifespan of marine organisms. Seahorses are unique owing to the occurrence of brood pouches, male pregnancy, and loss of gut-associated lymphatic tissues and spleen, which lead to increased sensitivity to environmental changes. This study evaluated the changes in microbial diversity and immune responses within the gut and brood pouch in the lined seahorse Hippocampus erectus under chronic exposure to environmental levels of triclosan (TCS) and sulfamethoxazole (SMX), which are common antibiotics in coastal regions. The results showed that microbial abundance and diversity within the gut and brood pouch of seahorses were significantly changed following antibiotics treatment, with the expression of core genes involved in immunity, metabolism, and circadian rhythm processes evidently regulated. Notably, the abundance of potential pathogens in brood pouches was considerably increased upon treatment with SMX. Transcriptome analysis revealed that the expression of toll-like receptors, c-type lectins, and inflammatory cytokine genes in brood pouches was significantly upregulated. Notably, some essential genes related to male pregnancy significantly varied after antibiotic treatment, implying potential effects on seahorse reproduction. This study provides insights into the physiological adaptation of marine animals to environmental changes resulting from human activity.

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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.

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The unique male pregnancy in pipefishes and seahorses ranges from basic attachment (pouch-less species: Nerophinae) of maternal eggs to specialized internal gestation in pouched species (e.g. Syngnathus and Hippocampus) with many transitions in between. Due to this diversity, male pregnancy offers a unique platform for assessing physiological and molecular adaptations in pregnancy evolution. These insights will contribute to answering long-standing questions of why and how pregnancy evolved convergently in so many vertebrate systems. To understand the molecular congruencies and disparities in male pregnancy evolution, we compared transcriptome-wide differentially expressed genes in four syngnathid species, at four pregnancy stages (nonpregnant, early, late and parturition). Across all species and pregnancy forms, metabolic processes and immune dynamics defined pregnancy stages, especially pouched species shared expression features akin to female pregnancy. The observed downregulation of adaptive immune genes in early-stage pregnancy and its reversed upregulation during late/parturition in pouched species, most notably in Hippocampus, combined with directionless expression in the pouch-less species, suggests immune modulation to be restricted to pouched species that evolved placenta-like systems. We propose that increased foeto-paternal intimacy in pouched syngnathids commands immune suppression processes in early gestation, and that the elevated immune response during parturition coincides with pouch opening and reduced progeny reliance. Immune response regulation in pouched species supports the recently described functional MHC II pathway loss as critical in male pregnancy evolution. The independent co-option of similar genes and pathways both in male and female pregnancy highlights immune modulation as crucial for the evolutionary establishment of pregnancy.

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Neurohypophysial hormones regulate the reproductive behavior of teleosts; however, their role in the gestation and parturition of ovoviviparous fishes with male pregnancy (syngnathids) remains to be demonstrated. In the present study, the complementary DNA (cDNA) sequences of arginine vasotocin (VT) and isotocin (IT) from the lined seahorse (Hippocampus erectus) were cloned and identified. We observed that the mature core peptides of seahorse VT and IT were conserved among teleosts. In the phylogenic tree, seahorse VT and IT were clustered independently with teleost VT and IT. The tissue distribution patterns of VT and IT were similar, and both were highly expressed in the brain, gills, and gonads. Interestingly, they were also expressed to some extent in the brood pouch. In situ hybridization revealed that VT and IT messenger RNA (mRNA) signals in the brain were mainly located in the preoptic area region of the hypothalamus. Intraperitoneal administration of the VT core peptide to pregnant seahorses induced premature parturition, stimulated gonadotropin release, increased serum estrogen levels, and decreased prolactin secretion. Moreover, VT injection upregulated the mRNA expression of the membrane estrogen receptor in the brood pouch. In summary, neurohypophysial hormones promote premature parturition by regulating estrogen synthesis through the hypothalamus-pituitary-gonad axis.

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Viviparity has originated independently more than 150 times in vertebrates, while the male pregnancy only emerged in Syngnathidae fishes, such as seahorses. The typical male pregnancy seahorses have closed sophisticated brood pouch that act as both uterus and placenta, representing an excellent model system for studying the evolutionary process of paternal immune protection. Phylogenetic analysis indicated that the hampII gene family has multiple tandem duplicated genes and shows independent lineage-specific expansion in seahorses, and they had the highest ratio of nonsynonymous substitutions to synonymous substitutions (dN/dS) in the seahorse phylogenetic branch. The expression levels of hampIIs in the brood pouch placenta were significantly higher during pregnancy than non-pregnancy. Both LPS stimulation test in vivo and cytotoxicity test in vitro proved the immunological protection function of hampIIs against pathogen infection in seahorse. Besides, seahorse hampII peptides exhibit weaker antibacterial function, but stronger agglutination and free endotoxin inhibition. We assumed that the modified immunological function seemed to be a trade-off between the resistance to microbial attack and offspring protection. In brief, this study suggests that the rapid co-option of hampIIs contributes to the evolutionary adaption to paternal immune care during male pregnancy.

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INTRODUCTION: Fishes of the Syngnathidae family are rare in having male pregnancy: males receive eggs from females and egg development occurs in the male brood pouch that diverged during evolution. The family is divided into two subfamilies: Nerophinae and Syngnathinae. METHODS: We compared histologically five types of the brood pouch in Syngnathinae: an open pouch without skinfolds (alligator pipefish); an open pouch with skinfolds (messmate pipefish); a closed pouch with skinfolds (seaweed pipefish); and closed pouches with a sac-like pouch on the tail (pot-bellied seahorse) or within a body cavity (Japanese pygmy seahorse). RESULTS: Histological observations revealed that all the examined species possess vascular egg compartments during the brooding period. The present immunohistochemical study revealed that the pregnant egg compartment epithelium grows thin in both open and closed pouches. The placenta of open and closed pouches is composed of dermis and reticulin fibers, respectively. The closed pouch placenta is a flexible and moist tissue, suitable for substance transport between the father and embryos through the epithelium and blood vessels and responsible for supplying nutrition and removing waste. DISCUSSION: These results suggest that the basic egg incubation structures were established at an early stage of Syngnathinae evolution. On the other hand, it is likely that the innovation of tissue structure, where dermis was replaced with reticular fibers, occurred in closed brood pouches to regulate the pregnant pouch environment. The present study presents the morphological evolutionary pathway of the brood pouch in Syngnathinae, providing a basis for further molecular-level evolutionary studies.

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In the present study, we aimed to evaluate the effects of hatching enzymes on the egg envelope digestion during the hatching period in the male brooding seahorse. The complementary DNAs encoding two hatching-enzyme genes, high choriolytic enzyme (HCE) and low choriolytic enzyme (LCE), were cloned and functionally characterized from the lined seahorse (Hippocampus erectus). The genomic-synteny analysis confirmed that teleosts shared LCE gene synteny. In contrast, the genomic location of HCE was found to be conserved with pipefish, but not other teleosts, suggesting that translocation into a novel genomic location occurred. Whole-mount in situ hybridization showed that HCE and LCE mRNAs were expressed in hatching gland cells. To determine the digestion mechanisms of HCE and LCE in hatching, recombinant HCE and LCE were generated and their enzyme activities were examined using fertilized egg envelopes and synthetic peptides. Seahorse HCE and LCE independently digested and softened the egg envelopes of the lined seahorse. Although the egg envelope was digested more following HCE and LCE co-treatment, envelope solubilization was not observed. Indeed, both HCE and LCE showed similar substrate specificities toward four different synthetic peptides designed from the cleavage sites of egg envelope proteins. HCE and LCE proteins from other euteleostean fishes showed different specificities, and the egg envelope was solubilized by the cooperative action of HCE and LCE. These results suggest that the function of LCE was degenerated in the lined seahorse. Our results imply a digestion mechanism for evolutionary adaptation in ovoviviparous fish with male pregnancy.

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Seahorses, with brood pouch in adult males, are a bioindicator species that exhibit specialized reproductive strategy of "male pregnancy". Bisphenol A (BPA), one of the most pervasive endocrine-disrupting chemicals (EDCs), is hazardous for reproductive, immune, and neurological systems. However, no evidence of BPA toxicity to the male-pregnant animals is available. Herein, the reproductive toxicity of BPA was evaluated in lined seahorses (Hippocampus erectus) following exposure to environmentally relevant concentrations (10, 100, and 1000 µg/L) through physiological, histological, and transcriptional analyses. Our results indicated BPA bioaccumulation to be positively correlated with exposure doses in both sexes. Ovarian failure was only observed in the high-dose BPA treatment group, accompanied by the apoptosis of follicular cells and up-regulation of pro-apoptotic genes. However, brood pouches maintenance were surprisingly inhibited at low concentration, and transcriptomic analysis revealed disturbed profiles of genes involved in the extracellular matrix and cell-cell adhesion pathways. Interestingly, seahorse testes were less sensitive to BPA exposure than that in other teleosts. Thus, our study suggests that BPA at environmentally relevant concentrations might cause reproductive dysfunction in seahorses, potentially exerting adverse effects on the seahorse population since most of them inhabit shallow coastal areas with prevalent estrogenic contaminants.

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A fundamental problem for the evolution of pregnancy, the most specialized form of parental investment among vertebrates, is the rejection of the nonself-embryo. Mammals achieve immunological tolerance by down-regulating both major histocompatibility complex pathways (MHC I and II). Although pregnancy has evolved multiple times independently among vertebrates, knowledge of associated immune system adjustments is restricted to mammals. All of them (except monotremata) display full internal pregnancy, making evolutionary reconstructions within the class mammalia meaningless. Here, we study the seahorse and pipefish family (syngnathids) that have evolved male pregnancy across a gradient from external oviparity to internal gestation. We assess how immunological tolerance is achieved by reconstruction of the immune gene repertoire in a comprehensive sample of 12 seahorse and pipefish genomes along the "male pregnancy" gradient together with expression patterns of key immune and pregnancy genes in reproductive tissues. We found that the evolution of pregnancy coincided with a modification of the adaptive immune system. Divergent genomic rearrangements of the MHC II pathway among fully pregnant species were identified in both genera of the syngnathids: The pipefishes (Syngnathus) displayed loss of several genes of the MHC II pathway while seahorses (Hippocampus) featured a highly divergent invariant chain (CD74). Our findings suggest that a trade-off between immunological tolerance and embryo rejection accompanied the evolution of unique male pregnancy. That pipefishes survive in an ocean of microbes without one arm of the adaptive immune defense suggests a high degree of immunological flexibility among vertebrates, which may advance our understanding of immune-deficiency diseases.

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Exposure to environmental estrogens and progestins has contributed to adverse effects on the reproduction of many aquatic wildlife species. However, few reports have paid attention to fish species with specialized reproductive strategies, such as male-pregnancy seahorses. In this study, the potential effects on the behavior, gonad and brood pouch development, and transcriptomic profiles of lined seahorse Hippocampus erectus exposed to environmentally relevant concentrations of 17α-ethynyl estradiol (EE2, 5 ng/L, 50 ng/L, 10 ng/L, 100 ng/L) or progesterone (P4) for 60 days were examined. Both EE2 and P4 significantly inhibited male brood pouch development by disrupting the extracellular matrix and basement membrane pathways. In addition, both EE2 and P4 impaired the expression of genes associated with spermatogenesis in the testis, and even caused male feminization. We suggest that seahorses be regarded as a sensitive indicator for evaluating the potential effects of endocrine disrupting chemical (EDC) pollution on aquatic biotic communities.

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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.

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Within a species' distribution, populations are often exposed to diverse environments and may thus experience different sources of both natural and sexual selection. These differences are likely to impact the balance between costs and benefits to individuals seeking reproduction, thus entailing evolutionary repercussions. Here, we look into an unusual population (Baltic Sea) of the broadnosed pipefish, Syngnathus typhle, where males do not seem to select females based on size and hypothesize that this pattern may derive from a reduction in direct benefits to the male. We further hypothesize that if larger females do not persistently secure a higher reproductive success, either through pre- or postcopulatory sexual selection, a decrease in sexual size dimorphism in the Baltic population should be apparent, especially when contrasted with a well-studied population, inhabiting similar latitudes (Swedish west coast), where males prefer larger females. We found that, in the Baltic population, variation in female quality is low. We were unable to find differences in abortion rates or protein concentration in oocytes produced by females of contrasting sizes. Direct benefits from mating with large partners seem, thus, reduced in the Baltic population. We also found no evidence of any postcopulatory mechanism that could favor larger mothers as embryo development was unrelated to female size. While female size can still be selected through intrasexual competition or fecundity selection, the pressure for large female body size seems to be lower in the Baltic. Accordingly, we found a noticeable decrease in sexual size dimorphism in the Baltic population. We conclude that, although far from negating the significance of other selective processes, sexual selection seems to have a decisive role in supporting pipefish sexual size asymmetries.

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In animals with uniparental care, the quality of care provided by one sex can deeply impact the reproductive success of both sexes. Studying variation in parental care quality within a species and which factors may affect it can, therefore, shed important light on patterns of mate choice and other reproductive decisions observed in nature. Using Syngnathus typhle, a pipefish species with extensive uniparental male care, with embryos developing inside a brood pouch during a lengthy pregnancy, we assessed how egg size (which correlates positively with female size), male size, and water temperature affect brooding traits that relate to male care quality, all measured on day 18, approximately 1/3, of the brooding period. We found that larger males brooded eggs at lower densities, and their embryos were heavier than those of small males independent of initial egg size. However, large males had lower embryo survival relative to small males. We found no effect of egg size or of paternal size on within-pouch oxygen levels, but oxygen levels were significantly higher in the bottom than the middle section of the pouch. Males that brooded at higher temperatures had lower pouch oxygen levels presumably because of higher embryo developmental rates, as more developed embryos consume more oxygen. Together, our results suggest that small and large males follow distinct paternal strategies: large males positively affect embryo size whereas small males favor embryo survival. As females prefer large mates, offspring size at independence may be more important to female fitness than offspring survival during development.

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Seahorse is characterized by its male pregnancy and sex-role reversal. To better understand the sexual dimorphism of male and female seahorses based on essential genes, we performed systematic transcriptome studies for both genders. A total of 157,834,590 cleaned reads were obtained and assembled into 129,268 transcripts and 31,764 could be annotated. Results showed that 176 up-regulated and 391 down-regulated transcripts were identified in the male seahorses compared with those in females. Genes involved in sex differentiation, such as dmrt1, sox9, fem1 and vasa, were identified and characterized. Moreover, the essential genes involved in reproductive molecular pathway were identified and analyzed in seahorses. In conclusion, the present study provides an archive for the future systematic research on seahorse sex differentiation.

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In an old Gene Wilder movie, an attractive woman dressed in red devastated a man's current relationship. We have found a similar 'Woman in Red' effect in pipefish, a group of fish where pregnancy occurs in males. We tested for the existence of pregnancy blocks in pregnant male black-striped pipefish (Syngnathus abaster). We allowed pregnant males to see females that were larger and even more attractive than their original high-quality mates and monitored the survival and growth of developing offspring. After exposure to these extremely attractive females, males produced smaller offspring in more heterogeneous broods and showed a higher rate of spontaneous offspring abortion. Although we did not observe a full pregnancy block, our results show that males are able to reduce investment in current broods when faced with prospects of a more successful future reproduction with a potentially better mate. This 'Woman in Red' life-history trade-off between present and future reproduction has similarities to the Bruce effect, and our study represents, to our knowledge, the first documentation of such a phenomenon outside mammals.

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The kisspeptin/GPR54 system plays a crucial role in the regulation of the reproductive axis in vertebrates. Male pregnancy and ovoviviparity are special reproductive phenomena among vertebrates. To better understand the neuroendocrine mechanisms of male pregnancy, cDNAs encoding kiss2 and GPR54 were cloned and functionally characterized from the lined seahorse, Hippocampus erectus, an ovoviviparous teleost with male pregnancy. The core mature peptide of seahorse Kiss2 is high conserved among seahorses, but unique among vertebrate Kiss orthologs. In the phylogenic analysis, the seahorse Kiss clustered with the teleost Kiss2 clade. The kiss2 transcripts were shown to be widely expressed in various tissues, notably in the brain and gonad of the seahorse, while GPR54-2 mRNA was expressed exclusively in the brain. In addition, kiss2 mRNA found in male seahorse brain tissue increased significantly at the early pubertal stage, and decreased significantly during pregnancy. Intraperitoneal administration of seahorse Kiss2-10 to sexual mature male seahorses demonstrated to stimulate lutropin ß (LHß) and follitropin ß (FSHß) release and increased serum testosterone levels. In summary, we first identified the kisspeptin/GPR54 system in an ovoviviparous fish with male pregnancy, which might be involved in the regulation of the reproductive functions of pubertal onset, gonadal development, and male pregnancy via regulating the synthesis of both gonadotropic hormone (GTH) and testosterone.