Potent Influence of Exogenous Melatonin on In Vitro Oocyte Maturation in the Longchin Goby, Chaenogobius annularis.

Effects of changes in photoperiod on the reproductive events in fish are suggested to be mediated mainly via the action of melatonin (MEL). Changing levels of plasma MEL throughout the day and year are suggested to influence the hypothalamus-pituitary-gonadal axis in fish. Therefore, in this study, we aimed to investigate the effects of MEL on oocyte maturation and germinal vesicle breakdown (GVBD) in the marine fish, Chaenogobius annularis, in vitro. Oocytes at three different stages (pre-, mid-, and late-vitellogenesis) were incubated with (a) only MEL (5, 10, 50, 100, 500, and 1,000 pg/mL) and (b) 50 pg/mL of 17α,20ß-dihydroxy-4-pregnen-3-one (17α20ßP), maturation-inducing hormone (MIH) of this species, and MEL (4-h incubation before addition of MIH). Any single MEL treatment did not significantly induce GVBD. However, treatment with 50 pg/mL MEL or MIH significantly induced GVBD. These results suggest that preincubation with MEL accelerates the effect of MIH on longchin goby oocyte maturation.

Dietary Sinapic Acid Alleviates Adiposity and Inflammation in Diet-Induced Obese Mice.

Sinapic acid (SA), a hydroxycinnamic acid, is known to confer protection against oxidative stress, inflammation, diabetes, and liver disease. However, the effectiveness of SA in improving obesity remains obscure. Therefore, this study evaluated anti-obesity efficacy of SA and to elucidate its mechanism of action. Male mice were maintained for 16 weeks on high-fat diet (HFD) alone or with SA (0.004%, w/w) and bodyweight, fat mass, adipocyte size, food intake, and biochemical and molecular markers were evaluated. SA-supplemented mice demonstrated markedly decreased fat mass and adipocyte size compared to unsupplemented group, without any changes in bodyweight and food intake between the two groups. Plasma adipocytokines levels including leptin, resistin, monocyte chemoattractant protein (MCP)-1 and interleukin-6 were also markedly reduced by SA supplementation. SA tended to lower plasma insulin level and improved homeostatic index of insulin resistance and intraperitoneal glucose tolerance test in HFD-induced obese mice. The anti-adiposity effect of SA was maybe owing to down-regulation of the mRNA expression of lipogenic genes, including acetyl coenzyme A (CoA) carboxylase, fatty acid synthesis, stearoyl-CoA desaturase 1, and phosphatidate phosphatase, and peroxisome proliferator-activated receptor γ, a transcription factor responsible for governing lipid metabolism, in adipose tissues. SA significantly down-regulated pro-inflammatory nuclear factor kappa B, MCP-1, tumor necrosis factor-α, and Toll-like receptor 4 mRNA expression in adipose tissue. Thus, SA could be beneficial for the development of functional foods or herbal medications to combat obesity.

In Vitro Sex Steroid Metabolism in Red Spotted Grouper, Epinephelus akaara during Oocyte Maturation.

We studied steroid metabolites produced from red-spotted grouper ovarian follicles during maturation. Oocytes with 350-500 µm diameter were in vitro incubated in the presence of [3H] 17α-hydroxyprogesterone as a precursor. Steroid metabolites were extracted from incubated media and oocytes. The extracts were separated and identified using thin layer chromatography, high performance liquid chromatography and gas chromatography-mass spectrometry. The identified metabolites were androstenedione (A4), testosterone (T) and estrone (E1). The metabolites of A4 was dominant in all size of oocytes and it was the highest in 480 µm diameter oocytes. The metabolites of two progestins, 17α,20ß-dihydroxy-4-pregnen-3-one and 17α,20α-dihydroxy-4-pregnen-3-one were detected in the oocytes less than 480 µm diameter although they were not identified definitely. In the oocytes of 480 µm diameter, metabolite of progestin was the highest, and germinal vesicle (GV) was still in the middle of cytoplasm. In the oocytes of 500 µm diameter, GV was began to migrate and the major metabolites were A4 and E1. The metabolite of E1 was detected in all size of oocytes and it was higher than that of E2. These results suggest that oocytes of 480 µm diameter are the transitional stage involving steroidogenic shift to final oocyte maturation and potential function of E1 during maturation process.

Mature and Bi-Sexual Phase Gonad Occurrence in Cultured Red Spotted Grouper, Epinephelus akaara.

This study reports the presence of mature and bi-sexual phase gonads in red spotted grouper, Epinephelus akaara after less than a year of cultivation in a commercial indoor tank and a net cage. In December 2018, juveniles were placed in an indoor tank and cultured for five months. In June 2019, the fish were transferred to a net cage and cultured until September. The rearing temperatures ranged from 19.86°C-24.65°C in the indoor tank and 21.86°C-27.65°C in the net cage. During the net cage culture period, specimens were randomly selected for histological gonad examination. The highest gonadosomatic index (GSI) value was measured in July (3.38±2.53), and dramatically decreased in August (0.44±0.21) and September (0.42±0.30). In July, some mature fish showed signs of vitellogenic stage oocyte development (vitellogenic and oil droplet stage oocytes), but immature fish were in an early developmental stage containing peri-nucleolus stage (PNS) oocytes. Bi-sexual phase gonads containing spermatocytes and spermatids were observed in the lumen and several PNS oocytes. By August and September, most specimens showed early-stage ovary development. However, mature testis (in August) and bi-sexual phase gonads (in September) were also observed. These results provide evidence for early puberty and hermaphroditism in the red spotted grouper.

Prostaglandin affects in vitro ovulation and 17α, 20ß-Dihydroxy- 4-pregnen-3-one production in longchin goby, Chasmichthys dolichognathus oocytes.

This study focused on the association of prostaglandins and a progestin, 17α, 20ß-dihydroxy-4-pregnen-3-one (17α20ßP) during the ovulation process in longchin goby, Chasmichthys dolichognathus. We performed several in vitro experiments using 850-920 µm diameter oocytes which were at the migratory nucleus stage. With the 890-920 µm diameter oocytes, no significant difference in ovulation was observed in any of the prostaglandins (PGE1, PGE2, and PGF2α) treated groups although PGE2 and PGF2α at concentrations of 50 ng/mL increased ovulation slightly compared with controls; however, 17α20ßP production was stimulated with PGE1 alone at low concentrations (5 ng/mL). In 850 µm diameter oocytes, PGF2α at concentrations of 50 and 500 ng/ml resulted in a significant increase in ovulation. 17α20ßP (50 ng/ml) alone had no observable effect on ovulation, but in the combined of PGF2α 50 or 500 ng/ml it caused the greatest effect on ovulation. The sensitivity of oocytes to the induction of ovulation varies between 850 and 890-920 µm, it appeared to vary depending on the migration status of nucleus. These results suggest that PGF2α (or combined of 17α20ßP) was more potent in inducing ovulation of the longchin goby.

Expression Patterns of Growth Related Genes in Juvenile Red Spotted Grouper (Epinephelus akaara) with Different Growth Performance after Size Grading.

Fish shows great difference in growth rate between individuals during larval development and early growth. This difference seriously reduces the production efficiency in fish culture. Growth hormone (GH)/Insulin-like growth factor 1 (IGF1) system is said to play some pivotal roles in fish growth. In this study, we investigated differences of GH, IGF1 and GHR gene expressions in juvenile red spotted grouper (Epinephelus akaara) with different growth performance. Red spotted groupers were reared under the same environmental condition (water temperature 24±1℃, natural light) for 96 days after hatching. They were divided into 3 groups by size (fast growing, middle growing and slow growing groups: FGG, MGG, and SGG, respectively). RNA was extracted from the brain, liver and muscle tissues from each group, and target gene expression was examined by real-time PCR. In the brain with pituitary gland, expression of GH gene in FGG was significantly higher than the expression in SGG, but the expression of IGF1 and GHR genes in the muscle was highest in SGG. Difference of GHR and IGF1 mRNA in the liver between groups with different growth performance was less clear than that in other tissues, although level of IGF1 mRNA was higher in SGG than in MGG. These results suggest that hormonal governing of growth is not the same in fast growing and slow growing fish, and size grading could cause a shift of hormonal state and growth pattern in this species.

Evaluation of Erythrocyte Morphometric Indices in Juvenile Red Spotted Grouper, Epinephelus akaara under Elevated Water Temperature.

Higher thermal exposure can influence the blood cell morphology and count. Hence, based on the previous results (Rahman et al., 2019), the present study evaluated the morphometric indices of erythrocytes and their nucleus under different water temperatures (25°C, 28°C, 31°C, and 34°C) to investigate their use as an indicator of thermal stress in red spotted grouper, Epinephelus akaara. 180 healthy specimens of E. akaara were exposed to four temperature conditions (25°C as control, 28°C, 31°C, and 34°C) for 42 days, following 2 weeks of acclimation at 25°C. Erythrocyte major axis (EL), erythrocyte minor axis (EW), nucleus major axis (NL), and nucleus minor axis (NW) were examined from the blood smears on each sampling day (i.e., 2, 7, and 42 days of thermal exposure). EL and NL were significantly decreased, whereas EW and NW were increased at higher water temperature (31°C and 34°C). The major-minor axis proportions of erythrocytes and their nucleus (EL/EW; NL/NW) were decreased with increasing water temperature (31°C and 34°C). The strong relationships were observed among the morphometric indices of erythrocytes and their nucleus, especially in EL vs. NL and EW vs. NW. This study reveals that elevated water temperature (31°C and 34°C) can influence the major and minor axis morphometry of erythrocytes and their nucleus in red spotted grouper. These indices may be used as stress indicators to monitor the health status of E. akaara and probably for other fish species.

Correlation of Developmental Deformity with Calcium, Phosphorus, or Estradiol-17ß Levels in Reared Red Spotted Grouper, Epinephelus akaara Juveniles.

Skeletal deformities are significant problems that affect the growth and commercial value of fish reared in hatcheries. However, studies of bone metabolic process related to skeletal deformities are limited. We investigated the potential correlation between bone deformities and plasma calcium, phosphorus, and estradiol-17ß levels in reared red spotted grouper (Epinephelus akaara) juveniles. We collected E. akaara frys from private farms at 110, 140, 180 and 300 days after hatching (DAH), and classified the normal and deformed fish by observing their external shape and inner frame by soft X-ray. We also analyzed the calcium, phosphorous, and estradiol-17ß levels in their plasma. A comparison between normal and deformed fish, indicated that calcium and estradiol-17ß levels were higher in deformed fish than in the normal at 180 and 300 DAH. The level of phosphorus was also higher in deformed individuals than in normal fish, but only at 300 DAH. These results suggest that skeletal deformities are associated with increases in plasma calcium, phosphorus, and estradiol-17ß levels.

Cathepsin B & D and the Survival of Early Embryos in Red Spotted Grouper, Ephinephelus akaara.

Survival of embryos largely depends on yolk processing during early development. Proteolytic enzymes, cathepsin B & D (ctsb & ctsd) are known to have some important roles in yolk processing of various fish species. Mature female red spotted groupers were injected with human chorionic gonadotropin (HCG) to induce ovulation. The fertilized eggs and embryos were sampled at 0, 4 and 24 HPF (hours post fertilization). Survivals of each groups of embryos were checked at 24 and 48 HPH (hours post hatching). Transcripts of ctsb & ctsd showed the highest level at 0 HPF and relatively high at 4 HPF, but greatly decreased at 24 HPF. In bad egg quality group (BE, embryos survived until 24 HPH), transcript level of ctsb at 4 HPF were significantly lower than the transcript level at the same stage in good egg quality group (GE, embryos survived until 48 HPH) while no significant change of ctsb transcript level was observed at 0 or 24 HPF between BE and GE. Transcript level of ctsd was decreased at 24 HPF, but the difference was not as strong as the case of ctsb transcript. These results suggest that maternal ctsb transcript rather than ctsd transcript is likely to be involved in egg quality resulting in the difference of survival rate of embryos at early developmental period in this species.

Plasma Stress Responses in Juvenile Red-Spotted Grouper (Epinephelus akaara) exposed to Abrupt Salinity Decrease.

The objective of the current study was to determine acute plasma stress responses in two size groups of juvenile Epinephelus akaara (average body weight: 8.4±2.1 and 3.3±0.6 g; 150 and 120 days after hatch, respectively) exposed to abrupt salinity drops (from 34 practical salinity unit, PSU seawater to 18, 10 PSU (experiment 1) or 26, 18, 10 PSU (experiment 2), respectively). Plasma glucose, glutamic oxalate transaminase, glutamic pyruvate transaminase, red blood cell counts, and gill histology were determined during 72 h exposure. Significantly increased plasma glucose, glutamic oxalate transaminase levels, and red blood cell counts were observed in fish exposed to 18 or 10 PSU. Histological changes, such as hyperplasia and lifting of epithelium in the gill secondary lamellae, were also observed in fish exposed to 18 or 10 PSU at 72 h post-drop. E. akaara exposed to sudden salinity drops to 18 or 10 PSU still seems to undergo the primary adjustment phase before fish reaches a new homeostasis, whereas fish exposed to 26 PSU seems to mount osmotic changes. Therefore, the no observed adverse effect levels for 72 h acute salinity challenge was 26 PSU in our study, and salinity drop to 18 PSU and below can possibly cause acute adverse effect, in which fish could be vulnerable to additional stresses such as a temperature changes or handling stress.

Three cDNAs encoding vitellogenin homologs from Antarctic copepod, Tigriopus kingsejongensis: Cloning and transcriptional analysis in different maturation stages, temperatures, and putative reproductive hormones.

Three full-length cDNAs encoding lipoprotein homologs were identified in Tigriopus kingsejongensis, a newly identified copepod from Antarctica. Structural and transcriptional analyses revealed homology with two vitellogenin-like proteins, Tik-Vg1 and Tik-Vg2, which were 1855 and 1795 amino acids in length, respectively, along with a third protein, Tik-MEP, which produced a 1517-residue protein with similarity to a melanin engaging protein (MEP) in insects Phylogenetic analysis showed that Vgs in Maxillopods including two Tik-Vgs belong to the arthropod vitellogenin-like clade, which includes clottable proteins (CPs) in decapod crustaceans and vitellogenins in insects. Tik-MEP clustered together with insect MEPs, which appear to have evolved before the apoB-like and arthropod Vg-like clades. Interestingly, no genes orthologous to those found in the apoB clade were identified in Maxillopoda, suggesting that functions of large lipid transfer proteins (LLTPs) in reproduction and lipid metabolism may be different from those in insect and decapod crustaceans. As suggested by phylogenetic analyses, the two Tik-Vgs belonging to the arthropod Vg-like clade appear to play major roles in oocyte maturation, while Vgs belonging to the apoB clade function primarily in the reproduction of decapod crustaceans. Transcriptional analysis of Tik-Vg expression revealed a 24-fold increase in mature and ovigerous females compared with immature female, whereas expression of Tik-MEP remained low through all reproductive stages. Acute temperature changes did not affect the transcription of Tik-Vg genes, whereas Tik-MEP appeared to be affected by temperature change. Among the three hormones thought to be involved in molting and reproduction in arthropods, only farnesoic acid (FA) induced transcription of the two Tik-Vg genes. Regardless of developmental stage and hormone treatment, Tik-Vg1 and Tik-Vg2 exhibited a strong positive correlation in expression, suggesting that expression of these genes may be regulated by the same transcriptional machinery.

Gene Expression of Aromatases, Steroid Receptor, GnRH and GTHs in the Brain during the Formation of Ovarian Cavity in Red Spotted Grouper, Epinephelus akaara.

Red spotted grouper, Epinephelus akaara, is a popular aquaculture species and a protogynous hermaphrodite. Induction of artificial sex change at the time of primary sex differentiation is of interest but has not been successful due to the lack of necessary basic information. To find out the potential neuroendocrine influence on the primary sex differentiation, the expression of key genes in the brain was investigated during the formation of ovarian cavity. Expression of cyp19a1b, esr1, gnrhr1, fsh, lh and cga in the brain was positively associated with the formation of ovarian cavity, showing gradual increase as the formation proceeds. However, the expression of gnrh1 was suppressed during the early part of the ovarian cavity formation, signifying potential hypothalamic influence on the primary sex differentiation in this species.

Effects of Water Temperature Change on the Hematological Responses and Plasma Cortisol Levels in Growing of Red Spotted Grouper, Epinephelus akaara.

This study was conducted to determine the stress response [ethological (operculum movement number (OMN)), hematological (hematocrit and hemoglobin), biochemical (glucose, cortisol and glutamic oxaloacetic transaminase (GOT))] in red spotted grouper, Epinephelus akaara during exposure of different water temperature in winter season. This species (Total length, 18.56±0.34 cm) previously maintained in water temperature of 15°C were transferred to 15, 20 and 25°C. During experimental period (7 days), OMN, hematocrit (Ht), glucose and GOT values were significantly high in 15°C when compared to 20 and 25°C. Hemoglobin value was also increased at 15°C, but no significant differences. There was no differences in cortisol levels among the temperature groups. No fish mortality was observed during the experimental period. From these results, 15°C is likely more stressful to red spotted grouper than 20°C and 25°C. These observations confirm that red spotted grouper adapts better to temperatures between 20 and 25°C during the winter season.

Early Gonadal Differentiation of the Protogynous Red Spotted Grouper, Epinephelus akaara.

Red spotted grouper, Epinephelus akaara is a popular aquaculture species in many Asian countries. This species is a protogynous hermaphrodite that first differentiates into female and changes to male later. Due to this reproductive characteristic, stable supply of male and female gametes is a key to the success of seed production in this species. Thus, understanding early gonadal differentiation is required to develop effective sex control techniques. Red spotted grouper were reared in indoor tanks and sampled every 5 days from 40 days post-hatch (DPH) to 130 DPH. Changes of gonadal tissues were examined and analyzed by means of histology. A pair of gonadal primordium has already existed underneath the kidney in the posterior part of the body cavity at 38 DPH when this study began. Gonadal primordia of 38, 40 DPH consisted of germ cells surrounded by a few somatic cells. The blood vessel was observed in the gonadal primordium at 45 DPH. The number of somatic cells and size of gonadal primordium increased age-dependently up to 60 DPH. Formation of ovarian cavity was obvious by two protuberant aggregations of somatic cells at 65 DPH. Completed ovarian cavity and oogonia were first observed in the gonad of one fish sample at 105 DPH. Based on these histological observations, it can be suggested that induction of primary male differentiation could be more successfully applied at around 60 DPH in this species.

Histological Analysis of Early Gonadal Development and Sex Differentiation in Chameleon Goby, Tridentiger trigonocephalus.

This study describes the developmental process of gonads in chameleon goby, Tridentiger trigonocephalus from the stage of hatching to 100 days after hatching (DAH). Based on histological observation, the primordial germ cells were observed in mesentery between mesonephric duct and gut at 15 DAH (total length, TL: 6.8±0.2 mm). At 20 DAH (TL: 7.9±0.1 mm), the primordial gonad began to protrude into peritoneal cavity and developed between mesonephric duct and gut. Initial ovarian differentiation was identified by the presence of ovarian cavity and oogonia in the gonads at 55 DAH (TL: 21.1±1.3 mm). Testicular differentiation started at 65 DAH (TL: 23.7±0.9 mm) with appearance of spermatogonial cells in the gonads. These findings indicate that sex differentiation in T. trigonocephalus occurs earlier in females than males, suggesting that this species can be classified as an undifferentiated gonochorist.

Characterization of cholecystokinin-producing cells and mucus-secreting goblet cells in the blacktip grouper, Epinephelus fasciatus.

The characteristics and distributions of cholecystokinin (CCK)-producing cells and mucus-secreting goblet cells were investigated in the digestive tract of the blacktip grouper (Epinephelus fasciatus). CCK-producing cells were scattered throughout the digestive tract. The highest frequency of CCK-producing cells was observed in the anterior intestine portion and pyloric ceca, with a very small number of cells distributed as far as the rectum. Mucus-secreting goblet cells were found to differ remarkably in their regional distributions and relative frequencies. High frequencies of mucus-secreting goblet cells were found in the digestive tract, mainly in the anterior intestine portion and pyloric ceca, but not the esophagus; the frequency decreased slightly toward the rectum. Our result suggests that food digested by gastric acid in the stomach moves on the anterior (including the pyloric ceca) and mid intestine portion, thereby ensuring effective stimulation of the CCK-producing cells. In addition, the distribution pattern of the CCK-producing cells closely resembled that of mucus-secreting goblet cells. In E. fasciatus, CCK-producing cells and mucus-secreting goblet cells seem to be well adapted to promoting optimal control of the digestive process.

Reproductive Cycle of Chameleon Goby, Tridentiger trigonocephalus in the Southern Coastal Waters of Korea.

The objective of this study was to characterize the reproductive cycle of the chameleon goby, T. trigonocephalus. Gonadal development was investigated using a histological method. Specimens were collected monthly, from April 2009 to March 2010. The gonadosomatic index (GSI) of females began to increase in April, reaching the maximum in May, and declined sharply in August. In males, the GSI began to increase in April and reaching the maximum in July. The annual reproductive cycle of T. trigonocephalus can be divided into four successive stages in females: the growing (November-March), maturing (April-May), ripe and spawning (June-July), and recovery (August-October) stages. Males passed through growing (November-March), maturing (April-June), ripe and spermiation (July-August), and recovery (September-October) stages. These results indicate the spawning season is from June to July. The relationship between fecundity (Fc) and body length (BL) was Fc=86.1511BL(2.6506). Fecundity was ranged from 3,448-9,654 eggs in a BL of 4.8-7.2 cm and it was increased as BL increased.

In Vitro Steroidogenesis on Oocyte Development in the Starry Flounder, Platichthys stellatus.

In this study, oocyte steroidogenesis are investigated in relation to oocyte development in the starry flounder, Platichthys stellatus, a marine multiple spawner. Vitellogenic (0.52 and 0.55 mm oocyte diameter) and mature oocytes (0.63, 0.66 and 0.71 mm oocyte diameter) were incubated in vitro in the presence of [(3)H]17α-hydevrepoxyprogesterone ([(3)H]17α-OHP) as a precursor. Steroid metabolites were extracted from the incubated media and oocytes, the extracts were separated and identified by thin-layer chromatography (TLC), high performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS). The major metabolites produced from [(3)H]17α-OHP were andevrepogens [andevrepostenedione (A4) and testosterone (T)] and estrogens [17ß-estradiol (E2) and estrone (E1)] and progestins [17α,20α-dihydevrepoxy-4-pregnen-3-one (17α20αP) and 17α,20ß-dihydevrepoxy-4-pregnen-3-one (17α20ßP)] in vitellogenic and mature oocytes. The results from this study suggest the potential roles of E1 in the oocytes with diameter 0.52-0.71 mm, 17α20αP and 17α20ßP at the oocytes of 0.63, 0.66 and 0.71 mm.

Changes in Plasma Sex Steroid and Cortisol Levels during Annual Reproductive Cycle of Ribbed Gunnel, Dictyosoma burgeri.

We investigated the changes in plasma sex steroid hormones, testosterone (T), estradiol-17ß (E2), 17,20ß -dihydroxy-4-pregnen-3-one (17α20ßP), 11-ketotestosterone (11KT) and cortisol levels from ribbed gunnel, Dictyosoma burgeri in associated with annual reproductive cycle. The gonadosomatic index (GSI) of females increased from November, peaked in February and decreased rapidly from March. The GSI of males also increased from November, peaked in January and then decreased gradually. In females, E2 levels increased and remained high from December to February. The levels of T showed a similar tendency and correlated (rs =0.898, p<0.01) with E2 levels. The levels of 17α20ßP increased rapidly in February (4.78±1.01 ng/ml) and peaked in July (5.08±0.65 ng/ml). Cortisol level was peaked in March and correlated with 17α20ßP levels (rs =0.696, p<0.01). In males, the levels of T was peaked in January and then decreased rapidly. The levels of 11KT were remained high from October to January. On the other hand, the levels of 17α20ßP fluctuated during reproductive cycle. These results suggest that plasma sex steroids in ribbed gunnels have annual periodicity, and that cortisol may involve in maturation of females.

Effects of BPA and DES on longchin goby (Chasmichthys dolichognathus) in vitro during the oocyte maturation.

This study was performed in order to assess whether bisphenol (BPA) and diethylstilbestrol (DES) had agonistic or antagonistic effects on oocyte maturation using marine fish. We tested the effects of these chemicals on in vitro maturation, germinal vesicle breakdown (GVBD), assay using oocytes from the longchin goby, Chasmichthys dolichognathus. During the maturation process, low concentrations of BPA and DES triggered GVBD depending on the stage of oocyte development; BPA at 0.044 nM and DES at 0.037, 0.37, and 3.73 nM induced GVBD in 0.82-0.88 mm diameter oocytes (germinal vesicle located near the center of oocytes). In 0.76-0.80 mm diameter oocytes (fully vitellogenic oocytes), BPA induced GVBD at relatively higher concentrations (4.38, 43.8, and 438 nM). In 0.86-0.90 mm diameter oocytes, BPA and DES had no observable effect on GVBD at the concentrations tested. Oocytes with diameters between 0.82 and 0.88 mm appeared to be more sensitive to these chemicals. Moreover, our results showed that BPA and DES did not inhibit GVBD.