Internal Fertilization Research Articles

Developmental regulation of the male urogenital papilla in the male marine teleost black rockfish, Sebastes schlegelii (Hilgendorf, 1880).

The male external genitalia of the black rockfish (Sebastes schlegelii Hilgendorf, 1880) is a fleshy protrusion known as the urogenital papilla (UGP), which functions to deliver sperm into the female reproductive tract for internal fertilization. It is not known which genes regulate the development of the UGP. The aim of this study was to identify key genes that regulate the development of the UGP in black rockfish and to determine the distribution of androgen receptor gene (ar) in the UGP. A total of twenty-six adult males and 560 juvenile fish were used in the experiment, in which we divided all normally developing juveniles into normal development and androgen groups. We added methyltestosterone solution (100 ug/L) to the androgen group-treated fish tank, soaked for 2hours per day for 38days, and sampled 5 ~ 10 samples each time every 5days during the culture process. Gene expression changes related to urogenital papilla were analyzed with tissue specificity between control and androgen groups during sex differentiation, adult male maturation, and the copulation stage (September to December) using Real Time Quantitative PCR (RT-qPCR). The expression of ar (ara and arb) was also localized by two-color in situ hybridization in the UGP region of juvenile fish. Androgen treatment enhanced ar expression levels and the ar signal was stronger in the UGP region of both adult breeding fish and androgen-treated juvenile fish. This study provides insights into the regulation of the external genitalia of black rockfish and presents vital information for the artificial breeding of viviparous fish.

An oviduct glycan increases sperm lifespan by diminishing the production of ubiquinone and reactive oxygen species†.

Sperm storage by females after mating for species-dependent periods is used widely among animals with internal fertilization to allow asynchrony between mating and ovulation. Many mammals store sperm in the lower oviduct where specific glycans on oviduct epithelial cells retain sperm to form a reservoir. Binding to oviduct cells suppresses sperm intracellular Ca2+ and increases sperm longevity. We investigated the mechanisms by which a specific oviduct glycan, 3-O-sulfated Lewis X trisaccharide (suLeX), prolongs the lifespan of porcine sperm. Using targeted metabolomics, we found that binding to suLeX diminishes the abundance of 4-hydroxybenzoic acid, the precursor to ubiquinone (also known as Coenzyme Q), 30min after addition. Ubiquinone functions as an electron acceptor in the electron transport chain (ETC). 3-O-sulfated Lewis X trisaccharide also suppressed the formation of fumarate. A component of the citric acid cycle, fumarate is synthesized by succinate-coenzyme Q reductase, which employs ubiquinone and is also known as Complex II in the ETC. Consistent with the reduced activity of the ETC, the production of harmful reactive oxygen species (ROS) was diminished. The enhanced sperm lifespan in the oviduct may be because of suppressed ROS production because high ROS concentrations have toxic effects on sperm.

Vaginas

What is the vagina? This seemingly simple question has a rather complex answer, depending on whether we use a functional or a developmental definition. The terminal portion of the female reproductive tract that opens to the environment initially served as a conduit for eggs to be laid, and in species with external fertilization the distal oviduct may be specialized for oviposition but there is no vagina. In animals with internal fertilization, this terminal section of the oviduct interacts with the sperm and the intromittent organ leading to functional specialization of this region that we often call a vagina in insects and some vertebrates. Here we address the evolution, morphology and diverse functions of the vagina and some of the unknown questions that remain to be addressed in the study of this remarkable structure.

Structural specializations of the sperm tail

Sperm motility is crucial to reproductive success in sexually reproducing organisms. Impaired sperm movement causes male infertility, which is increasing globally. Sperm are powered by a microtubule-based molecular machine-the axoneme-but it is unclear how axonemal microtubules are ornamented to support motility in diverse fertilization environments. Here, we present high-resolution structures of native axonemal doublet microtubules (DMTs) from sea urchin and bovine sperm, representing external and internal fertilizers. We identify >60 proteins decorating sperm DMTs; at least 15 are sperm associated and 16 are linked to infertility. By comparing DMTs across species and cell types, we define core microtubule inner proteins (MIPs) and analyze evolution of the tektin bundle. We identify conserved axonemal microtubule-associated proteins (MAPs) with unique tubulin-binding modes. Additionally, we identify a testis-specific serine/threonine kinase that links DMTs to outer dense fibers in mammalian sperm. Our study provides structural foundations for understanding sperm evolution, motility, and dysfunction at a molecular level.

Effect of ovarian fluid on sperm performance in teleost with internal and external fertilization strategies

Ovarian fluid is essential for successful fertilization by maintaining the viability, motility, and velocity of sperm. The organic compounds and inorganic ions in ovarian fluid significantly influence spermatozoa's motility, velocity, and longevity. However, the effect of ovarian fluid on sperm performance is limited in teleost fish. In this study, the effect of ovarian fluid on sperm performance and its components in external fertilization species (Scophthalmus maximus, turbot) and internal fertilization species (Sebastes schlegelii, black rockfish) was investigated using computer-assisted sperm analysis, high-performance liquid chromatography, and metabolome analysis. The ovarian fluid had a distinct and species-specific effect on both species. In the black rockfish, the ovarian fluid from turbot significantly increased sperm motility (74.07% ± 4.09%), as well as VCL (45 ± 1.67 μm/s), VAP (40.17 ± 1.6 μm/s), and VSL (36.67 ± 1.86 μm/s), and longevity (352 ± 11.31 min) (P < 0.05). In the turbot, only the longevity (71.33 ± 5.69 min) and fertilization rate (65.27% ± 11.59%) showed significantly improvement (P < 0.05). The ovarian fluid was rich in organic compounds, suggesting enrichment in the glycolysis/gluconeogenesis pathways. The results suggest that glycometabolism plays a crucial role in improving sperm performance in teleost with internal fertilization. Thus, incorporating ovarian fluid into the sperm activation medium can enhance artificial fertilization in fish breeding.

Piezo-mediated mechanosensation contributes to stabilizing copulation posture and reproductive success in Drosophila males

In internal fertilization animals, reproductive success depends on maintaining copulation until gametes are transported from male to female. In Drosophila melanogaster, mechanosensation in males likely contributes to copulation maintenance, but its molecular underpinning remains to be identified. Here we show that the mechanosensory gene piezo and its' expressing neurons are responsible for copulation maintenance. An RNA-seq database search and subsequent mutant analysis revealed the importance of piezo for maintaining male copulation posture. piezo-GAL4-positive signals were found in the sensory neurons of male genitalia bristles, and optogenetic inhibition of piezo-expressing neurons in the posterior side of the male body during copulation destabilized posture and terminated copulation. Our findings suggest that the mechanosensory system of male genitalia through Piezo channels plays a key role in copulation maintenance and indicate that Piezo may increase male fitness during copulation in flies.

Identification and characterization of sperm motility-initiating substance-2 gene in internally fertilizing Cynops species.

Sperm motility-initiating substance (SMIS) is an oviductal protein critical for internal fertilization in urodeles. It contributes to the establishment of various reproductive modes in amphibians and is thus a unique research model for the gene evolution of gamete-recognizing ligands that have diversified among animal species. In this study, a paralogous SMIS gene, smis2, was identified via the RNA sequencing of the oviduct of the newt, Cynops pyrrhogaster. The base sequence of the smis2 gene was homologous (˃90%) to that of the original smis gene (smis1), and deduced amino acid sequences of both genes conserved six cysteine residues essential for the cysteine knot motif. Furthermore, smis2 complementary DNA was identified in the oviduct of Cynops ensicauda, and the base substitution patterns also suggested that the smis gene was duplicated in the Salamandridae. Nonsynonymous/synonymous substitution ratios of smis1 and smis2 genes were 0.79 and 2.6, respectively, suggesting that smis2 gene evolution was independently driven by positive selection. Amino acid substitutions were concentrated in the cysteine knot motif of SMIS2. The smis2 gene was expressed in some organs in addition to the oviduct; in contrast, SMIS1 was only expressed in the oviduct. The SMIS2 protein was suggested to be produced and secreted at least in the oviduct and redundantly act in sperm. These results suggest that smis1 plays the original role in the oviduct, whereas smis2 may undergo neofunctionalization, which rarely occurs in gene evolution.

Insights into a putative polychaete-gastropod symbiosis from a newly identified annelid worm that predates upon Conus ermineus eggs

Abstract Cone snails are a diverse group of venomous marine gastropods that have dioecious reproduction and internal fertilization resulting in egg deposition inside capsules. However, the observational studies conducted on their spawning behaviour and egg masses have left many open questions. Here, we analyse egg masses from a specimen of Conus ermineus kept in captivity for over 17 years. We present the first detailed description of the morphological features of the egg capsules and eggs (e.g., egg shape, size, and colour). The analysis of these capsules led us to the identification of a dinophilid worm (Polychaeta: Dinophilidae) living inside the snail egg capsules and likely feeding upon the snail eggs. This is the first report of such behaviours among dinophilids. Our analysis suggests that these worms belong to a new species, here described as Dimorphilus oophagus sp. nov., supported by both molecular and morphological data. Finally, we discuss the possibility of a putative symbiotic relationship between the worm and the snail.

Evidence of the Genetic Mating System in Viviparity White-Edged Rockfish, &lt;i&gt;Sebastes taczanowskii&lt;/i&gt; Steindachner, 1880

Internal fertilization and viviparity are characteristic of some species of the genus Sebastes. Polyandry has also been reported for some species of this genus. We used five microsatellite markers and three main statistical approaches to estimate the level of polyandry in a wild population of white-edged rockfish (Sebastes taczanowskii). In some clutches, we detected multiple paternity. This study is the first record of multiple paternity in white-edged rockfish from the Far East. Polyandry is probably quite a widespread strategy in this species inhabiting the Far Eastern seas, and also an important factor in the regulation of genetic diversity and productivity.

Some Observations on the Reproductive Biology of the Mediterranean Pennatulacean Pteroeides Spinosum (Ellis and Solander, 1786) (Cnidaria: Octocorallia: Pennatulacea)

Sea pens are some of the most important structural species in soft bottom benthic communities. Pteroeides spinosum is a shallow-water pennatulacean (Cnidaria: Octocorallia: Pennatulacea) habiting NE Atlantic-Mediterranean bottoms. In this study, sixteen colonies of Pteroeides spinosum collected from NW Mediterranean Sea (Cap de Creus, Spain) at 99–106 m depth in July (2012), were used to acquire information of their reproductive biology, and to enable a comparison with other pennatulaceans. Our colonies of Pteroeides spinosum are gonochoric showing a bimodal oocyte frequency distribution and an unimodal spermatocyst diameter distribution. All polyp leaves contained oocytes and spermatocysts in different size classes, with those small and translucent spermatocysts/ oocytes (size-class I) often found near the base or lower end of the gastrovascular cavities of polyps. The largest observed diameter was 668 μm for spermatocysts and 687 μm for oocytes. Mean potential relative fecundity (PRF) ranged between 7–17 oocytes per polyp. The effective reproductive effort (ERE) reached a value of ca. 30,000 large oocytes per colony. As was observed for all pennatulaceans previously studied, no sign of hermaphroditism or internal fertilization was detected in our colonies of Pteroeides spinosum.

Morphology of the urogenital papilla of the male marine teleost Black Rockfish, Sebastes schlegelii (Hilgendorf, 1880), and its role in internal fertilization.

There are few detailed descriptions of the morphology of the male external genitalia, the urogenital papilla (UGP), of the Black Rockfish (Sebastes schlegelii Hilgendorf, 1880). The purpose of this study was to evaluate this organ histologically and to determine the time of development of the UGP in Black Rockfish. Twelve adult males, three adult females and around 500 juveniles were used in the experiment. The juveniles were divided into normal developmental and androgen groups. The androgen group was exposed to methyltestosterone (100 μg/L) for 2 h daily for 38 days. Samples (N = 10 per sampling) were randomly selected for analysis every 5 days from 30 to 116 days after birth. Parameters assessed included the type of epithelium, composition of connective tissue, muscular tissue, and the timing of UGP development. Differences in these parameters between normal developmental and androgen groups were evaluated. The results indicated that the UGP of the adult fish contains the sperm duct and ureter, which have the function of transporting sperm and urine, respectively. The androgen-treated juvenile fish developed the UGP earlier than the normal development group. This study provides a reference for understanding the external genitalia of other viviparous fishes by studying the UGP of the male Black Rockfish.

Female control of sperm ejection and retention in the cornsilk fly Euxesta eluta (Diptera: Ulidiidae).

Promiscuous mating systems provide the opportunity for females to bias fertilization toward particular males. However, distinguishing between male sperm competition and active female sperm choice is difficult for species with internal fertilization. Nevertheless, species that store and use sperm of different males in different storing structures and species where females are able to expel all or part of the ejaculates after copulation may be able to bias fertilization. We report a series of experiments aimed at providing evidence of female sperm choice in Euxesta eluta (Hendel), a species of ulidiid fly that expels and consumes ejaculates after copulation. We found no evidence of greater reproductive success for females mated singly, multiply with the same male, or mated multiply with different males. Female E. eluta possesses two spherical spermathecae and a bursa copulatrix for sperm storage, with a ventral receptacle. There was no significant difference in storing more sperm in spermathecae 24 h after copulation than immediately after copulation. Females mated with protein-fed males had greater reproductive success than similar females mated to protein-deprived males. Protein-fed females prevented to consume the ejaculate, retained more sperm when mated to protein-fed males than when mated to protein-deprived males. Our results suggest that female E. eluta can exert control of sperm retention of higher quality males through ejaculate ejection.

Fertilization modes and the evolution of sperm characteristics in marine fishes: Paired comparisons of externally and internally fertilizing species.

Fertilization mode may affect sperm characteristics, such as morphology, velocity, and motility. However, there is little information on how fertilization mode affects sperm evolution because several factors (e.g., sperm competition) are intricately intertwined when phylogenetically distant species are compared. Here, we investigated sperm characteristics by comparing seven externally and four internally fertilizing marine fishes from three different groups containing close relatives, considering sperm competition levels. The sperm head was significantly slenderer in internal fertilizers than in external fertilizers, suggesting that a slender head is advantageous for swimming in viscous ovarian fluid or in narrow spaces of the ovary. In addition, sperm motility differed between external and internal fertilizers; sperm of external fertilizers were only motile in seawater, whereas sperm of internal fertilizers were only motile in an isotonic solution. These results suggest that sperm motility was adapted according to fertilization mode. By contrast, total sperm length and sperm velocity were not associated with fertilization mode, perhaps because of the different levels of sperm competition. Relative testis mass (an index of sperm competition level) was positively correlated with sperm velocity and negatively correlated with the ratio of sperm head length to total sperm length. These findings suggest that species with higher levels of sperm competition have faster sperm with longer flagella relative to the head length. These results contradict the previous assumption that the evolution of internal fertilization increases the total sperm length. In addition, copulatory behavior with internal insemination may involve a large genital morphology, but this is not essential in fish, suggesting the existence of various sperm transfer methods. Although the power of our analyses is not strong because of the limited number of species, we propose a new scenario of sperm evolution in which internal fertilization would increase sperm head length, but not total sperm length, and change sperm motility.

In vitro and in vivo gene transfer in the cloudy catshark Scyliorhinus torazame.

Cartilaginous fishes have various unique physiological features such as a cartilaginous skeleton and a urea-based osmoregulation strategy for adaptation to their marine environment. Also, because they are a sister group of bony vertebrates, understanding their unique features is important from an evolutionary perspective. However, genetic engineering based on gene functions as well as cellular behavior has not been effectively utilized in cartilaginous fishes. This is partly because their reproductive strategy involves internal fertilization, which results in difficulty in microinjection into fertilized eggs at the early developmental stage. Here, to identify efficient gene transfer methods in cartilaginous fishes, we examined the effects of various methods both in vitro and in vivo using the cloudy catshark, a candidate model cartilaginous fish species. In all methods, green fluorescent protein (GFP) expression was used to evaluate exogenous gene transfer. First, we examined gene transfer into primary cultured cells from cloudy catshark embryos by lipofection, polyethylenimine (PEI) transfection, adenovirus infection, baculovirus infection, and electroporation. Among the methods tested, lipofection, electroporation, and baculovirus infection enabled the successful transfer of exogenous genes into primary cultured cells. We then attempted in vivo transfection into cloudy catshark embryos by electroporation and baculovirus infection. Although baculovirus-injected groups did not show GFP fluorescence, electroporation successfully introduced GFP into muscle cells. Furthermore, we succeeded in GFP transfer into adult tissues by electroporation. The in vitro and in vivo gene transfer methods that worked in this study may open ways for genetic manipulation including knockout experiments and cellular lineage analysis in cartilaginous fishes.

Interpopulation variation in inbreeding is primarily driven by tolerance of mating with relatives in a spermcasting invertebrate.

The degree to which individuals inbreed is a fundamental aspect of population biology shaped by both passive and active processes. Yet, the relative influences of random and non-random mating on the overall magnitude of inbreeding are not well characterized for many taxa. We quantified variation in inbreeding among qualitatively accessible and isolated populations of a sessile marine invertebrate (the colonial ascidian Lissoclinum verrilli) in which hermaphroditic colonies cast sperm into the water column for subsequent uptake and internal fertilization. We compared estimates of inbreeding to simulations predicting random mating within sites to evaluate if levels of inbreeding were (1) less than expected because of active attempts to limit inbreeding, (2) as predicted by genetic subdivision and passive inbreeding tolerance, or (3) greater than simulations due to active attempts to promote inbreeding via self-fertilization or a preference for related mates. We found evidence of restricted gene flow and significant differences in the genetic diversity of L. verrilli colonies among sites, indicating that on average colonies were weakly related in accessible locations, but their levels of relatedness matched that of first cousins or half-siblings on isolated substrates. Irrespective of population size, progeny arrays revealed variation in the magnitude of inbreeding across sites that tracked with the mean relatedness of conspecifics. Biparental reproduction was confirmed in most offspring (86%) and estimates of total inbreeding largely overlapped with simulations of random mating, suggesting that interpopulation variation in mother-offspring resemblance was primarily due to genetic subdivision and passive tolerance of related mates. Our results highlight the influence of demographic isolation on the genetic composition of populations, and support theory predicting that tolerance of biparental inbreeding, even when mates are closely related, may be favoured under a broad set of ecological and evolutionary conditions.

Dynamic cellular and molecular characteristics of spermatogenesis in the viviparous marine teleost Sebastes schlegelii†.

Spermatogenesis is a dynamic cell developmental process that is essential for reproductive success. Vertebrates utilize a variety of reproductive strategies, including sperm diversity, and internal and external fertilization. Research on the cellular and molecular dynamic changes involved in viviparous teleost spermatogenesis, however, is currently lacking. Here, we combined cytohistology, 10 × genomic single-cell RNA-seq, and transcriptome technology to determine the dynamic development characteristics of the spermatogenesis of Sebastes schlegelii. The expressions of lhcgr (Luteinizing hormone/Choriogonadotropin receptor), fshr (follicle-stimulating hormone receptor), ar (androgen receptor), pgr (progesterone receptor), and cox (cyclo-oxygen-ase), as well as the prostaglandin E and F levels peaked during the maturation period, indicating that they were important for sperm maturation and mating. Fifteen clusters were identified based on the 10 × genomic single-cell results. The cell markers of the sub-cluster were identified by their upregulation; piwil, dazl, and dmrt1 were upregulated and identified as spermatogonium markers, and sycp1/3 and spo11 were identified as spermatocyte markers. For S. schlegelii, the sperm head nucleus was elongated (spherical to streamlined in shape), which is a typical characteristic for sperm involved in internal fertilization. We also identified a series of crucial genes associated with spermiogenesis, such as spata6, spag16, kif20a, trip10, and klf10, while kif2c, kifap3, fez2, and spaca6 were found to be involved in nucleus elongation. The results of this study will enrich our cellular and molecular knowledge of spermatogenesis and spermiogenesis in fish that undergo internal fertilization.

Fertilization mode differentially impacts the evolution of vertebrate sperm components

Environmental change frequently drives morphological diversification, including at the cellular level. Transitions in the environment where fertilization occurs (i.e., fertilization mode) are hypothesized to be a driver of the extreme diversity in sperm morphology observed in animals. Yet how fertilization mode impacts the evolution of sperm components—head, midpiece, and flagellum—each with different functional roles that must act as an integrated unit remains unclear. Here, we test this hypothesis by examining the evolution of sperm component lengths across 1103 species of vertebrates varying in fertilization mode (external vs. internal fertilization). Sperm component length is explained in part by fertilization mode across vertebrates, but how fertilization mode influences sperm evolution varies among sperm components and vertebrate clades. We also identify evolutionary responses not influenced by fertilization mode: midpieces evolve rapidly in both external and internal fertilizers. Fertilization mode thus influences vertebrate sperm evolution through complex component- and clade-specific evolutionary responses.

Macroevolutionary patterns in marine hermaphroditism.

Most plants and many animals are hermaphroditic; whether the same forces are responsible for hermaphroditism in both groups is unclear. The well-established drivers of hermaphroditism in plants (e.g., seed dispersal potential, pollination mode) have analogues in animals (e.g., larval dispersal potential, fertilization mode), allowing us to test the generality of the proposed drivers of hermaphroditism across both groups. Here, we test these theories for 1153 species of marine invertebrates, from three phyla. Species with either internal fertilization, restricted offspring dispersal, or small body sizes are more likely to be hermaphroditic than species that are external fertilizers, planktonic developers, or larger. Plants and animals show different biogeographical patterns, however: animals are less likely to be hermaphroditic at higher latitudes-the opposite to the trend in plants. Overall, our results suggest that similar forces, namely, competition among offspring or gametes, shape the evolution of hermaphroditism across plants and three invertebrate phyla.

Transcriptome Analysis Provides Insights into Copulation, Fertilization, and Gestation in Sebastes schlegelii.

Among the viviparous marine teleosts of China, the black rockfish (Sebastes schlegelii Hilgendorf) is one of the most economically important. In addition to copulation and internal fertilization, it features lengthy sperm storage in the female ovary as well as a high rate of abortion. A network of gene regulation is necessary for these processes. To elucidate the mechanisms of copulation, fertilization, and gestation, it is essential to determine the genetic basis of viviparous teleost oogenesis and embryogenesis. In this study, we analyzed the transcriptome of the ovary during different developmental phases to investigate the dynamic changes that occur. We constructed 24 ovary transcriptomes. In order to investigate the regulation of embryogenesis, differentially expressed genes (DEGs) with specific expression patterns were subjected to gene ontology annotation, pathway analyses, and weighted gene co-expression network analysis (WGCNA). The up-regulated genes were significantly enriched in focal adhesion, regulation of the actin cytoskeleton, Wnt, and ECM-receptor interaction signaling pathways. As a result of our study, we provide omics evidence for copulation, fertilization, and gestation in viviparous marine teleosts. Decoding the S. schlegelii gene regulation network, as well as providing new insights into embryogenesis, is highly valuable to researchers in the marine teleost reproduction sciences.

The evolution of parental care in salamanders

Complex parenting has been proposed to contribute to the evolutionary success of vertebrates. However, the evolutionary routes to complex parenting and the role of parenting in vertebrate diversity are still contentious. Although basal vertebrates provide clues to complex reproduction, these are often understudied. Using 181 species that represent all major lineages of an early vertebrate group, the salamanders and newts (Caudata, salamanders henceforth) here we show that fertilisation mode is tied to parental care: male-only care occurs in external fertilisers, whereas female-only care exclusively occurs in internal fertilisers. Importantly, internal fertilisation opens the way to terrestrial reproduction, because fertilised females are able to deposit their eggs on land, and with maternal care provision, the eggs could potentially develop outside the aquatic environment. Taken together, our results of a semi-aquatic early vertebrate group propose that the diversity and follow-up radiation of terrestrial vertebrates are inherently associated with a complex social behaviour, parenting.