Vertebrate-type Steroids Research Articles

Testosterone maintains male longevity and female reproduction in Chrysopa pallens

Vertebrate testosterone, an androgen present in the testes, is essential for male fertility. Vertebrate-type steroid hormones have been identified in insects, but their function remains unknown. Insect vitellogenin (Vg) is usually a female-specific protein involved in reproductive processes. However, males of some species, such as the green lacewing Chrysopa pallens, have Vg. Here, we demonstrated that the knockdown of C. pallens male Vg by RNAi significantly shortened the lifespan of males, suppressed the reproduction of post-mating females, and strikingly reduced the abundance of several immune-related compounds, including testosterone. LC-MS/MS revealed that C. pallens male testosterone had the same structure and molecular mass as vertebrate testosterone. Topical testosterone application partially restored the lifespan of Vg-deficient males and the reproduction of post-mating females. These results suggest that vertebrate-type testosterone maintains male longevity and female reproduction under the control of the male Vg in C. pallens.

Insect Sterols and Steroids.

Insects are incapable of biosynthesising sterols de novo so they need to obtain them from their diets or, in certain cases, from symbiotic microorganisms. Sterols serve a structural role in cellular membranes and act as precursors for signalling molecules and defence compounds. Many phytophagous insects dealkylate phytosterols to yield primarily cholesterol, which is also the main sterol that carnivorous and omnivorous insects obtain in their diets. Some phytophagous species have secondarily lost the capacity to dealkylate and consequently use phytosterols for structural and functional roles. The polyhydroxylated steroid hormones of insects, the ecdysteroids, are derived from cholesterol (or phytosterols in non-dealkylating phytophagous species) and regulate many crucial aspects of insect development and reproduction by means of precisely regulated titres resulting from controlled synthesis, storage and further metabolism/excretion. Ecdysteroids differ significantly from vertebrate steroid hormones in their chemical, biochemical and biological properties. Defensive steroids (cardenolides, bufadienolides, cucurbitacins and ecdysteroids) can be accumulated from host plants or biosynthesised within the insect, depending on species, stored in significant amounts in the insect and released when it is attacked. Other allelochemical steroids serve as pheromones. Vertebrate-type steroids have also been conclusively identified from insect sources, but debate continues about their significance. Side chain dealkylation of phytosterols, ecdysteroid metabolism and ecdysteroid mode of action are targets of potential insect control strategies.

Sex steroid profile during oocyte development and maturation in the intertidal worm Marphysa madrasi (Polychaeta: Eunicidae) from the east coast of India

Marphysa madrasi is a commercially valuable maturation diet in crustacean aquaculture. This study presents the first detailed investigation of oogenesis in the intertidal polychaete worm M. madrasi and reports the steroid profile during oocyte growth and development. Oogenesis is extraovarian type I, originating from coelomic epithelial cells, with four stages of development - primary growth, early vitellogenic, late vitellogenic, and maturation. The primary growth phase contains oogonial cells and previtellogenic oocyte clusters in the early, mid, and late stages of development form a dispersed ovary attached to blood vessels. The late previtellogenic oocytes detach from the ovary at the onset of vitellogenesis. The detached oocytes complete vitellogenesis and final maturation in the coelomic fluid as solitary free-floating cells without any connection with follicle cells. The worms display asynchronous reproduction with a heterogeneous population of developing oocytes. Steroid extracts from the polychaete homogenates in different stages of oogenesis were identified by HPLC and confirmed by LC-MS/MS. In M. madrasi, two vertebrate-type steroids, pregnenolone (P5) and 17α-hydroxyprogesterone (17-OHP) were detected and quantified. The P5 levels were low in immature worms but increased significantly by ∼ 8.3-fold in the previtellogenic stage and peaked during oocyte maturation. 17-OHP levels were low in immature worms but gradually increase as the oogenesis progress to the primary growth and early vitellogenic phase, with a significant increase (p < 0.001) during the late vitellogenic phase. Although an increase in the concentration of P5 and 17-OHP during vitellogenesis and maturation of oocytes points to a possible role in reproduction, the absence of other vertebrate-type steroids in the investigated polychaete signifies a plausible uptake of P5 and 17-OHP from the environment.

Early embryonic exposure of freshwater gastropods to pharmaceutical\xa05-alpha-reductase inhibitors results in a surprising open-coiled \u201cbanana-shaped\u201d shell

In vertebrates, the steroidogenesis enzyme 5α-reductase converts testosterone to the more potent androgen 5α-dihydrotestosterone. Homologues of 5α-reductase genes have been identified in molluscs. However, recent findings suggest that vertebrate-type steroid androgens are not utilised in molluscan reproductive development. Genomic searches have revealed that molluscs do not possess many of the steroidogenic enzymes required to make testosterone, nor a nuclear androgen receptor. Consequently, the role of 5α-reductase in molluscs presents a mystery. Here, developmental exposures of Biomphalaria glabrata to selective pharmaceutical 5α-reductase inhibitors elicited a strong, highly reproducible phenotypic response characterised by the development of elongated “banana-shaped” shell morphology. In comparison to untreated snails, the shells are open-coiled and the whorls are unattached. Dutasteride (5α-reductase inhibitor) is approximately 10-times more potent at provoking the banana-shaped shell phenotype than finasteride, paralleling the pharmaceuticals’ efficacy in humans. Other enzyme inhibitors with different modes of action were tested to investigate the specificity of the phenotype. However, only the pharmaceutical 5α-reductase inhibitors provoked the response. Dutasteride elicited the same phenotype in a second gastropod, Physella acuta. In the absence of evidence for de novo androgen steroidogenesis in molluscs, these findings suggest that novel substrates for 5α-reductase exist in gastropods, lending support to the contention that molluscan endocrinology differs from the well-characterised vertebrate endocrine system.

Vertebrate-Type Steroid Profile in Different Tissues of Wild and Endocrinologically Manipulated Female Brood Stocks of &lt;i&gt;Penaeus monodon&lt;/i&gt;

The endocrine mechanism regulating reproduction of tiger shrimp, Penaeus monodon , is a field of topical interest, which is to be addressed in detail as control of reproduction in captivity is conditional for domestication. In this study, the titres of estradiol and progesterone in hemolymph, ovary and hepatopancreas during various reproductive phases of females were quantified using ELISA. Circulating concentration of hemolymph estradiol and progesterone in the endocrinologically-induced (eyestalk ablated) females was also quantified. Additionally, the evidence for estradiol and progesterone receptors was provided using immunohistochemistry. Levels of progesterone in hemolymph and ovary were highest in the vitellogenic and ripe phases (P > 0.05). Conversely, in hepatopancreas significantly higher progesterone level was detected at immature phase (184.3 ± 2.3 pg/ml). Estradiol was highest in hemolymph (65.97 ± 0.8 pg/ml) and ovary (58.47 ± 0.7 pg/ml) at the early vitellogenic phase. In hepatopancreas, however, lowest level of estradiol was detected at the early vitellogenic phase (121 ± 0.38 pg/ml). Progesterone and estradiol receptor protein signals were predominantly found in the pre vitellogenic oocytes. These results suggest the possible involvement of vertebrate-type steroid in the reproductive maturation of P. monodon .

Steroids and genes related to steroid biosynthesis in the female giant freshwater prawn, Macrobrachium rosenbergii

The giant freshwater prawn, Macrobrachium rosenbergii, is important to many Asian countries due to its high economic value as an aquaculture product. With demand increasing, there is requirement for a better understanding of the biosynthetic components that regulate its growth and reproduction, including steroids, in order to help increase production. Vertebrate-type steroids and their receptors were identified in crustaceans and implicated in reproduction. In this study, we presented the sex steroids estradiol and progesterone by LC–MS/MS in female M. rosenbergii, and reveal steroidogenic-related genes by in silico analysis of de novo assembled transcriptomes. Comparative analysis with other species was performed to confirm their putative role, as well as tissue-specific and quantitative gene expression. We reveal 29 transcripts that encode for steroidogenic-related proteins, including steroidogenic enzymes, a nuclear steroid hormone receptors, and a steroidogenic factor. Moreover, we identified for the first time the presence of steroidogenic factor 1, StAR-related lipid transfer protein, estradiol receptor- and progesterone-like protein in M. rosenbergii. Those targeted for gene expression analysis (3 beta-hydroxysteroid dehydrogenase, 17 beta-hydroxysteroid dehydrogenase, estrogen sulfotransferase and progesterone receptor-like) showed widespread expression within many tissues, and at relatively high levels in the central nervous system (CNS) during ovarian maturation. In summary, we provide further evidence for the existence of steroidogenic pathways in crustaceans, which may be useful for advancing prawn aquaculture.

The variations of vertebrate-type steroid hormones in the freshwater narrow-clawed crayfish Astacus leptodactylus (Eschscholtz, 1823) (Decapoda, Astacidae) during oocyte development

In this study, the levels of 17β-oestradiol (E2) and 17α-hydroxyprogesterone in the haemolymph, ovary and hepatopancreas of the freshwater narrow-clawed crayfish, Astacus leptodactylus (Eschscholtz, 1823) were analysed in different stages of oocyte development. In recent years, the occurrence of vertebrate-type steroid hormones, E2 and 17α-hydroxyprogesterone, has been reported in a few decapods, although their precise role in female reproduction is not fully elucidated. Thirty specimens of A. leptodactylus were caught at Aras Dam (Western Azerbaijan, Iran) and maintained under experimental conditions (at 25°C with a photoperiod of 14 : 10 hours light/dark) from November to March 2010. For steroid extraction, 3 ml haemolymph was withdrawn from the pericardial cavity with a syringe, and frozen at −20°C. The pieces of ovary and hepatopancreas were homogenized with phosphate buffer (pH 7.8) and, centrifuged at 3000 rpm for 15 min at 4°C as well. It was observed that the level of E2 increased in haemolymph and ovary during vitellogenesis, and decreased in the hepatopancreas. The level of 17α-hydroxyprogesterone was enhanced in haemolymph and ovary during the maturation stage. Maximum levels of E2 and 17α-hydroxyprogesterone were present in the ovary. The results point out the possible involvement of vertebrate-type steroids in the endocrine regulation of the ovarian cycle of A. leptodactylus.

Preliminary identification and quantification of steroid hormones in the red palm weevil, Rhynchophorus ferrugineus

In the present preliminary study, we used a gas chromatography–mass spectroscopy (GC-MS) method to identify and quantify steroid hormones, their precursors and metabolites in whole body of red palm weevil (RPW) R. ferrugineus adults. We identified a total of seven steroids by single ion monitoring mode (SIM) analysis and compared them to the National Institute of Standards and Technology (NIST) library. The steroids include: dehydroepiandrosterone (DHEA), estrone, estradiol-17β, testosterone, progesterone, cortisol and cholesterol, whereas pregnenolone, pregnan-20-one-17-hydroxy and corticosterone were not detected. This study shows that some invertebrate groups seem to use partially or totally comparable hormones to vertebrates and produce vertebrate-type steroids with functional roles. However, some steroids of the endocrine system of invertebrates are still lacking or yet to be identified in most phyla.

Detection of vertebrate-type steroid hormones and their converting activities in the neogastropod Thais clavigera (Kuster, 1858)

Knowledge of the regulation of molluscan reproductive systems is limited. Many studies have described how the development and maturation of molluscan gonads are regulated by oestrogen. Vertebrate-type oestrogen, 17b-oestradiol, has been detected in molluscs and its concentration in the gonads has been suggested to change in conjunction with reproductive cycles. However, it is unclear if molluscan gonadal tissues are relatively high in aromatase activities. Thais clavigera has been shown to possess a preferential expression of the oestrogen receptor gene in the ovary and ganglia, suggesting an association with gonadal maturation and expression of the oestrogen receptor gene. Therefore, it is interesting to explore the existence of vertebrate-type sex steroid hormones and aromatase activity in T. clavigera. We applied gas chromatography–mass spectrometry techniques to detect steroids in the gonadal tissues of T. clavigera and identified testosterone, androstenedione, 17b-oestradiol and oestrone. The aromatase activities converting androstenedione and testosterone into oestrone and 17boestradiol were much higher in gonads than in digestive glands. These data provide insights into the endocrinological system of neogastropods, prompting molecular evidence for the aromatase gene.

Testosterone, progesterone, and FSH levels in Pimpla turionellae L. (Hymenoptera: Ichneumonidae) and its host Galleria mellonella L. (Lepidoptera: Pyralidae)

Various vertebrate-type steroid and gonadotropin hormones have been identified in invertebrates, including insects; however, studies in this area are not sufficient to presume a generalized role for these hormones. We identified testosterone, progesterone, and follicle-stimulating hormone (FSH) in the whole-body homogenates of Galleria mellonella L. (Lepidoptera: Pyralidae) and its parasitoid Pimpla turionellae L. (Hymenoptera: Ichneumonidae). Testosterone concentration significantly decreased from early instars to last instars and pupal stages of G. mellonella; however, no significant difference was observed in progesterone levels at different stages. FSH concentration reached the highest level at the pupal stage of G. mellonella. No significant difference was observed in testosterone, progesterone, and FSH concentrations in young and mature adults of the parasitoid P. turionellae for both sexes. We also monitored the changes in testosterone, progesterone, and FSH levels over 2, 6, and 24 h in parasitized G. mellonella pupae. During the experimental periods no significant difference in hormone levels was found between controls and parasitized G. mellonella pupae.

Changes in progesterone levels and distribution of progesterone receptor during vitellogenesis in the female mud crab (Scylla paramamosain)

Vertebrate-type steroids, such as progesterone, have been identified in crustaceans. The physiological activity of progesterone during vitellogenesis is still not well understood. In this study, progesterone levels in the female mud crab, Scylla paramamosain, were determined by enzyme-linked immunosorbent assay. Peak levels of progesterone were detected during the previtellogenic stage in the hemolymph, ovary, and hepatopancreas, whereas the progesterone level decreased significantly in vitellogenic stage I. During vitellogenic stage II, progesterone levels rose again in the hemolymph and ovary, but continued to decrease in the hepatopancreas. By using western blotting, progesterone receptor (PR), with an apparent molecular weight of 70 kDa, was identified in the ovary during both vitellogenic stages I and II. By means of immunohistochemistry, PR was detected mainly in the follicle cells during vitellogenic stage I and in the nuclei of oocytes in vitellogenic stage II. Our results strongly suggest that progesterone promotes vitellogenesis in the mud crab, S. paramamosain via a classical genomic mechanism.

Gonadal ecdysteroidogenesis in arthropoda: occurrence and regulation.

Ecdysteroids are multifunctional hormones in male and female arthropods and are stored in oocytes for use during embryogenesis. Ecdysteroid biosynthesis and its hormonal regulation are demonstrated for insect gonads, but not for the gonads of other arthropods. The Y-organ in the cephalothorax of crustaceans and the integument of ticks are sources of secreted ecdysteroids in adults, as in earlier stages, but the tissue source is not known for adults in many arthropod groups. Ecdysteroid metabolism occurs in several tissues of adult arthropods. This review summarizes the evidence for ecdysteroid biosynthesis by gonads and its metabolism in adult arthropods and considers the apparent uniqueness of ecdysteroid hormones in arthropods, given the predominance of vertebrate-type steroids in sister invertebrate groups and vertebrates.

Detection and Changes in Levels of Testosterone During Spermatogenesis in the Freshwater Planarian Bdellocephala brunnea

It was reported recently that vertebrate-type steroids exist and control reproduction in several groups of invertebrates, including molluscs. Sexually reproductive freshwater planarians of the species Bdellocephala brunnea have a limited breeding season in their natural habitat. This phenomenon suggests that some endogenous reproductive hormones might play a role in vivo. However, to date, sex steroids such as androgen, estrogen, and progesterone have not been found in planarians. The goal of the present study was to determine whether androgen is present in sexual planarians such as B. brunnea. The presence of testosterone was detected by high-pressure liquid chromatography and, in sexually reproductive individuals in which no seminal vesicles were visible, the level of testosterone was about twice than that in individuals with visible seminal vesicles. An enzyme-linked immunosorbent assay revealed that the levels of testosterone during terminal spermatogenesis were three times higher than during the spermatocyte-building phase. Our results indicate that sexually reproductive freshwater planarians such as B. brunnea might have vertebrate-type steroids and show variation in testosterone levels during spermatogenesis.

Steroids in aquatic invertebrates

Steroid molecules are present in all invertebrates, and some of them have established hormonal roles: this is the case for ecdysteroids in arthropods and, to a lesser extent, for vertebrate-type steroids in molluscs. Steroids are not only hormones, they may also fulfill many other functions in chemical communication, chemical defense or even digestive physiology. The increasing occurrence of endocrine disruption problems caused by environmental pollutants, which interfere in particular with reproductive physiology of vertebrates but also of invertebrates has made necessary to better understand the endocrine physiology of the latter and the role of steroids in these processes. So many attempts are being made to better understand the endocrine roles of steroids in arthropods and molluscs, and to establish whether they also fulfill similar functions in other invertebrate phyla. At the moment, both the precise identification of these steroids, the determination of their origin (endogenous versus exogenous) and of their mechanism of action are under active investigation. This research takes profit of the development of genome sequencing programs on many invertebrate species, which allow the identification of receptors and/or biosynthetic enzymes, when related to their vertebrate counterparts, but the story is not so simple, as will be exemplified by estrogen receptors of molluscs.

Reproductive Cycle, Ovarian Development, and Vertebrate-Type Steroids Profile in the Freshwater Prawn Macrobrachium Rosenbergii

Abstract This study is intended to characterize the ovarian development and hemolymph vertebrate-type steroids concentration during the reproductive cycle of the freshwater prawn, Macrobrachium rosenbergii. A five-stage classification based on the external observation of the ovary's size and color as seen through the tegument was used. The results showed the existence of a direct correspondence between the ovarian stages and the gonadosomatic index, the oocyte diameter, and the characteristics of ovarian histology. In each stage total bleeding of the prawns was conducted and the hemolymph concentrations of 17β-estradiol (E2), testosterone (T) and 17α-hydroxyprogesterone (17-OHP; conjugated and unconjugated) were determined by solid-phase radioimmunoassay (RIA). High levels of unconjugated 17-OHP, relatively constant concentrations of unconjugated T, and null concentration of unconjugated E2 were found throughout the five stages considered. Nonetheless, low levels of E2 were determined in all stages, in co...

Effects of exposure to ED contaminants (TPT-Cl and Fenarimol) on crinoid echinoderms: comparative analysis of regenerative development and correlated steroid levels

Regenerative phenomena reproduce developmental processes in adult organisms and are regulated by neuro-endocrine mechanisms. They can therefore provide sensitive tests for monitoring the effects of exposure to endocrine disrupter contaminants (EDs) which can be bioaccumulated by the organisms causing dysfunctions in steroid hormone metabolism and activities and affecting reproduction and development. Echinoderms are prime candidates for this new ecotoxicological approach, since (1) they offer unique models to study physiological regenerative processes and (2) in echinoderms vertebrate-type steroids can be synthesized and used as terminal hormones along the neuro-endocrine cascades regulating reproductive, growth and developmental processes. We are currently exploring the effects on the regenerative potential of echinoderms of different classes of compounds that are well known to have ED activity. The present paper focuses on the possible effects of well-known compounds with suspected androgenic activity such as TPT-Cl (Triphenyltin-chloride) and Fenarimol [(±)-2,4-dichloro-α-(pyrimidin-5-yl) benzhydryl alcohol]. The selected test-species is the crinoid Antedon mediterranea, a tractable and sensitive benthic filter-feeding species which represents a valuable experimental model for investigation on the regenerative process from the macroscopic to the molecular level. The present investigation employs an integrated approach which combines exposure experiments and biological analysis utilizing microscopy, immunocytochemistry and biochemistry. The experiments were carried out on experimentally induced arm regenerations in semistatic controlled conditions with exposure concentrations comparable to those of moderately polluted coastal zones. The bulk of results obtained so far provide indications of significant sublethal effects from exposure to TPT-Cl and Fenarimol and mechanisms of toxicity related to developmental physiology, which are associated with variations in steroid levels in the animal tissues. The results indicate that these two substances (1) affect growth and development by interfering with the same basic cellular mechanisms of regeneration, such as cell proliferation, migration and differentiation/dedifferentiation, which are possibly controlled by steroid hormones; and (2) can induce a number of significant modifications in the timing, modalities and pattern of arm regeneration, which may involve the activation of cell mechanisms related to steroid synthesis/metabolism.

Hemolymph levels of vertebrate-type steroid hormones in female kuruma prawn Marsupenaeus japonicus (Crustacea: Decapoda: Penaeidae) during natural reproductive cycle and induced ovarian development by eyestalk ablation

: The occurrence of vertebrate-type steroid hormones in the hemolymph and ovaries has been reported in several crustacean species; however, their physiological roles are not sufficiently understood. In the present study, correlations between hemolymph levels of the vertebrate-type steroid hormones and ovarian development in female kuruma prawn Marsupenaeus japonicus were examined. In the first experiment, hemolymph samples were taken in November (non-reproductive period) and June (maturing and spawning period) and the hemolymph levels of estradiol-17β, estriol, progesterone, testosterone, and 11-ketotestosterone were found not to be significantly related to the ovarian development (gonadosomatic index, 0.80–9.39). In the second experiment, ovarian development was induced by bilateral eyestalk ablation in the non-reproductive period. The hemolymph levels of estradiol-17β, estriol, and progesterone did not significantly differ between the control female prawn and the eyestalk-ablated female prawn, which underwent vitellogenesis. If the examined steroid hormones were vitellogenesis-stimulating hormones as in fish, their hemolymph levels would be expected to show significant correlations with ovarian development. However, the present study found no significant correlations between hormone levels and ovarian development. This suggests that based on the available data, the examined vertebrate-type steroid hormones do not play an important role in ovarian development in M. japonicus.

Perspectives on Hormonal Manipulation of Shrimp Reproduction

Shrimp aquaculture in the world has developed remarkably, however, to enable further development, new technological advances in hormonal manipulation of shrimp reproduction are increasingly important for effective stock enhancement. To develop hormonal manipulation techniques, progress in shrimp endocrinology is necessary. In this article, I review my work and related studies on shrimp endocrinology. For female reproduction, eyestalk hormones, ecdysteroids, and vertebrate-type steroid hormones were examined. Eyestalk ablation induced ovarian development, indicating the occurrence of the eyestalk hormone, vitellogenesis-inhibiting hormone. Hemolymph levels of ecdysteroids and vertebrate-type steroid hormones were not correlated to ovarian development, suggesting that these hormones are not involved in the regulation of ovarian development. For male reproduction, the androgenic gland showed distinct structural changes in relation to male reproductive activity, suggesting that the hormone has a role in the regulation of male reproductive activity. For control of shrimp reproduction, at present, no hormones are available, and only eyestalk ablation is practically used. This is mainly because endocrinology of shrimp reproduction is not yet sufficiently understood. Only a few hormones have been detailed so far, and many hormones are still unidentified. For new technological advancements in hormonal manipulation, it is necessary to characterize these unknown hormones and to synthesize useful ones for administration.

Alternative to vertebrate animal experiments in the study of metabolism of illegal growth promotors and veterinary drugs

The continuous production of new illegal veterinary drugs and related products requires residue laboratories to initiate research into developing fast and accurate extraction and detection methods for the identification (and/or quantification) of the major analyte or metabolites of these compounds. In practice, animal experiments are carried out in which vertebrate animals (bovine, porcine, …) are treated orally or intramuscularly with the illegal compound. Different matrices (urine, faeces, blood) are collected over 2 or 3 weeks until the animal is sacrificed. Edible matrices (meat, liver, kidney,…) are collected. Because of the complexity of the animal experiment and the method development, a lot of valuable time and money is consumed. Recent studies have shown that some of these vertebrate experiments can be replaced by invertebrates metabolism studies. Vertebrate-type steroids such as testosterone have been used as substrates to study enzyme systems (cytP450) for the oxidative metabolism in invertebrates. Results from these studies provide information on the degree of similarity to the enzyme systems in vertebrates. These findings are of great importance to the research of illegally used substances but also to the downscaling of vertebrate animal experiments and their considerable cost factors. The invertebrate Neomysis integer (Crustacea, Mysidacea) has been used as an alternative model for the partial replacement of vertebrate animals in metabolism studies with illegal growth promotors and veterinary drugs. The principle of this assay and some examples are described.

Crustacean Vitellogenesis: Its Role in Oocyte Development1

One of the major changes that occurs during the maturation of oocytes is the accumulation of yolk protein, or vitellin (Vn). To better understand how this process is regulated, we characterized the Vn of the ridgeback shrimp, Sicyonia ingentis (Penaeoidea). This Vn is a 322 kDa molecule composed of three subunits. Using purified Vn, we developed an anti-Vn antiserum and used it to characterize vitellogenin by Western blot analysis. The antiserum was also used in an ELISA to measure hemolymph levels of vitellogenin. Previous studies suggested the presence of vertebrate-type steroids might stimulate reproductive processes in decapod crustaceans. Treatment of sexually quiescent female shrimp with progesterone, hydroxyprogesterone, and estradiol did not increase hemolymph levels of yolk protein precursor. The absence of a response to these steroids may reflect the presence of other hormones (such as the gonad-inhibiting hormone) that prevent oocyte development. To examine the molecular basis for the regulation of vitellogenesis, ovarian and hepatopancreas expression cDNA libraries were screened using the anti-Vn antiserum. A 2.9 kilobase clone was isolated from both cDNA libraries suggesting that both tissues are sites of vitellogenin synthesis. These molecular tools should be useful for in vitro studies of vitellogenin synthesis.