GENERAL

AND

COMPARATIVE

ENDOCRINOLOGY

87, 443-450 (1992)

Detection of the mRNA Encoding Vitellogenesis Inhi.biting Hormone in Neurosecretory Cells of the X-Organ in Homarus americanus by in Situ Hybridization A-M. LAVERDURE,” M. BREUZET,~ D. SOYEZ,* AND J. BECKER~ “Laboratoire de Biochimie et Physiologie du D&veloppement, Ecole Normale Supt+ieure, Paris, France; fLaboratoire de Physiologie des Insectes, Universitt Pierre et Marie Curie, Paris, France; and #Laboratoire de Diffkenciation Mole’culaire et Cellulaire du Dtveloppement, Universitt! Pierre et Marie Curie, Paris, France

Accepted February 17, 1992 Vitellogenesis

inhibiting hormone (VIH)-mRNA in secretory cells of the eyestalk of was detected by nonradioactive in situ hybridization (ISH) using two digoxigenin-tailed oligonucleotide probes deduced from the peptide sequence. Two distinct clusters of positive cells were observed in the medulla terminalis ganglionic X-organ (MGTX). Only one of them gave a strong immunoreaction after incubation with a specific polyclonal anti-VIH serum and corresponded to the conventionally described VIH producing cells. The significance of the cells reacting positively in ISH but not in immunocytochemistry (ICC) is discussed. Northern blot analysis using 32P-Iabeling confirms the specificity of the probes and indicates an approximate size of 2.5 kb for VIH mRNA. Q 1992 Homarus

Academic

americanus

Press, Inc.

In decapod crustaceans, there are many neuropeptide regulatory factors synthesized by the X-organ/sinus gland complex located in the eyestalks (see review by Webster and Keller, 1988). Thus, pigmentation is controlled by the pigment dispersing hormone (PDH) and the red pigment concentrating hormone (RPCH), and blood glucose by the widely studied crustacean hyperglycemic hormone (CHH). There are also inhibitory factors such as the molt inhibiting hormone (MIH) and the vitellogenesis inhibiting hormone (VIH). Amino acid sequences of some neuropeptides are also known for a few species. In Carcin~s menus structure of CHH and putative MIH were described by Kegel et al. (1989) and Webster (1991), respectively. In H~W~UYUS americanus, the primary structure of a peptide with both molt-inhibiting and hyperglycemic activities was established by Chang et al. (1990) and the amino acid sequence of VIH by Soyez et al. (1991). In the same

species, cDNAs encoding two CHHs were recently cloned and sequenced by Tensen et al. (1991a). In situ hybridization (ISH) was first used in arthropods for studying the, accumulation and spatial distribution of poly(A)+ RNAs in oocytes and early embryos of Drosophila melanogaster (Kobayashi et al., 1988). ISH facilitates the localization, of mRNAs coding for neuropeptides and has been applied, for example, to bombyxin in the silkmotb Bombyx mori (Kawakami et al., 1990). In crustaceans, ISH detected mRNA encoding the crustacean hyperglycemic hormone (CHH) in eyestalks of Orconectes limosus (Tensen et al., 199lb). To’elucidate the mechanism and regulation of prohormone synthesis in H. americanus, two synthetic oligonucleotidic probes deduced from the peptide sequence have been,used to locate VIH-encoding mRNA. The partial sequences selected for constructing the probes have no homologies with other crus-

443 00166480192 $4.00 Copyright Q 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

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LAVERDURE

tacean neuropeptides described so far (see above). VIH secreting cells have been studied using ISH coupled with immunocytochemistry (ICC) and Northern blot analysis. MATERIAL

AND METHODS

Tissue Processing for Histological Reactions All tissues were dissected from adult American lobsters H. americanus originating from the coast of New Brunswick (Canada). For ICC and ISH, the eyestalks were sectioned at their bases from living females and immediately dissected out and the nervous tissue was immersed in the modified Bouin’s fixative Tensen et al. (1991a). The fixed organs were dehydrated and embedded in paraffin. One 7+m section out of eleven was used for ICC. Ten others were mounted in a 0.2% gelatin (Sigma)/O.OS% chrome-alum autoclaved solution on slides treated with 2.5 p,g/ml poly-L-lysine (Sigma) and used for ISH. After drying for 24 hr at 37”, the sections were deparaffinized in xylene (twice for 5 min each), hydrated, and used for ICC or ISH.

Immunocytochemistry A polyclonal antiserum raised against purified lobster VIH was produced in guinea pigs. In control tests of specificity showing a light cross-reactivity with CHH, the antiserum was absorbed by this peptide to improve specificity (Meusy and Soyez, 1991). Immunocytochemical detection of VIH was performed by a procedure modified from Kallen and Meusy (1989). After hydration in Tris buffer solution (TBS) (50 mM Tris/200 mM NaCl, pH 7.6) for 15 min, sections were treated with absolute methanol containing 0.3% H,O, to eliminate endogenous peroxidase. For the blocking reaction, slides were placed for 30 min in a 3% solution of normal rabbit serum (NRS) and 0.5% Triton X-100, then rinsed in TBS for 15 min. Incubation with specific antiserum was carried out overnight at 4” in TBS containing 1% NRS and I:250 anti-VIH absorbed serum. Rabbit anti-guinea pig peroxidase (Sigma) was used diluted 1:SOOwith 1% NRS in TBS at 37” for 75 min. 3,3’-Diaminobenzidine-4HCl (DAB) (Sigma) was used as a substrate (sol. A, 10 mg DAB in 40 ml 50 mM Tris/HCl, pH 7.6; sol. B, 150 mg ammonium-nickel sulfate in 5 ml 50 mM Tris/HCl, pH 7.6); 40 ul 30% H,O, was added to 12 ml sol A + 4 ml sol B. Distilled water was used to remove excess substrate before mounting slides without additional staining.

In Situ Hybridization All the solutions used in the course of these exper-

ET AL. iments were prepared with diethyl pyrocarbonate (DEPC, Sigma) treated distilled water before sterilization (Maniatis et al., 1989). After hydration in 100 mM phosphate buffer, pH 7.2, sections were postfixed with 2% paraformaldehyde in 10 mM Na phosphate/O.l% KCl/O.I% NaCl, pH 7.4 (PBS), then rinsed in PBS containing 1% hydroxylammonium chloride for 4 min, 15 min in PBS, and quickly in distilled water before dehydration and drying for 1 hr. The digoxigeninlabeled VIH probes (see below) were diluted to a final concentration of 5 ng/pl (~0.5 pJ4) in hybridization buffer containing 50% deionized formamide, 4X saline sodium citrate (SSC) (SSC 20x: 3M NaCII0.3 M sodium citrate, pH 7) 1x Denhardt’s [Denhardt’s 100x: 1% Ficoll (Sigma), 1% polyvinylpyrrolidon (Sigma), 1% bovine serum albumin (Sigma)], 10% dextran sulfate, 100 &ml tRNA (Boehringer-Mannheim) prepared according to Maniatis (1989), 0.8% sarcosyl in DEPC distilled water. Twenty microliters of this mixture was placed on each section and the slides were protected by a coverslip. Incubation was carried out for 16 hr (overnight) at 42” in a moist chamber. All the following steps were performed at room temperature unless otherwise specified. Slides were washed with decreasing concentrations of SSC (2~ for 1 hr; IX for 1 hr 0.5~ at 37” for 30 min; 0.5~ for 30 min) before immunological reaction using the Boehringer-Mannheim Detection Kit with respect to the standard procedure. After washing the slides in PBS for 5 min, sections were incubated with 2% normal sheep serum (NSS), 0.3% Triton X-100 in PBS for 30 min. The antibody conjugate, diluted 1:500 with PBS containing 1% NSS and 0.1% Triton X-100 was applied to sections. After 16 hr incubation in a moist chamber, the slides were rinsed with PBS for 10 min, then treated with 100 mMTris/lSO mMNaC1, pH 7.5, for 10 min. After washing in 100 mM TrisllOO r&f NaCI, pH 9.5, a solution containing 0.45% nitroblue tetrazolium (NBT) solution and 0.34% 5-bromo-4-chloro-3-indolyl phosphate, toluidinium salt in dimethylformamide (XPhosphate) solution was applied to the slides and kept in darkness for 6 hr. The reaction was stopped by 0.5% of the blocking reagent in PBS, pH 7.5. Slides were conventionally dehydrated, placed in xylene (twice for 5 min each), and mounted in Eukitt (Prolabo). Northern Blot Analysis. For the Northern blot analysis, nervous tissue including the sinus gland was dissected from freeze-dried eyestalks. All the solutions used were prepared with distilled water and autoclaved. RNA extraction. Total RNA was extracted from 50 lyophilized eyestalks by the 3 M LiCl/6 M urea procedure (Aufmy and Rougeon, 1979; Roskam and Rougeon, 1979). After homogenization in 1 ml of a cold solution of 10 m&4 Tris/S mM EDTA, pH 7.5, containing 1% SDS, 6 ml of the LiCVurea solution was progressively added. After 10 hr at 4”, the homogenate was centrifuged at 20,OOOgfor 30 min in a refrigerated

VIH-mRNA

IN Homarus

Sorvall RC 28 centrifuge and kept at -20” for 1 hr. After discarding the supematant, the tube was immersed in a water-bath at 68” and the pellet was extracted with an equal volume of 100 mM Tris/O.S% SDS, pH 9, and phenol saturated in distilled water. The aqueous phase was collected and reextracted. The phenol phase was retreated in Tris/SDS, pH 9. After centrifugation, the aqueous phases were combined and the RNA precipitated overnight at -20” in 2.5 vol of cold absolute ethanol/O.1 vol of 3 M sodium acetate, pH 4.8. After two similar precipitations in ethanol/ acetate, and dissolutions in sterile water, the RNAs were quantified by spectrophotometry, aliquoted, and stored at - 20” until use. After each RNA extraction, a conuol electrophoresis in 1% agarose slab gel in denaturing conditions was performed. RNA blot analysis. Total RNA (150 pg) and total RNase A-treated RNA (150 pg) (Maniatis et al., 1989) were run simultaneously overnight under denaturing conditions in a 1% agarose formaldehyde gel. After washing in a 50 mM NaOHilO mM NaCl for 45 min, the gel was successively treated for 45 min by 2 mh4 Tris and for 1 hr in 20x SSC, then transferred by contact to a nitrocellulose membrane (Gelman Sciences) which was baked for 1 hr 15 min at SO”. Hybridization was performed followed Maniatis ef al. (1989). The filter was first prehybridized at 42” under weak shaking for 4 hr in a solution containing 50% deionized formamide, 5X SSC, 10X Denhardt’s 20% 500 mMphosphate buffer, pH 6.5, and 20% DNA from herring sperm (5 mg/ml) (Boehringer-Mannheim). After prehybridization, the membrane was incubated at 42” for 24 hr in a buffer containing 50% deionized formamide, 5~ SSC, lx Denhardt’s, 8% 500 mM phosphate buffer, pH 6.5,40% competitor DNA, 10% dextran sulfate and the probes were prepared as described below. The filter was washed three times for 10 min in 2X SSC/O.l% SDS at room temperature, then twice for 20 min in 0.2~ SSC/O.l% SDS at 5.5”. All washings were performed under weak shaking. Finally, the dried membrane was subjected to autoradiography (Amersham, Hyperfilm-MP, RPN 6). After exposure (4 days), the fiim was developed in Kodak solution LX24.

Probe Labeling Two partial amino acid sequences selected along the VIH sequence (from amino acid residue 7 to 15 and 46 to 53) were used to deduce oligonucleotidic structures A and B, respectively (Fig. 1). The probe A (26-mer, 16-fold degenerated) presents a molecular weight of 8643 Da and a GC content of 15. Probe B (23-mer, ldfold degenerated) has a MW of 8114 Da and a GC cantent of 11. The two oligonucleotides were synthesized in the Organic Chemistry Unity, Department of Biochemistry and Molecular Genetics, Pasteur Institut. Paris (Pr. Igolen).

Probe

445

americanus

A

Protein

46

53

-Asp-Cys-Phe-His-Thr-Met-Tip-PhemRNA

5’

GAU-“~U-~U-~‘-~~-~“~-~~~-~ c c c c

3’

FIG. 1. Partial sequences of Homarus americanus VIH, the corresponding segments of the mRNA, and the two synthetic oligonucleotides: Probe A (26-mer, 16-fold degenerated) and probe B (23-mer, 16fold degenerated) were used in this study. For ISH, oligonucleotides were labeled at the 3’ end with digoxigenine-1ldUTP using Klenow enzyme, according to the standard procedure (BoehringerMannheim). Briefly, to 1.1 yg oligonucleotide (=130 pM probe A and ~135 pM probe B) dissolved in 5 ~1 distilled water were added 2 )11 hexanucleatide mixture, 2 pl dNTP labeling solution, 10 pl sterile distilled water, and 2U Klenow enzyme. The reaction was performed for 2 hr at 37” and stopped by addition of 2 ~1 200 mM EDTA, pH 8. The labeled oligonucleotides were used without purification and gently dissolveti in the hybridization buffer at a concentration of 2 ng/wl. Of this buffer, 50 l.~l was applied per section. Control DNA, 1 pg of pBR328 linearized with EcoRI, was Pabeled in the same manner. The labeled control bNA was purified by precipitation in alcohol/acetate and centrifuged before solubilization in hybridization buffer at a concentration of 2 rig/ml, denahuated by heating at 95” for 2 min, and applied to contra1 sections as described above. For Northern blot analysis, oligonucleotides were labeled at the 5’ end with 32P-ATP using T4 polynucleotide kinase (Boehringer-Mannheim). To 300 ng oligonucleotide (-35 pit4 probe A and -40 pM’ probe B) dissolved in 1 pl sterile distilled water were added.5 p,l of 10x 500 mM TrisllOO mM MgCl,/SO mM dithiothreitol, pH 7.8, 20 &i, 3ZP-ATP (3000 CiimM), 2U enzyme for a total volume of 50 ~1. After incubation for 3 hr at 37”, the reaction was stopped by heatizlg at 75” for 5 min, and 10 p+l of the color solution (TE IX : 10 mM T&/IO mM EDTA, pH 8, containing O&J% bromphenol blue and 5% dextran blue) was added. Radiolabeled probes were separated from unincorpo-

446

LAVERDURE

rated ATP by gel filtration (Biogel P-10) and eluted in TE IX with the dextran blue. Radiolabeling was determined on a 1 ~1 aliquot and the specific activity was calculated to be close to 3.6 X 10’ dpming. Total activity used was approximately lo6 dpmiml hybridization mixture.

RESULTS Immunocytochemistry Immunological characterization of the VIH secreting cells is illustrated in Fig. 2a. Staining with CHH-adsorbed anti-VIH antibody demonstrates an immunopositive reaction in a group of cells located in the MTGX. A total of 20 +- 2 cells can be counted in each eyestalk nervous tract. Intensely stained axons are present close to the cells and can be traced toward the sinus gland. The perikarya are strongly stained while the nuclei appear free of a reaction product (Fig. 2b). Between and close to the positive cells, there are large unstained cells with large nuclei. The sinus gland contains intensely stained material located at the periphery of the nervous tract. In Situ Hybridization Immunoreactive cells observed hybridize strongly with the two probes, as demonstrated in Fig. 2c with probe B. Using separately the two nonradioactive tailed probes, the label is located over the cytoplasm and the nucleolus (Figs. 2d and 2e). Neither the axons nor the sinus gland is stained. When the cells are incubated without probe in hybridization buffer, or with a

ET AL.

random control probe (Fig. 2f), or after RNase treatment before applying the probes (Fig. 2g), no hybridization signal was observed. Another cluster of cells reacts positively with the both probes, distinct from the immunoreactive cells (Fig. 2h and Fig. 3). The specificity of the hybridization is supported by the absence of labeling after control treatments. Northern

Blot Analysis

For Northern blot analysis, a mixture of the two radioactive labeled probes was used. As shown in Fig. 4, one hybridization band was detected in total RNA extracted from lobster eyestalks. By comparison with migration distance of standard DNA, an approximate size of 2.5 kb could be attributed to the radioactive spot. DISCUSSION A specific polyclonal antiserum produced in a guinea pig against H. americanus VIH allows the description of immunoreactive cells in the MTGX. These cells correspond to the VIH cells described by Kallen and Meusy (1989) who used a mouse polyclonal antiserum to demonstrate more cells than in the present study. This difference may be due to antisera reactivities or to the physiological stages of the lobsters. On our slides, some unstained cells were observed close to the VIH cells. They likely correspond to the CHH secreting cells described by Kallen and Meusy (1989).

FIG. 2. (a) Immunohistochemical localization of VIH producing cells (VIH). Ax: intensely stained axons. Transverse section of the eyestalk at the MGTX level. x250. (b) Immunohistochemical localization of VIH cells (VIH). CHH: putative CHH producing cells. x600. (c) In situ hybridization with the cDNA (probe B) encoding part of VIH (arrows). {a) and (c) present two successive sections. x250. (d) Partial enlargement of Fig. 2c. Most label is located over the cytoplasm (C). In the nucleus (N), nucleolar material appears stained. X600. (e) In situ hybridization in the same cells as in Fig. 2d using probe A. x600. (f) Control experiment with a random tailed cDNA (pBR328) No label occurs. x250. (g) In situ hybridization with probe B after RNase treatment: no staining can be observed. x600. (h) In situ hybridization with probe B showing a labeled group of cells which do not react with the anti-VIH antiserum. They are distinct from the cells shown in Figs. 2c, d, e, but located at the same eyestalk level. x 600.

VIH-mRNA

IN Homarus

americanus

447

448

LAVERLIURE

FIG. 3. Diagram of a transverse section of the medulla terminalis. Circles indicate VIH-immunoreactive and in situ hybridized cells. Triangles indicate the opposite group of cells which react only in ISH. NF, nervous fibers; SG, sinus gland; Ax, VIH axons.

Radioactive labeling is currently used but several nonradioactive labeling techniques have been recently described and especially use digoxigenin-1 l-dUTP as a probemarker (Heiles et al., 1988; Tensen et al., 1991b). By ISH, the two oligonucleotides tagged with digoxigenin-1 l-dUTP generate

2,5kb

FIG. 4. Northern hybridization analysis of eyestalk mRNA. Total RNA RNase-treated (150 (18) (lane 1) and total RNA (150 pg) (lane 2) were electrophoresed and blotted for hybridization with “P-labeled synthetic oligonucleotides. Specific activity: 3.6 10’ dpmi pg. Total radioactivity: 10.9 lo6 dpm/ml hybridization buffer. kb, kilobases.

ET AL.

specific hybridization signals on thf perikarya of the VIH cells as demonstratec by ICC. The interesting observation of E nucleolar labeling indicates that the nucle. olus may play a role in the metabolism 01 mRNAs as suggested previously (Meyer er al., 1986). A difference in the intensity 01 the reaction was observed between the two cDNA probes, probably due to a difference in the labeling efficiency of the probes. The specificity of the probes has been checked by control experiments: use of a randomly tagged probe did not produce any staining reaction. RNase treatment of the sections before hybridizations abolished the labeling, and absence of probe in the buffer did not result in any hybridization signal. In Northern blot analysis, relatively high RNA concentrations were necessary in order to obtain a detectable spot. This can be explained by a low abundance of VIHmRNA in the cells resulting from a low transcription rate. Presence of a single spot suggests the existence of only one population of mRNA encoding the peptide. The estimated size of the VIH-mRNA (2.5 kb) agrees with the value (3 kb) obtained for CHH cDNA by Weidemann et al. (1989). In the MTGX, another group of hybridizing cells, distinct from the VIH secreting cells, were revealed in the ISH experiments. This may be a nonspecific reaction. Short cDNA probes although presenting a good penetration into the neurosecretory cells (Bloch, 1985) are less specific than the larger ones. On the other hand, formamide has been considered an unstable reagent sometimes giving adverse reactions including nonspecific staining (Koji and Nakane, 1990). However, in this case, staining should be widespread and not located just in a particular group of cells. These cells do not correspond to the CHH cells localized close to the VIH ones as described by Kallen and Meusy (1989). Several hypotheses may explain this observation: (1) These cells may contain little or no translated VIH mRNAs. A low accumulation of

VIH-mRNA

IN Homarus americanus

VIH in the cytoplasm, below the detection level, was not seen by ICC analysis. (2) They can synthesize a peptide as yet not sequenced that shares at least two sequence homologies with VIH. This hypothetical peptide would not present the epitope(s) allowing the immunological recognition by anti-VIH antibodies, since these cells were not immunoreactive in the present experiments, but the mRNAs coding for this molecule would be recognized by the cDNAs in the in situ hybridization experiments. The two probes used strictly hybridize with parts of VIH mRNA sequence. In the H. americanus X-organ, neurosecretory cells stained with both anti-VIH and antiCHH have been described by Kallen and Meusy (1989). This result suggests a synthesis of both peptides in the same perikarya, encoded by one primary RNA transcript or originated from a common precursor. It will be of interest to determine if such a colocalization can be reproduced at the nucleic acid level, using a CHHderived probe coupled with VIH-derived probes. ACKNOWLEDGMENTS We thank Dr. D. Sellos for his help in the preparation of the probes, Dr. J-G. Fournier, Pr. R. Lafont, and Pr. I. W. Henderson for their critical reading of the manuscript.

REFERENCES A&fray, C., and Rougeon, F. (1979). Purification of mouse immunoglobulin heavy chain messengers RNAs from total myeloma tumor cells. Eur. J. Biochem. 107, 303-307. Bloch, V. (1985). L’hybridation in situ: Methodologie et applications a l’analyse des phenomenes d’expression genique dam les glandes endocrines et le systeme nerveux. Ann. Endocrinol. 45, 253-261. Chang, E. S., Prestwich, G. D., and Bruce, M. J. (1990). Amino acid sequence of a peptide with both molt-inhibiting and hyperglycemic activities in the lobster, Homarus americanus. Biochem. Biophys. Res. Commun. 171, 818-826.

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Heiles, H. B. J., Genersch, E., Kessler, C., Neumann, R., and Eggers, H. 3. (1988). In situ hybridization with digoxigenin-labeled DNA of human papillomaviruses (HPV 16118) in HeLa and SiHa Cells. Biotechniques 6, 978-980. Kallen, J., and Meusy, .I. J. (1989). Do the neurohormones VIH (vitellogenesis inhibiting hormone) and CHH (crustacean hyperglycemic hormone) of crustaceans have a common precursor? Immunolocalization of VIH and CHH in the x-organ sinus gland complex of the lobster, Nomarus americanus. Invert. Reprod. Dev. 16, 43-52. Kawakami, A., Kataoka, H., Oka, T., Mizoguchi, A., Kimura-Kawakami, M., Adachi, T., Iwami, M., Nagasawa, H., Suzuki, A., and Ishizaki, H. (1990). Molecular cloning of the Bombyx mopi prothoracicotropic hormone. Science 247, 13331335. Kegel, G., Reichwein, B., Weese, S., Gaus, G., PeterKatalinic, J., and Keller, R. (1989). Ammo acid sequence of the crustacean hyperglycemic hormone (CHH) from the shore crab, Carcinus mcenas. FEBS Lett. 255, 10-14. Kobayashi, S., Mizuno. H., and Okada, M. (I%$). Accumulation and spatial distribution of poly(A)+RNA in oocytes and early embryos of Drosophila melanogaster. Dev. Growth D@er. 30, 251-260. Koji, T., and Nakane, P. K. (1990). Use of nucleotides as an alternative to formamide in non-radioactive in situ hybridization. Acta Histochem. Cytochem. 23, 327-334. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1989). “Molecular Cloning. A Laboratory Manual,” 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Meusy, J. J., and Soyez, D. (1991). Immunological relationships between neuropeptides from the sinus gland of the lobster Homarus americanus, with special references to the vitellogenesis inhibiting hormone and crustacean hyperglycemic hormone, Gen. Comp. Endocrinol. 81, 410-418. Meyer, J. L., Fournier, J. G., and Bouteille, M. (1986). Expression of integrated hepatitis B virus DNA in PLC/PFR/S, Hep 3B, and L6EC3 cell lines detected by in situ hybridization. Med. iSol. 64, 367-371. Roskam, W. G., and Rougeon, F. (1979). Molecular cloning and nucleotide sequence of the human growth hormone structural gene. Nucleic Acids Res. I, 305-320.

Soyez, D., Le Caer, J. P., Noel, P. Y I, and Rossier, J. (1991). Primary structure of two isoforms of the viteilogenesis inhibiting hormone from the lobster Homarus americanus. Neuropeptides 20, 25-32. Tensen, C. P., de Kleijn, D. P. W., and Van Herp, F.

STEROIDS

IN Asterias

(Licht, 1972; Pieau et al., 1982), and may have similarly affected steroidogenic processes in Asterias vulgaris. Therefore, we will discuss the 1988-1989 gametogenic cycle as representing the “typical” cycle. Transient, seasonal increases in steroid levels observed in the present study coincided with specific transitions in germinal cell populations. The beginning of a new spermatogenic cycle in Asterias vulgaris was characterized by an increase in the testicular index (Lowe, 1978; Walker, 1980; Smith and Walker, 1986; Watts et al., 1990a) and by changing levels of testicular sex steroids. Estradiol increased in the fall concomitantly with mitotic proliferation of previously quiescent spermatogonia. Testosterone increased approximately 1 month after the apparent proliferative response of spermatogonia to estradiol. These increases in testosterone coincided with the beginning of differentiation of spermatogonia into columns of primary spermatocytes. Approximately 1 month after the increases in testosterone, an increase in progesterone occurred and was coincident with the onset of spermiogenesis. During the winter and early spring all steroid concentrations remained low in the testes while testicular mass continued to increase as spermatozoa differentiated and were stored in the testicular lumen. These data suggest that increased concentrations of steroids, which may have been important for the initiation of spermatogonial proliferation, differentiation, and spermiogenesis, may not be required for maintenance of spermiogenesis. Later in the spring increases in the concentrations of steroids in the testes of A. vulgaris (most notably progesterone and testosterone) occurred just prior to spawning. The increased concentrations of steroids may be important for the preparation of testes for spawning as most spermatozoa are fully differentiated at that time. In the late spring spawning occurred, all steroids decreased, and the testes returned to an inactive state. The low,level of steroids together

vulgaris

457

with the relative inactivity of the testes during this aspermatogenic ‘period support the hypothesis that transient increases in steroid concentrations may regulate spermatogenie events in A. vulgaris. The levels of testicular sex steroids in two other sea stars also change during the annual gametogenic cycle. Similar to Asterias vulgaris, increases in the levels of estrone and progesterone, determined by RIA, marked the beginning of a new gametogenic cycle in male S. mollis (Xu, 19901, In A. rubens (Voogt and Dieleman, 19841, highest levels of progesterone and estrone, also determined by RIA, occurred after the onset of testicular growth. However, a significant increase in testicular growth ensued, suggesting that these hormones were important for gametogenesis. The function of sex steroids in the testes of sea stars may be comparable with that of vertebrates where testicular sex steroids are associated primarily with the regulation of spermatogenesis (Mainwaring et ail., 1988). In particular, increases in the levels of testosterone in testes of Asterias vulgaris coincide with the beginning of testicular growth, indicating a potential regulatory (or stimulatory) function similar to the testes of vertebrates (Mainwaring et al., 1988). Holmes et al. (1986) demonstrated that Sertoli cells in the rat secrete a potent mitogen which is regulated by testosterone. Since somatic cells are present in testes of A. vdgaris, and are in many ways similar to Sertoli cells of vertebrates (Walker, 19881, increased levels of testosterone at the begjnning of spermatogenesis may similarly influence germinal cell proliferation in the sea star. The onset of gametogenesis in female Asterias vulgaris occurred in the fall ‘with an increase in ovarian indices accompanied by increased levels of sex steroids. In: partieular, increases in the levels of estradioi l~oincided with the apparent proliferation of oogonia and early stages of vitellogenesis. Testosterone levels increased also at t,his

Detection of the mRNA encoding vitellogenesis inhibiting hormone in neurosecretory cells of the X-organ in Homarus americanus by in situ hybridization.

Vitellogenesis inhibiting hormone (VIH)-mRNA in secretory cells of the eyestalk of Homarus americanus was detected by nonradioactive in situ hybridiza...
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