Oocyte growth, follicular complex formation and extracellular-matrix remodeling in ovarian maturation of the imperial zebra pleco fish Hypancistrus zebra

Abstract

This contribution describes the growth of oocytes, addressing the formation of structures that compose the follicular complex, as well as the remodeling of the extracellular matrix, specifically laminin, fibronectin and type IV collagen during gonadal maturation. Thirty-seven females of the Acari zebra fish, Hypancistrus zebra were captured and the ovaries were submitted to histological processing for light and electron microscopy and immunohistochemistry techniques. Oogonia and four stages (I - IV) of oocytes were distinguished, and structures such as the postovulatory follicle and atretic oocytes (initial and advanced atresia) were observed. The follicular complex consists of the mature oocyte, zona radiata (Zr1, Zr2 and Zr3), follicular cells, basement membrane and theca. During oocyte growth, proteins of the extracellular matrix showed different intensities of staining. Based on these observations, a model of oocyte growth is proposed to define specific characteristics of the oocyte and the remodeling of the extracellular matrix in the ovary of H. zebra. This model of oocyte growth can be extended to other species of ornamental fishes. This study contributes data for induced fertilization and eventual conservation of this species.

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Oocyte growth, follicular complex
formation and extracellular-matrix
remodeling in ovarian maturation
of the imperial zebra pleco sh
Hypancistrus zebra
Ivana Kerly S. Viana, Liziane A. B. Gonçalves, Maria Auxiliadora P. Ferreira, Yanne A. Mendes
& Rossineide M. Rocha
This contribution describes the growth of oocytes, addressing the formation of structures that compose
the follicular complex, as well as the remodeling of the extracellular matrix, specically laminin,
bronectin and type IV collagen during gonadal maturation. Thirty-seven females of the Acari zebra
sh, Hypancistrus zebra were captured and the ovaries were submitted to histological processing
for light and electron microscopy and immunohistochemistry techniques. Oogonia and four stages
(I – IV) of oocytes were distinguished, and structures such as the postovulatory follicle and atretic
oocytes (initial and advanced atresia) were observed. The follicular complex consists of the mature
oocyte, zona radiata (Zr1, Zr2 and Zr3), follicular cells, basement membrane and theca. During oocyte
growth, proteins of the extracellular matrix showed dierent intensities of staining. Based on these
observations, a model of oocyte growth is proposed to dene specic characteristics of the oocyte and
the remodeling of the extracellular matrix in the ovary of H. zebra. This model of oocyte growth can be
extended to other species of ornamental shes. This study contributes data for induced fertilization and
eventual conservation of this species.
Oocyte growth in teleost shes has been widely investigated in order to elucidate the various morphological
changes that occur during reproduction. is reproductive process consists of the multiplication of the oogonia
and the dierentiation and release of mature oocytes1–5.
During gonadal development the oocytes gradually produce yolk globules and the cell volume increases, the
nucleus moves toward the cell periphery and the micropyle is formed. e follicular cells undergo molecular
changes with the extracellular matrix reorganization and the theca appears6–8. ese modications occur in the
layers that surround the oocyte, for example the zona radiata, follicular cells, basement membrane and theca, and
together with the mature oocyte form the follicular complex, FC9. e FC is important for reproduction and is
observed in dierent orders, Labriformes10, Perciformes11 and Siluriformes12. However, information concerning
the emergence of the layers that form this complex is limited for teleost shes.
e extracellular matrix is composed of brillar proteins, glycoproteins, and proteoglycans that provide struc-
tural support for the cells and control the transport of nutrients, hormones and other signaling substances from
the extracellular environment13,14. Among the extracellular-matrix components, laminin and collagen type IV
are important; these are specic to the basement membrane or external layer of cells and provide cell-matrix
adhesion. e glycoprotein bronectin also provides adhesion and support for the matrix. All components are
involved in growth, dierentiation and cell migration, i.e., the extracellular matrix aids in maturation of the ovar-
ian germ cells13,15. Although oocyte growth is important, information on this process in ornamental shes of the
family Loricariidae remains limited.
e imperial zebra pleco, Hypancistrus zebra16, commonly known as the Acari zebra or commercial code L46
is a small loricariid endemic to the Xingu River basin. Hypancistrus zebra is widely used in the ornamental-sh
Institute of Biological Sciences, Universidade Federal do Pará, Belém, Pará, Brazil. Correspondence and requests for
materials should be addressed to I.K.S.V. (email: ivanakerly@hotmail.com)
Received: 7 March 2018
Accepted: 16 August 2018
Published: xx xx xxxx
OPEN
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trade, although its capture and marketing are illegal since this overexploited loricariid has been added to the
Brazilian list of endangered species, and since 2016 because of its vulnerability was added to the list of the
Convention on International Trade in Endangered Species (CITES). Study of this species is particularly critical
because its habitat is located in an area where a hydroelectric dam was constructed. Our study investigated the
oocyte growth and follicular complex formation in H. zebra, through structural, ultrastructural, morphometric
and immunohistochemistry analyses during oocyte maturation.
Results
Gonadal Anatomy. e ovary of H. zebra (Fig.1A) consists of a pair of ovarian cyst-type sacculiform
organs, located dorsally in the abdominal cavity and extending toward the tail, joining in the distal portion of the
body to form a single gonoduct where the oocytes are released. e large mature ovary occupies almost the entire
abdominal cavity, and is yellowish and highly vascularized (Fig.1B).
Characterization of oocyte types. In the ovarian tissue, germ cells are surrounded by ovigerous lamellae
that support the oogonia and oocytes (Fig.1C). Oogonia may be individual (OgI) or grouped into nests (OgII)
(Fig.2A,B), and have a mean diameter of 5.97 ± 1.55 µm. Compared to the cytoplasm, oogonia have a large
nucleus with only one nucleolus, which may be located in the center or at the periphery of the nucleus.
Stage I oocytes have a mean diameter of 44.07 ± 11.72 µm, the cytoplasm is homogeneous and basophilic,
and the nucleus is central with a single nucleolus and is surrounded by squamous follicular cells. Granulations
are visible in the cytoplasm and nucleus (Fig.2D,E). Stage II oocytes have a mean diameter of 103.71 ± 23.32 µm
and a basophilic cytoplasm that contains cortical alveoli immediately below the plasma membrane (MC); the
nucleus is evident, with nucleoli scattered at the periphery of the nuclear membrane (Fig.2G). e zona radiata
(Zr) is located between the plasma membrane of the oocyte and the follicular cells (Fig.2H). A brillar network
surrounds stage I and II oocytes (2 C, F).
Stage III oocytes have a mean diameter of 212.20 ± 83.33 µm and a dense brillar network (Fig.2I). e
nucleus is central, with acidophilic cytoplasm containing yolk globules, and cortical alveoli at the periphery of
the cytoplasm (Fig.2J). ese oocytes have a thick zona radiata, cuboid follicular cells and the theca (Fig.2K).
Stage IV oocytes have a mean diameter of 487.81 ± 275.85 µm. e cytoplasm, lled with yolk globules and
cortical alveoli, is located below the plasma membrane, and the nucleus migrates to the periphery of the cell
(Fig.3A,B). ese cells are surrounded by the zona radiata, follicular cells and theca (Fig.4C). e oocyte types
show signicant dierences, except stages I and II (F = 87.26, df = 4, p < 0.0001) (Fig .5).
Figure 1. (A) Photograph of specimen of Hypancistrus zebra. (B) Mature ovary containing oocytes of dierent
sizes. (C) ovigerous lamellae in maturing stage.
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e presence of postovulatory follicles (POFs) indicates that spawning has occurred. ey are formed of
follicular cells, basement membrane and theca. POFs are irregular in form, with a lumen, and remain in the
ovary until subsequent reabsorption (Fig.3C). e atresia was classied as initial and advanced. Initial atresia
Figure 2. Morphology of oocyte types in Hypancistrus zebra. (A,B) Oogonia nest. (C,F,I) SEM of the surface
of stage I, II and III oocytes, respectively. (D,E) Stage I oocytes with granules in the cytoplasm and nucleus.
(G) Stage II oocyte. (H) Formation of the layers that surround the stage II oocyte. (J) Stage III oocyte. (K)
Layers surrounding the stage III oocyte. Stains: B,D,G,H - Methylene blue. E, K - PAS/hematoxylin. A, J - HE.
N: nucleus, Nu: nucleolus, OgI: Oogonia nest, OgII: Isolated oogônia, Ca: cortical alveolus, BM: basement
membrane, F: follicular cell, Zr: zona radiata, Y: yolk globules, T: theca layer.
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is characterized by the irregular form of the oocyte and the fragmentation of the nucleus (Fig.3D). In advanced
atresia, the nucleus is not observed, the follicular cells and theca are hypertrophied and the oocyte is totally
deformed (Fig.3E).
Structure of the follicular complex. e formation of the follicular complex begins with the stage I oocyte
and is characterized by a thin layer of squamous follicular cells. Stage II oocytes have a zona radiata, follicular
cells, a basement membrane, and fusiform cells organizing outside the lamina, characterizing the emergence of
the theca (Fig.2H). Stage III and IV oocytes have the zona radiata, follicular cells, theca and basement membrane,
which together form the follicular complex. ese structures are thicker in stage IV oocytes (Fig.3B).
Ultrastructurally, stage IV oocytes contain larger numbers of yolk globules in the cytoplasm (Fig.4B). e
zona radiata is subdivided into the zona radiata 1 (Zr1), zona radiata 2 (Zr2) and zona radiata 3 (Zr3). e Zr1 is
in contact with the follicular cell layer through membrane projections, and is less electron-dense than the other
layers. e intermediate Zr2 layer has smaller, rounded pore canals. e Zr3 layer is in contact with the oocyte
membrane (MC), with larger and irregularly shaped pore canals. Zr2 and Zr3 have microvilli inside the pore
canals (Fig.4A,C,E). ere is also a layer of cuboid follicular cells that have a cytoplasm lled with mitochondria
and an irregularly shaped nucleus with condensed chromatin (Fig.4D). e theca layer is subdivided into a theca
interna composed of pavement cells and a theca externa composed of collagen bers (c) (Fig.4F).
Immunohistochemical analysis. Immunolabeling for the extracellular-matrix protein was seen in dier-
ent components of the follicular complex. Fibronectin was observed mainly in the basement membrane of types
I and II oocytes (Fig.6A), the theca layer of stage III oocytes (Fig.6B) and the follicular cells and theca of stage
IV oocytes (Fig.6C). Laminin was observed in the follicular cells, basement membrane and theca (interna and
externa), beginning with stage II oocytes and observed in the other oocyte stages (Fig.6D,E). Collagen type IV
was observed in the theca and was more evident in stage IV oocytes (Fig.6F).
Discussion
We characterized the structure, ultrastructure, morphometry and follicular complex formation, as well as the
expression and organization of the extracellular matrix in H. zebra. e ovary of loricariids is generally large and
yellowish as found in H. zebra. In this species the oocytes are large but relatively few, as in other loricariids, e.g.
Rhinelepis aspera17, Neoplecostomus microps18 and Harttia torrenticola19. Generally, the presence of larger but
fewer oocytes is related to the mode of reproduction and parental care of the species, which suggests a higher
survival rate of the brood19,20.
Figure 3. Dierent structures in the ovary of Hypancistrus zebra. (A) Stage IV oocyte. (B) e layers (Zr, F
and T) that surround the stage IV oocyte. (C) Postovulatory follicle, showing the lumen. (D) Oocytes in initial
atresia. (E) Oocyte in nal atresia. Stains: A,C,D,E - Methylene blue, B - PAS/hematoxylin. N: nucleus, Ca:
cortical alveolus, F: follicular cell, Zr: zona radiata, Y: yolk globules, T: theca layer, L: lumen.
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Oogonia of H. zebra were found individually or in nests. is organization pattern is the same as in Laetacara
araguaiae10, Pseudotocinclus tietensis12 and Astyanax altiparanae21. is arrangement in nests occurs through
projections of follicular cells that separate the nests of other oogonia scattered through the germinal epithelium22.
Later, the oogonia enter meiosis, initiating oocyte development. A similar appearance was found in Oryzias latipes,
where the germ cells, oogonia and oocytes are organized in a germinal cradle23,24.
e early stages of oocytes of H. zebra were characterized by the presence of granulations in the cytoplasm and
nucleus (Stage I) and the emergence of the zona radiata (Stage II). Ribonucleic-acid and protein synthesis start in
the early stage of the cell25–28. Yolk globules and cortical alveoli appear and increase in stage III and IV oocytes.
Five species of ostariophysan shes show a similar sequence19, suggesting that these morphological characteristics
are conserved in teleost species.
Figure 4. Follicular complex formation in Hypancistrus zebra. (A,B) SEM of surface of stage IV oocyte,
showing membrane of oocyte and zona radiata (MC, Zr). (C) Detail of the layers that forms the follicular
complex. (D) Follicular cell with irregular cuboid nucleus and condensed chromatin. (E) Subdivision of zona
radiata. (F) eca layer, showing internal theca with theca cell, and external theca with collagen. BM: basement
membrane, F: follicular cell, Zr: zona radiata, Zr1: zona radiata 1, Zr2: zona radiata 2, Zr3: zona radiata 3, Y:
yolk globules, c: collagen bers, T: theca, Ti: internal theca, Te: external theca.
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Figure 5. Mean diameters (µm) of cell types in Hypancistrus zebra.
Figure 6. Immunohistochemistry reaction in oocytes of Hypancistrus zebra. (A–C) Immunolocalization
of bronectin (Fn), demonstrating reaction in basement membrane, follicular cells and theca. (D,E)
Immunostaining of laminin (La) in basement membrane and theca. (F) Immunolocalization of collagen type
IV(C IV) in theca. I: stage I oocyte, II: stage II oocyte, III: stage III oocyte, IV: stage IV oocyte, N: nucleus, BM:
basement membrane, F: follicular cell, Zr: zona radiata, T: theca layer.
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In the gonadal development of H. zebra, the POF had an irregular shape and central lumen. Other stud-
ies have reported the existence of a post-ovulation complex (POC), which forms immediately aer ovulation,
where some structures of the follicular complex such as the follicular cells, basement membrane and theca are
clearly dened11,29. However, in H. zebra no distinction between the layers was observed. In addition to the POF,
we observed follicular atresia in which we were able to distinguish initial and advanced stages; in contrast, in
Pimelodus maculatus three stages, initial, intermediate and nal atresia can be distinguished30. Our study estab-
lished the existence of the initial and advanced atresia stages. is classication is based on the fragmentation
of the nucleus and the hypertrophy of the follicular cells and theca, which occur at the same time in the follicle.
e oocytes of H. zebra vary in size (diameter 44.07–487.81 µm). e increase in the diameter of the oocytes
is similar to that found in Siluriformes, including Loricariichthys platymetopon, Loricariichthys sp., Loricaria sp.,
Hypostomus ternetzi, Megalancistrus aculeatus31 and Hypostomus francisci32. is size variation may be related to
the reproductive strategy: numerous small eggs are common in migratory species, whereas non-migratory species
tend to have fewer but large eggs. Investigators distinguish between migratory species that produce more eggs to
attempt to ensure the perpetuation of the species, and non-migratory species that usually have parental care and
invest in the size of the eggs rather than in their quantity20,33. H. zebra is a non-migratory ornamental sh; hence
we believe that this type of oocyte pattern suggests that the animal invests in oocyte quality with increased yolk
production, ensuring nutritional reserves for the larval stage.
e emergence of FC in H. zebra begins in the stage I oocyte with the presence of the basal membrane and
follicular cells, similarly to the sequence in Sciaenops ocellatus9. e subdivision of the Zr (Zr1, Zr2 and Zr3)
agrees with the ndings of some studies9,34 but diers from another19 that reported only two layers for the zona
radiata. In H. zebra the subdivision of the zona radiata is related to the variation in the size of the pore canals
(Zr1 and Zr2) and the presence of microvilli (Zr3). Some investigators have established that this layer functions
to transport substances and provide resistance to abrasion10,31. H. zebra is a sedentary species that lives in stream
rapids, suggesting that the thick zona radiata and its chemical components favor adhesion of the eggs to dierent
substrates.
e follicular cells are squamous to cuboidal and have an irregular nucleus. Follicular cells have been found in
other teleosts: cylindrical in Trachelyopterus galeatus, Lophiosilurus alexandri and Rhinelepis áspera17, cuboid in
Iheringichthys labrosus35 and cuboid and prismatic in Loricariichthys spixiiz36. e morphological variation of the
follicular cells depends on the maturation phase of the oocyte, since the follicular cells are one of the rst elements
to organize in the germinal epithelium, which is directly related to the growth and maintenance of oocytes during
the reproductive cycle26,37.
e theca is derived from ovary cells; initially it is a thin layer, but as the oocyte grows it is possible to dis-
tinguish the internal and external theca11. In H. zebra the theca appears in stage II oocytes and reaches its maxi-
mum development in stage IV oocytes. is structure is associated with the follicular cells, and together they are
responsible for the production of hormones that promote the maturation of the oocyte38. In H. zebra the theca
layer also contains laminin, collagen type IV and bronectin, suggesting that these elements have a strong inu-
ence on the organization of this layer beginning with the emergence of the follicular complex.
During gonadal maturation in H. zebra, a brillar network was observed around the oocyte. We believe that
this arrangement supports the cells of the ovarian parenchyma during oocyte development. Some authors have
reported that matrix elements are commonly associated with the growth of ovarian cells and transmit signals
regulating adhesion, migration, proliferation, apoptosis, survival and dierentiation13,39–41.
Based on these morphological and immunohistochemical analyses, we present a model of formation of the
follicular complex in H. zebra (Fig.7), highlighting the extracellular matrix components (laminin, bronectin
and collagen type IV).
Materials and Methods
Study area and preparation of specimens. A total of 37 females of H. zebra were caught in bimonthly
collections in 2012 and 2013 from the Xingu River in northern Brazil (03°12′52″S, 52°11′23″W). e specimens
were transported to the laboratory, anesthetized with benzocaine hydrochloride (0.1 g.L–1) and euthanized with
sodium pentobarbital solution (60–100 mg/Kg). e gonads were removed through a ventral incision and clas-
sied into three stages: maturing, mature and spawned. e classication of oocyte development was adapted
from the literature42. All animal experiments were approved by the National Council for Control of Animal
Experimentation (CONCEA) and were performed in accordance with approved guidelines.
Light microscopy and immunohistochemistry. Fragments of H. zebra ovary were xed in Bouin’s solu-
tion for 24 h. e samples were then dehydrated in increasing concentrations of ethanol, cleared in xylene and
inltrated and embedded in paran43. Sections 5 μm thick were cut and stained with hematoxylin and eosin
(HE) solution and periodic acid-Schi (PAS). For immunohistochemistry, replicates of previously identied
slides were deparanized in xylene, washed in phosphate-buered saline (PBS) and immersed in sodium-citrate
buer heated for 25 min. Subsequently, the slides were incubated in 3% hydrogen peroxide in methanol for
30 min, blocked with 10% normal goat serum for 1 h, incubated in anti-rabbit bronectin polyclonal primary
antibody (1:200), anti-rabbit laminin (1:60) and anti-rabbit collagen type IV (1:100) for 12 h, and post-incubated
in anti-rabbit IgG secondary antibody conjugated with peroxidase for 2 h. e samples were developed in DAB
(3,3′diaminobenzidine) for 5 min, washed in distilled water, counterstained with hematoxylin and examined
under a Carl Zeiss optical microscope (AxioStar Plus 1169151).
Morphometry of oocytes. Samples from 100 oogonia and 100 oocytes of each type (I, II, III and IV) were
analyzed. Only cells that contained a nucleus were measured. Serial sections were cut and the slides were evalu-
ated under a photomicroscope with the soware NIS-elements BR (4.00.07-bit) and measurements were made at
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40X magnication. Each cell was overlaid with two dashed lines crossing at right angles in the middle of the cell,
and the length of the segment of the line over that diameter of the cell was measured. e mean length of the two
measurements was taken as the approximate diameter of the cell (oocyte and oogonia). is method of measuring
the cell diameter was adapted from the literature42. In our analysis, we measured the cells in two dimensions (2d),
as in several previous studies44–46. e data were tested through one-way analysis of variance (ANOVA), followed
by a posteriori Tukey test with 5% signicance level (α).
Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM). Fragments
of ovaries were xed in Karnovsky’s solution (4% paraformaldehyde, 2% glutaraldehyde in 0.1 M sodium caco-
dylate buer, pH 7.4) for 24 h. Aer xation, the fragments were washed in 0.1 M sodium cacodylate buer, pH
7.4 and post-xed in 1% osmium tetroxide in 0.1 M sodium cacodylate buer, pH 7.4 for 2 h. For TEM analysis,
the fragments were dehydrated in an ascending acetone series and embedded in Epon 812. Semi-thin 1 μm-thick
sections were cut in a microtome, stained with 1% methylene blue and then analyzed under a Zeiss optical micro-
scope (AxioStar Plus 1169151). Ultra-thin sections were contrasted with uranyl acetate and lead citrate and exam-
ined in a JEOL (JEM -100CX II) electron microscope. For SEM analysis, the specimens were dehydrated in a
graded ethanol series (30% to 100%) and critical-point dried using CO2. Specimens were mounted on stubs,
coated with gold and examined using a LEO 1430 SEM.
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from stage III oocytes, accentuated in stage IV.
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SCIentIFIC REPORtS | (2018) 8:13760 | DOI:10.1038/s41598-018-32117-7
Acknowledgements
We are grateful to the National Council of Technological and Scientic Development (CNPQ) for the scholarship
granted, to FAPESPA Agreement 070/2008 for nancial support, the Pró-Reitoria de Pesquisa e Pós-Graduação –
PROPESP/UFPA for the support for the publication of the article, andthe Technician José Augusto of Universidade
Paulista (USP) of Ribeirão Preto for obtaining the electron micrographs.
Author Contributions
I.K.S.V., M.A.P.F. and R.M.R. conceived and designed the manuscript. Y.A.M. and L.A.B.G. conducted the statistic
procedure and revising it critically, nal approval of the version to be published. I.K.S.V. interpretation of data for
the work. M.A.P.F. and R.M.R. contributed reagents/materials/analysis tools. All authors wrote the paper.
Additional Information
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