JOURNAL OF EXPERIMENTAL ZOOLOGY 289:33–47 (2001)

© 2001 WILEY-LISS, INC.

Sex Steroid Hormone Fluctuations andMorphological Changes of the ReproductiveSystem of the Female of Octopus vulgarisThroughout the Annual Cycle

ANNA DI COSMO,1* CARLO DI CRISTO,1 AND MARINA PAOLUCCI2

1Department of Zoology, University of Naples, Federico II, 80134Napoli, Italy

2Faculty of Science, University of Sannio, 82100 Benevento, Italy

ABSTRACT Sex steroids (17β-estradiol and progesterone) and morphological variations of thereproductive system of the female of Octopus vulgaris from the Bay of Naples were followed over aperiod of 2 years. The increase in the ovary weight was independent of body weight as demon-strated by the gonado-somatic index (GSI). Both 17β-estradiol and progesterone have been de-tected in the ovary of O. vulgaris, and their concentrations changed in correlation with the ovariandevelopment. No 17β-estradiol or progesterone was found in the hemolymph. 3β-Hydroxysteroiddehydrogenase activity has been detected in the ovary, indicating that in the female of O. vulgaristhe reproductive system is a source of sex steroid hormones. According to the morphological changesof the ovary, the ovarian cycle can be divided into the following phases: previtellogenesis; earlyvitellogenesis, full vitellogenesis and late vitellogenesis. The morphological changes of the ovi-ducts and oviducal glands throughout the reproductive cycle were in accordance with their role inthe transport and secretion of gelatinous coat covering the eggs, as well as in sperm storage andsperm reactivation during fertilization. J. Exp. Zool. 289:33–47, 2001. © 2001 Wiley-Liss, Inc.

The reproductive strategy of the female of Oc-topus vulgaris, like many cephalopods, consists ofa single spawning over a short period of time nearthe end of the life (Mangold, ’87). The ovary, theoviducts, and the oviducal glands enlarge assexual maturity approaches. The ovary is a singlestructure, which contains oocytes at different ma-turity stages (Wells, ’78). Ovarian maturation hasbeen demonstrated to be under the control of theoptic glands, which are essential for the develop-ment and maturation of the oocytes during thelast phase of ovarian maturity, when yolk is pro-duced by follicle cells and taken up by growingoocytes (Wells and Wells, ’75). Eggs are attachedindividually to shelters by means of oviducalglands secretion and are protected by females. Asa rule, females die after eggs hatch (Boletzky, ’81),showing sexual behavior of high complexity, as isobserved with other cephalopods (Sauer et al., ’97).Although there are several reviews on the struc-ture of the reproductive system in the main or-ders of cephalopods (Fields, ’65; Arkhipkin, ’83),a detailed description of the reproductive system,its development, and function has been reportedonly for decapods such as the squid Loligo opale-

scens and Thysanoteuthis rhombus and cuttlefishSepia officinalis (Thompsett, ’39; von Boletsky, ’83;Kueta et al., ’93). Among octopuses, a general de-scription of the male and female reproductive sys-tem is available only for O. vulgaris (Froesch andMarthy, ’75; Guerra, ’75; Wells, ’78; Smale andBuchan, ’81; Arkhipkin, ’92), Octopus dofleini mar-tini (Mann et al., ’70), and Octopus bimaculoides(Peterson, ’59), while more extensive informationon maturity stage determination is available onlyfor Eledone cirrhosa (Boyle and Chevis, ’91, ’92),Octopus mimus (Olivares et al., ’94), and Eledonemoschata (Ezzeddine-Najai, ’97).

Recently, sex steroid hormones (17β-estradiol,progesterone, androgens), sex binding proteins,and 3β-hydroxysteroid dehydrogenase, a key en-zyme in steroidogenesis, have been reported in thereproductive system of the male of O. vulgaris (Di

Grant sponsor: MURST.*Correspondence to: Anna Di Cosmo, Department of Zoology, Uni-

versity of Naples, Federico II, via Mezzocannone 8, 80134 Napoli,Italy. E-mail: dicosmo@unina.it

Received 9 January 2000; Accepted 7 June 2000

34 A. DI COSMO ET AL.

Cosmo and Di Cristo, ’96; D’Aniello et al., ’96).17β-Estradiol and progesterone receptors havebeen characterized and immunolocalized in thereproductive system of the female of O. vulgaris(Di Cosmo et al., ’98a,b). Such findings supportthe hypothesized parallelism with the known hor-monal regulation system operating in vertebrates(Wells and Wells, ’59, ’75). This view is also sus-tained by the observation that, in the male of O.vulgaris, gonad may be a source of sex steroid hor-mones (D’Aniello et al., ’95). The parallel betweenthe reproductive physiology of O. vulgaris and ver-tebrates is not a recent discovery. Indeed, back in1959, Wells and Wells identified the optic glandsin O. vulgaris as the endocrine structures involvedin the gonadal maturation and hypothesized thatthey are under the control of the central nervoussystem (CNS). In favor of such a hypothesis, twoneuropeptides, the gonadotropin releasing factor(GnRH), known to be involved in the modulationof pituitary in vertebrates (King and Millar, ’95),and the FMRF amide, a molluscan neuromodu-lator (Cottrel et al., ’83, ’84), have been localizedin the nervous lobes that control the activity ofthe optic gland in O. vulgaris (Di Cosmo and DiCristo, ’98).

In this paper we show the presence of 17β-es-tradiol, progesterone, and 3β-hydroxysteroid de-hydrogenase in the ovary of the O. vulgaris. Ourfindings suggest a possible correlation between sexsteroid hormones and the morphological variationsof the reproductive system (ovary, oviducts, andoviducal glands) in this species during the repro-ductive cycle.

MATERIALS AND METHODSAnimals

About 100 females of O. vulgaris of the harborof Naples were collected from October to July dur-ing 2 years (1996–1998). In O. vulgaris from theharbor of Naples, eggs are laid in June and fe-males die about a month after the deposition. To-tal body weight and fresh gonad weight weremeasured in all specimens. The ratio between theovary weight and the body weight is referred toas gonadal somatic index (GSI). After capture, fe-males were maintained in aquarium tanks witha recirculating seawater system as described byLickey et al. (’70) and Moliner and Cakaza (’84)and under natural photoperiod (8:16, L:D) for amaximum of 5 days. Water temperature was setat a value similar to the one of the water in theharbor of Naples (13–15°C during winter months

and 19–20°C during summer months). Animalswere anesthetized on ice. Soon after the sacrificethe ovary, the proximal and distal regions of theoviducts, and oviducal glands were extracted andprocessed separately. Hemolymph was drawn bya 1-mm capillary inserted into the dorsal aortaand centrifuged at 800g for 15 min at 4°C. Thepellet was discharged, and the supernatant wascentrifuged at 105,000g for 4 hr at 4°C to elimi-nate the hemocyanin. At the end of centrifuga-tion the supernatant appeared to be composed ofthree layers of different color. Only the upperlayer, which was transparent, was collected andstored at –70°C until use.

Tissue preparation for histologyThe ovaries, the proximal and the distal portion

of the oviducts, and the oviducal glands were fixedin 4% paraformaldehyde in sea water, dehydrated,embedded in Histowax (Reichert-Jung, GmbH, Ger-many) and sectioned at 7 µm, mounted on coatedslides, and stained with hematoxylin/eosin.

From females caught in October and in July,a small piece of the ovary was collected andsoaked in 30% sucrose in sea water at 4°C, fro-zen, and sectioned by cryostat in slices of 10–15µm. Slices were coated on slides pre-treated withBio-Bond (Biocell) and dried for 5 min at roomtemperature.

Enzymatic histochemistry of 3b-hydroxysteroid dehydrogenase

Slices of ovary were washed in 0.1 M cacody-late buffer, pH 7.4, for 10 min at 4°C and then for5 min at room temperature. Slices were incubatedat 37°C for 1–3 hr in a reaction mixture consist-ing of 0.1 M cacodylate buffer, pH 7.4, containing0.2 µg/ml of steroid DHA, dissolved in diethylether, 0.5 mM βNAD, and 0.15 mM nitro-BT tet-razolium salt. Sections were then washed in wash-ing buffer, fixed for 10 min in formaldehydevapors, and mounted in glycerin. Controls wereroutinely run in all experiments omitting DHA,βNAD, or both.

Tissue extraction for radioimmunoassayOvaries were weighed and extracted with a mix-

ture of methanol and distilled water (70–30%). Tis-sues were homogenized with a glass potter andincubated at 75°C overnight. The next day sampleswere centrifuged at 2,000g for 30 min at room tem-perature, and supernatants collected and storedat 4°C. The radioimmunoassay was performedwithin 24 hr.

SEX STEROID LEVELS AND SEXUAL CYCLE IN O. VULGARIS 35

RadioimmunoassaySteroid (17β-estradiol and progesterone) concen-

trations in hemolymph and ovary extracts weremeasured by radioimmunoassay. The followingsensitivities were registered: 17β-estradiol, 5 pg(intrassay variability, 9%; interassay variability,13%); progesterone, 7 pg (intrassay variability, 8%;interassay variability, 10%).

The antisera were provided by G. Bolelli (Phys-iopathology of Reproduction Service, University ofBologna, Italy). Tritium-labeled steroids were pur-chased from Amersham International (Bucking-ham, England), and unlabeled steroids wereobtained from Sigma (St. Louis, MO).

STATISTICSAll data are expressed as mean ± SD. Data were

statistically analyzed by one-way ANOVA followedby the Duncan’s test.

RESULTS17b-Estradiol and progesterone in the

ovary and hemolymphThe concentrations of 17β-estradiol and proges-

terone in the ovary fluctuated throughout the year

and showed parallel profiles (Fig. 1). 17β-Estra-diol levels were always lower than those of proges-terone. Both steroids showed the lowest levelsfrom October to April and reached a peak in May(P < 0.01), which was followed by a decrease inJune. Neither 17β-estradiol nor progesterone wasdetected in the hemolymph throughout the repro-ductive cycle.

General morphology of thereproductive system

Female O. vulgaris have a single ovary with twooviducts opening on either side of the midlineabout halfway along the mantle cavity. The ovi-ducal gland lies about halfway along the oviductwhich is divided into two regions: the proximaloviduct, stretching from the ovary to the oviducalgland and the distal region, from the oviducalgland to the opening into the mantle cavity (Wells,’60; Wells and Wells, ’75; Di Cosmo et al., ’98).

Annual variations of the ovaryIn the months of October, November, and De-

cember the ovary contained small oocytes with adiameter between 80 and 110 µm developing fromthe germinal epithelium which was much en-

specimens analyzed was as follows: October = 4; November =4; December = 3; January = 3; February = 3; March = 3;April = 3; May = 8; June = 5; July = 3. Asterisks over barsindicate statistically significant values.

Fig. 1. 17β-Estradiol (9) and progesterone (9) levels inthe ovary and ovary weight (—) of O. vulgaris throughoutthe annual cycle. Values are reported as mean ± SD. Theovary weight values are reported as mean. The number of

36 A. DI COSMO ET AL.

folded. The oocytes remained attached by stalksto the underlying connective tissue, hanging inbunches from the anterior wall of the ovisac (Fig.2A). Oocytes were either spherical or ovoidal, andthe nucleus was spherical and centrally placedinto the dense, finely granular ooplasm and con-tained an eccentric nucleolus. Rarely was theremore than one nucleolus. Oocytes were sur-rounded by a double layer of follicle cells: the in-ner layer was cuboidal and secretory, the outerlayer was thin and flattened epithelium (the oo-cyte and the associated follicle cells are definedas follicle) (Fig. 2B). In the months of Januaryand February, the nucleus in the oocyte wasspherical and centrally placed, with a scarcely vis-ible nucleolus. Oocytes were surrounded by cuboi-

dal follicle cells. However, in this period the fol-licle cells start enfolding into the ooplasm. At thisstage 70% of oocytes did not show any fold, 3%showed three folds, 20% showed four folds, and4% and 3% were respectively characterized by fiveand six folds. Oocytes with folds showed a diam-eter of between 110 and 200 µm. The ooplasm wascompact in those oocytes without folds and withthree folds and was vacuolated in the oocytes withfour, five, and six folds. An extensive blood capil-lary system enveloped the follicle (Fig. 3A,B). Inthe months of March and April oocytes showed adiameter between 200 and 400 µm. Within theoocyte the nucleus was long and narrow. The fol-licle cells were columnar, with prominent and elon-gated nuclei. It is likely that in this period a phase

Fig. 2. Transverse section of the ovary of the month ofNovember (October and December gave similar results). A:ovary contains small oocytes developing from the germinalepithelium (original magnification ×100). B: transverse sec-tion of follicles at a higher magnification, showing the layerof follicle cells surrounding the oocytes (the oocyte and theassociated follicle cells are defined as follicle) (arrows); ge,germinal epithelium (original magnification ×280).

Fig. 3. Transverse section of the ovary of the month of Janu-ary (February gave similar results), A: showing oocytes sur-rounded by follicle cells starting to enfold: f0 = follicles withoutfolds; f3 = follicles with three folds; f4 = follicles with four folds(original magnification ×150). B: transverse section at a highermagnification of a follicle with four folds and one fold startingto enfold (original magnification ×320). Blood capillaries run-ning at the base of the folds are visible (arrow).

SEX STEROID LEVELS AND SEXUAL CYCLE IN O. VULGARIS 37

of synthesis probably of yolk proteins begins, withan increase of the basophilia of the nucleus withinthe follicle cells (Fig. 4). At this stage 35% of oo-cytes were without folds, 50% showed four folds,and 15% of oocytes showed five folds or six folds.In the month of May oocytes showed an averagediameter between 400 and 600 µm. Within theoocyte the nucleus was long and narrow. The fol-licle cells were columnar. At this stage folds werenarrower and extended less deeply into the oo-cyte with respect to the previous period (Fig. 5);20% of the oocytes did not show any fold, 70%showed four folds, and 10% had five and six folds.The ooplasm was strongly vacuolated in those oo-cytes with folds. In the month of June the oocyteswith four folds underwent a profound transforma-tion with a total regression of the folds, and thewhole ooplasm was filled with yolk (Fig. 6A). Atthis stage the diameter of the oocytes was between600 and 1000 µm. Oocytes with five and six foldsshowed strongly vacuolated ooplasm (Fig. 6B).

Annual variations of the oviductsIn the months of October, November, and De-

cember, the proximal and distal regions of the ovi-ducts were characterized by thin and thick walls,respectively, and a single cuboidal epithelium. Themucosa was arranged in irregular folds, whichwere finger-like with an irregular shape in theproximal region and extended deeply into the lu-men, whereas in the distal region folds wereshorter and protruded less deeply into the lumen(Fig. 7A,B). The epithelial cells of both regionswere ciliated and had basophilic, small, and round,

Fig. 4. Transverse section of the ovary of the month ofMarch (April gave similar results), showing follicles with fourfolds and vacuolated ooplasm filled with yolk; y, yolk; (origi-nal magnification ×170).

Fig. 5. Transverse section of the ovary of the month ofMay, showing follicles with four folds narrower and less deepthan during the previous period; y, yolk (original magnifica-tion ×200).

Fig. 6. Transverse section of a follicle with four folds ofthe month of June, showing in A: detail of a regressed fold(arrows) and ooplasm filled with yolk (original magnification×400). B: follicle with five folds and one fold starting to en-fold and strongly vacuolated ooplasm; y, yolk (original mag-nification ×150).

38 A. DI COSMO ET AL.

centrally located nuclei. In addition, few secretorynonciliated cells could be seen in the distal re-gion of the oviduct. In both regions of the oviductthe mucosa was very thin and scarcely vascular-ized. Blood vessels were located between the mu-cosa and the muscle layer. The muscle layer ofthe proximal region consisted of circular musclefibers thinner than those of the muscle layer ofthe distal region which, in addition, had thickouter longitudinal muscle fibers. In the monthsof January, February, March, and April the proxi-mal region was characterized by a thick wall andexhibited an increased number of folds, still inthe shape of a finger, but with a more indentedprofile (Fig. 7C). The cubical epithelium consistedof ciliated cells with a central basophilic nuclei.Scattered blood vessels were visible within thecompact connective tissue of the folds. The circu-lar muscle layer was thicker than in the previous

period. The distal region of the oviduct showed athick wall and an increase in folds (Fig. 7D). Theepithelium was composed of columnar nonciliatedsecretory cells, with prominent, strongly baso-philic, basal nuclei interspaced between ciliatedcells with central nuclei. Blood vessels were scat-tered within the thick wall. The circular and lon-gitudinal muscle fibers of the distal region werethicker than in the previous period. In May theproximal region of the oviduct showed a thinnerwall than in the previous period. Folds appearedshorter and larger with a branched profile (Fig.8A). The epithelium was characterized by ciliatedsecretory cells. Blood vessels were scattered withinthe connective tissue. The muscle layer wasformed by circular muscle fibers. The distal re-gion of the oviduct showed a reduced lumen dueto the thickening of the wall and the folds (Fig.8B). The epithelium was formed by ciliated co-

Fig. 7. Transverse section of the proximal and distal ovi-duct of the month of November (A, B) (October and Decem-ber gave similar results) and March (C, D) (JanuaryFebruary, and April gave similar results). A: proximal ovi-duct with the mucosa arranged in irregular, finger-like folds(original magnification ×50). B: distal oviduct with the mu-

cosa arranged in short and little protruding folds (originalmagnification ×50). C: proximal oviduct with the mucosa ar-ranged in irregular, finger-like folds with indented profile(original magnification ×50). D: distal oviduct with the mu-cosa arranged in thick and little protruding folds (originalmagnification ×50).

SEX STEROID LEVELS AND SEXUAL CYCLE IN O. VULGARIS 39

lumnar cells with basal nuclei. The muscle layerwas thicker than in the previous period and wascharacterized by inner circular fibers and outer lon-gitudinal fibers. In June the proximal region ofthe oviduct showed two different types of folds.Some were enlarged and dramatically irregular inshape, interspaced by little finger-like folds, andboth were surrounded by fluid mucous material(Fig. 8C). The epithelium was formed by ciliatedand nonciliated secretory cells. The muscle layerwas formed by circular muscle fibers, and blood ves-sels were dispersed within the connective tissue.The distal region exhibited maximal development,showing a thick wall and long folds, protrudingwithin the lumen, which appeared to be filled withdense mucous material (Fig. 8D). The epitheliumwas formed by empty secretory nonciliated cells.The morphological variations of the oviducts areschematically reported in Fig. 9.

Annual variations of the oviducal glandsThe oviducal glands are arranged halfway along

the oviducts. The analysis of the serial transversesections allows us to divide the oviducal glandsinto two regions: the former stretched from theproximal part of the oviduct to the central cavityconstituted by an enlargement of the lumen of theoviduct and extended for about two-thirds of thegland. It contained the spermathecae embeddedin compact connective tissue crossed by muscularfibers that were actually the wall of the oviductitself and that were surrounded by a glandularcompartment divided into an inner and an outerregion, with respect to the lumen of the oviduct.The latter region stretched from the central cav-ity to the distal part of the oviduct and extendedfor about one-third of the gland. Within this re-gion, the spermathecae were lacking and the glan-dular components converged and opened with

Fig. 8. Transverse section of the proximal and distal ovi-duct of the months of May (A, B) and June (C, D). A: proxi-mal oviduct with the mucosa arranged in irregular, finger-likefolds with short (small arrow), large (medium arrow), andbranched profile (large arrow) (original magnification ×50).B: distal oviduct with the mucosa arranged in thick and pro-truding folds (original magnification ×50). C: proximal ovi-

duct with the mucosa arranged in two different kinds of folds,one large and irregular in shape and the other little and fin-ger-like profile (arrows) (original magnification ×50). D: dis-tal oviduct with the mucosa arranged in maximally enlargedfolds, protruding into a lumen filled with mucous material(original magnification ×50); mm, mucous material (originalmagnification ×120).

40 A. DI COSMO ET AL.

ducts into the lumen of the central cavity. In themonths of October, November, and December thespermathecae showed simple ciliated cubical epi-thelium with basophilic elongated basal nuclei.The cytoplasm was weakly eosinophilic. Abundantsperm were crypted and immobilized in the sper-mathecae epithelium, with the heads insertedwithin the mucosa, while the tails were not clearlyvisible due to their being crowded together witha dense and weakly eosinophilic material, ob-served in the central part of the lumen of the sper-mathecae (Fig. 10A). The glandular compartmentlooked like a composite tubular gland with a com-pact texture and narrow lumens. The two regions(inner and outer regions) were not separated by a

basal lamina. The cytoplasm of the cells of theinner region was weakly eosinophilic, rich in vacu-oles, and showed elongated basophilic and basalnuclei. In the strongly eosinophilic cytoplasm ofthe cells of the outer region, vacuoles were rarelyseen. Basophilic, elongated nuclei were basallyplaced (Fig. 10B). In the months of January, Feb-ruary, March, and April no changes could be seenin the morphology of spermathecae or in the dis-tribution of the spermatozoa. On the other hand,the glandular component appeared less compactthan in the previous period, with wide lumensamong tubular components. A thick basal laminaseparated the inner from the outer region. Theeosinophilic cytoplasm of the cells of the inner re-

Fig. 9. Schematic drawing of the morphological variationsof the proximal and distal oviduct and the oviducal gland,from October to July. On the left is represented the oviductand the oviducal gland as they lay within the body cavity.On the right, transverse sections of the proximal and distaloviduct and the oviducal gland are represented, showing their

morphological changes. Drawings of the transverse sectionswere obtained by camera lucida at the level indicated by thelines on the oviduct and the oviducal gland on the left; sp,spermathecae; ir, inner region; or, outer region; l, lumen; w,oviduct wall. Drawings are to scale.

SEX STEROID LEVELS AND SEXUAL CYCLE IN O. VULGARIS 41

Fig. 10. Transverse section of the oviducal gland in themonths of November (October and December gave similar re-sults), showing in A: the spermathecae with sperm heads (ar-rows) inserted into the mucosa (arrows indicate sperm heads)(original magnification ×30). B: transverse section of the glan-dular compartment showing the inner weakly eosinophilic andthe outer region strongly eosinophilic (original magnification×40). C: transverse section of the oviducal gland in the monthof February (January, March, and April gave similar results).A thick basal lamina separated the inner from the outer re-

gion (original magnification ×40). D: transverse section of theoviducal gland in the month of May. The area occupied byspermatechae increased. Arrows indicate sperm heads (origi-nal magnification ×50). E: the glandular cells of the outerregion had the same columnar aspect of the glandular cellsof the inner region and their cytoplasm show the presence ofsecretory droplets of intensely eosinophilic substance; s,sperm; or, outer region; ir, inner region; bl, basal lamina (origi-nal magnification ×150).

42 A. DI COSMO ET AL.

gion showed less elongated basophilic nuclei andmore vacuoles than in the previous period. How-ever, the cytoplasm of the outer region with scarcevacuoles appeared less strongly eosinophilic thanin the previous period and with basophilic, elon-gated nuclei (Fig. 10C). In May, the area occupiedby spermathecae increased and the epitheliumappeared strongly eosinophilic with basal andsmall spherical basophilic nuclei. Spermatozoawere arranged in the same position with respectto the previous period, while the material in thelumen of the spermathecae appeared strongly eosi-nophilic (Fig. 10D). The glandular compartmentalso increased in size. The inner region epithe-lium, weakly eosinophilic, became columnar withsmall, basophilic nuclei, basally placed. The epi-thelial cells showed fewer vacuoles than in theprevious period and were filled with a transpar-ent substance. The glandular cells of the outerregion had the same columnar aspect of the in-ner region cells and their cytoplasm, which wasnot vacuolated, showed the presence of secretorydroplets of intensely eosinophilic substance. Thesmall, basophilic nuclei were basally arranged(Fig. 10E). In June, no changes in the morphol-

ogy of either spermathecae or the glandular com-partment could be seen. The whole oviducal gland,however, appeared to be larger. The morphologi-cal variations of the oviducal gland are schemati-cally reported in Fig. 9.

GSIGSI increased steadily from October to Febru-

ary. The starting value was 0.175% in October andreached 0.502% in February. Between March andApril, GSI showed a sudden increase, attainingthe value of 0.475% in April. From April to June,GSI increased, reaching the value of 9.810% inJune. The values of body weight, ovary weight,and GSI during the annual cycle are shown inFig. 11. GSI fluctuations paralleled those of theovary weight, while changes in the body weightwere not related to either the GSI or the ovaryweight.

3b-HSDUsing an enzymatic histochemistry method we

found 3β-HSD activity in the ovary of O. vulgaris.The analysis was carried out during two periodsof the annual cycle: November and June. The en-

Fig. 11. Body weight, ovary weight, and GSI values during the annual cycle. Error barsare SD. Five animals were measured for each month

SEX STEROID LEVELS AND SEXUAL CYCLE IN O. VULGARIS 43

Fig. 12. Histochemistry of 3β-hydroxysteroid dehydroge-nase. A: cryostat transverse slice of the ovary in the monthsof November (original magnification ×120) and B: showingthe enzymatic activity confined in the follicular cells surround-ing and enfolding the oocytes (arrows) in the month of June(original magnification ×400). C: control experiment cryostattransverse slice of the ovary in the month of November show-ing nonspecific products of reaction (large arrows) due to theprecipitation of NBT salts (see Materials and Methods) (origi-nal magnification ×150).

zymatic activity was localized, in both periods, inthe follicle cells (Fig. 12A,B). Control experimentswere performed in both periods. Nonspecific reac-tion products were observed in the shape of scat-tered granules of variable size and intensity ofcolor, due to the precipitation of NBT salts dur-ing the incubation time (Fig. 12C).

DISCUSSIONIn this paper we present data on 17β-estradiol

and progesterone levels, and morphological changesof the reproductive system (ovary, oviducts, andoviducal glands) of the female of O. vulgaris,throughout the reproductive cycle. The reproduc-tive system of the female of O. vulgaris increasedin size and weight regularly from October to June,and the increase does not seem to be correlatedto the weight of the animal, as indicated by theGSI values. Our data are in agreement with theobservations carried out by Buckley (’76) on O. vul-garis. As a common feature in cephalopods, folliclecells proliferate during the annual cycle, resultingin deep enfolding into the oocyte (Boucaud-Camouet al., ’91; Ikeda et al., ’94). We observed that in O.vulgaris, from October to June, oocytes increasedin both diameter and in number of folds. Althoughthe maximum number of folds present in a singleoocyte was six, the highest percentage of oocyteshad four folds. Moreover, oocytes with four foldswere the only ones that reached a maturationalstage such that the whole ooplasm was occupiedby yolk, with a contemporary regression of theheight and width of the enfolding. On the con-trary, oocytes with five and six folds never attainedsuch a maturational stage, but always showed deepfolds and scarce yolk, showing the typical aspectof atresia. This suggests that a morphological fea-ture is indicative of an achieved functional matu-rity. On the grounds of such observations, wepropose to divide the ovarian cycle of O. vulgarisinto four main phases: (i) previtellogenesis, whenthe majority of oocytes does not show any fold,and the ooplasm is compact; (ii) early vitellogen-esis, when the follicle cells start enfolding in theooplasm of the oocyte and yolk granule deposi-tion within the ooplasm begins: oocytes withoutfolds are still present in the ovary; however, themajority of oocytes that undergo the vitellogen-esis show four folds; (iii) full vitellogenesis, whenthe majority of oocytes has four folds and the oop-lasm is almost completely filled with yolk gran-ules; and (iv) late vitellogenesis, when the diameterof the oocyte is unchanged with respect to the pre-

44 A. DI COSMO ET AL.

been postulated and sustained by experimentalobservations (Perez and Callard, ’89). Among in-vertebrates 17β-estradiol and progesterone seemto play less clearly differentiated roles in repro-duction. It seems that 17β-estradiol is involved invitellogenesis in crustaceans (Couch et al., ’87; VanBeek and De Loof, ’88; Quinitio et al., ’91), and astimulatory effect of 17α-hydroxyprogesterone onvitellogenin secretion has been demonstrated inthe shrimp Penaeus japonicus (Yano, ’87). Proges-terone stimulates ovarian development in theprawn Parapenaeopsis hardwickii (Kulkarni et al.,’79) and the shrimp Metapenaeus ensis (Yano, ’85).In the snail Helix pomatia, progesterone andoestone-acetate stimulate ovogenesis (Csaba andBierbauer, ’79). Differently from crustaceans andinsects, where vitellogenin is synthesized into thefat bodies and transported through the hemo-lymph to the ovary (Engelman, ’94), in Octopus ithas been clearly established that the follicle cellssynthesize and transfer yolk proteins into the oop-

Fig. 13. Schematic drawing of the morphological changesin follicles of O. vulgaris. Follicles of the previtellogenic phase(PV; A, B); follicles of the early vitellogenic phase (EV; B, C);

follicles of the full vitellogenic phase (FV; D, E); follicles oflate vitellogenic phase (LV; E, F).

vious period, but folds are now dramatically re-gressed and the ooplasm is completely filled withyolk (Fig. 13).

In the ovary of O. vulgaris 17β-estradiol andprogesterone concentrations changed in relationto the ovarian development during the reproduc-tive cycle. Both steroids were low during theprevitellogenesis and increase at the onset of vi-tellogenesis, reaching a peak during the full vi-tellogenesis. Their increase prior to vitellogenesisseems to suggest their involvement in yolk syn-thesis, an aspect also sustained by their decreasewhen the vitellogenesis has come to an end. Invertebrates, the main steroid involved in yolk syn-thesis is the 17β-estradiol (Wallace, ’85), althoughother factors have been proved to play an activerole (Ho et al., ’81, ’85). Progesterone is regardedas the hormone of viviparity, due to its action inpreparing the genital tract to receive and shelterthe embryo (Callard et al., ’92). Recently, a novelrole in suppressing the vitellogenin synthesis has

SEX STEROID LEVELS AND SEXUAL CYCLE IN O. VULGARIS 45

lasm (O’Dor and Wells, ’73, ’75). In O. vulgaristhe factor(s) controlling the onset of vitellogenesisappear to be associated with increased secretionof a yet unidentified gonadotropic hormone fromthe optic gland (Wells and Wells, ’59, ’72, ’75).However, we believe that a local effect, exertedon follicle cells, in the modulation of vitellogen-esis might be carried out by either 17β-estradiolor progesterone or both, as suggested by the pres-ence of such steroids within the ovary when thevitellogenesis is active. 17β-Estradiol and proges-terone may act through a specific receptor, whosepresence within the follicle cells has been alreadyreported in this species (Di Cosmo et al., ’98a,b).Both an autocrine and paracrine role is sustain-able in light of 17β-estradiol and progesterone ab-sence from the hemolymph. It is important to notethat gonads are not only a target organ but alsoa source of steroids, as proved by the presence of3β-HSD in the ovary (present data) and testis ofO. vulgaris (D’Aniello et al., ’96).

Morphological changes in the oviduct and ovi-ducal gland observed throughout the reproductivecycle in O. vulgaris, along with 17β-estradiol andprogesterone fluctuations, suggest that both ste-roids might work in synergy to sustain the growthand differentiation of the female reproductivesystem in this species. In oviparous vertebrates17β-estradiol promotes the proliferation and dif-ferentiation of the epithelial cells, while progest-erone is active in albumen secretion and shellproduction (Ratia, ’79; Joensuu, ’90; Joensuu et al.,’92). In O. vulgaris, during the egg-laying period,the oviducal glands produce two different sub-stances which, when combined together, surroundthe eggs in a gelatinous covering. The polymer-ization of such gelatinous material is preventedby a mucous substance secreted by the proximaltract of the oviduct that, in this phase of the re-productive period, increases both the number andthe surface area of epithelial folds (Wells, ’78). Aswe report here, both the oviducts and the ovidu-cal glands undergo profound changes that may berelated to the functions of this structure. Indeed,the oviduct increases in number and surface offolds and secretes substances which are likely tobe involved in facilitating eggs’ descent. We havealready demonstrated that the proximal and thedistal tracts of the oviduct are target organs of17β-estradiol and progesterone, as demonstratedby the presence of specific receptors (Di Cosmo etal., ’98a,b). Progesterone might be responsible forthe production of lubricating material and formuscle contractions as sustained by the observa-

tion that the oviduct in this species is richly in-nervated (Young, ’67). Froesch and Marty (’75)suggest that in octopuses, when the eggs reachthe central cavity of the oviducal glands, they areretained and wait for sperm to be mobilized andreleased from spermathecae. The morphologicalchanges of the oviducal glands in O. vulgaris arein agreement with their role in sperm mobiliza-tion. We may hypothesize that the oviducal glandshave complex functions, as suggested by the twoglandular compartments which are characterizedby different staining properties, in agreement withthe supposed twofold role played by these glandsin secreting substances which harden into an egg-shell and store the sperm (Wells, ’78). We observedthat the difference between the two compartmentsbecomes evident during the full vitellogenesis,when sperm begins to mobilize, sustaining thatthe central cavity of the glands may be the site offertilization (Froesch and Marthy, ’75). Moreover,we cannot exclude that this phenomenon may behormonally controlled, as suggested by the pres-ence of progesterone receptors in the glandularregion (Di Cosmo et al., ’98b).

In conclusion, in this study we report that 17β-estradiol and progesterone fluctuations paralleledthe morphological changes of the reproductive sys-tem of the female of O. vulgaris during the repro-ductive cycle, suggesting that, in this species, thereproductive system is under hormonal control.

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