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Citation: Porcu, C.; Lai, E.; Bellodi,
A.; Carbonara, P.; Cau, A.; Mulas, A.;
Pascale, N.; Porceddu, R.; Follesa,
M.C. Investigating the Ovarian
Microstructure in the Genera
Helicolenus and Scorpaena (Teleostei,
Sub-Order Scorpaenoidei) with
Implications for Ovarian Dynamics
and Spawning. Animals 2022,12, 1412.
https://doi.org/10.3390/ani12111412
Academic Editor: Elena Chaves-Pozo
Received: 5 May 2022
Accepted: 27 May 2022
Published: 30 May 2022
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animals
Article
Investigating the Ovarian Microstructure in the Genera
Helicolenus and Scorpaena (Teleostei, Sub-Order Scorpaenoidei)
with Implications for Ovarian Dynamics and Spawning
Cristina Porcu 1,2, Eleonora Lai 1, Andrea Bellodi 1,2 , Pierluigi Carbonara 3, Alessandro Cau 1,2 ,
Antonello Mulas 1,2 , Noemi Pascale 1, Riccardo Porceddu 1,2 and Maria Cristina Follesa 1, 2, *
1
Dipartimento di Scienze della Vita e dell’Ambiente, Universitàdegli Studi di Cagliari, Via Tommaso Fiorelli 1,
09126 Cagliari, Italy; cporcu@unica.it (C.P.); elelai93@gmail.com (E.L.); abellodi@unica.it (A.B.);
alessandrocau@unica.it (A.C.); amulas@unica.it (A.M.); pascalenoemi3@gmail.com (N.P.);
riccardo.porceddu@unica.it (R.P.)
2Consorzio Nazionale Interuniversitario per le Scienze Mare (CoNISMa), Piazzale Flaminio 9,
00196 Roma, Italy
3COISPA Tecnologia & Ricerca, Stazione Sperimentale per lo Studio delle Risorse del Mare, Via dei Trulli 18,
70126 Bari, Italy; carbonara@coispa.it
*Correspondence: follesac@unica.it
Simple Summary:
The diversity of reproductive mechanism in bony fishes is greater than in any
other group of vertebrates. It ranges from oviparous, to several stages of viviparous forms. In this
context, scorpaenoid fishes belonging to the families Scorpaenidae and Sebastidae are of particular
interest, since they show extremely varied reproductive modes connected with ovarian structures.
We describe here the ovarian morphology of five rockfish species showing different reproductive
modalities, using histology. Specialized microscopic features were found during gametogenesis,
strictly related to the production of gelatinous mass surrounding the eggs, typical of these species.
Based on microscopic maturity stages here analyzed, we found that all species shed eggs more than
once through the spawning season, and were characterized by continuous oogenesis with multiple
oocyte deposition. Further ovarian dynamic observations supported the hypothesis that all species
had an indeterminate fecundity.
Abstract:
The sub-order Scorpenoidei appears to be particularly interesting due to the presence of
intermediate stages between oviparity and viviparity in several species. The present study aims to
describe the ovarian morphology, using a histological and histochemical approach, in four ovuli-
parous species belonging to Scorpaena genus compared with a zygoparous species, H. dactylopterus,
focusing also on the assessment of the ovarian dynamics in the populations of such species in Sar-
dinia waters (central–western Mediterranean). Ovarian sections of all species were examined using
light microscopy. All species showed a specialized ovary, cystovarian type II-3, strictly related to
the production of gelatinous matrices surrounding the eggs. Some microscopic peculiarities in the
oogenesis process were found: thin zona pellucida, small and low cortical alveoli, and a specialized
ovarian wall during the spawning period. All species analyzed were batch-spawners with an asyn-
chronous ovarian organization. A continuous recruitment of oocytes and the occurrence of de novo
vitellogenesis was also observed. During the spawning period, low atresia intensity was detected,
while a marked increase in this intensity found in the ovaries at the end of spawning season. Our
observations may support an indeterminate fecundity type for these species.
Keywords:
rockfish; gonad histology; morphology; ovarian dynamic; Central–Western Mediterranean
1. Introduction
The diversity of reproductive mechanisms in teleost fishes is greater than in any other
group of vertebrates. It ranges from oviparous, through several stages of lecithotrophic
Animals 2022,12, 1412. https://doi.org/10.3390/ani12111412 https://www.mdpi.com/journal/animals
Animals 2022,12, 1412 2 of 19
viviparity, to highly matrotrophic viviparous forms [
1
]. In this context, scorpaenoid fish
(Scorpaenoidei), belonging to the families Scorpaenidae and Sebastidae, are of particular
interest, since they show extremely varied reproductive modes [
2
,
3
]. Oviparity is common
to most genera, and includes three levels: ovuliparity, zygoparity, and embryoparity [
4
,
5
].
Ovuliparity refers to the release of eggs from females into the water column, which are then
fertilized by males (external insemination), and is known in the genera Pontinus,Pterois,
Scorpaena,Scorpaenopsis,Sebastapistes, and Scorpaenodes (e.g., [
6
,
7
]). Zygoparity refers to an
oviparous reproductive pattern in which the fertilization is internal, and early developed
embryos are released into the environment after a short period of time. It is known only in
the bluemouth Helicolenus dactylopterus (Delaroche, 1890) (family Sebastidae) [
8
,
9
], where
the female is able to store sperm in the ovary for a long time, within specialized structures
where spermatozoa are maintained in a viable state until oocyte maturation (e.g., [
10
–
13
]).
Sperm is then released towards the ovarian lumen to fertilize the mature oocytes. More
specifically, in H. dactylopterus, most of the embryos are in the early-celled stage, whereas
blastula and tail bud stages only represent a small percentage [
14
,
15
]. Embryoparity is an
oviparous mode in which the embryo is formed, the period of retention after fertilization
is prolonged, and embryos may develop within the maternal body to quite an advanced
state prior to their release (genus Sebastolobus, [
7
]). Consequently, the extreme limits of
embryoparity can overlap with those of viviparity (i.e., free embryos or larvae released
in the environment), observed in the genera Sebastes and Sebasticus and in the species
Helicolenus percoides (e.g., [6,7,16]).
The ovaries of teleosts can be generally classified into two major types according to
their structures: gymnovarian and cystovarian types [
17
]. In the first type, the ovary is
open to the abdominal cavity, the ovarian duct is absent, and their oocytes are released
directly into the coelomic cavity and carried to the outside through their genital papilla
(e.g., [
18
]). In the second, the ovary is enclosed with a muscular or non-muscular ovarian
membrane, and the ovarian duct is present, leading oocytes to the external environment.
The cystovarian type is further divided into four types based on histological characteristics:
II-1, II-2, II-3, and II-4 [
19
]. Generally, the ovarian type corresponds to the phylogenetic
classification comparatively well, and most species belonging to the same family have
the same type of ovary [
6
]. Overall, two ovarian variations have been identified in the
Scorpaenoidei [
19
]: (1) the ovary in which the lamella-like stroma develops from the
ovarian hilus located on the dorsal side, and the ovarian cavity is located on the ventral
side of the ovary, classified as cystovarian type II-1 (type II-1); (2) the ovary in which the
stroma develops radially around the blood circulatory system that traverses the center of
the ovary, and the ovarian cavity surrounds all the components around the ovary, classified
as cystovarian type II-3 (type II-3) [
19
]. It seems that there is a connection between these
two ovarian structures and the reproductive mode of scorpaenids [
6
]. Up until the present
time, the type II-1 ovary has been found in two viviparous genera, while the type II-3 ovary
has been identified in six oviparous ones (ovulipari) (e.g., [
11
,
20
]) and in Helicolenus genus
(zygoparous), but it is uncertain whether it can be found also in other scorpaenid species.
It is almost certain that the cystovarian type II-3 is an important structure able to form a
gelatinous egg mass specific for scorpaenids [
6
]. Several characteristics distinguish many
scorpaenids from the simpler ovuliparous species widely described in literature [
3
], but
they are not described in all species.
The sub-order Scorpenoidei is represented in the Mediterranean Sea by two fami-
lies [
21
,
22
]: Scorpaenidae and Sebastidae. Both families include two sub-families: Scorpaen-
inae and Pteroninae, and Sebastinae and Sebastolobinae, respectively. The Scorpeninae
sub-family includes three genera (Pontinus,Scorpaenodes, and Scorpaena) and nine species,
while the Pteroninae subfamily comprises a single genus and one species (Pterois miles). The
Sebastinae and Sebastolobinae sub-families are both represented by one genus and one sin-
gle species: Helicolenus dactylopterus and Trachyscopia cristulata 3evelop3, respectively [
21
,
22
].
All of these families represent an important component of commercial and recreational
fisheries distributed throughout the Mediterranean Sea. In particular, rockfish, belonging
Animals 2022,12, 1412 3 of 19
to the genus Scorpaena, are one of the most important and valuable captures for fisheries in
coastal areas around the Mediterranean Sea (e.g., [
23
–
28
]), and are mainly represented by
the black scorpionfish Scorpaena porcus Linnaeus, 1758, the small red scorpionfish S. notata
Rafinesque, 1810, and the red scorpionfish S. scrofa Linnaeus, 1758. The Sebastidae H. dacty-
lopterus, instead, is a benthic deep-water species caught mainly by bottom trawls [
29
,
30
],
playing an important ecological role in deep-sea fish communities [
31
], and is exploited in
deep-sea fisheries targeted at deep-water crustaceans [30].
Given its particular reproductive mode, several studies on the reproduction of H. dacty-
lopterus have been published (e.g., [
8
,
10
,
15
,
32
–
34
]), while this kind of information is still
scarce for Mediterranean species belonging to the genus Scorpaena [11,35–37].
For this reason, the present study aims to describe the ovarian morphology with a
histological and histochemical approach, including composition, morphology, and mor-
phometry of oocytes in four ovuliparous Mediterranean rockfish species (Scorpaena 3evelop3,
S. notata,S. porcus, and S. scrofa) in comparison with the same data obtained from the
Sebastidae H. dactylopterus, zygoparous. Since many characteristics of the reproductive
biology affecting their reproductive potential are unknown, this study is also focused in
assessing the ovarian dynamic in these species populations in central–western Mediter-
ranean examining, through histological analysis, the development pattern and the growth
of multiple oocyte groups and, finally, quantifying the atresia rate of the final number
of developed oocytes, which is useful for determining the oocyte recruitment process of
these species.
2. Materials and Methods
2.1. Sample Collection
Female specimens of four species belonging to the Scorpenidae family (Scorpaena elongata,
S. notata,S. porcus, and S. scrofa) and one Sebastidae species (Helicolenus dactylopterus) were
collected around Sardinian waters (central–western Mediterranean) during the Mediter-
ranean International Trawl Survey (MEDITS; [
38
]), along with data collected monthly from
commercial landings through the Data Collection Framework (European Union Regulation
199/2008). H. dactylopterus and S. elongata were sampled exclusively from bottom trawls,
while S. notata,S. porcus and S. scrofa were sampled from both bottom trawls and trammel
nets (Table 1). The collection and handling of animals strictly followed the ethical and
welfare considerations approved by the ethics committee of the University of Cagliari
(Sardinia, Italy).
Table 1.
List of the selected species: number of collected females (N), size (TL), and depth range at
which the individuals were caught are presented.
Species N Size Range (TL, cm) Depth Range (m)
Family Scorpaenidae
Scorpaena elongata Cadenat, 1943 45 19.5–47.5 60–478
Scorpaena notata Rafinesque, 1810 33 8.1–14.2 13–101
Scorpaena porcus Linnaeus, 1758 260 9.1–21.6 21–155
Scorpaena scrofa Linnaeus, 1758 206 9.5–36.9 30–155
Family Sebastidae
Helicolenus dactylopterus
(Delaroche, 1809) 436 7.8–34.2 60–651
For each specimen, the following parameters were measured: total length (TL, cm) to
the nearest half cm; and total weight (TW, g) to the nearest 0.01 g. Sex and ovary maturation
were recorded. The maturity status was assessed by dissection according to macroscopic
criteria established by [
39
,
40
], considering the dimension of the ovaries with respect to the
celomatic cavity, their degree of opacity, consistency and vascularization, oocyte visibility,
and overall coloration. All females were classified in seven stages as follows: 1, immature
virgin; 2A, developing virgin, 2B, recovering; 2C, maturing; 3, mature/spawner; 4A, spent;
Animals 2022,12, 1412 4 of 19
4B, resting. In H. dactylopterus, the stage 3 (mature/spawner) was modified ad hoc, and
subdivided in 3a (absence of embryos) and 3b (presence of embryos).
Ovaries were removed, weighed (OW, 0.1 g), and preserved in 5% buffered formalin
for histological examination (pH 7.4, 0.1 M).
2.2. Histological Preparation
A small piece of ovarian tissue (0.5 to 1 cm long) was processed for histological analysis.
The tissues were dehydrated and embedded in a synthetic resin (GMA, Technovit 7100, Bio-
Optica, Milan, Italy) following routine protocols, and sectioned at 3.5
µ
m with a rotating
microtome (ARM3750, Histo-Line Laboratories, Pantigliate, Italy). Slides were stained with
Gill hematoxylin, followed by eosin counterstain (H&E) for standard histology, and with
periodic acid–Schiff (PAS) and Alcian blue (AB) in combination to assess the production of
neutral and sulfated acid mucins [
41
]. Subsequently, sections were dehydrated in graded
ethanol (96–100%), cleared in Histolemon (Carlo Erba Reagents, Cornaredo, Italy), and
mounted in resin (Eukitt, Bio-Optica, Milano, Italy).
2.3. Ovarian Dynamics
Histological samples were examined to determine the developmental stage of oocytes,
the presence/absence of post-ovulatory follicles (POFs), and the prevalence and intensity
of atretic oocytes. Oocytes were assigned to stages in accordance with [
42
]; from least to
most developed stage, included primary growth (PG) and multiple secondary growth (SG)
stages consisting of: cortical alveoli (CA), primary vitellogenic (Vtg1, small granules of
yolk that first appear around either the periphery of the oocyte or the nucleus), secondary
vitellogenic (Vtg2, larger yolk globules throughout the cytoplasm.), tertiary vitellogenic
(Vtg3, numerous large yolk globules fill the cytoplasm, and oil droplets, if present, begin to
surround the nucleus), germinal migration (GVM), and hydration (H) stages. POFs were
classified based on their degree of degeneration: those having bigger size and thicker and
more convoluted epithelium composed of linearly arranged granulosa cells were classified
as new, whilst those with signs of degeneration were classified as old.
Concerning atresia, females were assigned to atretic states 0, 1, and 2, having 0%,
<50%, and
≥
50% SG oocytes with
α
-atresia, respectively [
43
]. All of these histological
markers were used to assign females to different spawning subphases [
42
]. In particular,
actively spawning (AS) females were categorized as those displaying markers of imminent
or recent spawning activity such as GVM, H, or POFs, while non-spawning (NS) was the
category assigned to all remaining SG females with no spawning markers.
The size composition of oocytes was obtained measuring only oocytes where the
nucleus was clear, and given that these oocytes are rarely perfectly spherical in shape,
in order to reduce the variance, the diameter of each oocyte was taken using TpsDig
software [
44
], and calculated as average of the major and minor axis [
45
–
47
]. For each
species, at the maturing and mature/spawner maturity stages, the thickness of zona
pellucida (
µ
m) of oocytes (n= 35 for each stage) from the cortical alveoli stage to the
germinal migration stage was taken. Differences in mean oocyte diameters and zona
pellucida thickness among maturity stages were tested using one-way ANOVA [48].
Ovarian dynamics were assessed in a subsample of females in each species. Oocytes
in each subsample were grouped into size classes of 50
µ
m in order to characterize the size
cohorts (oocyte size frequency distribution size, OSFD).
The relative intensity of atresia (RIA), i.e., the percentage of
α
- atretic oocytes in
relation to the total SG oocytes in an individual ovary, was also recorded from histological
sections of females. The co-occurrence of different spawning markers (e.g., POFs with
GVM oocytes) was utilized as an index of spawning interval, SI.
The seasonality of spawning was estimated for S. scrofa,S. porcus and H. dactylopterus
through an analysis of the seasonal or monthly distribution of the percentage of maturity
stages of females, as well as the evolution of the mean gonadosomatic index (GSI) estimated
Animals 2022,12, 1412 5 of 19
per sampling date and maturity stage, and calculated using the ovary free body weight
(OFW = TW −OW) [49]:
GSI = 100 ×OW ×(OFW)−1(1)
3. Results
3.1. Ovarian Morphology
All species analyzed showed paired, saccular ovaries, entirely separated from each
other, laying parallel in the dorsal part of the peritoneal cavity, and fused just before the
genital opening (Figure 1A–E in detail). The ovary was classified as cystovarian type II-3
(type II-3), in which the stroma develops radially around the blood circulatory system,
traversing the center of the ovary, and the ovarian cavity surrounds all components around
the ovary. The connective tissue, including the germ cells (oocytes), extends radially
from the central ovarian hilus to the surrounding ovarian wall, forming central stroma
(Figure 1). The ovarian lamellae are suspended from the rachis by a fibromuscular trunk
which contains blood vessels, and which has the surface covered with oocytes in different
stages of maturity. As the oocytes develop, they take up a position further away from the
rachis and closer to the ovarian lumen, so that the different stages are lined up in order
of development (Figure 1A–E). During the mature phase, eggs are embedded in a large,
pelagic gelatinous matrix in all species analyzed.
Holocrine glands, reacting positively to PAS staining, are present in all species ana-
lyzed (Figure 2A,B) (Table 2). The ovarian follicles are connected to the stroma by a narrow,
vascularized peduncle, formed by a smooth and mono-stratified lamellar epithelium, the
length of which increases as vitellogenesis advances (Figure 2C,D). Mature oocytes contain-
ing PAS positive ovarian fluid, which is particularly abundant during the spawning period,
are expelled into the lumen. The zona pellucida (Figure 2E,F), strongly PAS-positive, is
visible from the cortical alveoli stage (CA), and become thicker with the proceeding of the
maturation phase, reaching maximum dimensions during the GVM stage, being made up
of three layers, in all studied species (Figure 3). The thin outermost and the intermediate,
medium electron dense region form the outer zona pellucida, while the internal region is a
multilamellar striated region (inner zona pellucida) (Figure 2E–G).
Table 2.
Histochemical properties of the internal epithelium of the ovarian wall, ovarian fluid, and
holocrine gland in the rachis in Helicolenus dactylopterus (HD), Scorpaena elongata (SE); Scorpaena notata
(SN), Scorpaena porcus (SP) and Scorpaena scrofa (SS). PAS, periodic acid–Schiff; AB, Alcian blue; +,
presence; −, absence.
Structures PAS AB pH 2.5
HD SE SN SP SS HD SE SN SP SS
Ovarian wall + + + + + + + + + +
Ovarian fluid + + + + + − − − − −
Holocrine gland
+ + + + + − − − − −
The ovarian wall is made up of three layers (Figure 2H,I). The outer layer is composed
by a mesothelium and the middle layer has two sub-layers (an outer one formed by
a circular muscular system, and an inner one by much thicker longitudinal muscular
system), which contains smooth musculature and numerous blood vessels (Figure 2H).
The innermost layer (internal epithelium) of the ovary is covered with a simple cuboidal
epithelium, and, during the period of vitellogenesis and spawning, modifies its structure,
containing strongly PAS positive granules and cytoplasmic projections (Figure 2I) (Table 2).
In the zygoparous H. dactylopterus, cryptal structures packed with spermatozoa (here
called spermatic crypts) near the basal part of the stroma are observed (Figure 2J). Dur-
ing the storage period, the cryptal epithelium releases strongly PAS-positive granules
(Figure 2J).
Animals 2022,12, 1412 6 of 19
Animals2022,12,x6of20
Figure1.MorphologicalcharacteristicsofcystovariantypeII‐3ovaryofthefivespeciesstudied
(H&E).Correspondingmacroscopicovaries,locatedintheabdominalcavityabovethebladder,are
alsoshowndownontheleftofeachspecies.(A)Scorpaenascrofa(TL22.3cm);(B)Scorpaenanotata
(TL11.3cm);(C)Scorpaenaporcus(TL15.6cm);(D)Scorpaenaelongata(TL28.7cm);(E)Helicolenus
dactylopterus(TL18.2cm).bv,bloodvessel;ow,ovarianwall;r,rachis.Thedashedlineshowsthe
radialdevelopmentofoocytesfromthestromatosurroundingovarianwall.
Holocrineglands,reactingpositivelytoPASstaining,arepresentinallspecies
analyzed(Figure2A,B)(Table2).Theovarianfolliclesareconnectedtothestromabya
narrow,vascularizedpeduncle,formedbyasmoothandmono‐stratifiedlamellar
epithelium,thelengthofwhichincreasesasvitellogenesisadvances(Figure2C,D).
MatureoocytescontainingPASpositiveovarianfluid,whichisparticularlyabundant
duringthespawningperiod,areexpelledintothelumen.Thezonapellucida(Figure
2E,F),stronglyPAS‐positive,isvisiblefromthecorticalalveolistage(CA),andbecome
thickerwiththeproceedingofthematurationphase,reachingmaximumdimensions
duringtheGVMstage,beingmadeupofthreelayers,inallstudiedspecies(Figure3).
Thethinoutermostandtheintermediate,mediumelectrondenseregionformtheouter
zonapellucida,whiletheinternalregionisamultilamellarstriatedregion(innerzona
pellucida)(Figure2E–G).
Theovarianwallismadeupofthreelayers(Figure2H,I).Theouterlayeris
composedbyamesotheliumandthemiddlelayerhastwosub‐layers(anouterone
formedbyacircularmuscularsystem,andaninneronebymuchthickerlongitudinal
muscularsystem),whichcontainssmoothmusculatureandnumerousbloodvessels
(Figure2H).Theinnermostlayer(internalepithelium)oftheovaryiscoveredwitha
simplecuboidalepithelium,and,duringtheperiodofvitellogenesisandspawning,
modifiesitsstructure,containingstronglyPASpositivegranulesandcytoplasmic
projections(Figure2I)(Table2).
Figure 1.
Morphological characteristics of cystovarian type II-3 ovary of the five species studied
(H&E). Corresponding macroscopic ovaries, located in the abdominal cavity above the bladder, are
also shown down on the left of each species. (
A
)Scorpaena scrofa (TL 22.3 cm); (
B
)Scorpaena notata
(TL 11.3 cm); (
C
)Scorpaena porcus (TL 15.6 cm); (
D
)Scorpaena elongata (TL 28.7 cm); (
E
)Helicolenus
dactylopterus (TL 18.2 cm). bv, blood vessel; ow, ovarian wall; r, rachis. The dashed line shows the
radial development of oocytes from the stroma to surrounding ovarian wall.
Animals 2022,12, 1412 7 of 19
Animals2022,12,x7of20
InthezygoparousH.dactylopterus,cryptalstructurespackedwithspermatozoa(here
calledspermaticcrypts)nearthebasalpartofthestromaareobserved(Figure2J).Duringthe
storageperiod,thecryptalepitheliumreleasesstronglyPAS‐positivegranules(Figure2J).
Table2.Histochemicalpropertiesoftheinternalepitheliumoftheovarianwall,ovarianfluid,and
holocrineglandintherachisinHelicolenusdactylopterus(HD),Scorpaenaelongata(SE);Scorpaena
notata(SN),Scorpaenaporcus(SP)andScorpaenascrofa(SS).PAS,periodicacid–Schiff;AB,Alcian
blue;+,presence;−,absence.
StructuresPASABpH2.5
HDSESNSPSSHDSESNSPSS
Ovarianwall++++++++++
Ovarianfluid+++++−−−−−
Holocrinegland+++++−−−−−
Figure2.Photomicrographsofovarianhistology,depicting:(A)Scorpaenaelongata,crosssectionof
amaturingovaryinwhichholocrineglands,reactingpositivelytoPASstaining,arepresent
(AB/PAS).(B)Scorpaenaporcus,highmagnificationofanholocrineglandPAS‐positive(AB/PAS);
Figure 2.
Photomicrographs of ovarian histology, depicting: (
A
)Scorpaena elongata, cross section
of a maturing ovary in which holocrine glands, reacting positively to PAS staining, are present
(AB/PAS). (
B
)Scorpaena porcus, high magnification of an holocrine gland PAS-positive (AB/PAS);
(
C
) Scorpaena scrofa, cross-section of a mature/spawning ovary, in which ovarian follicles are
connected to the stroma by a branching, vascularized peduncle (H&E); (
D
)Helicolenus dactylopterus, a
long peduncle which encompasses the follicular cells of its vitellogenic oocytes (H&E); (
E
)Scorpaena
porcus, oocytes at different maturity stages (CA and Vtg3), where diverse thickness of the zona
pellucida, strongly PAS-positive, are evident (AB/PAS); (
F
)Scorpaena scrofa, high magnification of
inner and outer layers of zona pellucida in a vitellogenic oocyte. Striations are also visible (AB/PAS).
(
G
)Helicolenus dactylopterus, outer and inner zona pellucida with microvilli in evidence. Striations in
great detail (H&E). (
H
) Scorpaena notata, cross-section of an ovarian wall during developing stage
(H&E); (
I
) Scorpaena porcus, cross-section of the ovarian wall during spawning stage, where active
secretory epithelium is visible in greater detail (AB/PAS). (
J
)Helicolenus dactylopterus, spermatic
crypts packed with spermatozoa. PAS-positive granules are visible, realized by the epithelium
(AB/PAS). bv, blood vessel; CA, cortical alveoli; cp, cytoplasmatic projections; dl, dense line; ee,
external epithelium; ho, holocrine gland; ie, internal epiuthelium; m, microvilli; ml, muscular layer;
n, nucleus; p, peduncle; PG, primary growth oocyte; s, striation; sc, spermatic crypt; sp, spermatozoa;
Vtg1, primary vitellogenic oocyte; Vtg2, secondary vitellogenic oocyte; Vtg3, tertiary vitellogenic
oocyte; zp, zona pellucida; zpi, inner zona pellucida; zpo, outer zona pellucida.
Animals 2022,12, 1412 8 of 19
Animals2022,12,x8of20
(C)Scorpaenascrofa,cross‐sectionofamature/spawningovary,inwhichovarianfolliclesare
connectedtothestromabyabranching,vascularizedpeduncle(H&E);(D)Helicolenusdactylopterus,
alongpedunclewhichencompassesthefollicularcellsofitsvitellogenicoocytes(H&E);(E)
Scorpaenaporcus,oocytesatdifferentmaturitystages(CAandVtg3),wherediversethicknessofthe
zonapellucida,stronglyPAS‐positive,areevident(AB/PAS);(F)Scorpaenascrofa,highmagnification
ofinnerandouterlayersofzonapellucidainavitellogenicoocyte.Striationsarealsovisible
(AB/PAS).(G)Helicolenusdactylopterus,outerandinnerzonapellucidawithmicrovilliinevidence.
Striationsingreatdetail(H&E).(H)Scorpaenanotata,cross‐sectionofanovarianwallduring
developingstage(H&E);(I)Scorpaenaporcus,cross‐sectionoftheovarianwallduringspawning
stage,whereactivesecretoryepitheliumisvisibleingreaterdetail(AB/PAS).(J)Helicolenus
dactylopterus,spermaticcryptspackedwithspermatozoa.PAS‐positivegranulesarevisible,realized
bytheepithelium(AB/PAS).bv,bloodvessel;CA,corticalalveoli;cp,cytoplasmaticprojections;dl,
denseline;ee,externalepithelium;ho,holocrinegland;ie,internalepiuthelium;m,microvilli;ml,
muscularlayer;n,nucleus;p,peduncle;PG,primarygrowthoocyte;s,striation;sc,spermaticcrypt;
sp,spermatozoa;Vtg1,primaryvitellogenicoocyte;Vtg2,secondaryvitellogenicoocyte;Vtg3,
tertiaryvitellogenicoocyte;zp,zonapellucida;zpi,innerzonapellucida;zpo,outerzonapellucida.
Figure3.Box‐ andwhisker‐plotsshowingmeanvalueofzonapellucidathicknessatdifferent
maturitystages.Theboxrepresentsthe25thand75thquantile.Thepointsbeyondthewhiskersare
potentialoutliers.(A)Scorpaenascrofa;(B)Scorpaenanotata;(C)Scorpaenaporcus;(D)Scorpaena
elongata;(E)Helicolenusdactylopterus.CA,corticalalveoli;GVM,germinalvesiclemigration;VTG1,
primaryvitellogenic;VTG2,secondaryvitellogenic;VTG3,tertiaryvitellogenic.Foreachspecies,a
tablewithsignificantdifferences(p‐value<0.05,*)betweenmaturitystagesisprovided(NS,not
significantdifferences).
3.2.OvarianDynamics
Theprogressivechangeincellulardiametersofthefivestudiedspeciesisshownin
Figure4.Differencesamongdevelopmentalmaturitystageswerealwaysstatistically
significant(ANOVA,p<0.001).H.dactylopterusshowedthelargestoocytedimensions,
followedbyS.scrofa(Figure4A,E),whileS.notatathesmallestone(Figure4B).
Figure 3.
Box- and whisker-plots showing mean value of zona pellucida thickness at different
maturity stages. The box represents the 25th and 75th quantile. The points beyond the whiskers
are potential outliers. (
A
) Scorpaena scrofa; (
B
)Scorpaena notata; (
C
)Scorpaena porcus; (
D
)Scorpaena
elongata; (
E
)Helicolenus dactylopterus. CA, cortical alveoli; GVM, germinal vesicle migration; VTG1,
primary vitellogenic; VTG2, secondary vitellogenic; VTG3, tertiary vitellogenic. For each species, a
table with significant differences (p-value < 0.05, *) between maturity stages is provided (NS, not
significant differences).
3.2. Ovarian Dynamics
The progressive change in cellular diameters of the five studied species is shown
in Figure 4. Differences among developmental maturity stages were always statistically
significant (ANOVA, p< 0.001). H. dactylopterus showed the largest oocyte dimensions,
followed by S. scrofa (Figure 4A,E), while S. notata the smallest one (Figure 4B).
Photo identification of the microscopic female maturity scale for the five species
studied is presented in Figure 5.
Females in PG phase showed that only PG oocytes through the perinucleolar stage
in the ovaries (immature stages). As females move into secondary growth phase, they
can be histologically distinguished by the initial appearance of CA oocytes (recovering
stage, 2b). In these species, CA oocytes were characterized by very small and low in
number cortical alveoli (weakly PAS positive), as well as a lack of oil droplets. Females
in the maturing stage (stage 2C, NS) were distinguishable for the appearance of Vtg1 and
Vtg2 oocytes, with no evidence of POFs or Vtg3 oocytes. Vitellogenic oocytes observed
in species belonging to the genus Scorpaena were characterized by the complete absence
of oil droplets in the cytoplasm, which, instead, were visible in H. dactylopterus oocytes
(Figure 5E). Actively spawning females (AS, stage mature/spawner) were characterized
by the presence of the previous described stages, and Vtg3 and the early stages of oocyte
maturation (such as GVM oocytes), hydrated oocytes present simultaneously and POFs
(Figure 6A–C). In H. dactylopterus, the presence of embryos in the gelatinous mass were
recognizable with the appearance of hydrated oocytes, but with an undifferentiated mass
of cells. In the post-spawner phase (PS, spent/resting), females showed a great amount of
atresia of vitellogenic oocytes and PG oocytes (Figures 5and 6D–F).
Animals 2022,12, 1412 9 of 19
Animals2022,12,x9of20
Figure4.Box‐andwhisker‐plotsshowingthemeanoocytedimension(diameter,μm)ofdifferent
celltypesinthefivestudiedspecies.(A)Scorpaenascrofa,numberofmeasuredoocytes=1888;(B)
Scorpaenanotata,numberofmeasuredoocytes=1901;(C)Scorpaenaporcus,numberofmeasured
oocytes=2401;(D)Scorpaenaelongata,numberofmeasuredoocytes=1600;(E)Helicolenus
dactylopterus,numberofmeasuredoocytes=3140.Theboxrepresentsthe25thand75thquantile.
Thepointsbeyondthewhiskersarepotentialoutliers.Aboveorundereachbox,therelativeimage
ofthecelltypeforeachspeciesinvestigatedisshown.
Figure 4.
Box- and whisker-plots showing the mean oocyte dimension (diameter,
µ
m) of dif-
ferent cell types in the five studied species. (
A
)Scorpaena scrofa, number of measured oocytes
= 1888; (
B
)Scorpaena notata, number of measured oocytes = 1901; (
C
)Scorpaena porcus, num-
ber of measured oocytes = 2401; (
D
)Scorpaena elongata, number of measured oocytes = 1600;
(
E
)
Helicolenus dactylopterus
, number of measured oocytes = 3140. The box represents the 25th and
75th quantile. The points beyond the whiskers are potential outliers. Above or under each box, the
relative image of the cell type for each species investigated is shown.
Animals 2022,12, 1412 10 of 19
Animals2022,12,x10of20
Photoidentificationofthemicroscopicfemalematurityscaleforthefivespecies
studiedispresentedinFigure5.
FemalesinPGphaseshowedthatonlyPGoocytesthroughtheperinucleolarstage
intheovaries(immaturestages).Asfemalesmoveintosecondarygrowthphase,theycan
behistologicallydistinguishedbytheinitialappearanceofCAoocytes(recoveringstage,
2b).Inthesespecies,CAoocyteswerecharacterizedbyverysmallandlowinnumber
corticalalveoli(weaklyPASpositive),aswellasalackofoildroplets.Femalesinthe
maturingstage(stage2C,NS)weredistinguishablefortheappearanceofVtg1andVtg2
oocytes,withnoevidenceofPOFsorVtg3oocytes.Vitellogenicoocytesobservedin
speciesbelongingtothegenusScorpaenawerecharacterizedbythecompleteabsenceof
oildropletsinthecytoplasm,which,instead,werevisibleinH.dactylopterusoocytes
(Figure5E).Activelyspawningfemales(AS,stagemature/spawner)werecharacterized
bythepresenceofthepreviousdescribedstages,andVtg3andtheearlystagesofoocyte
maturation(suchasGVMoocytes),hydratedoocytespresentsimultaneouslyandPOFs
(Figure6A–C).InH.dactylopterus,thepresenceofembryosinthegelatinousmasswere
recognizablewiththeappearanceofhydratedoocytes,butwithanundifferentiatedmass
ofcells.Inthepost‐spawnerphase(PS,spent/resting),femalesshowedagreatamountof
atresiaofvitellogenicoocytesandPGoocytes(Figures5and6D–F).
Figure5.Femaledevelopmentalmaturitystagesofthefivestudiedspecies.(A)Scorpaenascrofa;(B)
Scorpaenanotata;(C)Scorpaenaporcus;(D)Scorpaenaelongata;(E)Helicolenusdactylopterus.ThePrimary
growthphase(PG)includedimmaturevirginandvirgin‐developingovaries,whilethemultiple
secondarygrowth(SG)phaseincludednon‐spawning(NS)females(withnospawningmarkers,
recoveringandmaturingfemales),activelyspawning(AS)females(withmarkersofimminentor
recentspawningactivity,spawner/maturefemales)andpost‐spawning(PS)females(agreatamount
ofatreticoocytesarepresent,spent,andrestingfemales).Atr,Atreticoocyte;CA,corticalalveoli;E,
embryo;GVM,germinalvesiclemigration;H,hydration;PG,primarygrowthoocyte;POF,post‐
ovulatoryfollicleVtg1,primaryvitellogenic;Vtg2,secondaryvitellogenic;Vtg3,tertiaryvitellogenic.
Figure 5.
Female developmental maturity stages of the five studied species. (
A
)Scorpaena scrofa;
(
B
)Scorpaena notata; (
C
)Scorpaena porcus; (
D
)Scorpaena elongata; (
E
)Helicolenus dactylopterus. The
Primary growth phase (PG) included immature virgin and virgin-developing ovaries, while the
multiple secondary growth (SG) phase included non-spawning (NS) females (with no spawning
markers, recovering and maturing females), actively spawning (AS) females (with markers of im-
minent or recent spawning activity, spawner/mature females) and post-spawning (PS) females (a
great amount of atretic oocytes are present, spent, and resting females). Atr, Atretic oocyte; CA,
cortical alveoli; E, embryo; GVM, germinal vesicle migration; H, hydration; PG, primary growth
oocyte; POF, post-ovulatory follicle Vtg1, primary vitellogenic; Vtg2, secondary vitellogenic; Vtg3,
tertiary vitellogenic.
Animals2022,12,x11of20
Figure6.Post‐ovulatoryfolliclesandatreticoocytesinScorpaenagenusandinHelicolenus
dactylopterus(AB/PASstaining).(A)ActivelyspawningfemalesofS.scrofa,(B)H.dactylopterusand
(C)S.elongatathathavealreadyspawnedforthepresenceofnewpost‐ovarianfollicles.Atretic
oocyteswithirregularcontourandzonapellucidawhicharePAS‐positiveandwithpartially
liquefiedyolkglobulesattheendofspawningin(D)Scorpaenascrofa,(E)Scorpaenanotata,and(F)
Scorpaenaporcus.Atr,atreticoocyte;GVM,germinalvesiclemigrationstage;of,ovarianfluid;POF,
post‐ovulatoryfollicle;zp,zonapellucida.
Theevolutionofthepercentageoftheovariansubphasesabovedescribed(Figure5),
togetherwiththetrendoftheseasonalGonadosomaticindex(GSI)(Figure7A,B)showed
thepresenceofimmatureS.scrofaandS.porcusfemaleswithexclusivelyPGoocytes
occurredinwintermonths.TheproportionofSGfemalesstartedinspring,wherethe
mostpartofthemwereattheCAstage,whiletheremainingwereatvariousstagesof
ovariandevelopmental,butnotexhibitedspawningmarkers,andwerethusidentifiedas
NS.ASfemalesoccurredduringthesummermonths,asconfirmedbythehighmean
valueofGSI(ASS.scrofafemales,GSI1.8–11.20%;ASS.porcusfemales,GSI1.11–14.18%).
PSfemaleswererecordedinlowproportioninsummerandintheautumn(mainlyin
October)(Figure7A,B).
InH.dactylopterus,ASfemaleswithembryosinearly‐celleddevelopmentalstage
withanintraovariangelatinousmatrix(GSI1.18–9.48%)wereobservedfromNovember
toMarch(Figures4Eand5E),withhighpercentagesinJanuary(Figure7C).FromApril
toOctober,PGandNSfemaleswereobservedindifferentpercentages(Figure7C).
Moreover,spermaticcryptswereobservedinfemalesfromAugust(attherecovering
stage,NS)toMarch(atspentstage,PS).
ThelownumberofS.elongataandS.notatafemalesstudieddidnotpermitan
estimationofaseasonalityofspawning.However,S.elongataASfemales(GSI2.54–3.76%)
wereobservedinJulyandSeptember,whileS.notataASfemales(GSI2.04–15.34%)in
AugustandSeptember.
Theoocytesizefrequencydistributions(OSFD)fromsubsamplesofmature/spawner
females(AS)oftheselectedspeciesarepresentedinFigure8.Duringtheseasonalityof
spawning,severalcohortsofoocyteinalldevelopmentstages(seeoocyte’sdimensionin
Figure4)wereobservedinallspecies,highlightinganasynchronousorganizationofthe
ovaries(Figure8A–E).TheOSFDwascontinuous,withoutanyevidenthiatusbetween
primaryandsecondarygrowthstageoocytes,withPGrepresentingalwaysthemost
abundantoocytes.CAoocytes,althoughrepresentedasmallfractionofthetotalnumber
oftheoocytepopulationinallspecies,werealwayspresentinallfemalesanalyzed,as
wellastheVtg1oocytes.Whenspawninghasstartedandatleastonebatchhasalready
beenreleased(ovarieswithPOFs),theOSFDshowedseveralcohortsconsistingofoocytes
inearly(Vtg1)andadvancedvitellogenesis(includingGVMoocytes).
Figure 6.
Post-ovulatory follicles and atretic oocytes in Scorpaena genus and in Helicolenus dactylopterus
(AB/PAS staining). (
A
) Actively spawning females of S. scrofa, (
B
)H. dactylopterus and (
C
)S. elongata
that have already spawned for the presence of new post-ovarian follicles. Atretic oocytes with
irregular contour and zona pellucida which are PAS-positive and with partially liquefied yolk
globules at the end of spawning in (
D
)Scorpaena scrofa, (
E
)Scorpaena notata, and (
F
)Scorpaena porcus.
Atr, atretic oocyte; GVM, germinal vesicle migration stage; of, ovarian fluid; POF, post-ovulatory
follicle; zp, zona pellucida.
Animals 2022,12, 1412 11 of 19
The evolution of the percentage of the ovarian subphases above described (Figure 5),
together with the trend of the seasonal Gonado somatic index (GSI) (Figure 7A,B) showed
the presence of immature S. scrofa and S. porcus females with exclusively PG oocytes
occurred in winter months. The proportion of SG females started in spring, where the
most part of them were at the CA stage, while the remaining were at various stages of
ovarian developmental, but not exhibited spawning markers, and were thus identified as
NS. AS females occurred during the summer months, as confirmed by the high mean value
of GSI (AS S. scrofa females, GSI 1.8–11.20%; AS S. porcus females, GSI 1.11–14.18%). PS
females were recorded in low proportion in summer and in the autumn (mainly in October)
(Figure 7A,B).
In H. dactylopterus, AS females with embryos in early-celled developmental stage
with an intraovarian gelatinous matrix (GSI 1.18–9.48%) were observed from November to
March (Figures 4E and 5E), with high percentages in January (Figure 7C). From April to
October, PG and NS females were observed in different percentages (Figure 7C). Moreover,
spermatic crypts were observed in females from August (at the recovering stage, NS) to
March (at spent stage, PS).
The low number of S. elongata and S. notata females studied did not permit an estima-
tion of a seasonality of spawning. However, S. elongata AS females (GSI 2.54–3.76%) were
observed in July and September, while S. notata AS females (GSI 2.04–15.34%) in August
and September.
The oocyte size frequency distributions (OSFD) from subsamples of mature/spawner
females (AS) of the selected species are presented in Figure 8. During the seasonality of
spawning, several cohorts of oocyte in all development stages (see oocyte’s dimension in
Figure 4) were observed in all species, highlighting an asynchronous organization of the
ovaries (Figure 8A–E). The OSFD was continuous, without any evident hiatus between
primary and secondary growth stage oocytes, with PG representing always the most
abundant oocytes. CA oocytes, although represented a small fraction of the total number
of the oocyte population in all species, were always present in all females analyzed, as well
as the Vtg1 oocytes. When spawning has started and at least one batch has already been
released (ovaries with POFs), the OSFD showed several cohorts consisting of oocytes in
early (Vtg1) and advanced vitellogenesis (including GVM oocytes).
In H. dactylopterus and in the species belonging to the genus Scorpaena, during the
seasonality of spawning, the new POFs (formed few hours before) co-occurred with GVM
oocytes in the late stage of development (to be spawned in the next hours) (Figure 6A,B),
providing a strong indication that SI (spawning interval) should not exceed two days.
Regarding the relative intensity of atresia (RIA), it was estimated only in the species
for which the spawning period was established (S. scrofa,S. porcus, and H. dactylopterus)
(Figure 9). SG oocytes with
α
-atresia were always present through the spawning period
in S. scrofa and S. porcus (Figure 9A), while in H. dactylopterus, no
α
-atresia (Atretic 0) was
observed in November (at the beginning of reproductive period) (Figure 9B). However,
atresia phenomenon was always relatively low (Atretic 1), with a strong increase towards
the end of spawning, mainly in September in S. scrofa and S. porcus, (Figure 8A), and in
March in H. dactylopterus (Figure 8B).
Animals 2022,12, 1412 12 of 19
Animals2022,12,x12of20
Figure7.ProportionsofScorpaenascrofa(A),Scorpaenaporcus(B),andHelicolenusdactylopterus(C)
femalesinrelationtotheirgonadalgrowthstage(PG,primarygrowth;NS,non‐spawning;AS,
activelyspawning;PS,post‐spawning).S.scrofaandS.porcusweredescribedperseason,whileH.
dactylopteruspermonth.Theevolutionofthegonado–somaticindex(GSI(%))isalsorepresented.
Thedouble‐headedarrowinFigure7Cindicatesthepresenceofspermaticcryptsinsidetheovaries.
Aboveeachseasonormonth,thenumberofanalyzedfemalesisreported.
Figure 7.
Proportions of Scorpaena scrofa (
A
), Scorpaena porcus (
B
), and Helicolenus dactylopterus
(
C
) females in relation to their gonadal growth stage (PG, primary growth; NS, non-spawning; AS,
actively spawning; PS, post-spawning). S. scrofa and S. porcus were described per season, while
H. dactylopterus per month. The evolution of the gonado–somatic index (GSI (%)) is also represented.
The double-headed arrow in Figure 7C indicates the presence of spermatic crypts inside the ovaries.
Above each season or month, the number of analyzed females is reported.
Animals 2022,12, 1412 13 of 19
Animals2022,12,x13of20
Figure8.Oocytesizefrequency(OSFD)distributionsfrom8Scorpaenascrofa(A),4Scorpaenanotata
(B),13Scorpaenaporcus(C),4Scorpaenaelongata(D),and20Helicolenusdactylopterus(E)females.
InH.dactylopterusandinthespeciesbelongingtothegenusScorpaena,duringthe
seasonalityofspawning,thenewPOFs(formedfewhoursbefore)co‐occurredwithGVM
oocytesinthelatestageofdevelopment(tobespawnedinthenexthours)(Figure6A,B),
providingastrongindicationthatSI(spawninginterval)shouldnotexceedtwodays.
Regardingtherelativeintensityofatresia(RIA),itwasestimatedonlyinthespecies
forwhichthespawningperiodwasestablished(S.scrofa,S.porcus,andH.dactylopterus)
(Figure9).SGoocyteswithα‐atresiawerealwayspresentthroughthespawningperiod
inS.scrofaandS.porcus(Figure9A),whileinH.dactylopterus,noα‐atresia(Atretic0)was
observedinNovember(atthebeginningofreproductiveperiod)(Figure9B).However,
atresiaphenomenonwasalwaysrelativelylow(Atretic1),withastrongincreasetowards
theendofspawning,mainlyinSeptemberinS.scrofaandS.porcus,(Figure8A),andin
MarchinH.dactylopterus(Figure8B).
Figure 8.
Oocyte size frequency (OSFD) distributions from 8 Scorpaena scrofa (
A
), 4 Scorpaena notata
(B), 13 Scorpaena porcus (C), 4 Scorpaena elongata (D), and 20 Helicolenus dactylopterus (E) females.
Animals2022,12,x14of20
Figure9.Relativeintensityofatresia(RIA)throughthespawningseasoninfemalesofScorpaena
scrofaandS.porcus(A)andHelicolenusdactylopterus(B).Atretic0:ovarieswith0%ofSGoocytes
withα‐atresia;Atretic1:ovarieswith<50%ofSGoocyteswithα‐atresia;Atretic2:ovarieshaving
≥50%SGoocyteswithα‐atresia.InScorpaenaspeciesdateareexpressedperseason,whileinH.
dactylopteruspermonth.
4.Discussion
TheovarianstructureofScorpenoidfishesanalyzedinthispaperdiffersfromthat
observedinthemajorityofteleosts.ThecystovariantypeII‐3,indeed,representsa
specializedstructurestrictlyrelatedtotheproductionofgelatinousmatricesthat
surroundtheeggs,characteristicsoftheScorpaenidaeandSebastidaefamilies(e.g.,[6]).
ComparingthehistologicalsectionsoftheScorpaenaspeciesovarieswiththoseofH.
dactylopterus,indeed,manyfeaturesresultedtobesimilarinvolvedinfacilitatingthe
productionofgelatinouseggmasses.
Ineachstudiedspecies,theoocytes(startingfromthoseatcorticalalveolistage)are
suppliedbyahighlyvascularizedpeduncle,whichbecomelongerwiththeincreaseofthe
oocytediameter.Thisspecializationisfoundinviviparous[50]andoviparousspecies[51],
aswellassomeothervertebrates,includingbirdsandreptiles[52].Severalfunctionsare
attributedtothisstructure.Inovuliparousoviparousspecies,suchasthefourstudied
speciesofthegenusScorpaena,peduncleshavethemainfunctiontopreventingoocyte
crowding[11,53]facilitatingtheovulationofmatureoocytesinthegelatinousmasses,
while,inspecieswithinternalfertilizationsuchasH.dactylopterus,thefunctionsof
pedunclearetoprovideaccessofspermatozoatotheoocytesduringfertilizationand
accesstonutrientsfortheembryos[11,54].
InthespeciesofthegenusScorpaena,hereanalyzed,despitethepelagicnatureofthe
eggs,thenumberofcorticalalveoliislowandoildropletsaretotallylackingalsoin
vitellogenicoocytes,makingtheswellingoftheeggsalmostabsentafterthecortical
reaction.Similarfeatureshavebeenregisteredintheoocytesofotherspeciesofthegenera
Scorpaena(notinvestigatedinthispaper)[55,56],andofthegeneraScorpaenopsisand
Sebastapistes[57,58],whileintheovuliparousspeciesbelongingtoPteroisgenus,a
proliferationofcorticalalveoliwasdisplaced,togetherwithaconsiderabledepositionof
lipiddropletsoccurredinthevitellogenicoocytes[20].Aphenomenonsimilartowhat
wasreportedforthePteroisgenushasalsobeenregisteredintheSebastidaeH.
dactylopterus,wherecorticalalveoliaresmallandlow,butlipiddropletsbeganto
accumulateintheoocytecytoplasmwithinthesecondarygrowthphaseatthesametime
ofcorticalalveoliprecursors,confirmingthepelagicphaseoftheeggs[11].
Anotherpeculiarityintheprocessofoogenesisinthestudiedspecies,strictlyrelated
withviviparity(e.g.,[59–62]),isthethicknessofthezonapellucida(zp).S.scrofaandS.
elongatashowedamaximumthicknessofzonapellucidaof5.8μm,whilethezygoparous
H.dactylopterusamaximumvalueof6.2μm,whichareconsiderablylessthanthatfound
inotherrelatedoviparousspecies,suchasTriglalyra(zp,19μm)andChelidonichthys
obscurus(zp,37μm)[63],butlargerthanthosefoundinotherScorpaenidaespecies,such
asDendrochiruszebra,zp,1μm;Scorpaenopsispossi,zp,1.4μm;Sebastapistescyanostigma,
Figure 9. Relative intensity of atresia (RIA) through the spawning season in females of Scorpaena scrofa
and S. porcus (
A
) and Helicolenus dactylopterus (
B
). Atretic 0: ovaries with 0% of SG oocytes with
α
-atresia; Atretic 1: ovaries with <50% of SG oocytes with
α
-atresia; Atretic 2: ovaries having
≥
50%
SG oocytes with
α
-atresia. In Scorpaena species date are expressed per season, while in H. dactylopterus
per month.
Animals 2022,12, 1412 14 of 19
4. Discussion
The ovarian structure of Scorpenoid fishes analyzed in this paper differs from that
observed in the majority of teleosts. The cystovarian type II-3, indeed, represents a special-
ized structure strictly related to the production of gelatinous matrices that surround the
eggs, characteristics of the Scorpaenidae and Sebastidae families (e.g., [
6
]). Comparing the
histological sections of the Scorpaena species ovaries with those of H. dactylopterus, indeed,
many features resulted to be similar involved in facilitating the production of gelatinous
egg masses.
In each studied species, the oocytes (starting from those at cortical alveoli stage) are
supplied by a highly vascularized peduncle, which become longer with the increase of the
oocyte diameter. This specialization is found in viviparous [
50
] and oviparous species [
51
],
as well as some other vertebrates, including birds and reptiles [
52
]. Several functions are
attributed to this structure. In ovuliparous oviparous species, such as the four studied
species of the genus Scorpaena, peduncles have the main function to preventing oocyte
crowding [
11
,
53
] facilitating the ovulation of mature oocytes in the gelatinous masses, while,
in species with internal fertilization such as H. dactylopterus, the functions of peduncle are
to provide access of spermatozoa to the oocytes during fertilization and access to nutrients
for the embryos [11,54].
In the species of the genus Scorpaena, here analyzed, despite the pelagic nature of
the eggs, the number of cortical alveoli is low and oil droplets are totally lacking also
in vitellogenic oocytes, making the swelling of the eggs almost absent after the cortical
reaction. Similar features have been registered in the oocytes of other species of the
genera Scorpaena (not investigated in this paper) [
55
,
56
], and of the genera Scorpaenopsis
and Sebastapistes [
57
,
58
], while in the ovuliparous species belonging to Pterois genus, a
proliferation of cortical alveoli was displaced, together with a considerable deposition of
lipid droplets occurred in the vitellogenic oocytes [
20
]. A phenomenon similar to what was
reported for the Pterois genus has also been registered in the Sebastidae H. dactylopterus,
where cortical alveoli are small and low, but lipid droplets began to accumulate in the
oocyte cytoplasm within the secondary growth phase at the same time of cortical alveoli
precursors, confirming the pelagic phase of the eggs [11].
Another peculiarity in the process of oogenesis in the studied species, strictly related
with viviparity (e.g., [
59
–
62
]), is the thickness of the zona pellucida (zp). S. scrofa and
S. elongata showed a maximum thickness of zona pellucida of 5.8
µ
m, while the zygoparous
H. dactylopterus a maximum value of 6.2
µ
m, which are considerably less than that found
in other related oviparous species, such as Trigla lyra (zp, 19
µ
m) and Chelidonichthys
obscurus (zp, 37
µ
m) [
63
], but larger than those found in other Scorpaenidae species, such
as Dendrochirus zebra, zp, 1
µ
m; Scorpaenopsis possi, zp, 1.4
µ
m; Sebastapistes cyanostigma, zp,
0.7
µ
m [
7
,
64
]. The zona pellucida of teleosts oocytes is complex, usually consisting of layers
crossed by pores or channels [
63
], the morphology of which varies between species [
64
].
It is interrupted at the animal pole region by a specialized opening, the micropyle, which
allows the passage of sperm in fertilization [
65
]. Zona pellucida plays various roles during
oogenesis, egg deposition, fertilization, and embryogenesis. It is involved not only in the
nourishment of the theca and granulosa layers and of the embryo, but also in the secretion
of enzymes, the transportation of yolk material in early developmental stages, and the
fixation of a deposited egg to the substratum. It is also implicated in the sperm attraction
and prevention of polyspermy and in the antibacterial and mechanical protection during
spawning, fertilization, and post fertilization periods Selman [
63
]. For the developing
embryo, the egg envelope enables gas exchange, excretion, and transport of nutrients from
the external environment [66].
As a general rule, viviparous fishes are characterized by a very thin zona pellucida,
as recorded in viviparous scorpaenoids, such as Sebastes paucispinis, in which the zona
pellucida was 1
µ
m thick [
67
,
68
]. If, in H. dactylopterus, the thickness of zona pellucida
can be justified with its reproductive mode (e.g., nourishment of embryos), in a species of
the genus Scorpaena, the reduction of thickness could imply loss of mechanical protection
Animals 2022,12, 1412 15 of 19
(essential when spawning is pelagic), which should be alternatively provided by the
gelatinous matrix that encloses the eggs, as also reported by [
11
] in S. notata. In addition,
as reported in other Scorpenoidei [
11
], all analyzed species showed during the spawning
period a specialization and modification of the ovarian wall with internal epithelium
secreting polysaccharides (PAS+) and highly developed cytoplasmatic projections, which
are probably related to the great production of the ovarian fluid (always PAS+) present
in the ovary. This type of secretory activity also found in the holocrine glands of the
stroma could be responsible for the production of ovarian fluid, as already observed in
S. notata [11].
The structuration of the spermatic crypts shown in H. dactylopterus is surely more
complex than those described in viviparous species belonging to the same family, in which
free swimming sperm is present in the ovarian fluid or singly adhered to the ovarian
epithelium (e.g., [
69
,
70
]). Only recently, in the viviparous Sebastes schelegelii, swimming
spermatozoa were found in the ovarian fluid at the early storage stage, while most sperma-
tozoa were wrapped in the crypt structures between the multi-layered columnar epithelium
and follicular layer of oocytes [16].
The mean monthly values of gonadosomatic index and the relative fractions of var-
ious developmental stages and spawning phases allowed for an estimation of a reliable
spawning seasonality of S. scrofa,S. porcus, and H. dactylopterus in Sardinian waters (central–
western Mediterranean Sea), until now unknown. A narrow reproductive period was found
in the two species of Scorpaena. Actively spawning females of S. scrofa were observed mainly
during the summer months from July to September, as well as females of
S. porcus
. These
data are consistent with the few observations available for the Mediterranean Sea, which
reported a very narrow spawning season (in summer) for both species (for S. scrofa: [
36
,
69
];
for S. porcus: [
37
,
71
,
72
]). The few data here provided for S. notata (mature females in Au-
gust and September) were comparable with the only reported reproductive period in the
Mediterranean for the species (July–October [
37
,
71
,
72
]), while with regards to S. elongata,
our records (mature females in July and September) have represented the first information
on spawning period for its entire geographical distribution.
A longer spawning cycle (November–March) was, indeed, observed for the blue-
mouth H. dactylopterus, with a peak of actively spawning females having embryos inside the
ovaries in January. This period differs from the observations of the western Mediterranean
Sea (e.g., [
3
,
8
,
34
]), while it is consistent with data from Portugal and the Azores [
73
–
75
]. The
presence of spermatic crypts found in developing females from August to October (and then
from November to March in developing, non- and actively spawning specimens) confirmed
a specific reproductive strategy of the species, with a prolonged sperm storage period
(8 months) available for period with a low possibility of mating. Ref. [
33
] also described a
delay of 1 to 3 months between insemination and fertilization, and, as suggested by [
8
,
74
,
75
]
reported a long sperm storage.
All of the analyzed species were batch-spawners in association with an asynchronous
ovarian organization. Indeed, based on the OSFD in the ovaries of sexually mature females,
all species were characterized by continuous oogenesis with multiple oocyte deposition.
This type of oogenesis was found also in the tropical fishes of the family Scorpaenidae
(genera Scorpaenopsis and Scorpaena), and appears to be associated, in the literature, with
small GSI values in the females with mature ovaries (1.08–1.60% in Scorpaenopsis papuensis,
0.89–1.05% in Sc. possi, and 1.3–1.61% in S. cyanostigma [
57
,
58
,
76
]). Our results show a
completely different trend for the studied species with high values of GSI in mature females
(among Scorpaena species, 15.34% GSI maximum in S. notata, and 9.48% maximum GSI in
H. dactylopterus).
The stable number of cortical alveoli and vitellogenic oocytes observed throughout the
spawning season in all the species analyzed indicated a continuous recruitment of oocytes
and the occurrence of de novo vitellogenesis (i.e., process of producing vitellogenic oocytes
from previtellogenic oocytes during the spawning season, and consequent recruitment
into the standing stock of yolked oocytes). This result has been already observed in
Animals 2022,12, 1412 16 of 19
H. dactylopterus in Spanish and Portuguese waters [
75
,
77
], but not in the Scorpaena species.
The OSFD showed that each cohort of oocyte was at the same stage, and sequential
developmental stages co-occurred, ranging from the onset of secondary growth (early
cortical alveoli stage) to final oocyte maturation. This type of modal dynamic organization
is quite typical for fish populations with an indeterminate fecundity type [
49
,
78
]. Indeed, in
H. dactylopterus and in the four Scorpaena species analyzed, the co-occurrence of very early
SG oocytes with new POFs indicating that oocyte recruitment was taking place in actively
spawning females. In addition, although during the spawning season low atresia was
detected, a marked increase in the relative intensity of
α
-atresia (up to 50%) was found in the
ovaries at the end of spawning season. This pattern, already found in H. dactylopterus [
74
,
77
],
has never been observed in the Scorpaena species here analysed, thus, it seems to corroborate
the hypothesis of an indeterminate fecundity, clearly distinguishing these species from fish
characterized by determinate fecundity, in which very low values of incidence of atresia
are expected even at the end of the spawning season (e.g., [
79
,
80
]). Usually, fish with
indeterminate fecundity, such as Merluccius merluccius [
47
],
Trachurus trachurus
[
81
], and
Scomber scombrus [
82
], show a continuous oocyte recruitment throughout the spawning
period exhibiting high fraction of early secondary growth oocytes, such as that observed in
our species. Currently, the fecundity type designation of H. dactylopterus is not unanimous:
ref. [
77
], suggested a determinate fecundity, whereas [
75
] suggested an indeterminate one.
Our observations may support an indeterminate fecundity type for the blue-mouth and
for the rockfish species. However, our study lacks either laboratory or field assessment of
POF degeneration rate, and needs to be validated with a more precise evaluation of the
time-period of full POF resorption.
5. Conclusions
Information reported here represents a step towards the expansion of the knowledge
concerning the reproductive strategies of the ovuliparous species belonging to Scorpaena
genus and the zygoparous Helicolenus dactylopterus. The specialized ovarian morphology
observed and the histological findings attest to specialized modes of oviparity of these
species, strictly related to the viviparity. Since many characteristics of their reproduc-
tive biology affecting the reproductive potential are unknown, the assessment ovarian
dynamic of such species in Sardinian populations allowed for the determination of the
ovarian modality, the oocyte recruitment process of the species, and the establishment, in a
preliminary way, of the type of fecundity to better comprehend their reproductive strategy.
Author Contributions:
Conceptualization, C.P. and M.C.F.; methodology, C.P. and E.L.; software,
A.B., C.P. and R.P.; validation, C.P. and R.P.; formal analysis, C.P. and E.L.; investigation, A.M., A.C.,
C.P., E.L. and N.P. resources, M.C.F.; data curation, C.P., E.L.; writing—original draft preparation, C.P.
and M.C.F.; writing—review and editing, C.P., A.B., A.M., M.C.F., P.C. and R.P.; visualization, A.C.,
A.M., N.P., P.C. and A.B.; supervision, M.C.F.; project administration, M.C.F.; funding acquisition,
M.C.F. All authors have read and agreed to the published version of the manuscript.
Funding:
Funding for this research was provided by the European Union and the Italian Ministry for
Agriculture and Forestry grant Number “199/2008” on the framework of the Italian Work Plan for
data collection in the Fishery and Aquaculture sectors.
Institutional Review Board Statement:
Ethical review and approval was not required for the animal
study because the vertebrate animals we worked with for this study were all dead before research
began. We can assure that all the animals analyzed in this study were already dead when they came
on board.
Informed Consent Statement: Not applicable.
Data Availability Statement:
The data presented in this study are available on request from the
first author.
Acknowledgments:
We were grateful to Editor and the Reviewers for their constructive comments
and suggestions, which greatly helped to improve the manuscript.
Animals 2022,12, 1412 17 of 19
Conflicts of Interest: The authors declare no conflict of interest.
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