BIODIVERSITY IN ENCLOSED SEAS

Gametogenesis and maturity stages scale of Raja asteriasDelaroche, 1809 (Chondrichthyes, Raijdae) from the SouthLigurian Sea

M. Barone Æ S. De Ranieri Æ O. Fabiani ÆA. Pirone Æ F. Serena

� Springer Science+Buisness Media B.V. 2007

Abstract This stu\dy was aimed at the acquisition

of basic life-history information on Raja asterias

through an objective approach in the assignment of

maturity stage, using both histological techniques

and a multivariate analysis aimed at macroscopic

evaluations of the reproductive system. The sam-

ples examined were collected from landings of

commercial trawl vessels from the port of Viareg-

gio. From July 2001 to August 2002 a total of 351

specimens, 166 females and 155 males, were

purchased. A description of the main stages of

oogenesis and spermatogenesis is presented here.

Moreover, on the basis of the maturity stage

definition, maturity ogives were constructed.

Length at 50% maturity was estimated to be

51.7 cm and 56.1 cm total length for males and

females respectively. Finally an informal table for

the maturity stage assignment, which can be useful

for a quick and easier definition both on board and

in the laboratory, is formulated.

Keywords Shark fisheries � Sexual maturity �Oogenesis � Spermatogenesis

Introduction

The starry ray Raja asterias Delaroche, 1809 is

common in the whole Mediterranean excluding

probably the Black Sea (Fischer et al., 1987;

Relini et al., 2000). Although along the Atlantic

coasts of northern Morocco and in southern

Portugal it has been recorded (Whitehead et al.,

1984), R. asterias may be considered in general

terms a Mediterranean endemic species. It is a

benthic species generally found in shallow waters

on muddy or sandy soft bottoms, mostly concen-

trated at depths up to 150 m (Serena et al., 1988).

In the southern Ligurian Sea port of Viareggio,

R. asterias is among the most important compo-

nents of the fish assemblages caught all year round

with beam-trawling. Elasmobranchs are particu-

larly vulnerable to fishing pressure, because of

their biological characteristics: low growth rates,

late sexual maturation, low fecundity, and a long

reproductive cycle. The decline of many species as

a consequence of removals that went beyond the

Guest editors: G. Relini & J. RylandBiodiversity in Enclosed Seas and Artificial MarineHabitats

M. Barone � F. Serena (&)Agenzia Regionale per la Protezione Ambientaledella Toscana, Via Marradi, 114, 56100 Livorno, Italye-mail: f.serena@arpat.toscana.it

S. De RanieriCentro Interuniversitario di Biologia Marina,Livorno, Italy

O. Fabiani � A. PironeSezione di anatomia – Dipartimento di produzionianimali, Universita di Pisa, Pisa, Italy

123

Hydrobiologia (2007) 580:245–254

DOI 10.1007/s10750-006-0448-x

sustainable levels has been stressed by many

authors (Musick, 1999; Stevens et al., 2000).

However, it seems that this did not occur for R.

asterias. On the grounds where the fishery oper-

ates, data on catch trends for the last 15 years

suggest that the biomass of the stock is in a steady

state (Serena et al., 1988; Abella & Serena, 2005).

Almost nothing is known about the reproduc-

tive biology of the starry ray in the South Ligurian

Sea. Lo Bianco (1908) reported observation of

specimens from the Golfo di Napoli, Tortonese

(1956) of specimens from markets of Livorno,

Napoli and the Adriatic Sea, Capape (1977) also

studied the biology of R. asterias in the Tunisian

waters. This species is oviparous, mature females

being present throughout the year (Capape, 1977)

and the egg-cases are usually deposited at a depth

of about 30–40 m. The only available data on

rates of egg laying and incubation were obtained

under experimental conditions. The rate of egg

lying estimated by Lo Bianco (1908) was one egg

capsule every 4 days; the incubation period lasted

about 5–6 months (Capape, 1977). As newborn

juveniles of about 8 cm TL have been found

distributed primarily in the northern Tyrrhenian

Sea at depth of 8–12 m, this area is very likely to

be a nursery (Abella et al., 1997).

This study was aimed at the acquisition of basic

life-history information on R. asterias through an

objective approach in the assignment of maturity

stage, using both histological techniques and a

multivariate analysis aimed at macroscopic evalu-

ations of the reproductive system. A description of

the main stages of oogenesis and spermatogenesis,

the definition of a maturity scale and an estimate of

length at first maturity are showed here.

Materials and methods

The samples examined were collected from land-

ings of commercial vessels utilizing a variant of

the beam-trawl called rapido and operating from

the estuary of the Arno River to the Isle of Tino.

From July 2001 to August 2002, monthly pur-

chases amounted to a total of 351 specimens, 166

females and 155 males. At the laboratory, total

length (nearest 0.5 cm below), from tip of snout

to terminal point of the caudal fin, disc width and

sex were recorded for each specimen. In addition,

in females the maximum nidamental gland width

(nearest 0.1 mm) was measured.

Methodology for definition of maturation

stages

In order to determine maturity stages, for

females, the size of the ovarian eggs, the condi-

tion of the oviducts, the measurements of the

nidamental gland, and the presence of egg-cases

were considered; for males the measurements and

the consistency of the claspers, the development

of the sperm ducts and the gonads (testes) were

observed. Each characteristic was associated with

a score. As a result, each specimen was allocated

a code made of four scores. In order to group

together specimens with the same macroscopic

features, data of reproductive apparatus were

analyzed with multivariate techniques carried out

through the option ‘‘agglomerative hierarchical

clustering’’ of S-Plus program (MathSoft, 1999).

Determination of the length at maturity

The length at 50% maturity (L50) for both sexes

was estimated. Females were classified as mature

when they were in stages up to III (vitellogenic

follicles, well differentiated oviducts, nidamental

gland of large size, including specimens with egg

capsules), conferred by the previous method;

males when they were in stage IV (claspers

longer than tips of posterior pelvic fin lobes and

with skeleton hardened, sperm ducts filled,

gonads wide, rose-coloured, with seminiferous

follicles filling the whole volume).

The proportion of mature females and males at

length was described through a logistic function

as follows:

Y ¼ 1=1þ eaþbX

where Y is the estimated mature proportion, a

and b are the estimated coefficients of the logistic

equation and X is the total length. The length at

maturity can be estimated as the ratio of the

coefficients (a/b) by substituting Y = 0.5 in equa-

tion. The curve fitting was done using the specific

tool included in Microsoft Excel.

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Histological techniques

The ovaries of 34 females and the testes of 7

males, representing all stages of morphological

maturity, were extracted in their entirely and

fixed in 10% formaldehyde 0.1 M phosphate

buffer, pH 7.4. Small fragments of each gonad

were extensively washed in fresh water for one

day, dehydrated in a graded ethanol series

(30 min at 30%, 50%, 70%; 1 h at 80%; 2 h at

95%), then embedded in glycol methacrylate

resin (JB-4, Polysciences Inc.). Microtomed sec-

tions of 5 lm were collected on glass slides coated

with 0.5% gelatine containing 0.05% chrome

alum. Sections were subsequently stained with

toluidine blue, methylene blue and PAS reaction,

cleared in xylene, mounted with DPX and exam-

ined with a light microscope (·10–·1000 magni-

fication).

Methodology for ovarian egg counts

One of the ovaries of 166 female specimens was

fixed in modified Gilson’s solution (Simpson,

1951), which composition was: 100 ml ethanol

60%, 15 ml nitric acid 80%, 18 ml acetic acid,

20 g mercury chloride, 800 ml distilled water. The

total number of eggs in each ovary was counted

and their diameter was measured with the eye-

piece micrometer of a stereoscope.

Results

Developing follicles of various sizes were present

in the ovary of R. asterias. In small previtellogenic

follicles ~50 lm in diameter, lying immediately

below the ciliated peritoneal epithelium, follicular

and theca layers were very thin, both composed

by a single layer of squamous cells (Figs. 1, 2). In

follicles ~150 lm in diameter the follicular epi-

thelium showed two kinds of cells: small and large

cells (Fig. 3). Small cells were localized both

under the basal lamina, surrounding the whole

follicle, and along the vitelline envelope. Large

cells were cubic and occupied the centre of the

follicular epithelium. A zona pellucida was evi-

dent between the oocyte and the granulosa cells.

Large follicles, up to 1500 lm, characterized by

the presence of a third follicle cell type, pyriform

cells, spanned the whole follicular epithelium

(Fig. 4). They showed an elongated apex pointing

toward the oocyte surface. Cell protrusions,

linking pyriform cells with oocyte, were often

observed inside the zona pellucida (Fig. 5). The

nucleus of an oocyte of ~1600 lm contained

chromosomes showing a lampbrush appearance

(Fig. 6). Yolk droplets, positive to the PAS

reaction, first appeared in follicles up to

3000 lm. Initially, very small yolk droplets were

evident in the cortical regions of the oocyte; then

their size increased and they filled the entire

ooplasm (Figs. 7, 8). Atretic previtellogenic and

postovulatory follicles were also found (Figs. 9,

Fig. 1 Small previtellogenic follicles ~50 lm in diameter. Thefollicular epithelium is single layered and made up to smallsquamous cells (SC). TC, theca cells; N, nucleus; Oo, oocyte

Fig. 2 Small previtellogenic follicles ~50 lm in diameter. Thefollicular epithelium is single layered and made up to smallsquamous cells (SC). TC, theca cells; N, nucleus; Oo, oocyte

Hydrobiologia (2007) 580:245–254 247

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10). The former were characterized by follicle and

theca cells that later become hypertrophic. In

postovulatory follicles the basal lamina, positive

to the PAS reaction, appeared collapsed and

invaded the central lumen.

In the testes, six stages of spermatocyst devel-

opment were observed. At the beginning Sertoli

cells lined the lumen and a lesser number of

spermatogonia were in a peripherical position in

the spermatocyst (Fig. 11). In the second stage

the Sertoli cells were also found in peripherical

position just inside the basement membrane

(Fig. 12). Successively, the primary spermatocytes

showed large nuclei and in the fourth stage

secondary spermatocytes appeared with nuclei

Fig. 4 Large previtellogenic follicles, up to 1500 lm indiameter. The follicular epithelium is multilayered andmade up to small (SC), large (LC) and pyriform (PC) cells.Oo, oocyte; ZP, zona pellucida; BL, basal lamina

Fig. 3 Previtellogenic follicle ~100 lm in diameter. Thefollicular epithelium is double layered and made up tosmall (SC) and large (LC) cells. ZP, zona pellucida

Fig. 6 Nucleus (N) of oocyte 1600 lm in diametercontained lampbrush chromosomes (LC)

Fig. 5 Intercellular bridges (IB) link pyriform (PC) cellswith oocyte (Oo)

Fig. 7 Follicles up to 3000 lm filled with yolk platelets (Y)

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containing condensed chromosomes (Figs. 13,

14). The fifth stage was characterized by sper-

matids with ellipsoidal nuclei and emerging

flagella. In this stage, spermatozoa began to

aggregate around the periphery of the spermato-

cyst to form tight bundles (Fig. 15). In the

terminal stage of spermatogenesis, spermatozoa

were present in the lumen (Fig. 16).

The diameter of oocytes contained in the

ovaries of 118 specimens were measured and

distributed in classes of 150 lm. The diameters

were correlated to the maturity stage previously

assigned. Table 1 shows the presence of oocytes

up to 3 mm in diameter, in which it was supposed

that vitellogenesis had begun. The mean number

Fig. 8 Follicles up to 3000 lm filled with yolk platelets (Y)

Fig. 9 Atretic previtellogenic follicle. Granulosa cells (C)and theca cells (TC) are hypertrophic

Fig. 10 Postovulatory follicle. The basal lamina (BL)appears collapsed and invades the central lumen (L)

Fig. 11 Sertoli cell nuclei (Sc) migrating toward theperiphery

Fig. 12 Sertoli cells (Sc) in peripherical position justinside the basement membrane

Hydrobiologia (2007) 580:245–254 249

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of eggs up to 3 mm in each ovary increased in

stage II (Maturing) and reached a maximum in

stage III (Mature) (Table 1).

The cluster analysis in the case of the males

(n = 121) showed 18 groups and for females

(n = 150) 12 groups of specimens with the same

features. These groups were further aggregated

on the basis of their proximity in the cluster and,

for females, considering also the results of micro-

scopic observations. Finally, to each of the five

groups, for both males and females, a maturity

stage was assigned as shown in Tables 2 and 3.

On the basis of the morphological determina-

tion of maturity, maturity ogives were constructed

for males (n = 136) and females (n = 165)

(Fig. 17). Males mature by 45 cm TL. Males

smaller than 45 cm TL (n = 49) were immature,

specimens 46–55 cm TL (n = 85) were maturing

and all those longer than 55 cm TL (n = 4) were

sexually mature. Females may mature by 53 cm

TL. Females 21–53 cm TL (n = 94) were imma-

ture, those 54–59 cm TL (n = 50) were maturing.

All females longer than 59 cm TL (n = 13) were

sexually mature. Total length at 50% maturity

was estimated to be 51.7 cm and 56.1 cm for

males and females respectively.

The monthly percentages of mature female

rays containing egg capsules during the sampling

period are presented in Fig. 18. Most (40%) of

the females with egg capsules, including those

with capsules not totally developed, were cap-

tured in May.Fig. 15 Spermatozoa (sp) associated in linear arrays in theSertoli cells (Sc)

Fig. 14 Secondary spermatocytes (Ss) with nuclei con-taining condensed chromosomes

Fig. 13 Primary spermatocytes (Ps) showing large nucleiFig. 16 Spermatozoa (sp) within the lumen

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Table 1 Number ofoocytes in females ofR. asterias at differentmaturity stages

Stage No. offemales

No. ofoocytes

Mean numberoocytes <3 mm/female

Mean numberoocytes >3 mm/female

I 71 6371 86 6II 19 1794 74 21III 11 1852 135 33IV 11 1599 117 27V 6 637 84 16

Table 2 Maturity scale for females of R. asterias

Stage State Description

Macroscopic Microscopic

I Immature Oocytes uniformly small and white. Ovary verysmall. Oviducts thread-like and nidamental glandabsent or little differentiated.

The anterior region of the ovary containingnumerous follicles in first phase ofprevitellogenesis, up to 150 lm in diameter.Atretic follicles present.

II Maturing Ovary small, oocytes differentiatedto various small sizes but all white. Oviducts andnidamental gland well developed but small(<2 mm maximum width).

Sections appear filled with previtellogenic folliclesof various sizes.

III Mature Ovary walls more transparent. Ovary filled withyellow Oocytes, some large. Oviducts welldeveloped and nidamental gland large(>2 mm maximum width).

Sections of oocytes of 3–7 mm show yolk droplets.

IV Extruding Ovary filled with large yellow oocytes. Egg capsulesmore or less formed in one or both oviducts.

Only the follicular walls were embedded because oflarge dimension of oocytes.Yolk droplets evident in the cortical region.

V Resting Ovary walls transparent. Oocytes ofdifferent sizes, white or yellow. Oviducts emptybut much enlarged and vascularized.Nidamental gland small (<2 mm).

Postovulatory follicles observed.

Table 3 Maturity stagesfor males of R. asterias

Stage State Description

I Immature Clasper shorter or as long as the extreme tips of posterior pelvic finlobes. Ducti deferentes not differentiated or narrow. Testes small andwhite occupying up to the half of abdominal cavity.

II Virgin Clasper longer than tips of posterior pelvic fin lobes but skeleton stillflexible. Ducti deferentes well developed. Testes occupying over thehalf of abdominal cavity with walls transparent but seminiferousfollicles not fill the whole testes.

III Maturing Clasper longer than tips of posterior pelvic fin lobes, skeleton hardenedbut axial cartilages still soft. Ducti deferentes whitish. Testes enlarged,not entirely filled with seminiferous follicles.

IV Mature Clasper longer than tips of posterior pelvic fin lobes, skeleton hardenedwith axial cartilages hardened and pointed. Ducti deferentes full.Testes wide, rose-coloured, with seminiferous follicles filling wholevolume.

V Resting Clasper longer than tips of posterior pelvic fin lobes, skeleton hardenedwith axial cartilages hardened and pointed. Testes scarcely occupyinghalf of abdominal cavity.

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The monthly abundances of females of R.

asterias at different stages of maturity is presented

in Fig. 19. The greatest number (80%) of females

at stage I (Immature) was observed in August.

This percentage diminished from September to

December, when the specimens at stage II increased

until the 50%. Stage III (Mature) stays almost

constant throughout the year, but stage IV (Extrud-

ing) were mainly observed in May and June, when it

reached the 20 %, decreasing in autumn.

Discussion

The analysis of histological maturity in female of

R. asterias indicates that both ovaries contain

developing follicles of various sizes. In all the

ovaries examined, the great majority of follicles

was in the phase of previtellogenesis, character-

ized by the progressive growth of the oocytes and

the increase in complexity of the follicular

epithelium. The change observed in the follicular

epithelium, during the early previtellogenic stage,

corresponds to that described by Andreuccetti

et al. (1999) in the same species. In addition, an

evidence of the phase of previtellogenesis is the

presence of chromosome with lampbrush appear-

ance in the nucleus of oocytes 50–1500 lm in

diameter. References to lampbrush chromosomes

in elasmobranch oocytes have not been found,

although the lampbrush chromosomes were de-

scribed by Guraya (1986) in the ovary of several

teleosts, in which they appear during the first

meiotic prophase. Furthermore, vitellogenis starts

when the follicle measures about 3000 lm in

diameter and is indicated by the appearance of

minute platelets of yolk in the cortical region. We

have been unable to describe the successive

phases of oogenesis because of the large size of

the oocytes. Instead, the count of oocytes allowed

us to observe that only few follicles continue with

vitellogenesis; therefore, the ovary of R. asterias

seems to contain a reserve of oocytes, present

both at every maturity stage and throughout the

year.

A brief analysis of the histology of testes

indicates that spermatogenesis of R. asterias is

similar to that described by Hamlett (1999) in

Urolophus jamaicensis. In the testes spermato-

cysts at different stages of development were

present, in accordance with the statement that the

gonads of rajids continuously produce reproduc-

tive gametes (Dodd, 1983).

Fig. 17 Maturity ogives of R. asterias

Fig. 19 Running average (2ra3) of maturity stages inR. asterias

Fig.18 Percentage and running average (2ra3) of egg-capsules in females of R. asterias

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Gross morphology of the reproductive tracts of

females at any time, supported by the histology,

facilitate the assignment of maturity stages, and

indicate that the process of maturation is both

progressive and gradual. In males only qualitative

analysis of the gonads were done; however, the

assignment of maturity stages was based on

accurate observations not only of claspers, as

was done by Capape (1977), but also of gonads

and sperm ducts.

The maturity stages both for females and males

resulting from the cluster analysis were numer-

ous. However, this method was used to eliminate

the subjectivity of the current maturity scales

(Stehmann, 2002) postponing the classification

into different stages until separate examination of

each feature of the reproductive tracts had been

carried out. The first three groups resulting from

the clustering were joined together in stage I

(Immature) both in males and females. Between

females classified as immature, the differences

consisted in the appearance of oviducts and

nidamental gland during growth; in all cases the

oocytes are small and white. In males, the

appearance of sperm ducts and the start of the

development of the gonads were the reasons

determining the different groups in the cluster. In

addition, in females, stage III (Mature) is char-

acterized by two phases in which the oocytes grow

and become yellow, contemporary the nidamen-

tal gland reaches a large size. Another finding,

resulting from the application of cluster analysis,

was the stage of resting hypothesized for both

males and females and rarely described for

elasmobranchs.

In females, at the onset of maturity, the ovaries

contain vitellogenic oocytes and the nidamental

gland is of medium dimension; in males claspers

are longer than the tips of pelvic fins, axial

cartilages are pointed, and gonads contain sem-

iniferous ampullae filling the whole volume. The

estimation of size at first sexual maturity (Lm) for

males (51.7 cm) is somewhat discordant from that

estimated by Capape (1977) (54 cm); also in the

case of females the estimated value of Lm

(56.1 cm) is lower than that calculated by Capape

(1977) and Tortonese (1956) (60 cm). These

differences could be related to environmental

and/or geographical aspects (Frisk et al., 2001).

In evaluating the reproductive cycle, the low

number of specimens reaching lengths at 50%

maturity, 36 females and 46 males, was insuffi-

cient to estimate the gonado-somatic indexes for

each month. From the occurrence of mature

females and females with egg-capsules during the

sampling period, it is concluded that the main

period of spawning activity occurs from March to

July. On the basis of these results, the tables of

maturity stages provide a more reliable tool for

describing rajid life history data.

Acknowledgements We wish to express our thanks toProf. Giorgio Mancino (Sezione di Biologia cellulare edello sviluppo del Dipartimento di Fisiologia eBiochimica—Universita di Pisa) for his advice onhistological investigations. We are very grateful toAlvaro Abella and Romano Baino (Agenzia Regionaleper la Protezione Ambientale della Toscana) for theiruseful support in processing the data. Many thanks also toCecilia Mancusi for her help and encouragement. We owespecial thanks to Caroline Bennett (Fao-AdriaMed) forher assistance with the translation of this paper.

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