Monoaminergic and cholinergic mechanisms of reproduction control in marine bivalve molluscs and echinoderms: A review

The autoreproduction ability is one of the most important properties of living systems. Evolution of metazoans ensured their reproduction by means of such determinants as sexual cells developing in specialized organs, in gonads. In most marine invertebrates, the gonad produces tens of millions of gametes per reproductive cycle. This reproduction level guarantees the species resistance in the external environment and is provided by the whole organism, though in unfavourable ecological conditions the gonad may become a source of trophic and energy material and it can maintain the viability of organism by means or the mass, often total, lysis of sexual cells. This metabolic interaction of a part (gonad) and the whole (organism) presumes the existence of the strictly determined relations between them, on the one hand, and more or less pronounced autonomy, on the other hand. The isolated organs, including gonads, are capable of fulfiling specific functions for a short period of time by means of local regulation. However, there is no full autonomy between the gonad and the organism: the hierarchical relations set in between them.     

Ultrastructural events in horse gonadal morphogenesis.

The establishment and sexual differentiation of the gonads of horse embryos were studied using high-resolution techniques. The most dramatic observation is the early cytodifferentiation of the somatic cells into steroidogenic cells which takes place before sexual differentiation of the gonads. A unique morphogenetic pattern is established during this process: the seminiferous cords of the testis are completely segregated from the steroidogenic tissue by a basal lamina, while in the medulla of the ovary, steroidogenic cells differentiate inside the epithelial cords which contain germ cells. This early difference in the topographical distribution of steroidogenic cells favours the hypothesis that the interactions between somatic and germ cells vary with the genetic sex. The possibility of finding qualitative differences in steroidogenesis before and during sexual differentiation of the gonad suggests the horse gonad as a good model for the study of the role of the steroid hormones in the sexual differentiation of the mammalian gonad.     

Early segregation of germ and somatic lineages during gonadal regeneration in the annelid Enchytraeus japonensis

Although regeneration studies are useful for understanding how organs renew, little information is available about regeneration of reproductive organs and germ cells. We here describe the behavior of germ-cell precursors during regeneration of the oligochaete annelid worm Enchytraeus japonensis, which has the remarkable feature of undergoing asexual (by fission) and sexual reproduction . We first found that the gonad can regenerate from any body fragment yielded by fission during asexual reproduction. We then examined behavior of germ-cell lineage during this regenerative process, by using a homolog of the Piwi gene (Ej-piwi) as a marker. We found that in asexually growing animals, specialized cells expressing Ej-piwi are distributed widely in the body as single cells. These cells seem to serve as a reservoir of germ-cell precursors because during asexual propagation these cells migrate into the regenerating tissue, where they ultimately settle in the prospective gonads, and give rise to germ cells upon sexualization. These cells are distinct from the neoblasts, thought to be stem cells in other animals. This is the first report to directly show that the germ and somatic lineages are segregated in asexually growing animals and behave differently during regeneration.     

Seasonal changes in energy allocation to somatic and reproductive body components of the common cold temperature sea urchin <Emphasis Type="Italic">Loxechinus albus</Emphasis> in a Sub-Antarctic environment

Monthly variations in the somatic indexes and energetic content of organs were investigated in Loxechinus albus from the Beagle Channel. Samples were collected monthly from May 2004 to May 2005. Lantern and test indexes did not vary significantly. A major peak of gonad index (GI) was observed in winter (sexual maturation period), with a strong declination in November suggesting a spawning period in spring. In coincidence a shortening of feeding activities was expressed by the lower values of gut index (Gut I), suggesting that the gut is a storage organ. The values of gonad energetic content (GEC) and total gonad energetic content (TGEC) showed minimum values in winter (ripe stage) and the maximum in spring (spawned stage). The TGEC reached higher monthly average values (50–200 kJ) than total gut energetic content (TGuEC) (20–40 kJ). These differences indicate that the gonads constitute the most important store organs in L. albus. Moreover, organic stores are built up in the gonads after spawning, and then utilized during gamete production.     

Ultrastructure of sperm and male reproductive system in Lineus viridis (Heteronemertea, Nemertea)

Nemerteans possess serially arranged gonads that lie between the midgut pouches. In both sexes the gonads are lined with an epithelium. During maturity, they gain contact to the exterior by a ciliated duct, which is generally assumed to be a derivative of the gonad. Gonad lining and sperm ultrastructure are little known in heteronemerteans, a group of nemerteans belonging to the Anopla, one of the two large nemertean subgroups. Reproduction biology in heteronemertean Lineus viridis allows predicting a modified sperm type, so-called introsperm for this taxon. Nothing is known on the fate of the testes at the end of the reproductive period of this perennial species. In order to test the predictions and to broaden the data base, males of L. viridis were collected at different times of the year. Histological and ultrastructural data show that the gonad wall is lined with different aciliated endothelial cells and germ cells, while the gonoduct is formed by densely ciliated cells. The testes are completely filled with sperm cells during maturity; there is no hint at ongoing spermiogenesis at this time. The sperm consists of head, midpiece and tail. Externally, head and midpiece cannot be discriminated. The acrosome is cup-shaped and lies anterior to the nucleus which contains 6–8 lateral ridges. Three long mitochondria mark the midpiece. They line the posterior section of the nucleus and extend up to the level of the ciliary basal structures. The sperm morphology corroborates the predictions derived from the mode of reproduction. At the end of the reproductive period the male gonads change cellular composition, while the gonoduct degenerates. Provided that both sexes show the same growth rate, male offspring acquire sexual maturity earlier than female offspring, since L. viridis males are always smaller than the females. In contrast to the males, females keep their gonads and gonoducts during most time of the year. Since large males were never found within the studied population, these data indicate that L. viridis might be a consecutive hermaphrodite.     

Reproduction of the spiny puffer, Diodon holocanthus (Pisces: Diodontidae) in the continental shelf of Mexican Central Pacific

Diodon holocanthus is an important economic and ecological species of the demersal fish community, caught as bycatch from local shrimp fishery. The reproductive biology of this long-spine porcupinefish has not yet been described, and reproductive season, the sex ratio, length distribution, length at first gonad maturity, and the gonad macro and microscopic features are described. A total of 400 organisms, ranging from 5.0 to 40.3 cm (average 18.4 cm) total length, were caught from the continental shelf of the Central Mexican Pacific, from December 1995 and December 1998. Sex ratio was 1:0.86 females to males (n = 253). The length at which 50% of the individuals showed maturing gonads was 19.7 cm for females and 20.1 cm for males. Length of the smallest organism with ripe gonads was 12.2 cm for females and 13 cm for males. Four gonadal maturation stages were found in both sexes, and five oocyte development phases were identified. The oocyte development pattern is of asynchronous type, which means the species can reproduce several times a year. Testicle development is lobular type, as in most teleost fishes. Monthly mean values of the gonad-somatic index suggest the reproduction activity peaks in June, and September-December.     

Sexual phenotype of avian chimeric gonads with germinal and stromal cells of opposite genetic sexes

The respective roles of germinal and stromal cells in determining the sexual phenotype of the gonad were analyzed in chimeric gonads obtained by surgical recombination between young avian blastodiscs in ovo. Equivalent territories were exchanged between two blastodisc, in order that the germinal crescent and the gonad territory had a different origin (fig. 3). Embryos used for these experiments carried a sex linked pigment mutation, that made it possible to diagnose the genetic sexes of germ cells and stroma at the time when the gonad was retrieved for examination. On the basis of species, three types of combination were performed: chick germ cells in chick or quail stroma, quail germ cells in chick stroma. In each chimera, the genetic sexes of the two gonadal cell populations could be identical or opposite. However it appeared that the germ cell population was not always homogeneous. In some grafting schemes, ectopic germ cells, located outside the germinal crescent, contributed to the colonization of the experimental gonad. These germ cells were from the same territory as the stroma element of the gonad, i.e., they were of the same species and the same genetic sex. Whatever the case, in 87 chimeras that were studied, the sex phenotype of the gonads always corresponded to the genetic sex of the stroma. Thus the genetic sex of germ cells has no role in the sexual differentiation of the gonadal rudiments.     

Male-specific cell migration into the developing gonad is a conserved process involving PDGF signalling

Male-specific migration of cells from the mesonephric kidney into the embryonic gonad is required for testis formation in the mouse. It is unknown, however, whether this process is specific to the mouse embryo or whether it is a fundamental characteristic of testis formation in other vertebrates. The signalling molecule/s underlying the process are also unclear. It has previously been speculated that male-specific cell migration might be limited to mammals. Here, we report that male-specific cell migration is conserved between mammals (mouse) and birds (quail-chicken) and that it involves proper PDGF signalling in both groups. Interspecific co-cultures of embryonic quail mesonephric kidneys together with embryonic chicken gonads showed that quail cells migrated specifically into male chicken gonads at the time of sexual differentiation. The migration process is therefore conserved in birds. Furthermore, this migration involves a conserved signalling pathway/s. When GFP-labelled embryonic mouse mesonephric kidneys were cultured together with embryonic chicken gonads, GFP+ mouse cells migrated specifically into male chicken gonads and not female gonads. The immigrating mouse cells contributed to the interstitial cell population of the developing chicken testis, with most cells expressing the endothelial cell marker, PECAM. The signalling molecule/s released from the embryonic male chicken gonad is therefore recognised by both embryonic quail and mouse mesonephric cells. A candidate signalling molecule mediating the male-specific cell migration is PDGF. We found that PDGF-A and PDGF receptor-alpha are both up-regulated male-specifically in embryonic chicken and mouse gonads. PDGF signalling involves the phosphotidylinositol 3-kinase (PIK3) pathway, an intracellular pathway proposed to be important for mesonephric cell migration in the mammalian gonad. We found that a component of this pathway, PI3KC2alpha, is expressed male-specifically in developing embryonic chicken gonads at the time of sexual differentiation. Treatment of organ cultures with the selective PDGF receptor signalling inhibitor, AG1296 (tyrphostin), blocked or impaired mesonephric cell migration in both the mammalian and avian systems. Taken together, these studies indicate that a key cellular event in gonadal sex differentiation is conserved among higher vertebrates, that it involves PDGF signalling, and that in mammals is an indirect effect of Sry expression.     

Interference of asexual and sexual reproduction in the green hydra

The green hydra, Hydra viridissima, has three sexes: hermaphrodite, male, and female. I investigated the reproductive strategies of the green hydra and the relationship between asexual budding and sexual reproduction. The proportion of mature individuals in the asexually reproducing population increased with increasing temperature. Sexual reproduction did not interrupt asexual budding in hermaphrodites or males; sexual reproduction did interrupt asexual budding in females. Sexual reproduction also resulted in exponential population growth during the reproductive season. The number of asexual buds on each parental individual was positively correlated with the parental individual size in asexual individuals and in males. The same positive correlation was found between the number of testicles and the size of males. These correlations reflect a common tendency in asexual and sexual reproduction: larger parental individuals have a greater number of propagules or gametes. No correlation was found between size and buds or size and gonads in hermaphrodites; hermaphrodites had at most one asexual bud and were significantly larger than males, females, and asexual individuals. The larger size of hermaphrodites supports the hypothesis that producing both female and male gonads is more energetically costly than producing only one type of gamete (gonochorism).     

Temperature-dependent gonadal differentiation in the turtle Emys orbicularis: concordance between sexual phenotype and serological H-Y antigen expression at threshold temperature

As in many other turtles, the sexual differentiation of gonads in embryos of Emys orbicularis is temperature-sensitive, 100% phenotypic males being obtained below 27.5 degrees C and 100% phenotypic females above 29.5 degrees C. The expression of the serologically defined H-Y (SD-H-Y) antigen at both low and high temperatures has been shown to be different in gonads and in blood : in gonads, it is closely associated with ovarian structure, whereas in blood it is independent of sexual phenotype and appears to indicate sexual genotype. Both sexes differentiate at 28.5 degrees C, suggesting that at this intermediate (threshold) temperature, sexual differentiation of gonads conforms with sexual genotype. To test this hypothesis, the expression of SD-H-Y antigen has been carried out in blood cells of Emys individuals raised from eggs incubated at the threshold temperature (28.5 degrees C). All phenotypic males typed SD-H-Y negative, whereas most phenotypic females typed SD-H-Y positive. From this concordance between sexual phenotype of gonads and SD-H-Y phenotype of blood, we postulate that a ZZ male/ZW female mechanism of genotypic sex determination is revealed at the threshold temperature for gonad differentiation in Emys.     

Uncovering gene regulatory networks during mouse fetal germ cell development

The commitment of germ cells to either oogenesis or spermatogenesis occurs during fetal gonad development: germ cells enter meiosis or mitotic arrest, depending on whether they reside within an ovary or a testis, respectively. Despite the critical importance of this step for sexual reproduction, gene networks underlying germ cell development have remained only partially understood. Taking advantage of the W(v) mouse model, in which gonads lack germ cells, we conducted a microarray study to identify genes expressed in fetal germ cells. In addition to distinguishing genes expressed by germ cells from those expressed by somatic cells within the developing gonads, we were able to highlight specific groups of genes expressed only in female or male germ cells. Our results provide an important resource for deciphering the molecular pathways driving proper germ cell development and sex determination and will improve our understanding of the etiology of human germ cell tumors that arise from dysregulation of germ cell differentiation.     

Sexual maturation and related changes in aspartate transcarbamylase activity of gonad tissues in the soft shell clam (Mya arenaria)

The information on the relation between the gonadal aspartate transcarbamylase (ACTase) activity and the sexual maturation in molluscs is very fragmentary and is still absent in Mya arenaria. The determination of ATCase activity, energy reserves levels and maturation stages were done in gonads of clams. Results showed that the seasonal cycle of storage and utilization of energy reserves in gonads of clams are linked to the bimodal reproduction well known in this bivalve. ATCase activity was high in clams at development and ripe stages, while this activity was low for individuals found in indifferent, spawning or spent stages. This difference can be explained by the fact that during gonad development, gonadal synthetic activity increased following the mitotic events associated to the reproductive cycle. The results presented in this paper have confirmed the link between ATCase activity and gametogenesis in M. arenaria. Further work should be realized in order to assess if ATCase activity could be considered as a potential biomarker to evaluate the disruption of sexual maturation in clams collected in sites such as the St. Lawrence estuary.     

Genome-wide identification of Phytophthora sojae SNARE genes and functional characterization of the conserved SNARE PsYKT6

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are central components of the machinery mediating membrane fusion and key factors for vesicular trafficking in all eukaryotic cells. Taking advantage of the available whole genome sequence of the oomycete plant pathogen Phytophthora sojae, 35 genes encoding putative SNARE proteins were identified in the genome of this organism. PsYKT6, one of the most conserved SNARE proteins, was functionally characterized by homology-dependent gene silencing. The phenotype analysis showed that PsYKT6 is important for proper asexual development, sexual reproduction, and pathogenesis on host soybean cultivars.     

Timing of avian reproduction in unpredictable environments

Organisms living in periodically varying environments adjust their life history events to the changes in food availability. When these changes are not entirely predictable animals face a trade-off between maintaining physiological preparedness (which can be costly) and being unprepared (which decreases the chances of successful reproduction). To investigate this problem, we developed an optimal annual routine model of gonad regulation in birds. Most birds regress their reproductive organs during non-breeding periods, but to start breeding again they need to have functional gonads. Maintaining the gonads in this state is costly, but because it takes time to achieve this state, if gonads are not functional the bird may miss a possible breeding opportunity. We explore the resolution of this trade-off in environments where favorable periods can occur at any time of the year and variability in the length of good and bad periods can be altered. Consistent with empirical studies of reproductive behavior in unpredictable environments, we find that birds maintain the gonads partially activated during unfavorable conditions in many cases. However, gonad regulation may differ strikingly depending on the consistency of the good and bad periods. Furthermore, seasonal changes in food availability lead to the entrainment of reproduction and the segregation of the breeding and non-breeding season, even if the magnitude of seasonality is small compared to the degree of environmental fluctuations. These results indicate that several aspects of the environment need to be taken into account to understand reproductive behavior in unpredictable environments. Given that the trade-off between the costs and benefits of maintaining physiological preparedness is not limited to birds, our results have implications for understanding behavioral flexibility in other organisms as well.     

Cytological paradoxes in the developmental cycle of the coelenterate Polypodium hydriforme--an intracellular parasite from the oocytes of acipenserid fishes

Successive stages of the embryonic development of Polypodium hydriforme, occurring at the parasitic phase of its life cycle, are considered. The development of a new parasitic generation starts without fertilization, i. e. parthenogenetically. The embryo develops from aberrant binucleate gametes formed in the result of meiosis within entodermal gonads of free-living animals. This type of gametogenesis, earlier considered as spermatogenesis (Raikova, 1961), is now interpreted as oogenesis. A conclusion is drawn about a change of the sexual orientation of the male gonad which becomes a female one in the course of evolution of Polypodium. As to the gonads of free-living animals, which were formerly interpreted as female ones, they seem to be abortive rudimentary organs since they produce no mature sex cells. A long-lasting block of cytokinesis of the 2nd meiotic division, as well as utilization of the polar body of this division as a phorocyte and, later, as a trophamnion, are important adaptations of Polypodium to parasitism. It is the larger nucleus with a voluminous cytoplasm, rather than the smaller nucleus, that becomes here the 2nd polar body. Polypodium differs from other coelenterates by the presence of highly polyploid feeding cells at both the parasitic (the trophamnion, 500 c) and free-living phases of the life cycle (trophocytes in the rudimentary female gonad, 8c-32c).     

Mating in the box jellyfish Copula sivickisi—Novel function of cnidocytes

Within cubozoans, a few species have developed a sexual reproduction system including mating and internal fertilization. One species, Copula sivickisi, is found in a large area of the indo pacific. They have separate sexes and when mature males and females meet they entangle their tentacles and the males transfer a sperm package, a spermatozeugmata, which is ingested by the female fertilizing her eggs internally. After 2–3 days, the females lay an embryo strand that sticks to the substrate and after another 2–3 days, the fully developed larvae leave the strand. We have examined the ultrastructure of the gonads and spermatozeugmata to look for structural adaptations to this specialized way of reproduction and understand how the fertilization takes place. Surprisingly, we discovered that the male gonads were heavily packed with cnidocytes of the isorhiza type and that they are transferred to the spermatozeugmata. The spermatozeugmata does not dissolve in the female gastrovascular cavity but is attached to the female gonad probably using the isorhizas. Here, the sperm cells are partly digested and the nuclei are released. The actual fertilization seems to happen through phagocytosis of the released nuclei by the epithelial cells. The female gonads are likewise packed with cnidocytes but of the eurytele type. They do not mature inside the female and putatively serve to protect the developing larvae once the embryo strand is laid. This specialized way of fertilization is to our knowledge novel and so is this first account of cnidocytes being directly involved in cnidarian reproduction. J. Morphol. 276:1055–1064, 2015. © 2015 Wiley Periodicals, Inc.     

Cytoplasmic retention of the sex-determining factor SOX9 via the microtubule network

SOX9 is a sex-determining factor which induces Sertoli cell differentiation and subsequent testis cord formation. It is expressed both in male and female undifferentiated gonads in the cytoplasmic compartment of pre-Sertoli cells. At the time of sexual differentiation, SOX9 moves into the nucleus of male pre-Sertoli cells whereas in female, it remains in the cytoplasm and then its expression decreases. To study the cytoplasmic localization of SOX9, we have analyzed its interaction with the cytoskeleton components. By treatment of NT2/D1 and transfected NIH3T3 cell lines and embryonic gonads with nocodazole, a drug depolymerizing the microtubules, we show that cytoplasmic retention of SOX9 requires the integrity of the microtubule network. Using biochemical experiments, we demonstrated that SOX9 is able to interact with microtubules in vitro and in vivo. On the other hand, we observed a complete male-specific reorganization of the microtubule network in epithelial Sertoli cells of the male embryonic gonad at the time of sexual differentiation and testis cord formation.     

The Hydra viridis/Chlorella symbiosis. Growth and sexual differentiation in polyps without symbionts

To investigate interactions between the basal metazoan Hydra viridis and its symbiotic Chlorella algae, we generated aposymbiotic hydra lacking algae and compared them to symbiotic ones with regard to growth and sexual differentiation. Under standard feeding conditions aposymbiotic polyps proliferated similarly to symbiotic polyps. Under moderate and low feeding conditions asexual growth was reduced in polyps lacking algae, indicating that the symbionts supply nutrients to their hosts. In addition, the Chlorella symbionts had a strong influence on the sexual reproduction of Hydra viridis: in most cases female gonads were produced only when symbiotic algae were present. Spermatogenesis proceeded similarly in symbiotic and aposymbiotic polyps. Since during oogenesis symbionts are actively transferred from endodermal epithelial cells to the ectodermal oocytes, this oogenesis promoting role could indicate that the symbionts are critically involved in the control of sexual differentiation in green hydra.