Abstract
An HPLC method was used to tentatively identify progesterone (P4) and its metabolites (17-hydroxyprogesterone (17-P4) and 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P)), corticosteroids (cortisol and corticosterone) and testosterone in ovary/follicular preparations of the catfish Heteropneustes fossilis associated with in vivo or in vitro oocyte maturation/ovulation. A single i.p. injection of human chorionic gonadotrophin (100 IU/fish, sampled at 0, 8 and 16 h) induced oocyte maturation and ovulation, which coincided with significant and progressive increases in 17,20β-P, and P4 and 17-P4, the precursors of the former. Both cortisol and corticosterone also increased significantly. Conversely, testosterone decreased significantly and progressively over time. Under in vitro conditions, incubation of post-vitellogenic (intact) follicles or follicular envelope (layer) with 2-hydroxyoestradiol (2-OHE2, 5 μM for 0, 6 and 24 h) elicited a sharp significant increase in 17,20β-P, the increase being higher in the follicular envelope incubate. P4 and 17-P4 also registered significant increases over the time with the peak values at 24 h. Cortisol and corticosterone increased significantly in the intact follicle, but not in the follicular envelope incubate. Testosterone decreased significantly in the intact follicle, but increased significantly (24 h) in the follicular envelope incubate. Coincident with these changes, the percentage of germinal vesicle breakdown (GVBD) increased over the time in the intact follicle incubate (48.9% at 6 h and 79.8% at 24 h). Denuded oocytes on incubation with 2-OHE2 (5 μM) did not produce any significant change in the percentage of GVBD or in the steroid profile. While corticosterone and 17,20β-P were undetected, P4, 17-P4, cortisol and testosterone were detected in low amounts. The results show that the 2-OHE2-induced GVBD response seems to be mediated through the production of 17,20β-P and corticosteroids. It is suggested that hydroxyoestrogens seem to be a component in the gonadotrophin cascade of regulation of oocyte maturation/ovulation in the catfish.
Introduction
In teleosts, meiotic resumption is initiated by the production of an ovarian maturation-inducing substance (MIS) or hormone under a luteinizing hormone (LH) surge. In a majority of fishes, the most potent MIS is the progesterone (P4) derivative 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) (Goetz 1983, Scott & Canario 1987, Jalabert et al. 1991, Nagahama 1997) and its synthesis has been demonstrated during oocyte maturation both in vivo and in vitro. In perciform fishes, another C21 steroid, 17,20β,21-trihydroxy-4-pregnen-3-one, has been identified as the MIS (Trant et al. 1986, Thomas 1994, Garcia-Alonso et al. 2004). The catfish Heteropneustes fossilis has been used as a model for oocyte maturation studies since the 1960s (Sundararaj & Goswami 1977). In a series of investigations, Sundararaj and his coworkers have demonstrated that corticosteroids (11-deoxycortisol, 11-deoxycorticosterone, 21-deoxycortisol and cortisol) of interrenal (adrenal) origin are the MIS in the catfish and proposed a pituitary–interrenal–ovarian control of oocyte maturation and ovulation. The MIS activity of corticosteroids has been demonstrated in vitro in other teleosts as well (Goetz 1983, Jalabert et al. 1991). Subsequently, it has been demonstrated in vitro that, as in other teleosts, 17,20β-P is the most potent MIS in this species (Sundararaj et al. 1985). However, its occurrence in the ovary has not been demonstrated in this species up until this time.
We reported earlier that catfish ovary synthesizes catecholoestrogens, which show both seasonal and periovulatory changes (Mishra & Joy 2006a). Further, hydroxyoestrogens have been shown to stimulate oocyte maturation (Senthilkumaran & Joy 2001, Mishra & Joy 2006b). However, the mechanism of induction of oocyte maturation, direct or indirect, was not clearly understood. The objective of the present study was to determine whether the hydroxyoestrogen-induced in vitro oocyte maturation was mediated through follicular steroidogenesis. The changes in the steroid profile were then compared with those that occurred during in vivo human chorionic gonadotrophin (hCG)-induced oocyte maturation and ovulation. Through the latter paradigm, we also demonstrated that catfish ovary appeared to synthesize both 17,20β-P and corticosteroids, like other teleosts.
Materials and Methods
Chemicals
1,3,5,(10)-oestratriene-2,3,17β-triol (2-hydroxyoestradiol, 2-OHE2), 11β,17,21-trihydroxy-4-pregnene-3,20-dione (cortisol), 11β,21-dihydroxy-4-pregnene-3,20-dione (corticosterone), 17β-hydroxy-4-androsten-3-one (testosterone), 17-hydroxy-4-pregnene-3,20-dione (17-hydroxy-progesterone (17-P4)), 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P), 4-pregnene-3,20-dione (progesterone, P4) (steroid nomenclature after Kime 1987) and hyaluronidase (type IV) were purchased from Sigma Chemical Company. hCG (Corion; IBSA, Switzerland) was purchased from a local medical store. Other chemicals were of analytical grade and purchased locally. Methanol (HPLC grade) and degassed and filtered nanopure diamond water (Barnstead International, Dubuque, IO, USA) were used throughout chromatography.
Animal collection and maintenance
The experiments were performed in accordance to local/national guidelines for experimentation in animals and all care was taken to prevent cruelty of any kind.
Mature female Heteropneustes fossilis (30–40 g) were purchased from local fish markets in the pre-spawning phase (June) of the annual reproductive cycle. They were maintained in the laboratory under normal photoperiod (13 h light:11 h darkness) and temperature (25 ± 2 °C) until used for experiments. The fish were freely fed goat liver daily. Sample fish specimens were randomly checked for spontaneous ovulation by mild hand stripping. A few fish were sampled for checking the maturation stage of the ovary. Post-vitellogenic dark green rounded follicles (1.034 ± 0.01 mm, average diameter) were used for incubation studies.
Induction of ovulation
Thirty acclimatized fish were divided into two groups of 15 each. One group was injected i.p. with hCG at a dose of 100 IU/fish, the second was injected with normal saline (0.6% NaCl). Five fish each from both groups were killed at 0, 8 and 16 h and ovaries were weighed and immediately processed for extraction of steroids.
Preparation of incubation medium and test compounds
The incubation medium was prepared as follows (g): NaCl 3.74, KCl 0.32, CaCl2 0.16, NaH2PO4.2H2O 0.10, MgSO4.7H2O 0.16, glucose 0.40 and phenol red 0.008 were dissolved in 1 litre of triple-distilled water. The pH was adjusted to 7.5 with 1 M sodium bicarbonate and autoclaved. Penicillin (2,00 000 U) and streptomycin sulphate (200 mg) were added and filtered. The medium was stored at 4 °C and prepared fresh every week.
2-OHE2 was weighed and dissolved in 50 μl ethanol in a dark bottle and kept at 0 °C. Just before the incubation, the stock solution was diluted with the incubation medium to make a working concentration of 5 μM.
In vitro incubation of follicular preparations with 2-OHE2
The acclimatized, gravid female catfish were killed by decapitation and ovaries were transferred to a sterile Petri dish containing freshly cooled incubation medium. Post-vitellogenic follicles were separated from each other with the help of a fine brush and watchmaker’s forceps. The following preparations were used for the incubation study:
Intact follicles
About 120 follicles were incubated in duplicate (group size=5) with the medium containing 5 μM of 2-OHE2 for 0, 6 and 24 h. The incubation was further continued in fresh plain medium for 30 h. As control, the follicles were incubated with plain medium or the medium containing vehicle (ethanol). Percentage of germinal vesicle breakdown (GVBD) was scored. The follicles and incubation medium were collected for steroid extraction.
Follicular envelope (layer)
Batches of about 120 follicles each were treated with 0.03% hyaluronidase (Fan et al. 2002) in plain incubation medium for 2 min under mild agitation. (Hyaluronidase dissolves hyaluronic acid in extracellular matrix present between corona radiata and zona pellucida of mammalian oocytes (Talbot 1984)). The follicular envelope containing granulose and thecal cells was separated in toto by the enzyme treatment and was collected by forceps. They were washed with fresh medium and incubated with 2-OHE2 (5 μM) for 0, 6, or 24 h in duplicate, as described above. The denuded oocytes were also collected for incubation, as described below. The incubation continued further up to 30 h in fresh plain medium. As control, the follicular envelopes were incubated in plain medium or the medium containing the vehicle. The tissues and medium were collected for steroid extraction.
Denuded oocytes
Batches of about 120 denuded oocytes (see above) were washed in fresh medium and incubated with 2-OHE2 for 0, 6 or 24 h, as described above. The incubation continued further up to 30 h in fresh plain medium. As control, the denuded oocytes were incubated in plain medium or the medium containing the vehicle. GVBD was scored, and the oocytes and medium were collected for steroid extraction.
Extraction of steroids
The tissues (ovaries and follicular preparations) from different experiments were homogenized separately or group-wise in 4 volumes of cold PBS (0.02 M, phosphate-buffered saline, pH 7.4) with an ultrasonic homogenizer (XL-2000 Microson; Misonix, New York, NY, USA) at 0 °C for 5–10 s. The homogenate was centrifuged at 5000 g for 20 min at 4 °C and extracted with 3 volumes of diethyl ether, three times. The ether phase was collected and pooled, evaporated and dried under N2 and stored at −20 °C until chromatography. The incubation medium was directly extracted with diethyl ether, as described above. The ether phase was collected and pooled group-wise, evaporated and dried under N2 and stored at −20 °C until chromatography.
Chromatography
A Shimadzu (Kyoto, Japan) HPLC system with two pumps (LC-10 ATVP), system controller (SCL-10 AVP) and an ultraviolet detector (SPD-10 AVP) with a variable wavelength (190–370 nm) range was used for the chromatographic analysis of steroids. The system was operated with Shimadzu Class VP Series software. The analysis was made with a reversed phase C18 column (150 × 4.5 mm, i.d., 5 μm; Luna; Phenomenex, Torrance, CA, USA) and absorbance was taken at 240 nm. The mobile phase was 60% methanol in water at a flow rate of 1.5 ml/min (Nagahama & Adachi 1985). The run time was 30 min.
Preparation of standards and determination of retention time
Steroids (cortisol, corticosterone, testosterone, 17-P4, 17,20β-P, P4) were dissolved in methanol separately to prepare stock solutions. From the stock solutions, serial dilutions were made with methanol. The diluted solutions were filtered (0.2 μm) and injected into the 20 μl loop of the HPLC system with the help of a Hamilton microlitre syringe. The standards were tested individually at different concentrations to record detection limit, retention time and peak area under isocratic conditions. This was repeated three times with each standard to verify the concurrence of the assay parameters.
Validation of the assay
Response-linearity
Different concentrations of the standards in triplicate were injected into the column to set up a concentration vs peak area curve. The response was linear with the concentration ranges used (10–1000 ng/ml).
Recovery and sensitivity
Known concentrations of the standards in different dilutions were processed in the same manner as tissue samples (described below) and were injected into the column, after filtration. The recovery study was repeated three times for each dilution. Percentage recovery was calculated from the concentrations of the standards injected directly and that measured after the extraction. Recovery was 89–93%. The values were not corrected for loss. The minimum detection limit was 1 ng/ml of the standards in individual runs.
Inter- and intra-assay variations
Inter- and intra-assay variations were determined from five chromatograms each, using the same set or different sets of diluted standards. The inter- and intra-assay variations were, respectively, 10 and 8%.
Sample analysis
The ether-evaporated and dried ovary samples were reconstituted separately in 200 μl methanol. Similarly, the samples of the follicular preparations and incubation medium were pooled group-wise with 200 μl methanol. The reconstituted samples were filtered (0.2 μm) and 20 μl each of the samples were injected into the system and eluted for 30 min. The samples were analysed in triplicate. The samples were also co-chromatographed with known concentrations of the standards in a mixture and the elution pattern was compared with that of the respective standards in the mixture for identification of the steroids. Chromatograms for blanks were run with the vehicle (methanol and the mobile phase) to check any interference in the elution of the steroids. The blank eluted before the steroid peaks appeared. The differences in the peak area between the standard and the standard with sample in the chromatograms were recorded with the help of Class VP Series software and the concentrations were calculated.
Statistical analysis
The data were expressed as means±s.e.m. and were analysed by one-way ANOVA, followed by a Newman–Keuls test (P<0.05) for multiple group comparisons.
Results
Steroid separation and suggested identities
With the chromatographic conditions we adopted, cortisol eluted first, followed by corticosterone, testosterone, 17-P4, 17,20β-P and P4 (Fig. 1A). The retention times were 3.7, 5.5, 9.9, 11.7, 13.4 and 23.0 min respectively. However, in the sample runs, the retention times showed minor shifts (Figs 2 and 4). Therefore, the steroid peaks were authenticated by co-running the samples with known concentrations of the standards in a mixture (Fig. 1B and C), and compared with those of the samples or standards run alone. In this manner, the steroids were tentatively identified. The chromatograms of the ovary, intact follicles and follicular envelope showed three elution peaks each, the identity of which could not be confirmed because of the lack of standards (Figs 2 and 4).
Effects of hCG on ovulation and ovarian steroid dynamics
The administration of 100 IU hCG/fish induced 100% ovulation and eggs could be stripped out from 12 h onwards. The gonadotrophin treatment produced overall significant changes in the concentrations of P4, 17-P4, 17,20β-P, testosterone, cortisol and corticosterone (Figs 2 and 3; F=385.18, 846.37, 21636.43, 90.00, 71.23 and 41.66 respectively; P<0.001, one-way ANOVA). The steroid concentrations did not vary significantly in the vehicle groups at 0, 8 and 16 h intervals. The administration of hCG produced a strong stimulation of the progestin pathway involving P4, 17-P4 and 17,20β-P production associated with oocyte maturation and ovulation. P4 increased significantly about 2-fold at 8 h and 5-fold at 16 h (P < 0.05, Newman–Keuls test). 17-P4 increased significantly at 8 h of the hormone treatment but decreased to the control level at 16 h. The most dramatic change was noticed in the concentrations of 17,20β-P. The concentration was very low in the vehicle groups (1.5 pg/mg ovary) and increased sharply registering about an 88-fold increase at 8 h and about 125-fold at 16 h. Testosterone decreased consistently with the lowest concentration at 16 h (P<0.05). Cortisol showed a significant and progressive increase at 8 and 16 h but corticosterone increased only at 16 h (P<0.05).
In vitro effects of 2-OHE2 on follicular steroid profile
Intact follicle
The incubation of the intact follicles with 5 μM 2-OHE2 produced an overall significant effect on GVBD (48.9% at 6 h and 79.8% at 24 h) and steroid hormone levels: P4, 17-P4, 17,20β-P, testosterone, cortisol and corticosterone (Figs 4 and 5A; F=401.74, 282.04, 518.86, 12.00, 102.50 and 65.25 respectively; P<0.001, one-way ANOVA). The concentrations of both P4 and 17-P4 increased significantly at 6 and 24 h giving the peak levels at 24 h (P<0.05). 17,20β-P was not detected at 0 h, but its level increased sharply giving the peak at 24 h (P<0.05). Testosterone decreased significantly at 6 and 24 h (P<0.05). On the other hand, cortisol registered a significant increase with the peak level at 24 h but corticosterone increased at 6 h and declined to the control level at 24 h.
Follicular envelope
The hyaluronidase treatment could easily separate the follicular envelope from the oocyte. The incubation of the follicular envelope with 2-OHE2 registered overall significant effects on P4, 17-P4 and 17,20β-P levels (Fig. 5B; F=117.01, 144.68 and 2093.80 respectively; P<0.001, one-way ANOVA). The levels of P4 and 17-P4 followed a similar pattern of changes as in the intact follicle incubates but the concentrations were low at 6 and 24 h. On the other hand, the production of 17,20β-P was higher than that of the intact follicle at 6 and 24 h. Testosterone level was significantly high only at 24 h, while cortisol and corticosterone levels did not show any significant change.
Denuded oocytes
The incubation of denuded oocytes with 2-OHE2 did not produce any significant effect on GVBD. The denuded oocytes contained low concentrations of 17-P4, testosterone and cortisol, which did not vary significantly (Fig. 5C). P4 level was higher compared with the other steroids but did not vary either. 17,20β-P and corticosterone were not detected at any of the intervals.
Discussion
We identified tentatively steroid hormones in the ovary/follicle incubates and measured changes in their levels associated with oocyte maturation and/or ovulation by an HPLC method. The study also provided evidence for the ovarian origin of the MIS (17,20β-P and corticosteroids), which was a point of contention in this catfish species (Sundararaj & Goswami 1977).
hCG is used extensively to induce ovulation in fishes, including the catfish (Joy & Tharakan 1999). Administration of 100 IU of hCG induced 100% oocyte maturation and ovulation, fertilizable eggs could be stripped out at 12 h and fish ovulated spontaneously from 16 h onwards. The hCG administration resulted in the stimulation of the progestin pathway, as has been reported in other species (Fostier et al. 1983, Nagahama et al. 1994), and the significant increases in P4, 17-P4 and 17,20β-P levels can be explained by the precursor–product relationship. Of all the steroids measured, 17,20β-P, which co-migrated with the authentic standard, registered a sharp increase from 1.5 pg/mg ovary at 0 h to 89 (8 h) and 126 (16 h) pg/mg associated with the maturational activity and ovulation. The control fish, which did not ovulate maintained a basal level of the hormone throughout. Thus, the rise in 17,20β-P could be correlated with its MIS activity, as has been reported in a number of teleosts (references reviewed in Introduction).
In the catfish, P4 that co-migrated with the authentic standard did not elicit any MIS activity in vitro (Sundararaj & Goswami 1977) although it elicited a moderate to strong activity in other species (Goetz 1983, Jalabert et al. 1991). In Clarias gariepinus, 17-P4 has been shown to induce oocyte maturation and ovulation apparently due to its conversion into 17,20β-P (Richter et al. 1987). In the present study, P4 level peaked at 16 h apparently due to reduced conversion to 17-P4, which itself decreased to low values. Likewise, the pattern of changes in 17-P4 levels at 8 and 16 h suggests its metabolite status rather than a hormone function.
The ovarian concentration of testosterone that co-migrated with the standard decreased significantly after the hCG treatment with the lowest level detected at 16 h. A decrease in testosterone may imply a downregulation of the enzymes involved in the conversion of 17-P4 to androstenedione (17,20-lyase) or androstenedione to testosterone (17β-HSD), or its further conversion into ketotestosterone or glucuronide (Lambert & Van Den Hurk 1982, Scott & Baynes 1982). Scott & Baynes (1982) reported that 17,20β-P inhibited C21–C19 desmolase (lyase) activity, inhibiting testosterone production. The decrease in testosterone production might be a mechanism to inhibit oestradiol-17β (E2) synthesis, prior to oocyte maturation and ovulation. According to Nagahama et al.(1994), E2 synthesis is arrested by the inhibition of aromatase activity. The present data show that E2 inhibition could be achieved upstream of the aromatization step by limiting the supply of testosterone as the substrate (Sakai et al. 1988). In the catfish, the decrease in testosterone (present data) and E2 (Mishra & Joy 2006a) at 8 and 16 h suggests a comprehensive downregulation of the C19–C18 pathway, suggesting a shift towards the C21 steroid pathway. This results in the lowering of E2 to the nadir, a prerequisite for the resumption/sustenance of meiosis (Mishra & Joy 2006a, 2006b).
This study reports the occurrence of corticosteroids in the ovary of the catfish. The previous investigation by Ungar et al.(1977) did not identify any ovarian corticosteroids, strengthening the hypothesis that corticosteroids of interrenal origin alone acted as the MIS in this species (Sundararaj & Goswami 1977). In the present study, low levels of cortisol and corticosterone that co-migrated with the respective standards could be measured in the control fish. The administration of hCG increased corticosteroid production at 8 and/or 16 h associated with maturational activity. Teleost ovary has been demonstrated to synthesize corticosteroids such as 11-deoxycorticosterone and 11-deoxycortisol (Colombo et al. 1973, Theofan & Goetz 1983, Scott & Canario 1990, Kime et al. 1992). Cortisol production by ovary has been reported in human (Yong et al. 2000), frog (Gobbetti & Zerani 1993) and sturgeon (Webb et al. 2002). Colombo et al.(1973) reported that ovarian corticosteroids might act as local hormones mediating pituitary gonadotrophin-induced oocyte maturation and ovulation. In the catfish, cortisol, but not corticosterone, was shown to have MIS activity in vitro (Goswami & Sundararaj 1971). However, in Fundulus heteroclitus, corticosterone induced a high percentage (98%) of GVBD (Greeley et al. 1986). Whether the low levels of corticosteroids in the presence of high amounts of 17,20β-P do elicit any direct maturational activity is debatable. Since high levels of corticosteroids have deleterious effects, their presence in low levels may assume physiological significance. Cortisol may enhance the sensitivity of the oocytes to gonadotrophin or MIS (Fostier & Jalabert 1982). Since corticosteroids are involved in metabolic and osmoregulatory processes, they may play an important role in metabolism and hydration of the follicles associated with maturation and ovulation (LaFleur & Thomas 1991).
In vitro incubation of the intact follicles or follicular envelope with 2-OHE2 stimulated 17,20β-P synthesis, as in the hCG study. Further, the significant sharp increase in the 17,20β-P level (6 and 24 h) could be correlated with the GVBD response (48.9 and 79.8% respectively) in the intact follicle incubates. Conversely, the incubation of the denuded oocytes with 2-OHE2 did not induce GVBD. The latter observation appears to rule out a direct effect of the steroid on oocyte maturation, although further detailed studies are required to confirm it. It is not known whether the hyaluronidase treatment had affected the oocyte membrane structure and function. The present study further shows that the follicular envelope (theca–granulosa complex) is responsible for the secretion of 17,20β-P, since denuded oocytes did not elaborate the steroid. The follicular envelope incubate produced more 17,20β-P than the intact follicle incubate. This suggests that the oocyte within the follicle might have inhibited the synthesis rate in some manner or the steroid might be used for maturational activity (Petrino et al. 1989). A similar pattern was reported by Kagawa et al.(1982) with regard to the in vitro production of E2. However, the intact follicle incubates secreted (accumulated) high amounts of P4 or 17-P4 (24 h) than the follicular envelope incubates, which suggests that in the latter the steroids might have been converted to 17,20β-P due to higher 20β-hydroxysteroid dehydrogenase (20β-HSD) activity.
The pattern of the in vitro production of P4, 17-P4 and 17,20β-P in both intact follicle and follicular envelope preparations showed parallelism with that of the ovary following the hCG treatment. A similar parallelism, in the overall pattern of changes in the corticosteroids (cortisol and corticosterone) and testosterone was noticed between the in vivo and in vitro studies. The basal level of 17,20β-P was not detected in vitro but a low level was detected in vivo, implying that the steroid induction started with the addition of 2-OHE2. Steroid precursors and gonadotrophin have been shown to promote MIS production in vitro (Petrino et al. 1989). Nagahama et al.(1986) reported that 17,20β-P could not be measured in vitro unless gonadotrophin or 17-P4 (substrate) was added exogenously. Since 2-OHE2 is not a precursor of the steroid, it might have stimulated the activity of specific enzymes (3β-hydroxysteroid dehydrogenase (3β-HSD), 17α-hydroxylase and 20β-HSD), like gonadotrophin (LH) to elevate the steroid production (Jalabert et al. 1991, Nagahama et al. 1994). Like LH, 2-OHE2 stimulated P4 secretion in rat luteal cells by stimulating the cholesterol side-chain cleavage enzyme and 3β-HSD, and inhibiting 20α-HSD activity (Tekpetey & Armstrong 1994). A 4 day co-treatment with either follicle-stimulating hormone (FSH) or LH enhanced P4 production in porcine granulose cells, stimulated by 2-OHE2 (Spicer & Hammond 1988, 1989). On the other hand, the catecholoestrogen inhibited basal or LH-stimulated androstenedione and 17-P4 production in porcine thecal cells (Morley et al. 1989). In the catfish, FSHβ mRNA transcript is expressed but the protein is not yet demonstrated in the circulation (Swanson et al. 2003). LH is present throughout the reproductive cycle (Tharakan 1998) and may serve the FSH function.
In the catfish (H. fossilis and C. batrachus), it has been reported that the LH surge stimulates oestrogen-2-hydroxylase activity resulting in the synthesis of 2-hydroxyoestrogens (Senthilkumaran & Joy 2001). The present data in conjunction with the above study suggest that 2-OHE2 may be a part of the LH cascade stimulating progestin synthesis during oocyte maturation and ovulation in this species. According to our hypothesis (Fig. 6), the LH surge inhibits E2 by metabolizing it into hydroxyoestrogens (Mishra & Joy 2006a), which stimulate alone or in synergy with the gonadotrophin the steroid pathway at multiple sites, altering the activity of key enzymes leading to the synthesis of 17,20β-P. Further work is required to demonstrate the effects of 2-OHE2 alone or in synergy with LH on the activity of the key enzymes involved in steroid metabolism in fish ovary/follicles.
The denuded oocytes presented a steroid hormone content with a relatively high level of P4 compared with testosterone, cortisol or corticosterone but lacked 17,20β-P. The occurrence of steroid hormones (androgens, oestrogens, progestins and corticosteroids) and thyroid hormones has been reported previously within fish oocytes (Schreck et al. 1991, Tagawa 1996). The egg (yolk) hormones may represent a continuum between the mother and offspring and have a role in sexual differentiation, early development, metabolism or behaviour (Schreck et al. 1991).
The chromatograms showed the elution peaks of three unidentified compounds, the peak area of which increased with the progress of maturational activity. Ungar et al.(1977) characterized a major ovarian steroid, 3α-hydroxy-5β-pregnan-20-one (pregnanolone), that sensitized oocytes for increased maturational response by cortisol (Sundararaj et al. 1979). Further, 11-deoxycorticosterone, 11-deoxycortisol and 21-deoxycortisol were shown to have MIS activity in vitro (Sundararaj & Goswami 1977). The chemical identity and functional significance of the compounds may lead to the concept that multiple MISs may exist in the catfish ovary.
In conclusion, the present study suggested the occurrence of 17,20β-P in the catfish, which showed a significant increase associated with oocyte maturation, both in vivo and in vitro. Testosterone level registered a significant decrease, which may account for the decrease in E2 level (Mishra & Joy 2006a). The catfish ovary may synthesize cortisol and corticosterone, which increased with maturational activity. A comparison of the changes in the steroid profiles suggests that 2-OHE2 may be an important link in the hormonal cascade of LH stimulation of oocyte maturation and ovulation.
Funding
The work was funded by a research project of DST, New Delhi (SP/SO/C-13/2001) to K P J. A M is grateful to Banaras Hindu University for a research fellowship. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.
HPLC chromatograms showing separation of steroid standards on a C18 reversed phase column (Luna, 150 × 4.5 mm i.d., 5 μm); mobile phase: 60% methanol in water. Absorption was taken at 240 nm. (A) Separation profile of the standards applied in a mixture: peak 1, cortisol (retention time (Rt)=3.7 min); peak 2, corticosterone (Rt=5.5 min); peak 3, testosterone (Rt=9.9 min); peak 4, 17-P4 (Rt = 11.7 min); peak 5, 17,20β-P (Rt=13.4 min); and peak 6, P4 (Rt=23.0 min). (B) Elution profile of an ovarian sample extract (8 h after hCG injection) in the presence of known concentrations of the standards in a mixture. (C) Elution profile of an intact follicle sample extract (6 h after in vitro 2-OHE2 treatment) in the presence of known concentrations of the standards in a mixture.
Citation: Journal of Endocrinology 189, 2; 10.1677/joe.1.06686
Effects of a single i.p. injection of hCG (100 IU/fish) on ovarian elution profile of steroids at 0 (A), 8 (B) and 16 (C) h. Note the increased peak profile and production of 17,20β-P during the maturational process. Note also the increase in the peak characteristics of three unidentified (UI) compounds that increased with the maturational process.
Citation: Journal of Endocrinology 189, 2; 10.1677/joe.1.06686
Periovulatory changes in ovarian steroid levels after a single i.p. injection of hCG (100 IU/fish). Values are means±s.e.m. of five fish in each group. Data were analysed by one-way ANOVA (P<0.001) and a Newman–Keuls test (P<0.05). Asterisks show significant difference from the 0 h group. P4, progesterone; 17-P4, 17-hydroxyprogesterone; T, testosterone; F, cortisol; B, corticosterone; 17,20β-P, 17,20β-dihydroxy-4-pregnen-3-one.
Citation: Journal of Endocrinology 189, 2; 10.1677/joe.1.06686
Steroid production by intact follicle incubated with 2-OHE2 (5 μM) in vitro. (A) Chromatographic elution profile of a sample extract at 0 h. Note the absence of 17,20β-P peak. (B) Chromatographic elution profile of a sample extract at 6 h. (C) Chromatographic elution profile of a sample at 24 h. UI, unidentified compounds whose peak characteristics increased with the progress of maturational activity.
Citation: Journal of Endocrinology 189, 2; 10.1677/joe.1.06686
In vitro effects of 2-OHE2 (5 μM) on steroid production by intact follicle (A), follicular envelope (B) and denuded oocyte (C). Values are means±s.e.m. of 120 follicle preparations incubated in duplicate (group size=five fish). Data were analysed by one-way ANOVA (P<0.001) and a Newman–Keuls test (P<0.05). Asterisks show significant difference from the 0 h group. ND, not detected; P4, progesterone; 17-P4, 17-hydroxyprogesterone; T, testosterone; F, cortisol; B, corticosterone; 17,20β-P, 17,20β-dihydroxy-4-pregnen-3-one.
Citation: Journal of Endocrinology 189, 2; 10.1677/joe.1.06686
A schematic diagram showing multiple sites of action of hydroxyoestradiol (OHE2) on progestin pathway during oocyte maturation. (+) stimulation, (−) inhibition, (?) a direct action of OHE2 on maturational activity is suspected. C, cholesterol; P5, pregnenolone; P4, progesterone; 17-P4, 17-hydroxyprogesterone; 17,20β-P, 17,20β-dihydroxy-4-pregnen-3-one (MIS); MPF, maturation promoting factor; ME2, methoxyoestradiol. (1) oestrogen-2/4-hydroxylase; (2) catechol-O-methyltransferase; (3) 3β-hydroxysteroid dehydrogenase; (4) 17α-hydroxylase; (5) 20β-hydroxysteroid dehydrogenase; (6) P-450 17,20-lyase; (7) 17β-hydroxysteroid dehydrogenase. R, receptor; A, androstenedione; OM, oocyte maturation.
Citation: Journal of Endocrinology 189, 2; 10.1677/joe.1.06686
References
Colombo L, Bern HA, Pieprzyk J & Johnson DW 1973 Biosynthesis of 11-deoxycorticosteroids by teleost ovaries and discussion of their possible role in oocyte maturation and ovulation. General and Comparative Endocrinology 21 168–178.
Fan H-Y, Li M-Y, Tong C, Chen D-Y, Xia G-L, Song X-F, Schatten H, Sun X-F, Schatten H & Sun Q-Y 2002 Inhibitory effects of cAMP and protein kinase C on meiotic maturation and MAP kinase phosphorylation in porcine oocytes. Molecular Reproduction and Development 63 480–487.
Fostier A & Jalabert B 1982 Physiological basis of practical means to induce ovulation in fish. In Proceedings of the International Symposium on Reproductive Physiology of Fish, pp 164–173. Compilers CJJ Richter & HJTh Goos. Wageningen, The Netherlands; Center for Agricultural Publishing and Documentation (Pudoc).
Fostier A, Jalabert B, Billard R, Breton B & Zohar Y 1983 The gonadal steroids. In Fish Physiology, vol IXA, pp 277–372. Eds WS Hoar, DJ Randall & EM Donaldson. New York, NY, USA: Academic Press.
Garcia-Alonso J, Nappa A, Somoza G, Rey A & Vizziano D 2004 Steroid metabolism in vitro during final oocyte maturation in white croaker Micropogonias furnieri (Pisces: Sciaenidae). Brazilian Journal of Biology 64 211–220.
Gobbetti A & Zerani M 1993 Prostaglandin E2 and prostaglandin F2 alpha involvement in the corticosterone and cortisol release by the female frog Rana esculenta during ovulation. Journal of Experimental Zoology 267 164–170.
Goetz FW 1983 Hormonal control of oocyte final maturation and ovulation in fishes. In Fish Physiology, vol IXB, pp 117–170. Eds WS Hoar, DJ Randall & EM Donaldson. New York, NY, USA: Academic Press.
Goswami SV & Sundararaj BI 1971 In vitro maturation and ovulation of oocytes of the catfish, Heteropneustes fossilis (Bloch): effects of mammalian hypophyseal hormones, catfish pituitary homogenate, steroid precursors and metabolites, and gonadal and adrenocortical steroids. Journal of Experimental Zoology 178 467–478.
Greeley MS Jr, Calder DR, Taylor MH, Hols H & Wallace RA 1986 Oocyte maturation in the mummichog (Fundulus heteroclitus): effects of steroids on germinal vesicle breakdown of intact follicles in vitro. General and Comparative Endocrinology 62 281–289.
Jalabert B, Fostier A, Breton B & Weil C 1991 Oocyte maturation in vertebrates. In Vertebrate Endocrinology: Fundamentals and Biomedical Implications, vol 4A, pp 23–90. Eds PKT Pang & MP Schreibman. New York, NY, USA: Academic Press.
Joy KP & Tharakan B 1999 Induced spawning of the Indian catfish, Heteropneustes fossilis, by GnRH analogue alone or in combination with dopamine-affecting drugs. Journal of Applied Aquaculture 9 23–32.
Kagawa H, Young G, Adachi S & Nagahama Y 1982 Estradiol-17β production in amago salmon (Oncorhynchus rhodurus) ovarian follicles: role of the thecal granulose cells. General and Comparative Endocrinology 47 440–448.
Kime DE 1987 Structure of steroidogenic tissues and their modes of secretion: the steroids. In Fundamentals of Comparative Vertebrate Endocrinology, pp 3–56. Eds I Chester-Jones, PM Ingleton & JG Phillips. New York, NY, USA: Plenum Press.
Kime DE, Scott AP & Canario AVM 1992 In vitro biosynthesis of steroids, including 11-deoxycortisol and 5α-pregnane-3β,7α, 17,20β-tetrol, by ovaries of the goldfish Carassius auratus during the stage of oocyte final maturation. General and Comparative Endocrinology 87 375–384.
LaFleur GJ Jr & Thomas P 1991 Evidence for the role of Na+,K+-ATPase in the hydration of Atlantic croaker and spotted sea trout oocytes during final maturation. Journal of Experimental Zoology 258 126–136.
Lambert JGD & Van Den Hurk R 1982 Steroidogenesis in the ovaries of the African catfish, Clarias lazera, before and after an HCG-induced ovulation. In Proceedings of the International Symposium on Reproductive Physiology of Fish, pp 99–102. Compilers CJJ Richter & HJTh Goos. Wageningen, The Netherlands; Center for Agricultural Publishing and Documentation (Pudoc).
Mishra A & Joy KP 2006a HPLC-electrochemical detection of ovarian estradiol-17β and catecholestrogens in the catfish Heteropneustes fossilis: Seasonal and periovulatory changes. General and Comparative Endocrinology 145 84–91.
Mishra A & Joy KP 2006b Relative effects of estradiol-17β (E2), catecholestrogens and clomiphene citrate on in vitro oocyte maturation in the catfish Heteropneustes fossilis (Bloch) and E2 inhibition of 2-hydroxyestradiol-induced maturation. General and Comparative Endocrinology (In Press).
Morley P, Khalil MW, Calaresu FR & Armstrong DT 1989 Catecholestrogens inhibit basal and luteinizing hormone-stimulated androgen production by porcine thecal cells. Biology of Reproduction 41 446–453.
Nagahama Y 1997 17α,20β-dihydroxy-4-pregnen-3-one, a maturation-inducing hormone in fish oocytes: mechanisms of synthesis and action. Steroids 62 190–196.
Nagahama Y & Adachi S 1985 Identification of maturation-inducing steroid in a teleost, the amago salmon (Oncorhynchus rhodurus). Developmental Biology 109 428–435.
Nagahama Y, Goetz FW & Tan JD 1986 Shift in steroidogenesis in the ovarian follicles of the goldfish (Carassius auratus) during gonadotrophin-induced oocyte maturation. Development Growth and Differentiation 28 555–561.
Nagahama Y, Yoshikuni M, Yamashita M & Tanaka M 1994 Regulation of oocyte maturation in fish. In Fish Physiology, vol XIII, pp 393–439. Eds AP Farrell & DJ Randall. New York, NY, USA: Academic Press.
Petrino TR, Greeley MS Jr, Selman K, Lin Y-WP & Wallace RA 1989 Steroidogenesis in Fundulus heteroclitus II. Production of 17α-hydroxy-20β-dihydroprogesterone, testosterone and 17β-estradiol by various components of the ovarian follicle. General and Comparative Endocrinology 76 230–240.
Richter CJJ, Eding EH, Goos HJTh, De Leeuw R, Scott AP & Van Oordt PGWJ 1987 The effect of pimozide/LHRHa and 17α-hydroxyprogesterone on plasma steroid levels and ovulation in the African catfish, Clarias gariepinus. Aquaculture 63 157–168.
Sakai N, Iwamatsu T, Yamauchi K, Suzuki N & Nagahama Y 1988 Influence of follicular development on steroid production in the medaka (Oryzias latipes) ovarian follicle in response to exogenous substrates. General and Comparative Endocrinology 71 516–523.
Schreck CB, Fitzpatrick MS, Feist GW & Yeoh C-G 1991 Steroids: developmental continuum between mother and offspring. In Proceedings of the Fourth International Symposium on the Reproductive Physiology of Fish, pp 256–258. Eds AP Scott, JP Sumpter, DE Kime & MS Rolfe. FishSymp 91, Sheffield: University of East Anglia, Norwich, UK.
Scott AP & Baynes SM 1982 Plasma levels of sex steroids in relation to ovulation and spermiation in rainbow trout (Salmo gairdneri). In Proceedings of the International Symposium on Reproductive Physiology of Fish, pp 103–106. Compilers CJJ Richter & HJTh Goos. Wageningen, The Netherlands; Center for Agricultural Publishing and Documentation (Pudoc).
Scott AP & Canario AVM 1987 Status of oocyte maturation-inducing steroids in teleosts. In Proceedings of the Third International Symposium on the Reproductive Physiology of Fish, pp 224–234. Eds DR Idler, LW Crim & JM Walsh. Third Fish Symposium 1987: Memorial University of Newfoundland, St John’s, Newfoundland.
Scott AP & Canario AVM 1990 Plasma levels of ovarian steroids including 17α,21-dihydroxy-4-pregnene-3,20-dione and 3α,17α,21-trihydroxy-5β-pregnan-20-one, in female plaice (Pleuronectes platessa) induced to mature with human chorionic gonadotrophin. General and Comparative Endocrinology 78 286–298.
Senthilkumaran B & Joy KP 2001 Periovulatory changes in catfish ovarian oestradiol-17β, oestrogen-2-hydroxylase and catechol-O-methyltransferase during GnRH analogue-induced ovulation and in vitro induction of oocyte maturation by catecholoestrogens. Journal of Endocrinology 168 239–247.
Spicer LJ & Hammond JM 1988 Comparative effects of androgens and catecholoestrogens on progesterone production by porcine granulosa cells. Molecular and Cellular Endocrinology 56 211–217.
Spicer LJ & Hammond JM 1989 Mechanism of action of 2-hydroxyestradiol on steroidogenesis in ovarian granulose cells: interactions with catecholamines and gonadotropins involve cyclic adenosine monophosphate. Biology of Reproduction 40 87–95.
Sundararaj BI & Goswami SV 1977 Hormonal regulation of in vivo and in vitro oocyte maturation in the catfish, Heteropneustes fossilis (Bloch). General and Comparative Endocrinology 32 17–28.
Sundararaj BI, Goswami SV & Lamba VJ 1979 Some aspects of oocyte maturation in catfish. Journal of Steroid Biochemistry 11 701–707.
Sundararaj BI, Goswami SV & Lamba VJ 1985 Oocyte maturation in teleost fishes. In Current Trends in Comparative Endocrinology, pp 369–372. Eds B Lofts & WN Holmes. Hong Kong: Hong Kong University Press.
Swanson P, Dickey JT & Campbell B 2003 Biochemistry and physiology of fish gonadotropins. Fish Physiology and Biochemistry 28 53–59.
Tagawa M 1996 Current understanding of the presence of hormones in fish eggs. In Survival Strategies in Early Life Stages of Marine Resources, pp 27–38. Eds Y Watanabe, Y Yamashita & Y Oozeki. Rotterdam, The Netherlands: AA Balkema.
Talbot P 1984 Hyaluronidase dissolves a component in the hamster zona pellucida. Journal of Experimental Zoology 229 309–316.
Tekpetey FR & Armstrong DT 1994 Catecholoestrogen modulation of steroid production by rat luteal cells: mechanism of action. Molecular and Cellular Endocrinology 101 49–57.
Tharakan B 1998 Neuroendocrine control of gonadotropin secretion in female catfish Heteropneustes fossilis: Basic mechanisms and involvement of olfactory system. PhD Thesis. Banaras Hindu University, Varanasi.
Theofan G & Goetz FW 1983 The in vitro synthesis of final maturational steroids by ovaries of brook trout (Salvelinus fontinalis) and yellow perch (Perca flavescens). General and Comparative Endocrinology 51 84–95.
Thomas P 1994 Hormonal control of final oocyte maturation in sciaenid fishes. In Perspectives in Comparative Endocrinology, pp 619–625. Eds KG Davey, RE Peter & SS Tobe. Ottawa, Canada: National Research Council of Canada.
Trant JM, Thomas P & Shackleton CHL 1986 Identification of 17α,20β, 21-trihydroxy-4-pregnen-3-one as the major ovarian steroid produced by the teleost Micropogonias undulatus during final oocyte maturation. Steroids 47 89–99.
Ungar F, Gunville R, Sundararaj BI & Goswami SV 1977 Formation of 3α-hydroxy-5β-pregnan-20-one in the ovaries of catfish, Heteropneustes fossilis (Bloch). General and Comparative Endocrinology 31 53–59.
Webb MAH, Feist GW, Trant JM, Eenennaam JPV, Fitzpatrick MS, Schreck CB & Doroshov SI 2002 Ovarian steroidogenesis in white sturgeon (Acipenser transmontanus) during oocyte maturation and induced ovulation. General and Comparative Endocrinology 129 27–38.
Yong PY, Thong KJ, Andrew R, Walker BR & Hillier SG 2000 Development-related increase in cortisol biosynthesis by human granulose cells. Journal of Clinical Endocrinology and Metabolism 85 4728–4733.
