The Open Zoology Journal, 2008, 1, 29-36 29 Open Access Progesterone Affects Vitellogenesis in Octopus vulgaris Carlo Di Cristo1, Marina Paolucci1 and Anna Di Cosmo*,2 1Department of Biological and Environmental Sciences, University of Sannio; Via Port’Arsa, 11 82100 – Benevento, Italy 2Department of Structural and Functional Biology, University of Naples “Federico II”; Complesso Monte Sant’Angelo, Via Cintia. 80126 – Naples, Italy Abstract: The electrophoretic analysis of egg yolk proteins of the mollusc Octopus vulgaris by both SDS- and native- PAGE, showed two different patterns that mirror the two main periods characterizing the reproductive cycle of the female of Octopus vulgaris: the non-vitellogenic period (October-February) and the vitellogenic period (March-July). Since dif- ferent levels of progesterone also characterize these stages, we performed in vitro incubations of previtellogenic ovary of Octopus vulgaris with this steroid in order to clarify its possible involvement in the regulation of egg yolk protein synthe- sis. Treatment of Octopus eggs with progesterone induced the appearance of a 70 kDa protein which cross-reacted with antibodies raised against crab vitellogenin. This protein was further identified as a component of Octopus egg yolk pro- teins of the vitellogenic period. Bromodeoxyuridine (BrdU) incorporation in the nuclei of follicle cells after treatment with progesterone brought about follicle cell proliferation, suggesting that this hormone may be involved in vitellogeneisis in Octopus. Keywords: Cephalopods, vitellogenin, cell proliferation, progesterone, reproduction. INTRODUCTION 18] and the estrogen receptor cloned in Crassostrea gigas has been immunolocalized in the nuclei of follicle cells, the Several types of sex steroids have been detected in vari- site of vitellogenin synthesis [12]. ous species of invertebrates [1-3] and evidence has been ac- cumulating over the last decades that the so called “verte- Conversely, direct evidence regarding a role for proges- brate sex steroids” (progesterone, estrogens, androgens) are terone in regulating reproductive functions in invertebrates also present in invertebrates, and their levels change accord- are scant. Information is currently available only in crusta- ing to certain phases of the life cycle often coinciding with ceans about the role of progesterone in the endocrine control reproduction [4]. In fact, “vertebrate sex steroids” are ancient of vitellogenesis. Injections of progesterone and 17OH- molecules that are widespread in vertebrates, invertebrates progesterone in the shrimp Metapenaeus ensis [19] induced and plants [5]. It has been proposed that estrogens are the ovarian maturation, and stimulated vitellogenin secretion in ligands of an ancestral steroid receptor whose specificity Penaeus japonicus [20]. In Penaeus monodon the levels of may have changed through evolution [6, 7]. A role for “ver- progesterone, as well 17-estradiol, in the hemolymph, ova- tebrate sex steroids” in the regulation of invertebrate repro- ries and hepatopancreas were closely related to the stage of duction has been disputed recently in light of the discovery ovarian maturity [21]. In Procambarus clarkii, 17- that estrogen receptors from several molluscs do not bind hydroxyprogesterone produced a significant increase in the estradiol in in vitro binding assays [8-13]. However, there is gonadosomatic index and oocyte diameter [22]. Progesterone general agreement that “vertebrate sex steroids” play a role also stimulated vitellogenin gene expression in Metapenaeus in controlling gonadal growth and sex determination in in- ensis [23]. Finally immunological characteristics and tissue vertebrates [14, 15]. localization of a putative progesterone receptor in Austro- potamobius pallipes suggest an involvement in the modula- Many studies have been carried out on the role of estro- tion of reproductive functions by progesterone in this crusta- gens in reproduction in molluscs, particularly in the control cean [24]. of vitellogenesis: in bivalves 17-estradiol is involved in vitellogenin synthesis [16] and recent studies corroborate In cephalopods, the synthesis of yolk proteins takes place estrogen involvement in controlling immune capacity and within the ovary [25-27]. Octopus vulgaris eggs (oocytes energy metabolism related to vitellogenesis [17]. In vivo and and follicle cells) are contained in a single ovary at different in vitro administration of 17-estradiol brought about an states of maturation. As a common feature in cephalopods increase in vitellins in the ovary of the Pacific oyster Cras- (squid, cuttlefish and octopus), during the annual cycle, the sostrea gigas and in the scallop Patinopecten yessoensis [16, follicle cells enfold into the oocyte and synthesize yolk com- ponents [28, 29]. Recently, the sex steroids progesterone and estradiol and their specific receptors have been identified in *Address correspondence to this author at the Department of Structural and the cephalopod Octopus vulgaris [30-32]. Functional Biology, University of Naples “Federico II”; Complesso Monte Sant’Angelo, Via Cintia. 80126 – Naples, Italy; Tel: ++39-081-679058; Morphological changes in the oviduct and oviducal gland Fax: ++39-081-679233; E-mail: [email protected] observed throughout the reproductive cycle in Octopus vul- 1874-3366/08 2008 Bentham Open 30 The Open Zoology Journal, 2008, Volume 1 Di Cristo et al. garis, along with progesterone and estradiol fluctuations, nitrocellulose was blocked for 30 min at 37°C with 5% non- suggest that both steroids might work in synergy to sustain fat dry milk in Tris-HCl 50 mM/Tween 20 (TBS-T) buffer, the growth and differentiation of the female reproductive pH 7.6 and then incubated with primary antibody anti-crab system in this species [4]. Moreover, the localization of both vitellin (85kDa subunit, kindly provided by Dr. L. Pateraki) progesterone and 17-estradiol receptors in the nuclei of the or rabbit IgG as a control for two hours at 20°C. The incuba- follicle cells in the ovary of Octopus vulgaris, sustains the tion with the secondary antibody (goat anti-rabbit alkaline theory that there is a role for these steroids in the regulation phosphate conjugated) was carried out at 20°C for 4 h. Im- of the synthesis of yolk [31, 32]. munoreactive bands were revealed by incubation with NBT/BCIP (Sigma, St. Louis, MO). Marker proteins for The purpose of studies reported here was to investigate SDS/PAGE were from Sigma: Myosin (205 kDa); ß- the role played by progesterone in Octopus vulgaris vitello- genesis. To reach this goal we performed in vitro incubations galactosidase (116 kDa), Albumin (66 kDa), Ovalbumin (45 kDa), Glyceraldehyde-3-phosphate Dehydrogenase (36 of Octopus vulgaris eggs with progesterone. Our findings kDa), Carbonic anhydrase (29 kDa). Marker proteins for indicate that progesterone induces yolk protein synthesis in native PAGE were from Sigma: urease hexamer (545kDa), Octopus vulgaris eggs. Only one protein was identified with urease trimer (272 kDa), bovine serum albumin dimer (132 immunological methods by using antibodies raised against kDa), bovine serum albumin monomer (66kDa) and chicken crab vitellogenin that specifically recognises a 70kDa pro- tein. Incorporation of bromodeoxyuridine (BrdU) in the nu- egg albumin (45 kDa). Negative controls were performed by omission of primary antibody. clei of follicle cells after progesterone exposure showed that progesterone induced cell proliferation. Egg Yolk Protein Analysis MATERIALS AND METHODS About 5 g of strands of eggs from mature females were homogenized in ten volumes of 50mM Tris-HCl, pH 7.5, Animals containing 100 mM NaCl, 50 mM MgCl2, 10 mM CaCl2, 1 Females of Octopus vulgaris (n= 40; body weight be- mM PMSF, and 0.02% sodium azide. The homogenate was tween 1,0 and 2,5 kg) were captured in the harbor of Naples. centrifuged at 7500 x g for 20 min at 4°C. After centrifuga- Sampling started in October and ended in July (at least 4 tion, the supernatant, was freed from the debris and the fat animals per month) and was carried out for two years (2006- cap and desalted on a sephadex G25 column (8 x 0.6 cm) 2007). Based on a previous study [4], females collected from equilibrated with homogenisation buffer. October to February were considered previtellogenic, and A partial purification of the egg yolk proteins was females collected from March to July were considered vitel- achieved by ultracentrifugation using the procedure of [36]. logenic. Animals were maintained, for a maximum of two Two ml of the supernatant eluate from the sephadex G25 weeks, in aquarium tanks with a recirculating seawater sys- column were mixed with 0.9 g of KBr to a final density of tem as elsewhere described [33, 34] and fed on crabs Car- –1 1.28 g ml and placed into an SW41 Ti ultra clear centrifuge cinus moenas. Animals were kept under natural photoperiod. tube. The sample was overlaid with the following densities Water temperature was set at 13-15°C. Animals were anes- –1 –1 of KBr solutions: 2 ml of 1.23 g ml , 2 ml of 1.15 g ml , thetized on ice and ovary was removed and processed as and 2 ml of 1.063 g ml–1. Five ml of 0.9% (w/v) NaCl were follows. placed on top of the gradient, and the tubes were centrifuged Tissue Preparation and Steroid Treatment at 274,000 x g for 18 h at 4°C in a Beckman (Fullerton, CA) SW41 Ti rotor. After ultracentrifugation, the gradient was Strands of eggs from the ovary of females in the previtel- collected in 1 ml aliquots. Each aliquot was assayed for total logenic period (0,5-1 g) were excised, weighed, and incu- protein content by measuring the absorption at 280 nm, and bated for 30-60 min in artificial sea water (ASW) at 20°C analysed by native- and SDS-PAGE. containing progesterone (0.2 ng/ml) or without steroid (con- trol). The steroid concentration employed for the in vitro Purification of egg yolk proteins was performed by ion incubation is 10 times higher the physiological concentra- exchange chromatography. A 10 x 0.8 cm column of CM tions reported in [4].