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Chapter 4 Endocrine regulation of sea urchin reproduction
Edible Sea Urchins: Biology and Ecology Editor: John Miller Lawrence © 2007 Elsevier Science B.V. All rights reserved.
Chapter 4
Endocrine Regulation of Sea Urchin Reproduction Kristina M Wasson and Stephen A Watts Department of Biology, University of Alabama at Birmingham, Birmingham, AL (USA).
1. SEA URCHIN GONAD As the demands for sea urchin gonads increase and as natural populations decline, a need for sea urchin aquaculture increases (Keesing and Hall 1998; Robinson 2004). To maximize gonad production, it is essential to understand those processes that regulate gonad production. Sea urchin gonads consist of two major cell types: (1) germinal cells, which undergo mitotic proliferation and meiotic reductions to produce large quantities of gametes, and (2) nutritive phagocytes, which undergo cyclic depletion and renewal of macromolecules to provide the nutrients and energy required by the developing gametes (Chapter 2). In this chapter, we consider the putative chemical messengers and their potential regulatory mechanisms in the control of reproduction.
2. EXOGENOUS REGULATION OF REPRODUCTION 2.1. Environmental Factors Sea urchin reproduction is characterized by specific cycles of gametogenesis and nutritive phagocyte growth and depletion (Chapter 2). Correlative data indicate that seasonal changes in environmental factors, such as photoperiod, temperature, and salinity, influence reproduction within a population (reviewed by Pearse and Cameron 1991). Nutritional condition also affects gametogenesis (Bishop and Watts 1994). The environmental and nutritional effects on the physiology and morphology of the gonads suggest that these exogenous cues are transduced to endogenous factors that regulate reproduction in the adult sea urchin. 2.2. Endocrine Disruptors Endocrine disruptors are a diverse group of naturally occurring and synthetic compounds that mimic or antagonize the effects of endogenous chemical messengers. Naturally occurring endocrine disruptors include phytoestrogens as well as sex steroids, such as
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estrogens and androgens. Synthetic compounds include industrial chemicals, herbicides, and pesticides. Endocrine disruptors typically enter marine environments via effluents from sewage treatment plants, and from industry and agricultural runoff (Langston et al. 2005). Few studies have investigated the effects of potential endocrine disruptors on sea urchin reproduction. Lipina et al. (1987) reported an increase in the number of oogonia within the ovaries of Strongylocentrotus intermedius after a short exposure (15 days) to cadmium chloride; however, longer exposure (for up to 130 days) appeared to have toxic effects, including decreased number of germ cells, decelerated germ cell growth, and destroyed gametes and nutritive phagocytes. Khristoforova et al. (1984) reported similar effects on the gonads of S. intermedius due to cadmium sulfate. Böttger and McClintock (2002) found that chronic exposure of Lytechinus variegatus to either inorganic phosphates or organic phosphates inhibited gonadal growth and spawning activities as well as altered the biochemical composition of the gonads. Whether these reported toxicities directly or indirectly affected endocrine function is not known.
3. ENDOGENOUS REGULATION OF REPRODUCTION Although the endocrine and/or paracrine mechanisms that regulate physiological processes in sea urchins are poorly understood, several classes of putative chemical messengers have been identified. These include sex steroids (Fig. 1), proteins, peptidergic factors, and catecholaminergic and cholinergic factors (Fig. 2). The sex steroids, which are most likely synthesized from cholesterol, consist of progesterone (P4), testosterone (T), and estradiol-17 (E2). Peptidergic factors are small peptides that contain no secondary structure. Catecholaminergic or monaminergic factors that include noradrenaline and dopamine are amino acid derived trophic factors. The putative cholinergic factor in sea urchins is acetylcholine. Examination of these chemical messengers in the context of gonad production, gametogenesis, and nutrient translocation allows the generation of new hypotheses for research into the manipulation of gonad production and regulation of gametogenesis. 3.1. Steroids 3.1.1. Sex steroids in the gonads Mammalian models of reproduction demonstrate that both sex steroids and peptide hormones regulate gametogenesis. Early studies looked for the presence of sex steroids in sea urchin ovaries. Donahue and Jennings (1937) found that an oil-soluble substance extracted from the ovaries of L. variegatus induced vaginal growth in ovariectomized rats. They concluded that this substance was slightly estrogenic in nature, requiring over 4 g of sea urchin ovaries to produce the equivalent of 1 rat unit of estrogenic activity. Donahue (1940) also discovered that both L. variegatus and Echinometra sp. contained oil-soluble substances that induced estrogenic responses in rat vagina. Twenty years later, Botticelli et al. (1961) demonstrated that ovary extracts from S. franciscanus had E2 activity and estimated the E2 concentration to be approximately 100 pg g−1 of ovaries. Botticelli et al.
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Fig. 1. Pathway of sex steroid synthesis from cholesterol.
Fig. 2. Putative (A) catecholaminergic factors and (B) cholinergic factors in echinoids.
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(1961) also tentatively identified P4 in these ovaries. These early studies indicate that P4 and E2 are present in sea urchins but did not indicate their origin or biological functions. 3.1.2. Steroid converting enzymes in the gonads De novo synthesis provides the consistency required for the long-term regulation of physiological processes by steroids and requires the activity of steroid-converting enzymes. Colombo and Belvedere (1976) used radiotracers to demonstrate that the gonads of Paracentrotus lividus convert androstenedione (AD), the mammalian precursor to T and estrogens, into T, indicating the activity of 17-hydroxysteroid dehydrogenase (HSD) (required for the reduction of C17 to an alcohol group) in gonadal tissues (Fig. 3). 17-HSD activity has been localized in the ovaries and testes of sea urchins. Varaksina and Varaksin (1991) used histochemistry to demonstrate that auxiliary elements (presumed to be nutritive phagocytes), developing oocytes, and spermatids contain 17-HSD activity. They suggested that this enzyme is probably necessary for gonad function. Watts et al. (1994) used in vitro radiotracers to show that both the ovaries and the testes of L. variegatus synthesize T and the 5-reduced androgens, including 5-androstanedione (5-AO), 5-androstane-3, 17-diol, and 5-androstane-3, 17-diol (hereafter referred to as 5-adiols), from AD. The presence of these steroids indicates the activity of 17-HSD, 5-reductase (required for the reduction of the 4−5 double bond) and 3/-hydroxysteroid dehydrogenase (required for the reduction of C3 keto groups to an alcohol). Interestingly, only the testes synthesize 11-oxygenated androgens, suggesting that sex-specific synthesis of androgens exists in L. variegatus. Manipulation of the nutritional status of L. variegatus influences androgen metabolism in a sex-specific manner. Wasson et al. (1998) starved L. variegatus, collected from the field during the gonadal growth stage, for 48 days to inhibit gonadal growth and maturation (Bishop and Watts 1994). Total rates of AD conversion for both the ovaries and testes increased significantly with the onset of feeding. The ovaries and testes converted AD into T, 5-adiols, 5-AO, androsterone (AN), and epiandrosterone (EPIAN); however, the relative quantities of T and the 5-adiols differed significantly between females and males. On day 4 of feeding, ovarian T synthesis was not detectable but the synthesis of the 5-adiols increased threefold. In contrast, testicular synthesis of T and 5-adiols was maximal at this time. The sex-specific shift in the steroid metabolic pathways may be associated with the initiation of biosynthetic processes associated with oogenesis and spermatogenesis (Wasson et al. 1998). Periodic measurement of androgen metabolism during the annual reproductive cycle of L. variegatus also suggests a relation between steroid converting enzyme activity and reproduction (Wasson et al. 2000a). As found by Wasson et al. (1998), both ovaries and testes always synthesized T, 5-adiols, 5-AO, EPIAN, and AN. The ovaries and testes showed maximal accumulation of 5-adiols in April, when gonad indices started to decline, indicating initiation of the spawning season. Furthermore, the ovaries generally synthesized larger quantities of 5-adiols compared to T on all sampling days. Based on this sex-specific pattern of 5-adiols accumulation, Wasson et al. (2000a) suggested that the 5-adiols influence reproduction in L. variegatus. Progesterone is readily metabolized by the gonads, primarily to a variety of 5-reduced progestins (Fig. 4). Both the ovaries and testes of L. variegatus synthesized
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Fig. 3. Pathway of androstenedione metabolism in Lytechinus variegatus. Solid lines represent tentatively identified pathways. Dashed lines represent suggestively identified pathways. Diamond-shaped arrowheads represent pathways not identified in echinoids. 5-R = 5-reductase, 17-HSD = 17-hydroxysteroid dehydrogenase, 3/-HSD = 3/-hydroxysteroid dehydrogenase.
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Fig. 4. Pathway of progesterone metabolism in Lytechninus variegatus. Solid arrows represent tentatively identified pathways. Dashed arrows represent suggestively identified pathways. 5-R = 5-reductase, 3-HSD = 3-hydroxysteroid dehydrogenase, 3-HSD = 3-hydroxysteroid dehydrogenase, 20-HYD = 20-hydroxylase, 20-HYD = 20-hydroxylase.
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5-pregnane-3, 20-dione, 3-hydroxy-5-pregnan-20-one, 5-pregnane-3, 20-diol, 5-pregane-3, 20-diol, and 5-pregnane-3, 20-diol. Interestingly, the synthesis of androgens from progesterone and sex-specific differences in progesterone metabolism were not observed in the gonads (Wasson and Watts 2000). Since the majority of steroid metabolites synthesized from the gonads of L. variegatus are 5-reduced steroids, Wasson and Watts (1998) suggested that 5-reductase is an important enzyme in the steroidogenic pathway of sea urchin gonads. The specific 5-reductase inhibitor, finasteride (F; Proscar© ), inhibited 5-reductase activity in vitro in the gonads. Additionally, the gonads of sea urchins fed for 16 days on a formulated diet supplemented with F demonstrated in vivo inhibition of 5-reductase activity. Specific chemical ablation by F of 5-reductase could be used as tool to investigate the role of 5-reduced steroids in the gonads of sea urchins (Wasson and Watts 1998). It is interesting that the synthesis of estrogens was not observed in any of the studies examining androgen metabolism in sea urchins (Colombo and Belvedere 1976; Watts et al. 1994; Wasson et al. 1998, 2000a). However, Hines et al. (1994) showed that testes of L. variegatus converted E2 into an E2 ester, estrone, and aqueous soluble metabolites. Additionally, estrone was converted primarily into E2, and to a lesser extent E2 ester and aqueous soluble metabolites. These data indicate that 17-HSD is active in the interconversion of E2 and estrone and those estrogens are converted rapidly into aqueous soluble metabolites. Hines et al. (1994) suggested that radiotracer studies might not provide the sensitivity necessary to detect the rapidly metabolized estrogens. 3.1.3. Sex steroids in the gonads Wasson et al. (2000a) detected immunoreactive E2 in the gonads of L. variegatus using a commercial double antibody E2 radioimmunoassay (RIA). Measurement of T and E2 concentrations by RIA in the gonads during one reproductive season demonstrated no significant correlation between reproductive state (as determined by gonad index) and season, reportedly due to high levels of individual variations in steroid concentrations. These variations in steroid concentrations were presumably related to individual variations in specific stages of the gonads (Wasson et al. 2000a). Interestingly, comparison of steroid concentrations indicated significant differences with gonad size, suggesting a relation between gonad production and steroid levels. In both the ovaries and the testes, individuals with low gonad indices (0–3%) had significantly higher T and E2 concentrations than those with higher gonad indices (>61%). Whether or not these steroids are involved in the stimulation or maintenance of mitotic and meiotic events in gametes has not been established. The levels of 5-adiols, one of the major steroid metabolites in sea urchin gonads, are not known. Since the synthesis of the 5-adiols varied with gonad size (Wasson et al. 2000a), perhaps the 5-adiols may be more appropriate steroids to measure during the reproductive cycle. 3.1.4. Response to exogenous administration of sex steroids Varaksina and Varaksin (1987) injected E2 diproprionate into the coelomic fluid of S. nudus in November when the ovaries were beginning to grow. Individuals injected with E2 diproprionate produced a higher ovary index and a significantly larger number
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of mature oocytes than the control treatment. Similar results were obtained when individuals were injected with E2 diproprionate in April (when gonads were maturing). They suggested that E2 diproprionate induced ovarian growth via stimulating RNA and protein synthesis, enhancing vitellogenesis and leading to oocyte maturation. Wasson et al. (2000b) examined the effects of dietary administration of steroids during the gonadal growth stage. Sex- and steroid-specific effects occurred in the gonads of L. variegatus that were fed either E2, E2/ P4, P4, P4/F, T, or T/F for 36 days (Wasson et al. 2000b). Individuals fed E2 had ovary indices 54% larger than individuals fed control diets. Sea urchins fed E2, E2/P4, P4, or P4/F had significantly smaller oocytes than echinoids fed control diets. In addition, individuals fed E2/P4 or P4 alone had a significantly larger volume of the ascini occupied by nutritive phagocytes than control individuals. Since no ovarian growth occurred in individuals fed E2/P4, P4, or P4/F, they concluded that the daily dose of P4 administered was sufficient to inhibit any observable E2 stimulus of ovarian growth. These data suggest further that 5-reduced progestins may stimulate nutrient accumulation in nutritive phagocytes. Although individuals fed T had significantly smaller oocytes than individuals fed T/F, both treatments had significantly larger oocytes than control individuals. Finally, individuals fed either E2 or T had greater amounts of protein in the ovaries than individuals fed T/F, indicating that E2 and 5-reduced androgens influence protein accumulation in ovaries. Wasson et al. (2000b) concluded that E2 stimulates ovarian growth but does not stimulate oocyte growth and that E2 and 5-reduced progestin metabolites may promote nutrient allocation to nutritive phagocytes. Furthermore, T does not affect ovary growth but, upon removal of 5-reduced androgens (by feeding F), T does promote significant increases in oocyte diameters (Wasson et al. 2000b). The testes of L. variegatus fed P4 exhibited the most pronounced effects (Wasson et al. 2000b). Testis indices were 56% larger in individuals fed P4 than controls; however, no effects on testis growth were detected in individuals fed P4/F, T, or T/F, suggesting that 5-reduced progestins stimulated testicular growth. Individuals fed P4 also had a significantly larger volume of the ascini occupied by nutritive phagocytes and significantly smaller volumes occupied by spermatogenic columns. Furthermore, individuals fed E2/P4, P4/F, or T/F had greater amounts of carbohydrates in their gonads than control individuals, and individuals fed E2/P4, T, or T/F had significantly greater amounts of lipids in their testes than control individuals. Wasson et al. (2000b) concluded that 5-reduced progestins may regulate nutrient allocation into nutritive phagocytes and may also influence spermatogenic column formation. Corroboratively, low concentrations of P4 were measured during the early stages of spermatogenesis (as indicated by low testis indices) (Wasson et al. 2000a). Two studies have examined the effect of sex steroids in juvenile sea urchins. Unuma et al. (1996) demonstrated that juvenile Pseudocentrotus depressus fed estrone for 29 days had significantly higher body weights, food intake, and feed efficiencies than control individuals. Feeding of T or E2 had no effect on growth or food utilization of juveniles (Unuma et al. 1996), but juveniles fed AD or estrone for 30 days had significantly higher testis indices than control individuals (Unuma et al. 1999). In addition, estrone promoted spermatogenesis, suggesting that estrone may be important for the initiation of spermatogenesis (Unuma et al. 1999). The ovaries were not affected in the juveniles and
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Unuma et al. (1999) concluded the lack of an ovarian response to steroids was due to the lack of sexual maturation of the females. The role of sterols or steroids obtained from natural dietary sources has not been examined. It is unlikely that predictable levels of steroids could be extracted from the diet to support long-term control over reproductive processes. Anecdotal information suggests that soy, a natural phytoestrogen, inhibits gametogenesis in S. purpuratus (J Pearse, pers. comm.), suggesting further that sea urchins can respond to dietary endrocrine disruption, which may provide a potential, noninvasive mechanism to regulate gonad physiology and affect gonad production.
3.2. Protein and Peptidergic Factors Sea urchin coelomocytes synthesize the precursor to a major yolk glycoprotein (MYP; Chapter 2). Recognizing that estrogens regulate yolk production in vertebrates, Harrington and Ozaki (1986a) examined the response of sea urchin coelomocytes to E2 stimulation. Although they did not observe an effect on the synthesis of the MYP precursor, they did demonstrate the synthesis of a novel protein. This protein varied in size from 78 to 82 kDa depending on species. No other effects on the synthesis of total proteins were observed. This novel protein produced by the coelomocytes may act as a transcription factor to induce transcription of the MYP precursor gene (Harrington and Ozaki 1986b). No other studies have examined the role of other proteins or peptidergic factors on gametogenesis in sea urchins. Cochran and Engelmann (1972) isolated a spawn-inducing factor from radial nerve extracts of S. purpuratus. The supernatant of radial nerves boiled in seawater contained a heat-stable factor that induced spawning by testis fragments. The concentration of radial nerve factor paralleled the reproductive season of S. purpuratus (Cochran and Engelmann 1972). Digestion of radial nerve factor with pronase and pepsin destroyed all spawn-inducing activity, indicating that this factor is a peptide. The estimated molecular weight of the radial nerve factor is 5600 Da. This peptide was not glycosylated, but had a persistent yellowish-brown color that was associated with the spawn-inducing activity (Cochran and Engelmann 1976). The radial nerve factor induced the production of a gonad factor, which was not digestible with pronase. This gonad factor also induced spawning in testis fragments of S. purpuratus. Although this gonad factor co-migrated with 1-methyladenine in thin layer chromatography (Cochran and Engelmann 1976), this substance was not identified definitively. In starfish, 1-methyladenine was identified as the oocyte maturation inducing substance (reviewed by Kanatani 1979). Since oocyte production is continuous and asynchronous throughout the breeding season in S. purpuratus (Chatlynne 1969), Cochran and Engelmann (1976) suggested that the significant levels of this gonad factor must be chronically maintained to induce spontaneous oocyte maturation; however, no seasonal measurement of this gonad factor has been reported. This was the first demonstration of an “endocrine system” in sea urchins. A neuropeptide produced in radial nerves stimulated the release of a factor by the gonads that induce
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spawning. The mechanism of action and mode of transfer of this radial nerve factor is unknown. In starfish, radial nerve factor is released into the coelomic cavity and transported via coelomic fluid to the gonads (Cochran and Engelmann 1976); however, such mode of transfer in sea urchins would not account for the rapid (within 1 min) spawning response to radial nerve factor. In addition, the cell type that releases the gonad factor is not known. It is interesting to speculate that nutritive phagocytes would synthesize and store this gonad factor and, upon stimulation by the radial nerve factor, release it. The gonad factor would then stimulate the release of neighboring gametes or stimulate the contraction of muscular epithelium, forcibly releasing gametes from the gonad lumen. Most likely, the gonad factor is synthesized and stored within the neurons or epithelial cells of the peritoneum and, upon stimulation by the radial nerve factor, released to stimulate contraction of the muscular epithelium. Although Elphick et al. (1992) identified a class of putative neuropeptides, they suggested no physiological function for them. Using antisera raised against the asteroid SALMFamide, they isolated five immunoreactive peptides and putatively identified an amidated nonapeptide, FPVGRVHRFamide. SALMFamide immunoreactivity had widespread distribution throughout the gut of Echinus esculentus. Specifically, the pharynx–esophagus portion of the gut displayed extensive immunoreactivity associated with the basi-epithelial nerve plexus, which runs between secretory epithelium and the underlying layers of connective tissue and muscle. Although SALMFamide immunoreactivity was also found in association with the outer sub-coelomic epithelial nerve plexus (which is found between the outer connective tissue layer and coelomic epithelium), no immunoreactive epithelial endocrine or neuroendocrine cells were found (Elphick et al. 1992).
3.3. Catecholaminergic and cholinergic factors Sea urchin gonads are innervated by monaminergic, cholinergic, and peptidergic nerve fibers but do not contain adrenergic fibers (Cobb 1969). Both the ectoneural and endoneural systems contain the catecholamines noradrenalin and dopamine (Khotimchenko 1983). Thin nerve fibers interlace the ascini of the gonads and may contain biogenic amines (Khotimchenko and Deridovich 1991). Both noradrenalin and dopamine inhibited temperature-stimulated oogenesis in S. nudus by suppressing growth and maturation of oocytes (Khotimchenko 1983). Interestingly, the levels of these catecholamines varied with reproductive state. Concentrations were low during periods of minimal gametogenic activity and also before spawning. The initiation of spawning was accompanied by increases in catecholamine concentrations (reviewed by Khotimchenko and Deridovich 1991). Since noradrenaline and dopamine decreased the intensity of uptake of labeled uridine and leucine in large oocytes, Khotimchenko (1983) concluded that the suppression of oocyte growth was due to inhibition of RNA and protein synthesis. They hypothesized that these catecholamines influenced oogenesis via transport through the coelomic fluid. In contrast, acetylcholine was secreted in the regions of neuromuscular contacts of the gonads and was effective at inducing the reduction of muscle elements of ascini,
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suggesting that the act of gamete release may be regulated by cholinergic mechanisms (Khotimchenko 1983).
4. MECHANISMS OF REGULATION 4.1. Paracrine Analysis of paracrine mechanisms, in which a chemical messenger induces a response in a cell located in close proximity to the cell that synthesizes it, requires an understanding of gonad morphology. Although nutritive phagocytes lack direct physical contact with developing gametes, thin extensions of nutritive phagocytes surround developing gametes. In male sea urchins, the role of these cells has been equated to that of Sertoli cells in mammalian testes (Pearse and Cameron 1991), providing the necessary local environment to stimulate gamete proliferation and growth. The demonstration of endogenous steroid converting enzymes in nutritive phagocytes (Varaksina and Varaksin 1991) and the E2 and P4 effects on mature gonad growth and gametogenesis (Wasson et al., 2000b) suggest that steroids may be produced by the nutritive phagocytes and influence gametogenesis or nutrient translocation to developing gametes. Histochemical studies also indicate that developing oocytes and spermatids possessed 17-HSD activity (Varaksina and Varaksin 1991), suggesting that developing gametes also synthesize steroids that may act in paracrine or autocrine mechanisms. The secretion of steroids from either nutritive phagocytes or developing gametes would most likely occur by mechanisms similar to those in vertebrates. The promotion of gonad growth, gametogenic activity, and nutrient storage by sex steroids and/or their derivatives (Wasson et al. 2000b) suggests that sex steroid receptors are present in echinoid gonads. The presence of steroid receptors in either nutritive phagocytes or germinal cells has not been investigated in any echinoid. Shyu et al. (1987), however, identified an estrogen-responsive element upstream from the vitellogenin gene in S. purpuratus, suggesting that the vitellogenin synthesis may be regulated by estrogen. Direct contacts (tight junctions) are found between nutritive phagocytes (Pearse and Cameron 1991). Similar associations have been reported for mammalian follicle cells, which use these junctions to coordinate cellular communication throughout large numbers of follicle cells. Via these tight junctions, interconnected nutritive phagocytes may communicate the necessary factors to stimulate nutrient translocation to the developing gametes. In vertebrates, the peptides insulin and glucagon regulate nutrient deposition in storage cells, mobilization of nutrients, and translocation to appropriate sites for utilization. It would be of interest to determine whether similar proteins exist in sea urchins and have a role in paracrine regulation of the nutrient translocation activities of nutritive phagocytes. It is likely that protein or peptidergic factors act by paracrine mechanisms to regulate gonad growth. Mammalian models indicate that a variety of proteins, such as TGF- (Teerds and Dorrington 1995), inhibin (Hillier and Miro 1993), kit ligand (Joyce et al. 1999), and GDF-9 (Dong et al. 1996), produced by oocytes or follicle cells induce specific
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cellular responses in follicle cells or oocytes, respectively. Future research on similar proteins and peptidergic factors in sea urchin gonads will be of considerable interest. 4.2. Endocrine Cochran and Engelmann (1972, 1976) identified a radial nerve factor and a gonad factor that potentially represent a true endocrine system. If this gonad factor induces oocyte maturation (Kanatani 1974), then this system would be chronically stimulated since mature ova are present in the ovarian lumen prior to spawning (Pearse and Cameron 1991). It may be more likely that this gonad factor simply induces a rapid muscular response in the gonads to release gametes. No other studies have examined endocrine mechanisms of regulation in sea urchin reproduction. Several levels of endocrine regulation become apparent when examining sea urchin reproduction. First, the general synchrony of spawning among individuals within a population indicates that a factor (pheromone?) released into the water by an individual induces spawning in other individuals. The nature and identity of this factor is still unknown. Second, the dramatic influence of the environment on gametogenesis and nutrient storage indicates the existence of a communication system between the environment and the organs involved in reproduction. This should not be surprising since environmental factors have been shown to influence the hypothalamo-hypophyseal-gonadal axis in mammals and other vertebrates. Finally, coordination of gametogenic and nutrient translocation activities among and within each gonad indicates the existence of a physiologically controlled mechanism that coordinates these activities. Chemical messengers, whether of neuronal or non-neuronal origins, would most likely accomplish such communication. Movement of these chemical messengers could be accomplished by using the perivisceral coelom and direct diffusion across the gonad wall or by using the hemal system for direct transport to the gonads. Since the hemal system interconnects with each gonad via the aboral ring complex, this system may provide a more efficient and specific means of control.
5. GENE REGULATION IN REPRODUCTION Identification of the genes involved in reproduction of adult sea urchins is a vastly untapped area of research. Most of the research involved in sea urchin genomics has dealt primarily with developmental biology and embryogenesis. Recently, genome sequencing of S. purpuratus was initiated (http://www.hgsc.bcm.tmc.edu/projects/seaurchin/). A search of the NBCI Entrez gene database (http://www.ncbi.nlm.nih.gov/mapview/ map_search.cgi?taxid=7668) for “steroid” and “receptor” revealed only six annotated genes in S. purpuratus that resemble steroid hormone receptors. As of September 2005 none of these genes have been ascribed functions. Interestingly, one gene resembles the estrogen-related receptor- and another resembles the progesterone receptor membrane component 1. Molecular endocrinology will be a fruitful area of sea urchin research in the future, from both a functional and evolutionary standpoint.
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6. CONCLUSIONS Environmental cues influence seasonal changes in gonad morphology and physiology. Knowledge of the physiological mechanisms that stimulate growth and maintenance of nutritive phagocytes and gametes is essential to understand the reproductive biology and ecology of sea urchins in the field. This information is also essential for the development of technologies that will optimize the texture, quality, and quantity of gonads from sea urchins in aquaculture. Diet plays a vital role in stimulation and maintenance of gonad production and is one parameter that is easily manipulated in aquaculture. We have found that formulated feeds supplemented with steroids affect gametogenesis and nutrient allocation to nutritive phagocytes. In the future, phytosteroids may be used to influence gonad growth. For example, Pearse (pers. comm.) attributed the increase in nutritive cells relative to gametes in the gonad to the presence of soy meal in individuals fed a formulated feed. Soy-based diets promote estrogen-dependent as well as estrogen-independent processes (Barnes 1998), and may provide a natural method to adjust a physiological state. Further research is required to fully understand how the dietary administration of these and other specific micronutrients may stimulate or inhibit gonad growth. Sex steroids, proteins, catecholaminergic, and cholingeric factors all play roles in regulating various aspects of gonad function. No data, however, exist on the chemical messengers required to promote nutrient translocation from the gut to the gonads, the mobilization of nutrients within the nutritive phagocytes, or translocation of nutrients to developing gametes. Understanding the physiological control of these mechanisms will assist in the development of formulated feeds to specifically regulate gonad quality and quantity. Most of these studies have examined singular reproductive events (i.e. spawning, gonad growth, etc.) in only a few species of sea urchins. Large gaps are still present in our general understanding of the physiological mechanisms that control reproduction and provide direct links between environmental stimuli and gonad function. Studies using molecular biology, genomics, and bioinformatic tools are essential for elucidating the cellular pathways that regulate specific reproductive processes. Such tools will be extremely beneficial to our understanding of the role of chemical messengers and the influence of endocrine disruptors in sea urchin reproduction.
ACKNOWLEDGMENTS We thank IJ Blader and JM Lawrence for comments on this chapter. Preparation of this review was supported in part by the Mississippi-Alabama Sea Grant Consortium.
REFERENCES Barnes S (1998) Evolution of the health benefits of soy isoflavones. Proc Soc Exp Biol Med 217: 386–392 Bishop CD, Watts SA (1994) Two-stage recovery of gametogenic activity following starvation in Lytechinus variegatus Lamarck (Echinodermata: Echinoidea). J Exp Mar Biol Ecol 177: 27–36
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Böttger SA, McClintock JB (2002) Effects of inorganic and organic phosphate exposure on aspects of reproduction in the common sea urchin Lytechinus variegatus (Echinodermata: Echnoidea). J Exp Zool 292: 660–671 Botticelli CR, Hisaw FL, Wotiz HH (1961) Estrogens and progesterone in the sea urchin (Strongylocentrotus franciscanus) and Pecten (Pecten hericius). Proc Soc Exp Biol 106: 887–889 Chatlynne LG (1969) A histochemical study of oogenesis in the sea urchin Strongylocentrotus purpuratus. Biol Bull 136: 167–184 Cobb JLS (1969) The distribution of monamines in the nervous system of echinoderms. Comp Biochem Physiol 28: 967–971 Cochran RC, Engelmann F (1972) Echinoid spawning induced by a radial nerve factor. Science 178: 423–424 Cochran RC, Engelmann F (1976) Characterization of spawn-inducing factors in the sea urchin, Strongylocentrotus purpuratus. Gen Comp Endocrin 30: 189–197 Colombo L, Belvedere P (1976) Gonadal steroidogenesis in echinoderms. Gen Comp Endocrinol 29: 255–256 Donahue JK (1940) Occurrence of estrogens in the ovaries of certain marine invertebrates. Endocrinology 27: 149–152 Donahue JK, Jennings DE (1937) The occurrence of estrogenic substances in the ovaries of echinoderm. Endocrinology 21: 587–877 Dong J, Albertini DF, Nishimori K, Kuman TR, Lu N, Matzuk MM (1996) Growth differentiation factor-9 is required during early folliculogenesis. Nature 383: 531–535 Elphick MR, Parker K, Thorndyke MC (1992) Neuropeptides in sea urchins. Regul Peptides 39: 265 Harrington FE, Ozaki H (1986a) The effect of estrogen on protein synthesis in echinoid coelomocytes. Comp Biochem Physiol 84B: 417–421 Harrington FE, Ozaki H (1986b) The major yolk glycoprotein precursor in echinoids is secreted by coelomocytes into the coelomic plasma. Cell Different 19: 51–57 Hillier SG, Miro F (1993) Inhibin, activin, and follistatin. Potential roles in ovarian physiology. Ann NY Acad Sci 687: 29–38 Hines GA, Watts SA, McClintock JB (1994) Biosynthesis of estrogen derivatives in the echinoid Lytechinus variegatus Lamarck. In: David B, Guille A, Féral J-P, Roux M (eds) Echinoderms through time. Balkema, Rotterdam, pp 711–716 Joyce IM, Pendola FL, Wigglesworth K, Eppig JJ (1999) Oocyte regulation of kit ligand expression in mouse ovarian follicles. Dev Biol 214: 342–353 Kanatani H (1974) Presence of 1-methyladenine in the sea urchin gonad and its relation to oocyte maturation. Dev Growth Diff 16: 157–170 Kanatani H (1979) Hormones in echinoderms. In: Barrington EJW (ed.) Hormones and evolution. Vol. 1. Academic Press, New York, pp 273–307 Keesing JK, Hall KC (1998) Review of harvests and status of world sea urchin fisheries points to opportunities for aquaculture. J Shellfish Res 17: 1597–1604 Khotimchenko YS (1983) Effect of adrenotropic and cholinotropic preparations on growth, maturation, and spawning of the sea urchin Strongylocentrotus nudus. Biol Morya 2: 57–63 Khotimchenko YS, Deridovich II (1991) Monoaminergic and cholinergic mechanisms of reproduction control in marine bivalve molluscs and echinoderms: a review. Comp Biochem Physiol 100C: 311–317 Khristoforava NK, Gnezdilova SM, Vlasova GA (1984) Effect of cadmium on gametogensis and offspring of the sea urchin Strongylocentrotus intermedius. Mar Ecol Prog Ser 17: 9–14 Langston WJ, Burt GR, Chesman BS, Vane CH (2005) Partitioning, bioavailability and effects of oestrogens and xeno-oestrogens in the aquatic environment. J Mar Biol Ass UK 85: 1–31 Lipina IG, Evtushenko ZS, Gnezdilova SM (1987) Morpho-functional changes in the ovaries of the sea urchin Strongylocentrotus intermedius after exposure to cadmium. Ontogenez 18: 269–276 Pearse JS, Cameron RA (1991) Echinodermata: Echinoidea. In: Giese AC, Pearse JS, Pearse VB (eds) Reproduction of marine invertebrates. Vol. VI. Boxwood Press, Pacific Grove, pp 514–664 Robinson SM (2004) The evolving role of aquaculture in the global production of sea urchins. In: Lawrence J, Guzmán O (eds) Sea urchins: fisheries and ecology. DEStech Publications Inc., Lancaster, pp 343–357 Shyu AB, Blumenthal T, Raff RA (1987) A single gene encoding vitellogenin in the sea urchin Strongylocentrotus purpuratus: sequence at the 5 end. Nucleic Acid Res 15: 10405–10417
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Teerds KJ, Dorrington JH (1995) Immunolocalization of transformation growth factor-alpha and luteinizing hormone receptor in healthy and atretic follicles of the adult rat ovary. Biol Reprod 52: 500–508 Unuma T, Yamamoto T, Akiyama T (1996) Effects of oral administration of steroids on the growth of juvenile sea urchin Pseudocentrotus depressus. Suisanzoshoku 44: 79–83 Unuma T, Yamamoto T, Akiyama T (1999) Effects of steroids on gonadal growth and gametogenesis in the juvenile red sea urchins Pseudocentrotus depressus. Biol Bull 196: 199–204 Varaksina GS, Varaksin AA (1987) Effects of estradiol diproprionate on oogenesis of the sea urchin Strongylocentrotus nudus. Biol Morya 4: 47–52 Varaksina GS, Varaksin AA (1991) Localization of steroid dehydrogenase in testes and ovaries of sea urchins. Biol Morya 2: 77–82 Wasson KM, Gower BA, Hines GA, Watts SA (2000a) Levels of progesterone, testosterone, and estradiol and the androstenedione metabolism in the gonads of Lytechinus variegaus (Echinodermata: Echinoidea). Comp Biochem Physiol 126C: 153–165 Wasson KM, Gower BA, Watts SA (2000b) Responses of ovaries and testes of Lytechinus variegatus (Echinodermata: Echinoidea) to dietary administration of estradiol, progesterone and testosterone. Mar Biol 137: 245–255 Wasson KM, Hines GA, Watts SA (1998) Synthesis of testosterone and 5-androstanediols during nutritionally stimulated gonadal growth in Lytechinus variegatus Lamarck (Echinodermata: Echinoidea). Gen Comp Endocrinol 111: 197–206 Wasson KM, Watts SA (1998) Proscar® inhibits 5-reductase activity in the ovaries and testes of Lytechinus variegatus Lamarck (Echinodermata: Echinoidea). Comp Biochem Physiol 120C: 425–431 Wasson KM, Watts SA (2000) Progesterone metabolism in the ovaries and testes of the echinoid Lytechinus variegatus Lamarck (Echinodermata). Comp Biochem Physiol 127C: 263–272 Watts SA, Hines GA, Byrum CA, McClintock JB, Marion KR (1994) Tissue- and species-specific variations in androgen metabolism. In: David B, Guille A, Féral J-P, Roux M (eds) Echinoderms through time. Balkema, Rotterdam, pp 155–164
Chapter 4
Endocrine Regulation of Sea Urchin Reproduction Kristina M Wasson and Stephen A Watts Department of Biology, University of Alabama at Birmingham, Birmingham, AL (USA).
1. SEA URCHIN GONAD As the demands for sea urchin gonads increase and as natural populations decline, a need for sea urchin aquaculture increases (Keesing and Hall 1998; Robinson 2004). To maximize gonad production, it is essential to understand those processes that regulate gonad production. Sea urchin gonads consist of two major cell types: (1) germinal cells, which undergo mitotic proliferation and meiotic reductions to produce large quantities of gametes, and (2) nutritive phagocytes, which undergo cyclic depletion and renewal of macromolecules to provide the nutrients and energy required by the developing gametes (Chapter 2). In this chapter, we consider the putative chemical messengers and their potential regulatory mechanisms in the control of reproduction.
2. EXOGENOUS REGULATION OF REPRODUCTION 2.1. Environmental Factors Sea urchin reproduction is characterized by specific cycles of gametogenesis and nutritive phagocyte growth and depletion (Chapter 2). Correlative data indicate that seasonal changes in environmental factors, such as photoperiod, temperature, and salinity, influence reproduction within a population (reviewed by Pearse and Cameron 1991). Nutritional condition also affects gametogenesis (Bishop and Watts 1994). The environmental and nutritional effects on the physiology and morphology of the gonads suggest that these exogenous cues are transduced to endogenous factors that regulate reproduction in the adult sea urchin. 2.2. Endocrine Disruptors Endocrine disruptors are a diverse group of naturally occurring and synthetic compounds that mimic or antagonize the effects of endogenous chemical messengers. Naturally occurring endocrine disruptors include phytoestrogens as well as sex steroids, such as
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estrogens and androgens. Synthetic compounds include industrial chemicals, herbicides, and pesticides. Endocrine disruptors typically enter marine environments via effluents from sewage treatment plants, and from industry and agricultural runoff (Langston et al. 2005). Few studies have investigated the effects of potential endocrine disruptors on sea urchin reproduction. Lipina et al. (1987) reported an increase in the number of oogonia within the ovaries of Strongylocentrotus intermedius after a short exposure (15 days) to cadmium chloride; however, longer exposure (for up to 130 days) appeared to have toxic effects, including decreased number of germ cells, decelerated germ cell growth, and destroyed gametes and nutritive phagocytes. Khristoforova et al. (1984) reported similar effects on the gonads of S. intermedius due to cadmium sulfate. Böttger and McClintock (2002) found that chronic exposure of Lytechinus variegatus to either inorganic phosphates or organic phosphates inhibited gonadal growth and spawning activities as well as altered the biochemical composition of the gonads. Whether these reported toxicities directly or indirectly affected endocrine function is not known.
3. ENDOGENOUS REGULATION OF REPRODUCTION Although the endocrine and/or paracrine mechanisms that regulate physiological processes in sea urchins are poorly understood, several classes of putative chemical messengers have been identified. These include sex steroids (Fig. 1), proteins, peptidergic factors, and catecholaminergic and cholinergic factors (Fig. 2). The sex steroids, which are most likely synthesized from cholesterol, consist of progesterone (P4), testosterone (T), and estradiol-17 (E2). Peptidergic factors are small peptides that contain no secondary structure. Catecholaminergic or monaminergic factors that include noradrenaline and dopamine are amino acid derived trophic factors. The putative cholinergic factor in sea urchins is acetylcholine. Examination of these chemical messengers in the context of gonad production, gametogenesis, and nutrient translocation allows the generation of new hypotheses for research into the manipulation of gonad production and regulation of gametogenesis. 3.1. Steroids 3.1.1. Sex steroids in the gonads Mammalian models of reproduction demonstrate that both sex steroids and peptide hormones regulate gametogenesis. Early studies looked for the presence of sex steroids in sea urchin ovaries. Donahue and Jennings (1937) found that an oil-soluble substance extracted from the ovaries of L. variegatus induced vaginal growth in ovariectomized rats. They concluded that this substance was slightly estrogenic in nature, requiring over 4 g of sea urchin ovaries to produce the equivalent of 1 rat unit of estrogenic activity. Donahue (1940) also discovered that both L. variegatus and Echinometra sp. contained oil-soluble substances that induced estrogenic responses in rat vagina. Twenty years later, Botticelli et al. (1961) demonstrated that ovary extracts from S. franciscanus had E2 activity and estimated the E2 concentration to be approximately 100 pg g−1 of ovaries. Botticelli et al.
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Fig. 1. Pathway of sex steroid synthesis from cholesterol.
Fig. 2. Putative (A) catecholaminergic factors and (B) cholinergic factors in echinoids.
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(1961) also tentatively identified P4 in these ovaries. These early studies indicate that P4 and E2 are present in sea urchins but did not indicate their origin or biological functions. 3.1.2. Steroid converting enzymes in the gonads De novo synthesis provides the consistency required for the long-term regulation of physiological processes by steroids and requires the activity of steroid-converting enzymes. Colombo and Belvedere (1976) used radiotracers to demonstrate that the gonads of Paracentrotus lividus convert androstenedione (AD), the mammalian precursor to T and estrogens, into T, indicating the activity of 17-hydroxysteroid dehydrogenase (HSD) (required for the reduction of C17 to an alcohol group) in gonadal tissues (Fig. 3). 17-HSD activity has been localized in the ovaries and testes of sea urchins. Varaksina and Varaksin (1991) used histochemistry to demonstrate that auxiliary elements (presumed to be nutritive phagocytes), developing oocytes, and spermatids contain 17-HSD activity. They suggested that this enzyme is probably necessary for gonad function. Watts et al. (1994) used in vitro radiotracers to show that both the ovaries and the testes of L. variegatus synthesize T and the 5-reduced androgens, including 5-androstanedione (5-AO), 5-androstane-3, 17-diol, and 5-androstane-3, 17-diol (hereafter referred to as 5-adiols), from AD. The presence of these steroids indicates the activity of 17-HSD, 5-reductase (required for the reduction of the 4−5 double bond) and 3/-hydroxysteroid dehydrogenase (required for the reduction of C3 keto groups to an alcohol). Interestingly, only the testes synthesize 11-oxygenated androgens, suggesting that sex-specific synthesis of androgens exists in L. variegatus. Manipulation of the nutritional status of L. variegatus influences androgen metabolism in a sex-specific manner. Wasson et al. (1998) starved L. variegatus, collected from the field during the gonadal growth stage, for 48 days to inhibit gonadal growth and maturation (Bishop and Watts 1994). Total rates of AD conversion for both the ovaries and testes increased significantly with the onset of feeding. The ovaries and testes converted AD into T, 5-adiols, 5-AO, androsterone (AN), and epiandrosterone (EPIAN); however, the relative quantities of T and the 5-adiols differed significantly between females and males. On day 4 of feeding, ovarian T synthesis was not detectable but the synthesis of the 5-adiols increased threefold. In contrast, testicular synthesis of T and 5-adiols was maximal at this time. The sex-specific shift in the steroid metabolic pathways may be associated with the initiation of biosynthetic processes associated with oogenesis and spermatogenesis (Wasson et al. 1998). Periodic measurement of androgen metabolism during the annual reproductive cycle of L. variegatus also suggests a relation between steroid converting enzyme activity and reproduction (Wasson et al. 2000a). As found by Wasson et al. (1998), both ovaries and testes always synthesized T, 5-adiols, 5-AO, EPIAN, and AN. The ovaries and testes showed maximal accumulation of 5-adiols in April, when gonad indices started to decline, indicating initiation of the spawning season. Furthermore, the ovaries generally synthesized larger quantities of 5-adiols compared to T on all sampling days. Based on this sex-specific pattern of 5-adiols accumulation, Wasson et al. (2000a) suggested that the 5-adiols influence reproduction in L. variegatus. Progesterone is readily metabolized by the gonads, primarily to a variety of 5-reduced progestins (Fig. 4). Both the ovaries and testes of L. variegatus synthesized
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Fig. 3. Pathway of androstenedione metabolism in Lytechinus variegatus. Solid lines represent tentatively identified pathways. Dashed lines represent suggestively identified pathways. Diamond-shaped arrowheads represent pathways not identified in echinoids. 5-R = 5-reductase, 17-HSD = 17-hydroxysteroid dehydrogenase, 3/-HSD = 3/-hydroxysteroid dehydrogenase.
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Fig. 4. Pathway of progesterone metabolism in Lytechninus variegatus. Solid arrows represent tentatively identified pathways. Dashed arrows represent suggestively identified pathways. 5-R = 5-reductase, 3-HSD = 3-hydroxysteroid dehydrogenase, 3-HSD = 3-hydroxysteroid dehydrogenase, 20-HYD = 20-hydroxylase, 20-HYD = 20-hydroxylase.
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5-pregnane-3, 20-dione, 3-hydroxy-5-pregnan-20-one, 5-pregnane-3, 20-diol, 5-pregane-3, 20-diol, and 5-pregnane-3, 20-diol. Interestingly, the synthesis of androgens from progesterone and sex-specific differences in progesterone metabolism were not observed in the gonads (Wasson and Watts 2000). Since the majority of steroid metabolites synthesized from the gonads of L. variegatus are 5-reduced steroids, Wasson and Watts (1998) suggested that 5-reductase is an important enzyme in the steroidogenic pathway of sea urchin gonads. The specific 5-reductase inhibitor, finasteride (F; Proscar© ), inhibited 5-reductase activity in vitro in the gonads. Additionally, the gonads of sea urchins fed for 16 days on a formulated diet supplemented with F demonstrated in vivo inhibition of 5-reductase activity. Specific chemical ablation by F of 5-reductase could be used as tool to investigate the role of 5-reduced steroids in the gonads of sea urchins (Wasson and Watts 1998). It is interesting that the synthesis of estrogens was not observed in any of the studies examining androgen metabolism in sea urchins (Colombo and Belvedere 1976; Watts et al. 1994; Wasson et al. 1998, 2000a). However, Hines et al. (1994) showed that testes of L. variegatus converted E2 into an E2 ester, estrone, and aqueous soluble metabolites. Additionally, estrone was converted primarily into E2, and to a lesser extent E2 ester and aqueous soluble metabolites. These data indicate that 17-HSD is active in the interconversion of E2 and estrone and those estrogens are converted rapidly into aqueous soluble metabolites. Hines et al. (1994) suggested that radiotracer studies might not provide the sensitivity necessary to detect the rapidly metabolized estrogens. 3.1.3. Sex steroids in the gonads Wasson et al. (2000a) detected immunoreactive E2 in the gonads of L. variegatus using a commercial double antibody E2 radioimmunoassay (RIA). Measurement of T and E2 concentrations by RIA in the gonads during one reproductive season demonstrated no significant correlation between reproductive state (as determined by gonad index) and season, reportedly due to high levels of individual variations in steroid concentrations. These variations in steroid concentrations were presumably related to individual variations in specific stages of the gonads (Wasson et al. 2000a). Interestingly, comparison of steroid concentrations indicated significant differences with gonad size, suggesting a relation between gonad production and steroid levels. In both the ovaries and the testes, individuals with low gonad indices (0–3%) had significantly higher T and E2 concentrations than those with higher gonad indices (>61%). Whether or not these steroids are involved in the stimulation or maintenance of mitotic and meiotic events in gametes has not been established. The levels of 5-adiols, one of the major steroid metabolites in sea urchin gonads, are not known. Since the synthesis of the 5-adiols varied with gonad size (Wasson et al. 2000a), perhaps the 5-adiols may be more appropriate steroids to measure during the reproductive cycle. 3.1.4. Response to exogenous administration of sex steroids Varaksina and Varaksin (1987) injected E2 diproprionate into the coelomic fluid of S. nudus in November when the ovaries were beginning to grow. Individuals injected with E2 diproprionate produced a higher ovary index and a significantly larger number
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of mature oocytes than the control treatment. Similar results were obtained when individuals were injected with E2 diproprionate in April (when gonads were maturing). They suggested that E2 diproprionate induced ovarian growth via stimulating RNA and protein synthesis, enhancing vitellogenesis and leading to oocyte maturation. Wasson et al. (2000b) examined the effects of dietary administration of steroids during the gonadal growth stage. Sex- and steroid-specific effects occurred in the gonads of L. variegatus that were fed either E2, E2/ P4, P4, P4/F, T, or T/F for 36 days (Wasson et al. 2000b). Individuals fed E2 had ovary indices 54% larger than individuals fed control diets. Sea urchins fed E2, E2/P4, P4, or P4/F had significantly smaller oocytes than echinoids fed control diets. In addition, individuals fed E2/P4 or P4 alone had a significantly larger volume of the ascini occupied by nutritive phagocytes than control individuals. Since no ovarian growth occurred in individuals fed E2/P4, P4, or P4/F, they concluded that the daily dose of P4 administered was sufficient to inhibit any observable E2 stimulus of ovarian growth. These data suggest further that 5-reduced progestins may stimulate nutrient accumulation in nutritive phagocytes. Although individuals fed T had significantly smaller oocytes than individuals fed T/F, both treatments had significantly larger oocytes than control individuals. Finally, individuals fed either E2 or T had greater amounts of protein in the ovaries than individuals fed T/F, indicating that E2 and 5-reduced androgens influence protein accumulation in ovaries. Wasson et al. (2000b) concluded that E2 stimulates ovarian growth but does not stimulate oocyte growth and that E2 and 5-reduced progestin metabolites may promote nutrient allocation to nutritive phagocytes. Furthermore, T does not affect ovary growth but, upon removal of 5-reduced androgens (by feeding F), T does promote significant increases in oocyte diameters (Wasson et al. 2000b). The testes of L. variegatus fed P4 exhibited the most pronounced effects (Wasson et al. 2000b). Testis indices were 56% larger in individuals fed P4 than controls; however, no effects on testis growth were detected in individuals fed P4/F, T, or T/F, suggesting that 5-reduced progestins stimulated testicular growth. Individuals fed P4 also had a significantly larger volume of the ascini occupied by nutritive phagocytes and significantly smaller volumes occupied by spermatogenic columns. Furthermore, individuals fed E2/P4, P4/F, or T/F had greater amounts of carbohydrates in their gonads than control individuals, and individuals fed E2/P4, T, or T/F had significantly greater amounts of lipids in their testes than control individuals. Wasson et al. (2000b) concluded that 5-reduced progestins may regulate nutrient allocation into nutritive phagocytes and may also influence spermatogenic column formation. Corroboratively, low concentrations of P4 were measured during the early stages of spermatogenesis (as indicated by low testis indices) (Wasson et al. 2000a). Two studies have examined the effect of sex steroids in juvenile sea urchins. Unuma et al. (1996) demonstrated that juvenile Pseudocentrotus depressus fed estrone for 29 days had significantly higher body weights, food intake, and feed efficiencies than control individuals. Feeding of T or E2 had no effect on growth or food utilization of juveniles (Unuma et al. 1996), but juveniles fed AD or estrone for 30 days had significantly higher testis indices than control individuals (Unuma et al. 1999). In addition, estrone promoted spermatogenesis, suggesting that estrone may be important for the initiation of spermatogenesis (Unuma et al. 1999). The ovaries were not affected in the juveniles and
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Unuma et al. (1999) concluded the lack of an ovarian response to steroids was due to the lack of sexual maturation of the females. The role of sterols or steroids obtained from natural dietary sources has not been examined. It is unlikely that predictable levels of steroids could be extracted from the diet to support long-term control over reproductive processes. Anecdotal information suggests that soy, a natural phytoestrogen, inhibits gametogenesis in S. purpuratus (J Pearse, pers. comm.), suggesting further that sea urchins can respond to dietary endrocrine disruption, which may provide a potential, noninvasive mechanism to regulate gonad physiology and affect gonad production.
3.2. Protein and Peptidergic Factors Sea urchin coelomocytes synthesize the precursor to a major yolk glycoprotein (MYP; Chapter 2). Recognizing that estrogens regulate yolk production in vertebrates, Harrington and Ozaki (1986a) examined the response of sea urchin coelomocytes to E2 stimulation. Although they did not observe an effect on the synthesis of the MYP precursor, they did demonstrate the synthesis of a novel protein. This protein varied in size from 78 to 82 kDa depending on species. No other effects on the synthesis of total proteins were observed. This novel protein produced by the coelomocytes may act as a transcription factor to induce transcription of the MYP precursor gene (Harrington and Ozaki 1986b). No other studies have examined the role of other proteins or peptidergic factors on gametogenesis in sea urchins. Cochran and Engelmann (1972) isolated a spawn-inducing factor from radial nerve extracts of S. purpuratus. The supernatant of radial nerves boiled in seawater contained a heat-stable factor that induced spawning by testis fragments. The concentration of radial nerve factor paralleled the reproductive season of S. purpuratus (Cochran and Engelmann 1972). Digestion of radial nerve factor with pronase and pepsin destroyed all spawn-inducing activity, indicating that this factor is a peptide. The estimated molecular weight of the radial nerve factor is 5600 Da. This peptide was not glycosylated, but had a persistent yellowish-brown color that was associated with the spawn-inducing activity (Cochran and Engelmann 1976). The radial nerve factor induced the production of a gonad factor, which was not digestible with pronase. This gonad factor also induced spawning in testis fragments of S. purpuratus. Although this gonad factor co-migrated with 1-methyladenine in thin layer chromatography (Cochran and Engelmann 1976), this substance was not identified definitively. In starfish, 1-methyladenine was identified as the oocyte maturation inducing substance (reviewed by Kanatani 1979). Since oocyte production is continuous and asynchronous throughout the breeding season in S. purpuratus (Chatlynne 1969), Cochran and Engelmann (1976) suggested that the significant levels of this gonad factor must be chronically maintained to induce spontaneous oocyte maturation; however, no seasonal measurement of this gonad factor has been reported. This was the first demonstration of an “endocrine system” in sea urchins. A neuropeptide produced in radial nerves stimulated the release of a factor by the gonads that induce
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spawning. The mechanism of action and mode of transfer of this radial nerve factor is unknown. In starfish, radial nerve factor is released into the coelomic cavity and transported via coelomic fluid to the gonads (Cochran and Engelmann 1976); however, such mode of transfer in sea urchins would not account for the rapid (within 1 min) spawning response to radial nerve factor. In addition, the cell type that releases the gonad factor is not known. It is interesting to speculate that nutritive phagocytes would synthesize and store this gonad factor and, upon stimulation by the radial nerve factor, release it. The gonad factor would then stimulate the release of neighboring gametes or stimulate the contraction of muscular epithelium, forcibly releasing gametes from the gonad lumen. Most likely, the gonad factor is synthesized and stored within the neurons or epithelial cells of the peritoneum and, upon stimulation by the radial nerve factor, released to stimulate contraction of the muscular epithelium. Although Elphick et al. (1992) identified a class of putative neuropeptides, they suggested no physiological function for them. Using antisera raised against the asteroid SALMFamide, they isolated five immunoreactive peptides and putatively identified an amidated nonapeptide, FPVGRVHRFamide. SALMFamide immunoreactivity had widespread distribution throughout the gut of Echinus esculentus. Specifically, the pharynx–esophagus portion of the gut displayed extensive immunoreactivity associated with the basi-epithelial nerve plexus, which runs between secretory epithelium and the underlying layers of connective tissue and muscle. Although SALMFamide immunoreactivity was also found in association with the outer sub-coelomic epithelial nerve plexus (which is found between the outer connective tissue layer and coelomic epithelium), no immunoreactive epithelial endocrine or neuroendocrine cells were found (Elphick et al. 1992).
3.3. Catecholaminergic and cholinergic factors Sea urchin gonads are innervated by monaminergic, cholinergic, and peptidergic nerve fibers but do not contain adrenergic fibers (Cobb 1969). Both the ectoneural and endoneural systems contain the catecholamines noradrenalin and dopamine (Khotimchenko 1983). Thin nerve fibers interlace the ascini of the gonads and may contain biogenic amines (Khotimchenko and Deridovich 1991). Both noradrenalin and dopamine inhibited temperature-stimulated oogenesis in S. nudus by suppressing growth and maturation of oocytes (Khotimchenko 1983). Interestingly, the levels of these catecholamines varied with reproductive state. Concentrations were low during periods of minimal gametogenic activity and also before spawning. The initiation of spawning was accompanied by increases in catecholamine concentrations (reviewed by Khotimchenko and Deridovich 1991). Since noradrenaline and dopamine decreased the intensity of uptake of labeled uridine and leucine in large oocytes, Khotimchenko (1983) concluded that the suppression of oocyte growth was due to inhibition of RNA and protein synthesis. They hypothesized that these catecholamines influenced oogenesis via transport through the coelomic fluid. In contrast, acetylcholine was secreted in the regions of neuromuscular contacts of the gonads and was effective at inducing the reduction of muscle elements of ascini,
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suggesting that the act of gamete release may be regulated by cholinergic mechanisms (Khotimchenko 1983).
4. MECHANISMS OF REGULATION 4.1. Paracrine Analysis of paracrine mechanisms, in which a chemical messenger induces a response in a cell located in close proximity to the cell that synthesizes it, requires an understanding of gonad morphology. Although nutritive phagocytes lack direct physical contact with developing gametes, thin extensions of nutritive phagocytes surround developing gametes. In male sea urchins, the role of these cells has been equated to that of Sertoli cells in mammalian testes (Pearse and Cameron 1991), providing the necessary local environment to stimulate gamete proliferation and growth. The demonstration of endogenous steroid converting enzymes in nutritive phagocytes (Varaksina and Varaksin 1991) and the E2 and P4 effects on mature gonad growth and gametogenesis (Wasson et al., 2000b) suggest that steroids may be produced by the nutritive phagocytes and influence gametogenesis or nutrient translocation to developing gametes. Histochemical studies also indicate that developing oocytes and spermatids possessed 17-HSD activity (Varaksina and Varaksin 1991), suggesting that developing gametes also synthesize steroids that may act in paracrine or autocrine mechanisms. The secretion of steroids from either nutritive phagocytes or developing gametes would most likely occur by mechanisms similar to those in vertebrates. The promotion of gonad growth, gametogenic activity, and nutrient storage by sex steroids and/or their derivatives (Wasson et al. 2000b) suggests that sex steroid receptors are present in echinoid gonads. The presence of steroid receptors in either nutritive phagocytes or germinal cells has not been investigated in any echinoid. Shyu et al. (1987), however, identified an estrogen-responsive element upstream from the vitellogenin gene in S. purpuratus, suggesting that the vitellogenin synthesis may be regulated by estrogen. Direct contacts (tight junctions) are found between nutritive phagocytes (Pearse and Cameron 1991). Similar associations have been reported for mammalian follicle cells, which use these junctions to coordinate cellular communication throughout large numbers of follicle cells. Via these tight junctions, interconnected nutritive phagocytes may communicate the necessary factors to stimulate nutrient translocation to the developing gametes. In vertebrates, the peptides insulin and glucagon regulate nutrient deposition in storage cells, mobilization of nutrients, and translocation to appropriate sites for utilization. It would be of interest to determine whether similar proteins exist in sea urchins and have a role in paracrine regulation of the nutrient translocation activities of nutritive phagocytes. It is likely that protein or peptidergic factors act by paracrine mechanisms to regulate gonad growth. Mammalian models indicate that a variety of proteins, such as TGF- (Teerds and Dorrington 1995), inhibin (Hillier and Miro 1993), kit ligand (Joyce et al. 1999), and GDF-9 (Dong et al. 1996), produced by oocytes or follicle cells induce specific
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cellular responses in follicle cells or oocytes, respectively. Future research on similar proteins and peptidergic factors in sea urchin gonads will be of considerable interest. 4.2. Endocrine Cochran and Engelmann (1972, 1976) identified a radial nerve factor and a gonad factor that potentially represent a true endocrine system. If this gonad factor induces oocyte maturation (Kanatani 1974), then this system would be chronically stimulated since mature ova are present in the ovarian lumen prior to spawning (Pearse and Cameron 1991). It may be more likely that this gonad factor simply induces a rapid muscular response in the gonads to release gametes. No other studies have examined endocrine mechanisms of regulation in sea urchin reproduction. Several levels of endocrine regulation become apparent when examining sea urchin reproduction. First, the general synchrony of spawning among individuals within a population indicates that a factor (pheromone?) released into the water by an individual induces spawning in other individuals. The nature and identity of this factor is still unknown. Second, the dramatic influence of the environment on gametogenesis and nutrient storage indicates the existence of a communication system between the environment and the organs involved in reproduction. This should not be surprising since environmental factors have been shown to influence the hypothalamo-hypophyseal-gonadal axis in mammals and other vertebrates. Finally, coordination of gametogenic and nutrient translocation activities among and within each gonad indicates the existence of a physiologically controlled mechanism that coordinates these activities. Chemical messengers, whether of neuronal or non-neuronal origins, would most likely accomplish such communication. Movement of these chemical messengers could be accomplished by using the perivisceral coelom and direct diffusion across the gonad wall or by using the hemal system for direct transport to the gonads. Since the hemal system interconnects with each gonad via the aboral ring complex, this system may provide a more efficient and specific means of control.
5. GENE REGULATION IN REPRODUCTION Identification of the genes involved in reproduction of adult sea urchins is a vastly untapped area of research. Most of the research involved in sea urchin genomics has dealt primarily with developmental biology and embryogenesis. Recently, genome sequencing of S. purpuratus was initiated (http://www.hgsc.bcm.tmc.edu/projects/seaurchin/). A search of the NBCI Entrez gene database (http://www.ncbi.nlm.nih.gov/mapview/ map_search.cgi?taxid=7668) for “steroid” and “receptor” revealed only six annotated genes in S. purpuratus that resemble steroid hormone receptors. As of September 2005 none of these genes have been ascribed functions. Interestingly, one gene resembles the estrogen-related receptor- and another resembles the progesterone receptor membrane component 1. Molecular endocrinology will be a fruitful area of sea urchin research in the future, from both a functional and evolutionary standpoint.
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6. CONCLUSIONS Environmental cues influence seasonal changes in gonad morphology and physiology. Knowledge of the physiological mechanisms that stimulate growth and maintenance of nutritive phagocytes and gametes is essential to understand the reproductive biology and ecology of sea urchins in the field. This information is also essential for the development of technologies that will optimize the texture, quality, and quantity of gonads from sea urchins in aquaculture. Diet plays a vital role in stimulation and maintenance of gonad production and is one parameter that is easily manipulated in aquaculture. We have found that formulated feeds supplemented with steroids affect gametogenesis and nutrient allocation to nutritive phagocytes. In the future, phytosteroids may be used to influence gonad growth. For example, Pearse (pers. comm.) attributed the increase in nutritive cells relative to gametes in the gonad to the presence of soy meal in individuals fed a formulated feed. Soy-based diets promote estrogen-dependent as well as estrogen-independent processes (Barnes 1998), and may provide a natural method to adjust a physiological state. Further research is required to fully understand how the dietary administration of these and other specific micronutrients may stimulate or inhibit gonad growth. Sex steroids, proteins, catecholaminergic, and cholingeric factors all play roles in regulating various aspects of gonad function. No data, however, exist on the chemical messengers required to promote nutrient translocation from the gut to the gonads, the mobilization of nutrients within the nutritive phagocytes, or translocation of nutrients to developing gametes. Understanding the physiological control of these mechanisms will assist in the development of formulated feeds to specifically regulate gonad quality and quantity. Most of these studies have examined singular reproductive events (i.e. spawning, gonad growth, etc.) in only a few species of sea urchins. Large gaps are still present in our general understanding of the physiological mechanisms that control reproduction and provide direct links between environmental stimuli and gonad function. Studies using molecular biology, genomics, and bioinformatic tools are essential for elucidating the cellular pathways that regulate specific reproductive processes. Such tools will be extremely beneficial to our understanding of the role of chemical messengers and the influence of endocrine disruptors in sea urchin reproduction.
ACKNOWLEDGMENTS We thank IJ Blader and JM Lawrence for comments on this chapter. Preparation of this review was supported in part by the Mississippi-Alabama Sea Grant Consortium.
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