Crustacean Vitellogenesis: Its Role in Oocyte Development1

Abstract

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.

INTRODUCTION

Oogenesis is an energetically expensive reproductive process that can be divided into several phases. The latter phases of oogenesis, periods characterized by the accumulation of yolk proteins in the growing oocytes and by significant increases in oocyte diameter, are referred to as primary and secondary vitellogenesis (Meusy and Charniaux-Cotton, 1984; Meusy and Payen, 1988). Vitellin (Vn) is the common form of yolk stored in oocytes and a nutrient source for developing embryos. In many species, vitellogenin (Vg), the precursor molecule to Vn, is transported through the hemolymph to developing oocytes, where it is sequestered and modified with the addition of polysaccharides and lipids into Vn. Thus, the synthesis of yolk proteins is a good indicator of female reproductive activity in many species. Furthermore, the presence of yolk proteins has been frequently used to study the hormones involved in the control of reproduction.

One organism that seems particularly suitable for such endocrinological studies is the ridgebacked shrimp, Sicyonia ingentis (Decapoda: Dendrobranchiata: Penaeoidea). This species is distributed between Isla Maria Madre, Mexico and Monterey Bay, California (Perez-Farfante, 1985) and is found at depths of 55–82 m (Frey, 1971). This shrimp can be obtained from commercial fisheries, and has proven to be easily handled in captivity. Since these shrimps are reproductive between June and November when they broadcast spawn during the phase of the new moon (Anderson et al., 1985; personal observation), they are easy to study during the summer and autumn months. Before examining the effects of various hormones, we first characterized Vn, developed and characterized an anti-Vn antiserum, and used this antiserum to produce an ELISA (enzyme linked immunosorbant assay) to monitor changes in hemolymph levels of yolk protein precursor.

Characterization of vitellogenin and vitellin

Initiation of our investigations required the isolation, purification, and characterization of yolk proteins from S. ingentis. Vitellin was isolated from ovarian homogenates through differential centrifugation, and purified from other proteins by differential precipitation with increasing concentrations of saturated ammonium sulfate (Tsukimura et al., 2000). The molecular mass (MM) of S. ingentis Vn was found to be 322 kDa ± 3 kDa by gel filtration chromatography. In the Decapoda, the MM of Vn ranges from 283 kDa to over 600 kDa, with the Dendrobranchiata possessing lower Vn MM than the Pleocyemata (Table 1). The Vn MM of S. ingentis is consistent within the Dendrobranchiata and the rest of the Decapoda.

Under denaturing conditions on SDS-PAGE, S. ingentis Vn separates into three subunits with MM's of 182 kDa, 91 kDa, and 85 kDa (Tsukimura et al., 2000). The range in number of Vn subunits in the Dendrobranchiata and Pleocyemata is 2–8 (Table 2), most of which are between 2–4 subunits. The purified Vn was used to develop an anti-Vn antiserum. Western blot analysis was used to ascertain that the S. ingentis anti-Vn antiserum bound only the three Vn bands from oocytes and also cross-reacted with female hemolymph (Fig. 2). No bands were detected in male hemolymph. Most reports of hemolymph yolk proteins in crustaceans have used antisera developed against Vn and found that these proteins did not exist in males or in sexually quiescent females, and thus referred to the protein as Vg. This demonstrated that the Vn and Vg are immunologically related. Caution should be noted in examining results immunological results because lipid and carbohydrate moiety differences can cause proteins to be sufficiently dissimilar that antibody cross-reactivity is diminished or absent (Spaziani et al., 1995). We also used the anti-Vn antiserum to develop an enzyme-linked immunosorbant assay (ELISA) for vitellogenin, with which we determined hemolymph levels of Vg that peaked at about 1 mg/ml during the final stages of oocyte development (Fig. 1). This hemolymph level of Vg is consistent with levels reported for other decapod crustaceans that range from 0.03–10 mg/ml (Table 3).

Hormonal regulation of reproduction

Classical eyestalk ablation experiments have demonstrated that malacostracan reproduction is under sinus gland control (Panouse, 1943; Brown and Jones, 1947; Stephens, 1952; Carlisle, 1953; Gomez, 1965). These and more recent studies have been extensively reviewed (Adiyodi, 1985; Fingerman, 1987; Chang, 1992; De Kleijn and Van Herp, 1995). Briefly, the removal of the sinus gland, a neuralhemal organ that is situated at the peripheral end of the eyestalk allows simple removal by ablation techniques, has demonstrated that a gonad-inhibiting hormone (GIH) exists. This neuropeptide has been also referred to as the ovarian-inhibiting hormone (Klek-Kawinska and Bomirski, 1975; Kulkarni and Nagabhushnam, 1980), and the vitellogenin-inhibiting hormone (Gohar et al., 1984; Soyez et al., 1987; Aguilar et al., 1992). In addition, the sinus gland has been suggested to contain vitellogenin-stimulating compounds (Junéra et al., 1977; Picaud and Souty, 1980; Zerbib and Meusy, 1983; Charniaux-Cotton, 1985).

Eyestalk ablation can also result in molting (see reviews: Adiyodi, 1985; Chang, 1992). Thus, eyestalk ablation stimulates molting in some animals and reproduction in others. This suggests that intermediate factors may be involved in stimulating the reproductive system. Various tissues, including the brain, thoracic ganglion, and mandibular organ have been examined for substances that stimulate reproductive processes. The thoracic ganglia contain factors that stimulate or accelerate oocyte development (Otsu, 1960; Hinsch and Bennett, 1979; Kulkarni et al., 1981; Sarojini et al., 1982; Takayanagi et al., 1986). Stimulation of sexually quiescent shrimp by extracts of the thoracic ganglion suggests that stimulatory factors exist (Otsu, 1963). Likewise, implantation of brain tissue into the crab, Paratelphusa hydrodromous, has been shown to accelerate oocyte development (Gomez, 1965). Brain extracts also enhanced oocyte growth in Parapenaeopsis hardwickii, Macrobrachium kistnensis and Paratya compressa (Kulkarni et al., 1981; Sarojini et al., 1982; Takayanagi et al., 1986).

Another tissue that may be involved in the regulation of reproduction is the mandibular organ (MO). The histological appearance of the MO led to the suggestion that its activity increases during reproduction (LeRoux, 1974; Hinsch, 1980). Shortly thereafter, lipid based materials, either terpenoid or steroid, were shown to be produced by the MO (Laufer et al., 1987; Couch et al., 1987). Methyl farnesoate (MF), a terpenoid, will be discussed in detail in this volume (see: Borst et al., 2001; Laufer and Biggers, 2001). The role MF in reproduction was originally inferred by correlating oocyte size and MF levels in the hemolymph (Borst et al., 1987; Laufer et al., 1987; and Borst et al., 1995). More manipulative data has shown mixed results for stimulating oocyte development (Table 4). Incubation of ovarian tissue with MF and the addition of MF to the diet have both been shown to stimulate ovarian development of Penaeus vannamei and Procambarus clarkii (Tsukimura and Kamemoto, 1991; Laufer et al., 1998). However, no significant effects were detected in Homarus americanus and Macrobrachium rosenbergii when MF was injected into senescent females with MF (Tsukimura et al., 1993; Wilder et al., 1994). With the half-life of MF less than one hour, it is possible that the incidental presence of MF was insufficient to reinitiate reproduction. Conversely, MF incubations with fully active T. longicaudatus ovarian tissue might not be recognized because vitellogenesis was already near maximal capacity (Riley and Tsukimura, 1998).

One hypothesis (Laufer et al., 1987) suggested that MF may act as a juvenile hormone-like compound that, as in insects, maintains juvenile morphology and enhances reproduction in adults. We have recently reported that MF significantly reduced the number of developing oocytes when administered continuously to juvenile tadpole shrimp, T. longicaudatus (Linder and Tsukimura, 1999). Our Triops data seem to supports Laufer and colleagues (1987) initial ideas that MF may act as a juvenilizing agent in Crustacea.

The ultrastructure of the MO led some investigators to suggest that the MO might produce steroid compounds (Yudin et al., 1980; Hinsch, 1981). The presence of the vertebrate steroid progesterone was later reported in the MO (Couch et al., 1987). In addition, the presence of the non-ecdysteroid steroid hormones has been reported in other crustacean tissues (Donahue, 1940; Lisk, 1961). Three steroids (17β-estradiol, progesterone, and 17α-hydroxyprogesterone) have been extensively examined for their effects on reproduction and have produced variable results (Table 5). Many of these studies used increases in the Gonad/Somatic Indices or ovoposition as an endpoint that might obscure immediate effects from injected hormones. We have expanded the database on the effects of steroid compounds in S. ingentis using the hemolymph levels of yolk proteins. For three consecutive days, sexually quiescent female shrimp were injected with one μg injections of progesterone, 17α-hydroxyprogesterone, or 17β-estradiol. None of the three steroid hormones produced a significant change in hemolymph vitellogenin levels during any of the seven days following the first injection. Using a similar approach, we have also tested these steroids (50 μg) into the American lobster, H. americanus, and found them to be ineffective in elevating hemolymph vitellogenin levels (Tsukimura, Waddy and Borst, unpublished data). One explanation for these results it that the endocrine environment of sexually quiescent females, which probably involves high levels of GIH, may have made these animals unresponsive to the steroid compounds. Studies on isolated tissues, physically separated from GIH, would clarify the effects of steroids on vitellogenesis. However, the tissues in which vitellogenin is synthesized has not been clearly demonstrated in many species.

Biological markers to identify endocrine compounds

One difficulty in identifying compounds that stimulate crustacean reproduction is the lack of adequate biological markers for reproduction. As we stated earlier, Vn can easily be purified and characterized. However, a problem with using the presence of yolk proteins as indicators of reproduction is that their presence in tissues does not clearly distinguish between synthesis, storage, or degradation. Indeed, the location of Vg synthesis has produced much controversy. This uncertainty makes difficult the examination of direct hormone effects upon incubated tissues. Previous studies suggest that the site of decapod crustacean vitellogenin synthesis is the ovary, the hepatopancreas or both (Table 6). The primary assay for these reports has been the cross-reaction of anti-Vn antibodies to antigens located in tissues, such as the ovary and hepatopancreas. In addition, recent experiments have examined the incorporation of labeled amino acids into proteins that might co-migrate with native yolk proteins or immunoprecipitate with anti-Vn antisera. There is no clear division of Vg synthesis sites between Decapoda taxonomic suborders (Dendrobranchiata and Pleocyemata).

Reports suggesting that Vg is synthesized in the ovary extend over a number of taxonomic levels from the Brachyura, Astacidea, Caridea and Penaeoidea (Lui et al., 1974; Yano and Chinzei, 1987; Quackenbush, 1992; Browdy et al., 1990; Fainzibler et al., 1992; Khayat et al., 1994a; Lee and Walker, 1995; Lee and Watson, 1995; Sagi et al., 1995). Evidence indicating that Vg synthesis occurs in the hepatopancreas occurs from the same groups (Dehn et al., 1983; Paulus and Laufer, 1987; Khoo et al., 1990; Shafir et al., 1992; Khayat et al., 1994b; Sagi et al., 1995; Lee and Chang, 1997; Rani and Subramonium, 1997; Tuberty and Fingerman, 1997). Recent attempts at obtaining Vg-mRNA have thus far been inconclusive. A 1.1 kb mRNA was found to be abundant in both the hepatopancreas and ovary, however the product was never shown to be Vg-mRNA (Khayat et al., 1994b). Synthesis of Vg at an extra-ovarian site is corroborated by the discovery of Vg receptors (presumably receptors for endocytosis) in ovarian tissues of lobsters and crayfish (Jugan and Van Herp, 1989). Determining the site of Vg synthesis will permit a more precise examination of chemical mediators that stimulate and enhance Vg production.

To identify the site of Vg synthesis, we have used standard techniques (Chomczynski and Sacchi, 1987) to isolate Vg transcripts from both the ovary and hepatopancreas of S. ingentis (Pooyan, 1998; Pooyan et al., 1999). Purity of the total mRNA extraction was significantly increased by performing additional phenol:chloroform extractions (Pooyan, 1998). Reduction in the number of transcripts that were screened was accomplished by synthesizing a 30-mer degenerate probe (biotinylated), which was generated from a ten amino acid residue sequence (N-terminus—YITEANEVAF—C-terminus) of the 182 kDa subunit of vitellin. These ten amino acids that produced the least degenerate probe were selected from a longer peptide sequence. The hybrid of transcript and biotinylated-probe were recovered using streptavidin covered magnetic beads. To construct partial cDNA libraries, the first strand was synthesized using AMV reverse transcriptase, and was completed with DNA polymerase I (Young and Davis, 1982). Standard procedures were used to insert the cDNA into an expression vector, λ gt-11 phage. The insertion site was into Lac Z region (β-galactosidase) of the phage, which allowed blue/white screening for successful insertions.

The cDNA libraries were screened using standard protocols (Sambrook et al., 1989). We used the S. ingentis anti-Vn antiserum, which we developed earlier, to determine which of the positive colonies contained the Vg sequence. We found a 2.9 kilobase clone in both the hepatopancreas and ovary cDNA libraries, which suggests that both tissues are responsible for synthesizing Vg (Pooyan, 1998). These data are similar to that found in Penaeus vannamei using incorporation of label-amino acids (Quackenbush, 1989a). Future studies will be conducted to determine when Vg of these two tissues is expressed and if differential expression occurs during the reproductive cycles. We are currently in the process of determining the gene sequence of Vg and will compare the sequences between the hepatopancreas and ovary to determine their similarity. Obtaining the gene sequence will allow us to develop probes that will assist us in the assessment of hormonal control of vitellogenesis in S. ingentis and provide a model system with which other hormones can be tested in other crustaceans.

SUMMARY

Determining the regulation of crustacean vitellogenesis has been a difficult and protracted task. The increase in the number descriptions of vitellogenin and vitellin has increased the ability to comprehend the differences that exist in decapod crustaceans. Using S. ingentis, we have characterized the Vn as a 322 kDa molecule that separates into three subunits. Against the purified Vn, we developed an anti-Vn antiserum that we used to develop an ELISA to detect hemolymph levels of Vg. Injections of progesterone, hydroxyprogesterone and estradiol did not increase hemolymph levels of yolk proteins in sexually quiescent shrimp. A likely reason for this result is that gonad-inhibiting hormone may prevent any oocyte development.

The introduction of molecular techniques will augment the ability to study the regulation of yolk protein synthesis. As a start, through screening cDNA libraries, we have identified a 2.9 kilobase clone both the ovary and hepatopancreas as sites of Vg synthesis. We are currently obtaining a sequence for these clones, with which we will be able to screen a number of hormones in vitro to determine the efficacy on the stimulation of Vg transcription.

Table 1. Molecular mass of crustacean vitellin

Table 2. Number of vitellin subunits in crustacean yolk proteins

Table 3. Vitellogenin levels in crustacean hemolymph

Table 4. Effects of methyl farnesoate on crustacean reproduction

Table 5. Effects of steroids in Dendrobranchiata (Decapoda)

Table 6. Site of Crustacean Vitellogenin Synthesis

1

From the Symposium Recent Progress in Crustacean Endocrinology: A Symposium in Honor of Milton Fingerman presented at the Annual Meeting of the Society for Integrative and Comparative Biology, 4–8 January 2000, Atlanta, Georgia.

Fig. 1. Yolk protein concentrations by reproductive stage. The female Sicyonia ingentis were staged and hemolymph concentrations determined using the ELISA. The Vg levels significantly rise during stage 2–4, where it peaks before significantly decreasing in stage 5, which immediately precedes ovoposition. Means (±SEM) followed by the same letter are not significantly different (1-way ANOVA, Tukey's Test @ P < 0.05). From Tsukimura et al., 2000

Fig. 2. Western Blot of Sicyonia ingentis hemolymph and vitellin. SDS-PAGE separation of yolk proteins demonstrates the presence of three subunits in female hemolymph and purified vitellin that are immunoreactive to the anti-vitellin antiserum. No immunoreactivity is observed with male hemolymph. Vn indicates vitellin, “Female” and “Male” indicate source of hemolymph, and HPT is a hepatopancreas extract. Molecular mass prestained markers are in the left column (BioRad: myosin, 203 kDa; β-galactosidase, 116 kDa; β-galactosidase, 97 kDa; ovalbumin, 45 kDa). From Tsukimura et al., 2000

The author wishes to recognize his past and current graduate students for their assistance in compiling sufficient data that permitted me to be invited to participate in this symposium: J. S. Bender, C. J. Linder, J. Oldenkamp, K. Murphy, R. Pooyan, L. G. Riley, S. Stukovsky, and A. Toste. In addition, Drs. D. W. Borst and P. M. Hopkins need to be recognized for their efforts in organizing this symposium. This research was funded in part by a grant from the National Sea Grant College Program, National Oceanic and Atmospheric Administration, U.S. Department of Commerce, under grant number NA36R-G0-537, project number 68A through the California Sea Grant College System, the California State University Research and Creative Activity Award, and College of Science and Mathematics Research Award.