Zinc-binding property of the major yolk protein in the sea urchin - implications of its role as a zinc transporter for gametogenesis

Major yolk protein (MYP), a transferrin superfamily protein that forms yolk granules in sea urchin eggs, is also contained in the coelomic fluid and nutritive phagocytes of the gonad in both sexes. MYP in the coelomic fluid (CFMYP; 180 kDa) has a higher molecular mass than MYP in eggs (EGMYP; 170 kDa). Here we show that MYP has a zinc-binding capacity that is diminished concomitantly with its incorporation from the coelomic fluid into the gonad in the sea urchin Pseudocentrotus depressus. Most of the zinc in the coelomic fluid was bound to CFMYP, whereas zinc in eggs was scarcely bound to EGMYP. Both CFMYP and EGMYP were present in nutritive phagocytes, where CFMYP bound more zinc than EGMYP. Saturation binding assays revealed that CFMYP has more zinc-binding sites than EGMYP. Labeled CFMYP injected into the coelom was incorporated into ovarian and testicular nutritive phagocytes and vitellogenic oocytes, and the molecular mass of part of the incorporated CFMYP shifted to 170 kDa. Considering the fact that the digestive tract is a major production site of MYP, we propose that CFMYP transports zinc, essential for gametogenesis, from the digestive tract to the ovary and testis through the coelomic fluid, after which part of the CFMYP is processed to EGMYP with loss of zinc-binding site(s).     

The sea urchin major yolk protein is synthesized mainly in the gut inner epithelium and the gonadal nutritive phagocytes before and during gametogenesis

Major yolk protein (MYP), the predominant component of yolk granules in sea urchin eggs, is also contained in the coelomic fluid and nutritive phagocytes of the gonad in both sexes. MYP is stored in ovarian and testicular nutritive phagocytes prior to gametogenesis and is used during gametogenesis as material for synthesizing proteins and other components necessary for eggs and sperm. To reveal the expression profile and the main production site of MYP, we analyzed MYP mRNA expression in immature and maturing Pseudocentrotus depressus. Real‐time reverse‐transcribed polymerase chain reaction analysis showed that MYP mRNA was expressed predominantly in the digestive tract (stomach, intestine and rectum) and the gonad of both sexes. The total amounts of MYP mRNA in the whole digestive tract and in the whole gonad were at similar levels in both immature and maturing sea urchins. MYP mRNA was also detected in white morula cells and vibratile cells separated from the coelomic fluid by density gradient centrifugation, but the expression levels in these cells were very low compared with those in the digestive tract and the gonad. Using in situ hybridization analysis, MYP mRNA was detected in the inner epithelium of the digestive tract and in nutritive phagocytes of the ovary and testis, but was not detected in the germ cells. We conclude that the adult sea urchin has two predominant production sites for MYP regardless of sex and reproductive stage: the inner epithelium of the digestive tract and the nutritive phagocytes of the gonad. Mol. Reprod. Dev. 77: 59–68, 2010. © 2009 Wiley‐Liss, Inc.     

The major yolk protein is synthesized in the digestive tract and secreted into the body cavities in sea urchin larvae

Major yolk protein (MYP), a transferrin superfamily protein contained in yolk granules of sea urchin eggs, also occurs in the coelomic fluid of male and female adult sea urchins regardless of their reproductive cycle. MYP in the coelomic fluid (CFMYP; 180 kDa) has a zinc-binding capacity and has a higher molecular mass than MYP in eggs (EGMYP; 170 kDa). CFMYP is thought to be synthesized in the digestive tract and secreted into the coelomic fluid where it is involved in the transport of zinc derived from food. To clarify when and where MYP synthesis starts, we investigated the expression of MYP during larval development and growth in Pseudocentrotus depressus. MYP mRNA was detected using RT-PCR in the early 8-arm pluteus stage and its expression persisted until after metamorphosis. Real-time RT-PCR revealed that MYP mRNA increased exponentially from the early 8-arm stage to metamorphosis. Western blotting showed that maternal EGMYP disappeared by the 4-arm stage and that newly synthesized CFMYP was present at and after the mid 8-arm stage. In the late 8-arm larvae, MYP mRNA was detected in the digestive tract using in situ hybridization, and the protein was found in the somatocoel and the blastocoel-derived space between the somatocoel and epidermis using immunohistochemistry. These results suggest that CFMYP is synthesized in the digestive tract and secreted into the body cavities at and after the early 8-arm stage. We assume that in larvae, CFMYP transports zinc derived from food via the body cavities to various tissues, as suggested for adults.     

Ege A,a novel C2 domain containing protein,is essential for GPCR-mediated gene expression in dictyostelium

The major yolk protein (MYP) in sea urchins has historically been classified as a vitellogenin based on its abundance in the yolk platelets. Curiously, it is found in both sexes of sea urchins where it is presumed to play a physiological role in gametogenesis, embryogenesis, or both. Here we present the primary structure of MYP as predicted from cDNAs of two sea urchins species, Strongylocentrotus purpuratus and Lytechinus variegatus. The sequence from these two species share identity to one another, but bear no resemblance to other known vitellogenins. Instead the sequence shares identity to members of the transferrin superfamily of proteins. In vitro iron binding assays, including both (59)Fe overlay assays of MYP enriched coelomic fluid and immunoprecipitation of native iron-bound MYP from coelomic fluid, support this classification. We suggest that one of MYP's transferrin-like properties is to shuttle iron to developing germ cells.     

饥饿对中间球海胆MYP基因转录表达的影响

应用实时定量PCR技术对主要卵黄蛋白(Major yolk protein,MYP)基因在不同饥饿时期中间球海胆的体腔细胞、性腺、肠、胃中的转录表达差异进行了分析。结果表明,在正常状态下,MYP基因在体腔细胞、性腺、肠、胃等不同组织中的转录表达差异明显,肠中的表达量最高,其他组织中的表达量均较低。随饥饿时间的延长,MYP基因在体腔细胞中的表达量先迅速下降,而后稳定在较低水平,实验结束时下降至对照组的1.58%;在性腺中的表达量持续上升,实验结束时上升至对照组的679.75%;在肠中的表达量持续下降,实验结束时下降至对照组的33.33%;在胃中的表达量呈上升趋势,实验结束时上升至对照组的106.52倍。综合来看,饥饿状况下,中间球海胆肠中的MYP表达量持续下降,但仍是MYP的主要合成部位;性腺中MYP表达量持续上升,致使其MYP表达比重上升;胃、体腔细胞中表达量在饥饿过程中虽有变化,但总表达量很少,对MYP的整体表达影响不大。     

MYP基因在中间球海胆及杂交海胆生殖腺不同发育时期的转录表达差异

摘要: 应用实时荧光定量PCR技术对主要卵黄蛋白(Major yolk protein, MYP)基因在中间球海胆和杂交海胆(中间球海胆♀×光棘球海胆♂)的生殖腺不同发育时期的转录表达差异进行了分析。结果表明, MYP基因在海胆雌雄个体生殖腺中转录水平上的表达差异不明显; MYP基因在中间球海胆生殖腺发育的4个时期的表达呈快速下降的趋势, 而在杂交海胆呈缓慢下降的趋势。在分析的生殖腺4个发育期中, 中间球海胆雌雄个体 MYP 基因的表达量(以18S rRNA为参照)分别从44.55%和41.17%下降到9.59%和1.83%; 杂交海胆的雌雄个体分别从37.66%和36.66%下降到19.22%和12.55%。MYP基因的转录表达量差异与杂交后生殖腺产生的变异密切相关。     

Comparative biochemical analysis of the major yolk protein in the sea urchin egg and coelomic fluid

The major yolk protein (MYP) is localized to the egg and coelomic fluid of the adult sea urchin. While the egg‐localized MYP has been extensively studied, much less is known about the coelomic fluid‐localized protein. Therefore, we have conducted a comparative biochemical analysis of these proteins. Sucrose density gradient ultracentrifugation revealed unique elution profiles for the MYP species present in the egg, 170‐ and 240 kDa, and the coelomic fluid, 180‐ and 250 kDa. Fractionation in polyacrylamide gels revealed that under reducing conditions both species were present in each location. However, in the absence of reducing agent only one species was present in each fraction: 240 kDa in the egg and 250 kDa in the coelomic fluid. In addition, V8 peptide mapping indicated that all four species have very similar primary structures. Circular dichroic spectral analysis and endogenous tryptophan measurements of the purified 170‐ and 180 kDa species revealed distinctive secondary and tertiary structural features with notable differences in their responses to calcium: apparent Kds of 245‐ and 475 μmol/L were measured for the 170‐ and 180 kDa species, respectively. Further analysis revealed that both species have differing calcium requirements for binding to membranes as well as protein‐dependent, membrane‐membrane interaction. We discuss the functional implications arising from the structural differences which exist between the egg and coelomic fluid resident MYPs.     

Ultrastructure of the somatic portion of the gonads in asteroids,with emphasis on flagellated-collar cells and nutrient transport

Somatic portions of gonads in two phanerozonian sea-stars, Ctenodiscus crispatus and Hippasteria phrygiana, were similar in all aspects of gross structure and histology seen previously in both forcipulate and spinulosan asteroids. For the first time, detailed ultrastructural observations have been made of cells and tissues that reveal several features believed to be of universal occurrence in the gonads of asteroids. These include flagellated-collar cells in the visceral peritoneum and other coelomically derived epithelia, muscular-flagellated-collar cells in the visceral peritoneum and genital coelomic (perihaemal) sinus, the digestion of collagen fibers by cells in the connective tissue layer, and the intimate relationship of the genital haemal sinus and the entire germinal epithelium. Structural and functional compartmentalization are discussed in relation to major activities of the gonad throughout the annual reproductive cycle. The distinctive ultrastructure and current generation of flagellated-collar cells found in the visceral peritoneum are analyzed relative to their role in nutrient transport to gonadal tissues. The single flagellum of each flagellated-collar cell beats in coordination with those on neighboring cells to produce extremely rapid, oriented currents of coelomic fluid. The form of beating in an individual flagellum is planar, and the resulting synchronized activity of many adjacent flagella is non-metachronal; both of these characteristic aspects of current production have, thus far, been encountered together only in the Echinodermata. Flagellated-collar cells are efficient in generating currents which mix contents of the coelomic fluid, and they can presumably supply themselves with nutrients. It is concluded that nutrients so obtained are generally not passed through the wall of the gonad to the germinal epithelium and, as a result, have little to do with nutrition of somatic and germinal cells of the germinal epithelium. Alternatively, well-developed genital portions of the haemal system of the sea-star are advanced as the major channels supplying nutrients to germinal epithelia during gametogenesis.     

A histological investigation of the occurrence of non-reproductive female bluefin tuna Thunnus thynnus in the Mediterranean Sea

The presence of non-reproductive Atlantic bluefin tuna Thunnus thynnus females in the Mediterranean Sea was investigated through histological analysis of the gonads. Three hundred and twenty-six ovary samples were collected from adults captured at different locations in the Mediterranean Sea during the reproductive seasons between 1998 and 2008. Only three specimens were considered to be in a non-reproductive state: two of them were in a reabsorbing state showing ovaries with early vitellogenic oocytes and extensive α and β atresia of vitellogenic follicles; the third showed gonads with perinucleolar oocytes and was considered to be in a resting state. The low occurrence of non-reproductive individuals found in this study makes it unlikely that non-reproductive individuals aggregate with reproductive ones during their migration towards spawning grounds. Further research is suggested in order to investigate the potential presence of non-reproductive individuals on non-spawning grounds during the reproductive season.     

Gonadal development and diandric protogyny in two populations of Dascyllus reticulatus from Madang, Papua New Guinea

Two Dascyllus reticulatus populations from Madang, Papua New Guinea exhibited diandric protogyny. In both populations, gonads began as undifferentiated, and then developed oocytes in the primary growth stage and an ovarian lumen. From this ovarian state or from more developed ovaries containing oocytes beyond the primary‐growth stage, some gonads developed into testes. The first sign of testicular development was degeneration of oocytes, degeneration of oocytes in the primary growth stage in ovarian gonads and degeneration of oocytes of all growth stages present including the primary growth stage in ovaries, which was then followed by development of spermatogenic tissue. In both populations, most of the fish that had gonads with degenerating oocytes were smaller than the smallest mature females, indicating that development towards testes was mostly initiated in immature gonads containing only pre‐vitellogenic oocytes. On some occasions, however, females as large as other mature females also had gonads with degenerating oocytes, suggesting that development towards testes may have occurred in mature ovaries as well. This latter notion is further strengthened by the discovery of a fish having a gonad that contained both degenerating vitellogenic oocytes and developing spermatogenic tissue. Taken together, these results suggest that D. reticulatus can exhibit diandric protogyny, because testes in D. reticulatus developed from juvenile gonads as well as from mature ovaries.     

The gonads of Aegla platensis Schmitt (Decapoda, Anomura, Aeglidae): a macroscopic and histological perspective

The aim of this study is to characterize the gonads of Aegla platensis, relating aspects of colour and size of ovaries, testes and vasa deferentia (VD) to histological observations. The ovaries are H‐shaped, extending from behind the stomach and ending at the 1st or 2nd abdominal somite. The male has a pair of testes from which the VD issue and extend over the pereon. For males and females, macroscopic categories of gonad development are defined. Stages of the male gonad are defined as types 1, 2 and 3. Type 1 includes the less developed gonads, and type 3 includes extremely developed testes and completely differentiated VD. The ovaries are classified into four stages according to their colour: I (white), II (yellow), III (orange) and IV (red), beginning with the least developed gonads. The number and size of oogonia and oocytes depend on the stage of the ovary and coincide with the different degrees of development as indicated by ovary colour. In males, the presence or absence of spermatozoa in the testes and VD is determined only for the more developed gonads. Neither spermathecae (female) nor spermatophores (male) were found. The lack of spermatophore in the present species is a rare characteristic among the anomurans.     

Selective transport and packaging of the major yolk protein in the sea urchin

The major yolk protein of sea urchins is an iron-binding, transferrin-like molecule that is made in the adult gut. Its final destination though is the developing oocytes that are embedded in somatic accessory cells and encompassed by two epithelial layers of the ovary. In this study, we address the dynamics of yolk transport, endocytosis, and packaging during the vitellogenic phase of oogenesis in the sea urchin by use of fluorescently labeled major yolk protein (MYP). Incorporation of MYP into the accessory cells of the ovary and its packaging into yolk platelets of developing oocytes is visualized in isolated oocytes, ovary explants, and in whole animals. When MYP is introduced into the coelom of adult females, it is first accumulated by the somatic cells of the ovarian capsule and is then transported to the oocytes and packaged into yolk platelets. This phenomenon is specific for MYP and accurately reflects the endogenous MYP packaging. We find that oocytes cultured in isolation are endocytically active and capable of selectively packaging MYP into yolk platelets. Furthermore, oocytes that packaged exogenous MYP are capable of in vitro maturation, fertilization, and early development, enabling an in vivo documentation of MYP utilization and yolk platelet dynamics. These results demonstrate that the endocytic uptake of yolk proteins in sea urchins does not require a signal from their surrounding epithelial cells and can occur autonomous of the ovary. In addition, these results demonstrate that the entire population of yolk platelets is competent to receive new yolk protein input, suggesting that they are all made simultaneously during oogenesis.     

Drought leads to reproductive quiescence in smooth-billed anis: Phenotypic evidence for opportunistic breeding and reproductive readiness

Previous studies have suggested that the smooth-billed ani (Crotophaga ani, Linnaeus, 1758) breeds opportunistically following unpredictable rainfall in drought areas. To obtain proof of this phenomenon, the present study described and compared reproductive morphology and cell proliferation in the gonads of free-living smooth-billed anis during a wet season (April to June 2012) and the following dry season (July to September 2012) in a semiarid area using light and electron microscopy (transmission and scanning) and the AgNOR method. The morphological findings indicated distinct levels of reproductive activity related to seasonal changes. Morphological and morphometric analyses of the gonads confirmed intense gametogenic activity during the wet season, whereas gonadal involution occurred after rainfall ceased. The sizes of the testes and ovaries were significantly reduced compared to those in the wet season. The volumetric fraction of the seminiferous tubules in the testis decreased considerably, and no preovulatory follicles were detected in the ovary in the dry season. Moreover, the AgNOR count in the gonads revealed a significant decline in cell recruitment for gametogenesis after rainfall ceased. The histological findings indicated partial gonadal activation throughout the dry season. The analysis of the seminiferous epithelium confirmed the early testicular recrudescence phase, and sporadic postovulatory follicles indicated random ovulation during this time. The excurrent ducts and the oviduct also underwent remarkable involution in the dry season. Taken together, these findings confirm opportunistic breeding by smooth-billed anis in a semiarid habitat and suggest that gonadal recrudescence has been established as a reproductive strategy to cope with unexpected precipitation events.     

Juvenile bisexuality in the red sea bream,Pagrus major

The histology of the gonad of the red sea bream,Pagrus major, was examined in order to study the early gonadal development, sexual maturation and sex ratio in a natural population. A total of 1,117 fish between the ages of 4 months and 8 years were examined. Gonads of 4-month-old fish were either sexually undifferentiated with a central cavity, or ovarian in form. Gonads of 12- and 18-month-old fish were ovaries or bisexual gonads, while those of 2-year-old fish were ovaries, bisexual gonads or testes. Fish aged between 3 and 8 years had ovaries or testes, except for a few bisexual gonads found in 3- and 4-year-old fish. The chronological appearance of females, hermaphrodites and males in that order, and histological evidence, suggested that the testis originates from the ovary via a bisexual gonad in the juvenile stage. The sex ratio of females to males at the age of 2 years and over was about 1:1, suggesting that hermaphroditic red sea bream appear in about 50% of the juvenile population. The sexual pattern in this species, therefore, is concluded to be gonochorism with a bisexual juvenile stage.     

Morphological development of the gonads in zebrafish

Gonadogenesis of zebrafish Danio rerio was investigated by means of light microscopy to test the suitability of gonad histology as an endpoint in hazard assessment of endocrine‐active compounds. At age 2 weeks post‐fertilization (pf), primordial germ cells were found in a dorsocaudal position in the body cavity. At 4 weeks pf, the majority of the fish (86%) possessed paired gonads with meiotic germ cells; these gonads represented presumptive ovaries. At week 5 pf, 87% of the fish examined had ovaries with perinucleolar oocytes. Further development of the gonads in female zebrafish up to week 11 pf was characterized by an increase in gonad size as well as in the number and size of perinucleolar oocytes. Starting with week 5, some fish showed alterations of gonad morphology, including a decrease in the number and size of the oocytes, an enhanced basophilia and irregular shape of the oocytes, and finally their degeneration into residual bodies. With the decline in oocyte number, stromal cells became more numerous and they infiltrated the gonadal matrix. In several 7 week‐old zebrafish with altered gonadal morphology, enhanced numbers of gonial cells arranged in cyst‐like groups appeared. These gonads were interpreted as presumptive testes. In one fish out of 32 individuals examined, spermatocytes were detected, in addition to the gonial cells. During the subsequent weeks, the percentage of fish showing early testes with spermatogonia, spermatocytes and spermatids increased and reached 40% at 11 weeks pf. The sequence of gonadal alterations taking place in some of the individuals from week 5 pf onwards was interpreted to reflect the transition of protogynic ovaries into testes. The developmental pattern described identifies zebrafish to be a juvenile hermaphrodite. The results of this study are of relevance for the use of gonadal histopathology as endpoint in endocrine disruption testing, particularly in order to avoid false diagnoses of ‘intersex gonads’ in zebrafish.     

Gametogenesis in the cirratulid polychaetes Dodecaceria concharum and D. caulleryi

In Northumberland Dodecaceria concharum coexists in the same habitat as D. caulleryi . Morphologically both species are very similar but reproductively they are not. D. concharum is a parthenogenetic atoke while D. caulleryi has an asexual cycle that ends in an epitokous sexual phase. Histologically the gonad of both species had six cell types and the number of cells in each type were found during gametogenesis. In D. concharum the period over which gametogenesis occurs was followed from the increase in size of free coelomic oocytes and numbers of body segments. In D. caulleryi the period was followed by means of changes in the asexual cycle and metamorphosis into epitokes. In spite of the differences in the methods of reproduction of the two species gametogenesis of each was found to be almost identical. Only oogenesis could be compared since D. concharum does not have males. Prior to spawning in both species the production of oocytes by the ovary appeared to be inhibited. Possible hormonal mechanisms used to control atokous and epitokous spawning are discussed. From the histology of the gonads and oocytes D. concharum appeared to have half the number of chromosomes of D. caulleryi.