Oocyte development stages in the ovary of E. tetradactylum
The oocytes of
Eleutheronema tetradactylum were classified into five stages of development based on histological examination, oocyte size measurements and morphological characteristics. These stages are previtellogenic, vitellogenic, late-vitellogenic, post vitellogenic and follicular atresia (Table 1). Based on the percentage distribution of oocyte composition and size frequency, ovarian development was classified into five stages: immature, developing, maturing, mature and spent. In most teleosts, oogenesis is classified into five to eight stages (
Nagahama, 1983;
West, 1990). Similarly, a five-stage classification of oocyte development has been documented in other teleost species, such as
Barilius bendelisis (Saxena et al., 2018) and
Macrognathus pancalus (Borah et al., 2022), with staging determined by oocyte size, morphology and histological characteristics.
Immature stage
The immature stage of the ovary includes previtellogenic oocytes, which include the chromatin nucleolus stage, perinucleolar stage and late perinucleolar stage.
(a) Chromatin nucleolus stage
Oogonial cells undergo mitotic division, which results in the formation of the chromatin nucleolus stage. It represents the earliest phase of oocyte development in
E. tetradactylum. Oocytes are small and compact, with a centrally located prominent nucleus (Fig 1a). The size of the oocytes ranged from 50-80 µm. The cytoplasm appears homogenous and lacks yolk inclusions. This stage indicates the initiation of oocyte growth, with the proliferation of cells inside the ovary. This phase is observed in immature ovaries and is essential for subsequent maturation (
Brown-Peterson et al., 2011).
(b) Early perinucleolar stage
The early perinucleolar stage is characterized by the appearance of multiple nucleoli arranged at the periphery of the nucleus (Fig 1b). The size of the oocyte ranged from 80 to 120 µm. As the oocyte increased, the number of nucleoli multiplied, accompanied by a decrease in the nucleus-to-cell ratio. Multiple nucleoli show active ribosomal gene amplification
, which supports protein synthesis and is essential for oocyte growth
(Vincent et al., 1969; Vlad, 1976;
Monaco et al., 1981).
(c) Late perinucleolar stage
In the late peri-nucleolus stage, oocytes become more regularly shaped and large in size, with small lipid vacuoles beginning to appear within the cytoplasm (Fig 1c).
During the primary growth stage, the size of the oocyte increases in diameter from 50-60 µm to 120-150 µm. Similarly, a considerable increase in oocyte diameter in the primary growth phase of rainbow trout was observed from 10-20 µm to 100-200 µm (
Nagahama, 1983;
Sumpter et al., 1984).
Developing stage
The developing stage of the ovary consists of cortical alveolar oocytes. The cytoplasm shows the accumulation of lipid vesicles and cortical alveolus, along with presence of numerous nucleoli around the periphery of the nucleus. The follicular layers of the oocyte, zonaradiata, granulosa and theca are clearly visible at this stage (Fig 1d). The diameter of cortical alveolar oocytes ranged from 150 to 250 microns. This is the transition from primary growth to the vitellogenesis stage.The presence of cortical alveolar oocytes in the developing stage of the
E. tetradactylum ovary has also been reported by
(Pember et al., 2005; Shihab et al., 2017; Soe et al., 2023).
Maturing stage
The maturing stage of the ovary of
E. tetradactylum consists of vitellogenic and late vitellogenic oocytes, such as the primary yolk, secondary yolk and tertiary yolk stages.
Primary yolk vesicle oocytes were characterized by yolk globules between yolk vesicles in the peripheral region of the cytoplasm (Fig 1e). This indicates the initiation of vitellogenesis. Yolk granules are small, spherical in shape and basophilic. Increase in size and numbers of oil droplets in the central region of oocytes, which distributed around the nucleus. Increase in size and number of oil droplets in the central region of oocyte, distributed around the nucleus. The thickness of the follicular layer increased to 5 µ.
Secondary yolk stage oocytes measure 330-420 µm. This stage represents the active vitellogenic phase, where the oocyte increases in size due to the uniform accumulation of yolk globules and oil droplets throughout the cytoplasm. Oil globules and yolk vesicles surround the nucleus (Fig 1f). The follicular layer of the oocyte differentiates into two distinct layers: the zonaradiata externa and the zonaradiata interna.
The tertiary yolk stage oocytes measure (420-550 µm). The increased accumulation of yolk globules in the cytoplasm results in a significant increase in oocyte size. Oil droplets in the cytoplasm attach and fuse, forming a few large oil globules (Fig 1g). Further thickening of the zonaradiata is observed. Vitellogenesis is the vital mechanism in teleosts that causes the enormous growth of oocytes; it involves the uptake and incorporation of vitellogenin, a liver-derived plasma precursor, into the developing yolk proteins. This process can contribute up to 95% of the final egg size (
Wallace, 1978;
Tyler, 1991).
Ripe stage
In ripe oocytes, the number of oil droplets increases and they gradually merge to form a single large oil globule, which displaces the germinal vesicle to the cytoplasm periphery (Fig 1h).
Hydrated stage
The size of the oocyte increases to 720-750 µm due to excessive water intake and becomes translucent. Hydration is essential for buoyancy and fertilization. In many marine finfish, hydration facilitates the size of the oocyte during maturation (
Fulton, 1898;
Clemens and Grant, 1964;
Hirose and Ishida, 1974;
Hirose et al., 1976). The hydration of oocytes during the final maturation is crucial to producing buoyant pelagic eggs in species like sea bass, mummichog, cod and halibut (
Carnevali et al., 1991, 1992).
Spent stage
The spent stage of the ovary consists of post-ovulatory follicles, characterized by the presence of empty ovarian follicle and, the presence of primary oocytes like chromatin nucleolus stage, perinucleolar stages, cortical alveolar and primary stage oocytes can be seen (Fig 1i).
Percentage distribution and size of oocytes in different stages of ovarian development in Eleutheronema tetradactylum
The percentage frequency distribution of oocytes in different stages of ovarian development is depicted in (Fig 2 a to 2 e). In the Immature stage of the ovary, 80% of the oocytes were in the chromatin nucleolus (50-80 µm) stage, 20% were early-perinucleolar stage (80-100 µm) and 10 % were late-perinucleolar stage (120-150 µm) (Fig 2a). In developing stage of the ovary, indicated the presence of different stages of the ovary, dominated by the presence of a higher percentage of cortical alveolar stage (50 %) followed by late perinucleolar stages, early perinucleolar and chromatin nucleolus stage (Fig 2b). The maturing overy contains oocytes of all developmental stages, previtellogenic to vitellogenic. The ovary consists mainly of primary yolk stage oocytes, which comprise 25% with a diameter of 311 µm and secondary yolk stage oocytes, which constitute 25% with a diameter of 380 µm (Fig 2c). The ripe ovary consists of oocytes of all developmental phases, ranging from previtellogenic to hydrated (Fig 2d). The tertiary yolk stage oocytes comprise 23% and have a diameter of 445 µm. Oocytes in the germinal vesicle migration stage constitute 22% with a diameter of 550 µm, while hydrated oocytes constitute 15% with a diameter of 740 µm. The spent ovary primarily contains oocytes in the early stages of development (Fig 2e). Cortical alveolar stage oocytes account for 19% with a diameter of 220 µm, late perinucleolar stage oocytes represent 23% with a diameter of 120 µm and primary perinucleolar stage oocytes comprise 22% with a diameter of 90 µm. Cortical alveolar stage oocytes comprise 30% of the population, exhibiting a diameter of 73.12 µm. Oocytes at the primary and secondary yolk stages are diminished, representing 3%, with diameters measuring 311 µm and 380 µm, respectively. The absence of the tertiary yolk stage, germinal vesicle migration stage and hydrated oocytes was noted.
Based on histological sections, percentage of type and size of oocyte distributions in the ovary, we describe
E. tetradactylum is asynchronous multiple spawners with indeterminate fecundity.
The various ovarian maturity stages of many stages of oocytes confirm asynchronous oocyte growth and further, the pattern of continuous oocyte size frequency in all maturity stages from maturing to ripe stage, batch spawning strategy, where, oocytes are continuously recruited, matured and ovulated in batches during the spawning season. Similarly,
Pember et al., (2005) reported indeterminate fecundity in
E. tetradactylum, characterized by sustained oocyte recruitment and maturation during the spawning season in Australian waters.In ovaries characterized by asynchronous development, oocytes of various stages of development are present, with no single stage dominating the population (
Murua and Saborido-Rey, 2003;
Lubzens et al., 2010; Lowerre-Barbieri et al., 2011). Batch spawners with indeterminate fecundity show unrestricted secondary growth (SG) and continuously recruit oocytes for spawning throughout the season
(Ganias et al., 2017). In line with our observations, previous studies reported ova are released in batches, suggesting continuous spawning in
Eleutheronema tetradactylum, supported by the consistent presence of larvae and fry stages in the waters of Bombay (
Karandikar and Palekar, 1950) and the Hooghly estuary (
Sarojini and Malhotra, 1952;
Ravish, 1962). Similarly,
Soe et al., (2023) reported asynchronous pattern of ovarian maturation in
E. tetradactylum from the waters of Thailand.
