Fish Physiol Biochem (2015) 41:1173–1186 DOI 10.1007/s10695-015-0078-1
Changes in vasotocin levels in relation to ovarian development in the catfish Heteropneustes fossilis exposed to altered photoperiod and temperature Radha Chaube . Rahul Kumar Singh . Keerikattil P. Joy
Received: 27 September 2014 / Accepted: 16 May 2015 / Published online: 31 May 2015 Ó Springer Science+Business Media Dordrecht 2015
Abstract Photoperiod and temperature are the major proximate factors that activate the brain–pituitary– gonadal–endocrine axis stimulating gonadal recrudescence. Vasotocin (VT), the basic nonapeptide hormone, is secreted by the nucleus preopticus in the hypothalamus and released from the pituitary into circulation as a neurohormone for physiological actions. Additionally, VT is secreted de novo in the ovary of the catfish and has been implicated in ovarian functions. In the present study, we evaluated the changes in VT secretion during altered photoperiod and temperature exposure. The ovarian changes were monitored over gonadosomatic index (GSI) and plasma steroid hormone levels. Exposure of the catfish to long photoperiod (LP, 16L:08D) daily, alone or in combination with high temperature (HT, 28 ± 2 °C), for 14 or 28 days resulted in a decrease in brain– pituitary VT level with a concomitant increase in plasma and ovarian VT levels. The changes were greater in the LP ? HT group on day 28. Concurrently, the treatments stimulated the GSI and plasma estradiol-17b (E2), testosterone (T) and progesterone
(P4) levels with higher more responses in the LP ? HT group. Exposure of the catfish to short photoperiod (SP, 08L:16D) daily or total darkness (TD, 24L:00D) daily, with or without changing the ambient temperature, for 14 or 28 days produced a depressing effect on VT, GSI and steroid hormone levels, the range of the response varied with the temperature. The brain VT level was low except in the TD ? NT group. Plasma and ovarian VT levels decreased more in the SP and TD groups under ambient temperature than in the groups at the raised temperature. The GSI and plasma steroid hormones (E2, T and P4) responded in a similar manner. Plasma cortisol level registered a significant increase in all the groups compared to the initial control groups, and the increase was significantly higher on day 28. The simultaneous activation of VT secretion and ovarian recrudescence by photoperiod and temperature suggests the peptide’s involvement in the hormonal control of gametogenesis. Keywords Catfish Photoperiod Temperature Steroid hormones Vasotocin
R. Chaube R. K. Singh K. P. Joy (&) Department of Zoology, Centre of Advanced Study, Banaras Hindu University, Varanasi 221 005, India e-mail: [email protected]; [email protected]
Introduction
R. Chaube (&) Zoology Department, Mahila Mahavidyalaya, Banaras Hindu University, Varanasi 221 005, India e-mail: [email protected]
Photoperiod and temperature are the main proximate factors that control the annual or seasonal reproductive cycle. The seasonally changing day length
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(photoperiod) is considered responsible for cueing and timing (environmental zeitgeber) of reproduction in the majority of fishes (Lam 1983; Bromage et al. 2001; O’Brien et al. 2012; Borg 2010). The zeitgeber (s) influence the endogenous rhythms resulting in synchronous spawning within each population at approximately the same time of the year. The changes in photoperiod and temperature activate the brain– pituitary–gonad (BPG)–endocrine axis signalling initiation, maintenance and culmination of breeding activity. Many fishes rely on the increasing photoperiod–temperature or declining photoperiod–temperature as the cue for starting gonadal recrudescence (Sundararaj 1981; Lam 1983). Spawning is induced by warm or cool temperature conditions. Gonad growth and maturation in many carps, catfish and other cyprinids in tropical and subtropical waters are influenced by temperature (Sundararaj 1981; Munro 1990; Borg 2010; O’Brien et al. 2012). The manipulation of these factors can accelerate or decelerate gametogenic and growth activities, and the strategy has acquired aquaculture importance (Tyler and Sumpter 1996; Davies et al. 1999; Rodriguez et al. 2001; Gines et al. 2003). Heteropneustes fossilis is one of the most extensively studied fish related to environmental control of reproduction (Sundararaj1981). Joy and Senthilkumaran (1995) have demonstrated that altered photoperiod and temperature influence the BPG-endocrine axis in the catfish. Exposure to a long photoperiod regime (16L:8D) for 30 days, without altering the ambient temperature, stimulated plasma gonadotropin-II (LH), estradiol-17b (E2) and testosterone levels, and gonadosomatic index (GSI) in female catfish. Vasotocin is the basic nonapeptide hormone in teleosts, synthesized in the hypothalamic magnocellular neurosecretory neurons of the nucleus preopticus (NPO) and axons project to the neurohypophysis where the hormone is stored and/or released (Urano et al. 1994; Acher 1996). In the catfish, the distribution of VT in all components of the BPG axis: hypothalamus, pituitary and gonads, was demonstrated previously (Singh and Joy 2008). VT has been known to play a predominant regulatory/modulatory role in osmoregulation, cardiovascular activity, metabolism, reproduction and behavior (Balment et al. 2006; Goodson and Thompson 2010). In the central nervous system, VT may act as a neurotransmitter and/or neuromodulator controlling reproductive functions,
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social hierarchy and behavior (Foran and Bass 1999; Chaube et al. 2012; Singh et al. 2013). Daily or seasonal activity in VT neuron morphology and number, peptide level, or pro-VT and receptor gene expression has been reported in some fish species (Hontela and Lederis 1985; Gilchriest et al. 1998; Kulczykowska 1999; Ota et al. 1999a, b; Gozdowska et al. 2006; Marsuka et al. 2007; Rodriguez-Illamola et al. 2011). The functional significance of these rhythms is not clearly elucidated but may reflect, and influenced by, subtle changes in environmental factors like photoperiod and temperature. In the catfish, the reproductive role of VT was extensively investigated and the evidence indicates that VT is a reproductive hormone, similar to gonadotropin. VT showed seasonal changes in immunoreactivity in the neurons of the NPO and axonal terminals in the posterior neurohypophysis, and in its concentration in the brain– pituitary, plasma and ovary/testis, correlated with the annual reproductive cycle of the catfish (Singh and Joy 2008). Further, in vitro follicular culture studies have demonstrated a direct physiological role of VT in ovarian steroidogenesis, final oocyte maturation, oocyte hydration, prostaglandin secretion and ovulation (Singh and Joy 2009a, 2010, 2011; Joy and Singh 2013). VT secretion is regulated by E2 (Singh and Joy 2009b; Chaube et al. 2012; Singh et al. 2013). In view of its direct reproductive involvement in the catfish, it is hypothesized that the secretion of VT would be modulated by environmental photoperiod and temperature, similar to pituitary gonadotropin (s) and ovarian steroid hormones. This assumption was tested by increasing or decreasing the environmental conditions (photoperiod and temperature) to stimulate or inhibit ovarian development in the catfish during early ovarian recrudescence (preparatory phase). Under these conditions, the changes in VT levels were compared with those in the GSI and steroid hormones to draw functional parallelisms.
Materials and methods Collection and acclimatization Adult female H. fossilis (40–50 g) were collected from local fish markets in Varanasi in the first week of March, which is the preparatory phase of the annual reproductive cycle (gonadosomatic index, GSI-
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0.62 %, early vitellogenic stage). The fish were maintained in 60-L experimental plastic tanks (1 9 1 9 0.2 m) under natural photoperiod and temperature (12.5L:11.5D, 18 ± 2 °C) for at least 48 h prior to the start of the experiment, to overcome stress due to transportation, and fed daily with goat liver ad libitum. All experiments were performed in accordance with the guidelines of the Animal Ethical Committee, Banaras Hindu University, Varanasi. Chemicals (Arg8)-Vasotocin enzyme immunoassay kit (Catalogue No. S-1239, EIAH 8121) was purchased from Bachem Peninsula Laboratories, California, USA. Estradiol-17b (No.E-2031), testosterone (No. AA E-1300), progesterone (No. FR E-2500) and cortisol (No. MS E-5000) ELISA kits, Labor Diagnostika Nord GmbH & Co. KG, Germany, were purchased locally. Solid-phase extraction (SPE) C18 cartridge was purchased from Ranbaxy Fine Chemicals Ltd. Ghaziabad, India. Other chemicals used in the study were of HPLC grade and purchased from E. Merck, Mumbai, India. Degassed and filtered nanopure water (Barnstead International, Dubuque, IO, USA) was used throughout for ELISA. Experimental study The experiments were carried out in the preparatory phase of the gonad from March 5 to April 5, 2012. The acclimated fish were divided into nine groups of 15 fish each and exposed to the altered photoperiod and temperature conditions for 14 and 28 days, as described below. Group 1 (NP ? NT) Control, maintained under normal photoperiod and normal temperature (12.5L:11.5D, 18 ± 2 °C), sampled with other groups after 14 or 28 days. Group 2 (NP ? HT) Normal photoperiod (12.5L:11.5D) and high temperature (28 ± 2 °C) group. The water temperature was elevated with a thermostatically controlled aquarium heater.
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Group 3 (LP ? NT) Long photoperiod (16L:08D) and normal temperature (18 ± 2 °C) group. Light was on from 06 to 22 h. The light source was a 23-W compact fluorescent lamp fixed to the ceiling of the animal house. Group 4 (LP ? HT) Long photoperiod (16L:08D) and high temperature (28 ± 2 °C) group. Light was on from 06 to 22 h.
Group 5 (SP ? NT) Short photoperiod (08L:16D). Light was on from 10 to 18 h and normal temperature (18 ± 2 °C). Group 6 (SP ? HT) Short photoperiod (08L:16D). Light was on from 10 to 18 h and high temperature (28 ± 2 °C) group. Group 7 (TD ? NT) Total darkness (24D:00L). The fish were maintained in a dark chamber for 14 or 28 days under normal temperature (18 ± 2 °C). Group 8 (TD ? HT) Total darkness (24D:00L). The fish were maintained in a dark chamber for 14 or 28 days under high temperature (28 ± 2 °C). During the experiment, the fish were fed with goat liver twice daily ad libitum. At the end of the experiments, the fish were weighed and killed by decapitation between 0900 and 1100 hours. The fish maintained in total darkness were killed under red light. Blood was collected in heparinized tubes. Plasma was separated by centrifugation at 2800g for 15 min at 4 °C and stored at -80 °C till assayed for VT and steroid hormones. Brain and ovary were removed and collected in dry acetone and stored at -80 °C for VT assay. The GSI (%) was calculated as weight of ovary/weight of fish 9 100.
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VT extraction and assay Acetone-dried brains, ovarian pieces and plasma were processed for VT extraction, as described earlier in our laboratory (Singh and Joy 2008; Chaube et al. 2012). The tissue supernatant and medium were extracted through a solid-phase extraction column: first the incubation medium followed by the supernatant of the tissue of the same incubation. The samples were airdried and stored at -20 °C (3–4 days) for the EIA. The samples were reconstituted with acidified ethanol prior to the assay. The assay was performed according to the instruction manual (Bachem Peninsula Laboratories, California, USA, Catalogue No S-1239EIAH8121). The detection range was 0.01–10 ng/mL. The cross-reactivity of the kit for other peptides was VT—100 %, isotocin—0 %, oxytocin—0 %. The cross-reactivity with vasopressin and lysine vasopressin, according to the manual, was 100 and 0 %, respectively, which we did not check in the assay. The intra- and inter-assay variations were 7.4 and 6 %, respectively. Steroid extraction and assay The tissues were homogenized separately in 4 vol of cold PBS (0.02 M, phosphate-buffered saline, pH 7.4) with an ultrasonic homogenizer (XL-2000 Microson, Misonix, USA) at 0 °C for 5–10 s. The homogenate was centrifuged at 500g for 20 min at 4 °C and extracted with 3 vol of diethyl ether, three times. The ether phase was collected and pooled group-wise evaporated and dried under N2 gas and stored at -20 °C till estimation. Plasma and incubation medium were directly extracted with diethyl ether, as described above. The ether phase was collected and pooled group-wise, evaporated, dried under gas and stored at -20 °C till the assay.
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Validation of E2, testosterone, progesterone (P4) and cortisol assays The response–concentration curves for E2, testosterone, P4 and cortisol were linear over 20–3200 pg/mL, 0.1–20 ng/mL, 0.1–60 ng/mL and 0.5–60 lg/dL range, respectively. The sensitivity of the E2, testosterone, P4 and cortisol assay was 10 pg/mL, 0.022 ng/mL, 0.1 ng/mL and 0.4 lg/dL, respectively. Cross-reactivity of the E2 antibody was E2—100 %, estriol—1.6 %, estrone— 1.3 %, progesterone—0.1 %, cortisol—0.1 %, testosterone—0 %, deoxycorticosterone—0 % and 17, 20bDP—0 %. Cross-reactivity of the testosterone antibody was testosterone—100 %, 5a-DHT—5.2 %, androstenedione—1.4 %, androstanediol—0.8 %, P4—0.5 %, androsterone—0.1 %, E2—0.001 % and 17, 20b-DP—0 %. Cross-reactivity of the P4 antibody was P4—100 %, 11-OHP—100 %, 17-OHP—0.4 %, deoxycorticosterone—1.7 %, 17,20b-DP—4.6 %, E2—0 %, testosterone—0.0004 %, corticosterone— 0.3 %, pregnenolone—0.2 %, 5a-Androstan-3b, 17bdiol—0.3 % and cortisol—0 %. Cross-reactivity of the cortisol antibody was cortisol—100 %, prednisolone —13.6 %, corticosterone—7.6 %, deoxycorticosterone—7.2 %, P4—7.2 %, cortisone—6.2 %, deoxycortisol—5.6 %, prednisone—5.6 %, dexamethasone—1.6 %, 17-OHP—0 %, 17,20b-DP—7.6 %, E2—0 %, testosterone—0 % and pregnenolone— 0 %. Known concentrations of E2, testosterone, P4 and cortisol were processed in the same manner as samples to calculate percentage recovery. The percentage recovery was E2 (77.417 pg/mL added)— 98.2 ± 1 % (n = 5), testosterone (1.1 ng/mL added) —94 ± 3 % (n = 5), P4—97 ± 2 % (n = 3) and cortisol—93.8 ± 1.6 % (15.3 lg/dL added). The coefficients of inter and intra-assay variations were 9.2 and 9.9 % for E2, 7.7 and 7.3 % for testosterone, 10.4 and 11.4 % for P4, and 5.6 and 5.76 % for cortisol, respectively.
Steroid hormone assays E2, testosterone, progesterone and cortisol were assayed by specific ELISA kits (Labor Diagnostika Nord GmbH & Co. KG, Germany). Standards and samples were processed according to the manufacturer’s instructions. Absorbance was taken at 450 nM using a Multiscan microplate reader (Thermo Electron Corporation, USA). The details of the steroid assay were already described (Chaube et al. 2014).
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Statistical analysis Data were expressed as mean ± SEM and checked for homogeneity and normality. Levene’s test of equality of error variances found that the error variances of the dependent variables are equal across groups (P [ 0.05) and the data were homogeneous. The data followed normal distribution in Kolmogorov–
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Effects of altered photoperiod and temperature on brain, plasma and ovarian VT levels
significantly but the magnitude of decrease was low. The altered photoperiod (P) and temperature (T) showed significant overall effects on the brain VT levels (two-way ANOVA, P \ 0.001; FP = 108.61, FT = 112.60, and the interaction between the two, FP 9 T = 86.24 for 14 days; FP = 146.72, FT = 123.62, and the interaction between the two, FP 9 T = 48.61 for 28 days). The highest brain VT level was observed in the TD ? NT groups, followed by the SP ? NT group, and the lowest in the LP ? HT group.
Brain VT
Plasma VT
The treatments produced an overall significant effect on brain VT level (Fig. 1; two-way ANOVA, P \ 0.001; FTreatment = 9.74, FDuration = 8.8, and the interaction between the treatment and duration, FT 9 D = 6.16). The VT level decreased significantly compared to the control group, and the scale of reduction was in the order LP ? HT (Group 4), NP ? HT (Group 2) and LP ? NT (Group 3) groups (P \ 0.05). The reduction was the highest on day 28 in the LP ? HT (Group 4) group. In the TD ? NT (Group 7) group, the VT level increased over the control group. In other groups, the level decreased
The treatments produced an overall significant effect on plasma VT level (Fig. 2; two-way ANOVA, P \ 0.001; FTreatment = 23.8, FDuration = 16.2, and the interaction of the two, FT 9 D = 11.12). The VT level increased maximally in the LP ? HT groups, followed by NP ? HT groups at both times (P \ 0.05). In the LP ? HT (Group 4) group, the increase was higher more in the 28-day group. The VT level increased in the LP ? NT (Group 3) and SP ? HT (Group 6) groups at both times compared to the control group. In the TD groups, a significant decrease was noticed in the TD ? NT (Group 7)
Smirnov test. The data were analyzed by a two-way ANOVA (P \ 0.001), taking treatment and duration, and photoperiod and temperature, as independent variables. Multiple group comparisons were done by the Newman–Keuls test (P \ 0.05).
Results
Fig. 1 Effects of photoperiod and temperature treatments on brain VT level in the female catfish H. fossilis. Data were expressed as mean ± SEM and analyzed by two-way ANOVA (P \ 0.001), followed by Newman–Keuls test (P \ 0.05). The groups with different letters are significantly different, and those with the same letters are not significant. NP normal photoperiod, LP long photoperiod, SP short photoperiod, TD total darkness, NT normal (ambient) temperature, HT high temperature
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Fig. 2 Effects of photoperiod and temperature treatments on plasma VT level in the female catfish H. fossilis. Data were expressed as mean ± SEM and analyzed by two-way ANOVA (P \ 0.001), followed by Newman–Keuls test (P \ 0.05). The groups with different letters are significantly different, and those with the same letters are not significant. Other abbreviations as in Fig. 1
groups at both times; however, on day 28 the VT level increased significantly in the TD ? HT (Group 8) group. The altered photoperiod and temperature showed significant overall effects on the plasma VT levels (two-way ANOVA, P \ 0.001; FP = 46.82, FT = 52.13, FP 9 T = 98.10 for 14 days; FP = 73.12, FT = 68.12, FP 9 T = 115.32 for 28 days). The highest plasma VT level was noticed in the LP ? HT group and the lowest in the TD ? NT group.
was decreased in the SP ? NT (Group 5) group but increased in the SP ? HT (Group 6) group. In the TD ? HT (Group 8) group, a significant increase was noticed on day 28. The ovarian VT levels showed significant interaction with the altered photoperiod and temperature treatments (two-way ANOVA, P \ 0.001; FP = 102.02, FTe = 115.38, FP 9 T = 128.17 for 14 days and FP = 118.60, FT = 134.12, FP 9 T = 85.11 for 28 days). The ovarian VT levels were the highest in the LP ? HT group and lowest in the TD ? NT group.
Ovarian VT The treatments produced an overall significant effect on the ovarian VT level (Fig. 3; two-way ANOVA, P \ 0.001; FTreatment = 33.4, FDuration = 23.35, and the interaction of the two, FT 9 D = 9.04). Ovarian VT level showed a significant increase in the NP ? HT (Group 2) and LP ? HT (Group 4) groups at both times with the maximal increase in the LP ? HT (Group 4) group in comparison with the control (P \ 0.05). A significant increase was also noticed in the LP ? NT (Group 3) group on day 28. The VT level was inhibited in the TD ? NT (Group 7) groups strongly at both times, followed by the SP ? NT (Group 5) group. In the SP groups, the inhibition was seen on day 14 in both temperature groups. On day 28, however, the VT level
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Effects of altered photoperiod and temperature on GSI The treatments produced an overall significant effect on the GSI (Fig. 4; two-way ANOVA, P \ 0.001; FTreatment = 442.67; Fduration = 151.25, and the interaction between the two, FT 9 D = 36.20). The GSI did not vary between the NP ? NT (Group 1) groups on day 14 and day 28, and between the NP ? NT (Group 1) group (control), and SP ? NT (Group 5) and SP ? HT (Group 6) groups on day 14. In the NP ? HT (Group 2), LP ? NT (Group 3), LP ? HT (Group 4) and TD ? HT (Group 8) groups, a significant increase was noticed in comparison with the control groups at both times. The increase was
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Fig. 3 Effects of photoperiod and temperature treatments on ovarian VT level in the female catfish H. fossilis. Data were expressed as mean ± SEM and analyzed by two-way ANOVA (P \ 0.001), followed by Newman–Keuls test (P \ 0.05). The groups with different letters are significantly different, and those with the same letters are not significant. Other abbreviations as in Fig. 1
Fig. 4 Effects of photoperiod and temperature treatments on gonadosomatic index (GSI) of the female catfish H. fossilis. Data were expressed as mean ± SEM and analyzed by two-way ANOVA (P \ 0.001), followed by Newman–Keuls test (P \ 0.05). The groups with different letters are significantly different, and those with the same letters are not significant. Other abbreviations as in Fig. 1
maximal in the LP ? HT (Group 4) group, followed by the NP ? HT (Group 2) and LP ? NT (Group 3) groups. The TD ? NT group showed a decrease in the GSI at both times. In the short photoperiod exposure,
no significant effect was seen on day 14; however, on day 28, a decrease was noticed in the SP ? NT (Group 5) group and an increase in the SP ? HT (Group 6) group (P \ 0.05). The GSI level showed significant
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interaction between the altered photoperiod and temperature treatments (two-way ANOVA, P \ 0.001; FP = 15.84, FT = 12.17, FP 9 T = 28.17 for 14 days and FP = 21.63, FT = 18.09, FP 9 T = 21.66 for 28 days). The LP ? HT group showed the highest GSI increase, and the TD ? NT group showed the least increase. Effects of altered photoperiod and temperature on plasma steroid hormone levels
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(Group 7) groups at both intervals, and also in the TD ?HT (Group 8) group on day 14. The plasma E2 levels showed significant interaction between the altered photoperiod and temperature treatments (two-way ANOVA, P \ 0.001; FP = 110.08, FT = 86.12, FP 9 T = 96.18 for 14 days and FP = 115.02, FT = 96.17, FP 9 T = 103.26 for 28 days). The E2 level was the highest in the LP ? HT group and lowest in the SP ? NT and TD ? NT groups. Plasma T
Plasma E2 The treatments produced an overall significant effect on plasma E2 (Fig. 5; two-way ANOVA, P \ 0.001; FTreatment = 103.27, FDuration = 73.32, and the interaction between the two, FT 9 D = 101.12). The highest increase was noticed in the LP ? NT (Group 3) and LP ? HT (Group 4) groups at both intervals compared to the control group. A significant increase was also noticed on day 28 in the NP ? HT (Group 2) and SP ? HT (Group 6) groups. The highest reduction was noticed in the SP ? NT (Group 5) and TD ? NT
Fig. 5 Effects of photoperiod and temperature treatments on plasma E2 level in the female catfish H. fossilis. Data were expressed as mean ± SEM and analyzed by two-way ANOVA (P \ 0.001), followed by Newman–Keuls test (P \ 0.05). The
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The treatments produced an overall significant effect on plasma testosterone (Fig. 6; two-way ANOVA, P \ 0.001; FTreatment = 93.14, FDuration = 69.13, and the interaction between the two, FT 9 D = 58.14). Plasma T level increased in the control group and increased further in the LP ? HT (Group 4) at both intervals (P \ 0.05). A significant increase was also noticed on day 28 in the LP ? NT (Group 3) and NP ? HT (Group 2) groups. The highest reduction was noticed in the TD ? NT (Group 7) group, followed by SP ? NT (Group 5), TD ? HT (Group 8)
groups with different letters are significantly different, and those with the same letters are not significant. Other abbreviations as in Fig. 1
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Fig. 6 Effects of photoperiod and temperature treatments on plasma testosterone level in the female catfish H. fossilis. Data were expressed as mean ± SEM and analyzed by two-way ANOVA (P \ 0.001), followed by Newman–Keuls test (P \ 0.05). The groups with different letters are significantly different, and those with the same letters are not significant. Other abbreviations as in Fig. 1
and SP ? HT (Group 6) groups when comparisons were made with the control group. The plasma T level showed significant interaction between the altered photoperiod and temperature treatments (two-way ANOVA, P \ 0.001; FP = 83.41, FT = 62.17, FP 9 T = 52.73 for 14 days and FP = 80.32, FT = 58.08, FP 9 T = 48.17 for 28 days). The T level was the highest in the LP ? HT group and the lowest in the TD ? NT group.
photoperiod and temperature treatments (two-way ANOVA, P \ 0.001; FP = 102.17, FT = 74.32, FP 9 T = 63.24 for 14 days and FP = 107.24, FT = 68.92, FP 9 T = 58.90 for 28 days). The P4 level was showed the highest increase in the LP ? HT group and the least increase in the TD ? NT and TD ? HT groups.
Plasma cortisol Plasma P4 The treatments produced an overall significant effect on plasma P4 (Fig. 7; two-way ANOVA, P \ 0.001; FTreatment = 104.28, FDuration = 81.48, and the interaction between the two, FT 9 D = 43.24). Plasma P4 increased significantly in the LP ? NT (Group 3) and LP ? HT (Group 4) groups, compared to the control group. The increase was maximal in the LP ? HT (Group 4) group. In the NP ? HT (Group 2) group, the increase was noticed on day 28. In the SP ? NT (Group 5), TD ? NT (Group 7), SP ? HT (Group 6) and TD ? HT (Group 8) groups, the P4 level decreased significantly from the control group. The P4 level registered the lowest value in the TD ? NT (Group 7) group at both intervals. The plasma P4 level showed significant interaction between the altered
Plasma cortisol level showed an overall significant effect following the treatments (Fig. 8; twoway ANOVA, P \ 0.001; FTreatment = 104.18, FDuration = 81.2, and the interaction between the two, FT 9 D = 79.28). The cortisol level increased in all groups compared to the control groups, and the increase was higher on 28. The highest increase was noticed in the LP ? HT (Group 4) and TD ? HT (Group 8) groups. The plasma cortisol level showed significant interaction between the altered photoperiod and temperature treatments (two-way ANOVA, P \ 0.001; FP = 108.26, FT = 83.12, FP 9 T = 76.24 for 14 days and FP = 110.35, FT = 86.42, FP 9 T = 63.08 for 28 days). The cortisol level was the highest in the TD ? HT group, followed by LP ? HT group, and lowest in the SP ? HT group.
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Fig. 7 Effects of photoperiod and temperature treatments on plasma progesterone level in the female catfish H. fossilis. Data were expressed as mean ± SEM and analyzed by two-way ANOVA (P \ 0.001), followed by Newman–Keuls test (P \ 0.05). The groups with different letters are significantly different, and those with the same letters are not significant. Other abbreviations as in Fig. 1
Fig. 8 Effects of photoperiod and temperature treatments on plasma cortisol level in the female catfish H. fossilis. Data were expressed as mean ± SEM and analyzed by two-way ANOVA (P \ 0.001), followed by Newman–Keuls test (P \ 0.05). The groups with different letters are significantly different, and those with the same letters are not significant. Other abbreviations as in Fig. 1
Discussion To our knowledge, this is the first report describing effects of altered photoperiod and temperature on VT
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secretion in the BPG axis. The preparatory phase (early March) heralds the initiation of gonadal recrudescence in the catfish when both photoperiod and temperature start increasing after the winter/
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spring transition, and this period is ideal for studying the effects of photothermal manipulations on gonadal activity. The catfish were exposed to a summer-like condition (long photoperiod (16L) with or without raising the temperature or a winter-like condition (short photoperiod (8L) or totally deprived of light (24 h darkness) with or without raising the temperature. The high temperature chosen falls within the range of temperatures experienced by the catfish during summer months (May–June), and 8-h light regime used is shorter by 2 h what the fish experiences in the nature. The exposure of the catfish to long photoperiod, with or without changing the ambient temperature, stimulated gonadal activity as evident from the increase in the GSI and plasma levels of ovarian steroid hormones compared to the control groups. The exposure of the catfish to short photoperiod and total darkness, with or without changing the ambient temperature, had a decelerating effect on the BPG axis. The GSI was significantly low in the 14-day TD ? NT group (Group 7), and 28-day SP ? NT (Group 5) and TD ? NT groups. In the 14-day TD ? HT group (Group 8), a marginal but significant increase was noticed compared to the control group. The steroid hormone (E2, T and P4) levels except cortisol (see below) were significantly low or not changed compared to the control group. These results are similar to that reported by earlier workers in the catfish (Sundararaj 1981; Joy and Senthilkumaran 1995; Senthilkumaran and Joy 1995). A highly significant stimulatory effect on the GSI was seen in the LP groups and NP ? HT groups compared to the control groups. This shows that light and temperature can independently stimulate gonadal activity, but the combination produced the maximal response, as in the nature. Given alone, temperature had a greater effect on the GSI than photoperiod, as has been also reported previously in this species (Sundararaj 1981). The SP and TD had a depressing effect on the GSI, regardless of the temperature conditions. Taken together, an optimal photoperiod is important for gonadal recrudescence to start upon which temperature exerts the stimulatory actions. E2 is the functionally active estrogen in teleosts and stimulates vitellogenesis and oocyte growth (Fostier et al. 1983; Wallace 1985; Tyler and Sumpter 1996). P4 and T are intermediates in the biosynthesis of E2. Hence, we investigated the dynamics of the steroids after exposure to various treatments. Plasma E2 level
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increased significantly in the LP groups at both intervals and on day 28 in the NP ? HT group (Group 2). There is no significant variation between the LP alone and LP ? HT groups (Group 3 and 4). It appears that, like the GSI, light and temperature may act independently on E2 production but the photoperiod has an edge over the temperature. Like the GSI, the exposure to SP and TD inhibited the steroid level below the control group. However, the high temperature could maintain the E2 level (14-day group) or elevate it (28-day group) compared to the control groups. The changes in the GSI and E2 level are parallel under the altered conditions. The increase in the E2 level and GSI could be due to increased secretion of pituitary gonadotropin, as reported by Joy and Senthilkumaran (1995) in LP ? NT-maintained catfish. Photoperiod and temperature act at the level of the hypothalamo-hypophyseal system to modulate gonadotropin secretion (Joy and Senthilkumaran 1995; Senthilkumaran and Joy 1995). The environmental factors modulate monoaminergic activity differentially. Testosterone is the immediate precursor of E2, and its level did not vary greatly on exposure to the altered photoperiod and temperature perhaps due its conversion. Even then, a general trend can be seen with its level increased under normal or LP conditions with or without changing the temperature. Under SP or TD conditions, the T level was significantly low under ambient conditions or was maintained (SP ? HT Group 6 and 14-day TD ? HT Group 8). The P4 level showed a pattern of changes similar to E2 and was significantly high in the LP ? NT (Group 3), LP ? HT (Group 4) and NP ? HT (Group 2—day 28) groups. In the SP and TD groups, the steroid level was low or maintained compared to the parallel control group. The brain–pituitary–adrenal axis is crucial for vertebrates to cope up with various forms of stress. In teleosts, cortisol is the main corticosteroid secreted by the interrenal tissue (homologue of adrenal cortex) and functions as both glucocorticoid and mineralocorticoid (Norris and Carr 1997). The teleost ovary is also a site of corticosteroid synthesis (Mishra and Joy 2006), and corticoids like cortisol and deoxycorticosterone are implicated in final oocyte maturation (Sundararaj 1981; Rao and Haider 1992). The apparent increase in the cortisol level in all the treatment groups may be primarily a stress response, increasing carbohydrate
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metabolism related to yolk synthesis or general energy demand. The exposure to SP, TD and high temperature conditions may be more stressful than the LP conditions, judging from the higher increase in cortisol level. Parallel with the changes in ovarian recrudescence, the altered photothermal treatments modulated VT secretion in the BPG axis. In general, the exposure to high temperature with or without altering the photoperiod stimulated a greater secretion of VT (release). However, plasma and ovarian VT levels recorded the highest levels in the LP ? HT Group 4. There may be other factors regulating the hormone dynamics in the circulation. It is not clear whether the increase in ovarian VT level was due to a direct action of photoperiod and temperature on the ovary. It is likely that the increased secretion of gonadotropins may increase the ovarian level of VT (Singh and Joy 2009b). Under the SP and TD conditions, brain VT levels did not change from the control group levels or increased, and plasma and ovarian VT level decreased with the lowest level in the TD ? NT Group 7. The changes can be attributed to an inhibition of the hormone release from the brain. However, the high temperature exposure lowered the inhibitory effect of the SP and TD conditions. It is inferred that both photoperiod and temperature act synergistically to modulate the VT secretion under a threshold photoperiod. The results explain the seasonal changes in VT secretion in the BPG axis during the reproductive cycle of the catfish, reported by us earlier (Singh and Joy 2008). With the rise in photoperiod and temperature, plasma and ovarian VT level increased to the peak in the spawning phase (July) and declined thereafter. The brain VT level increased to the peak early in the preparatory phase (March–April), decreased subsequently due to increased release into the circulation. Though VT is involved in multiple functions (see Introduction), the increased secretion of VT can be attributed to its physiological role in ovarian functions, as demonstrated in this species. Under in vitro conditions, VT stimulates steroidogenesis, stimulating E2 in a biphasic manner and P4, 17-hydroxyprogesterone and 17, 20b-dihydroxy-4-pregnen-3-one, 17, 20b-DP (Singh and Joy 2009a). Further, it stimulates final oocyte maturation, oocyte hydration, prostaglandin secretion and ovulation (Singh and Joy 2010, 2011; Joy and Singh 2013). A functional interaction between VT and ovarian steroids has been demonstrated, implying feedback control of each other’s secretory
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activity. E2 modulated follicular VT secretion biphasically, while progestin steroids (P4 and 17,20b-DP) stimulated VT secretion positively (Singh and Joy 2009b). Ovariectomy decreased brain VT levels duration-dependently, and E2 replacement reversed the ovariectomy effects dose-dependently (Singh and Joy 2009b; Chaube et al. 2012). Working further on the mechanism of E2 regulation of VT, it was shown that the E2 action was mediated through the catecholaminergic system, dopamine (DA) inhibits, and noradrenaline (NA), and adrenaline stimulates brain VT (Chaube et al. 2012; Singh et al. 2013). Senthilkumaran and Joy (1995) have shown that photoperiod and temperature act differentially on hypothalamic catecholamines. Exposure of the catfish to LP and HT inhibited DA activity but stimulated NA and adrenaline activities. Exposure to SP and TD conditions had opposite effects on the catecholamines. Taken together, photoperiod and temperature act on the hypothalamic catecholaminergic system to regulate VT differentially. Estrogen-negative feedback acts on the catecholaminergic system to fine-tune the regulation of VT secretion. Since photoperiod and temperature-induced changes on the catecholaminergic system also regulate gonadotropin secretion and E2 exerts negative feedback effect on it through the catecholaminergic system (Senthilkumaran and Joy 1995), it is very likely that the external and internal signals converge on a common mechanism to regulate both VT and gonadotropin secretion. Such a regulatory coupling can bring functional coordination of the two systems on gonad functions over time. In conclusion, the present study demonstrated photoperiod and temperature modulation of VT secretion in the BPG axis. The simultaneous activation of VT secretion and ovarian recrudescence by long photoperiod and high temperature points to the peptide’s participation in the hormonal control of gametogenesis. Acknowledgments R.K.S. is a recipient of Senior Research Fellowship (CAS-Merged R-A/C Grant) of Department of Zoology, Banaras Hindu University. This work was partially supported by a UGC Research grant (SR/34-436/2008) to R.C, New Delhi.
References Acher R (1996) Molecular evolution of fish neurohypophysial hormones: neutral and selective evolutionary mechanisms. Gen Comp Endocrinol 102:157–172
Fish Physiol Biochem (2015) 41:1173–1186 Balment RJ, Lu W, Weybourne E, Warne JM (2006) Arginine vasotocin, a key hormone in fish physiology and behaviour: a review with insights from mammalian models. Gen Comp Endocrinol 147:9–16 Borg B (2010) Photoperiodism in fishes. In: Denlinger DL, Somers DE, Nelson RJ (eds) Photoperiodism: the biological calender. Oxford University Press, Oxford, pp 371–398 Bromage N, Porter M, Randall C (2001) The environmental regulation of maturation in farmed finfish with special reference to the role of photoperiod and melatonin. Aquaculture 197:63–98 Chaube R, Singh RK, Joy KP (2012) Estrogen regulation of brain vasotocin secretion in the catfish Heteropneustes fossilis: an interaction with catecholaminergic system. Gen Comp Endocrinol 175:206–213 Chaube R, Singh RK, Joy KP (2014) Effects of ovaprim, a commercial spawning inducer, on vasotocin and steroid hormone profiles in the catfish Heteropneustes fossilis: in vivo and in vitro studies. Gen Comp Endocrinol 195:190–200 Davies B, Bromage N, Swanson P (1999) The brain–pituitary– gonadal axis of female rainbow trout, Oncorhynchus mykiss: effects of photoperiod. Gen Comp Endocrinol 115:155–166 Foran CM, Bass AH (1999) Preoptic GnRH and AVT: axes for sexual plasticity in teleost fish. Gen Comp Endocrinol 116:141–152 Fostier A, Jalabert B, Billard R, Breton B, Zohar Y (1983) The gonadal steroids. In: Hoar WS, Randall DJ, Donaldson EM (eds) Fish Physiology, vol IXA. Academic Press, New York, pp 277–372 Gilchriest BJ, Tipping DR, Levy A, Baker BI (1998) Diurnal changes in the expression of genes encoding for arginine vasotocin and pituitary pro-opiomelanocortin in the rainbow trout (Oncorhynchus mykiss): correlation with changes in plasma hormones. J Neuroendocrinol 10:937–943 Gines R, Afonso JM, Arguello A, Zamorano MJ, Lopez JL (2003) Growth in adult gilthead sea bream (Sparus aurata L.) as a result of interference in sexual maturation by different photoperiod regimes. Aquac Res 34:73–78 Goodson JL, Thompson RR (2010) Nonapeptide mechanisms of social cognition, behavior and species-specific social systems. Curr Opin Neurobiol 20:784–794 Gozdowska M, Kleszczyn´ska A, Sokołowska E, Kulczykowska E (2006) Arginine vasotocin (AVT) and isotocin (IT) in fish brain: diurnal and seasonal variations. Comp Biochem Physiol B 143:330–334 Hontela A, Lederis K (1985) Diel variations in arginine vasotocin content of goldfish brain and pituitary: effects of photoperiod and pinealectomy. Gen Comp Endocrinol 57:397–404 Joy KP, Senthilkumaran B (1995) A serotonergic control of pituitary–gonadal activity in the female catfish, Heteropneustes fossilis, under normal and long photoperiods. In: Goetz F, Thomas P (eds) Reproductive physiology of fish, fishsymp 95. The University of Texas at Austin, Austin, pp 67–70 Joy KP, Singh V (2013) Functional interactions between vasotocin and prostaglandins during final oocyte maturation
1185 and ovulation in the catfish Heteropneustes fossilis. Gen Comp Endocrinol 186:126–135 Kulczykowska E (1999) Diel changes in plasma arginine vasotocin, isotocin, and melatonin in rainbow trout (Oncorhynchus mykiss). Fish Physiol Biochem 21:141–146 Lam TJ (1983) Environmental influences on gonadal activity in fish. In: Hoar WS, Randall DJ, Donaldson EM (eds) Fish Physiology, vol IXB. Academic Press, New York, pp 65–116 Maruska KP, Mizobe MH, Tricas TC (2007) Sex and seasonal co-variation of arginine vasotocin (AVT) and gonadotropin-releasing hormone (GnRH) neurons in the brain of the half spotted goby. Comp Biochem Physiol A 147:129–144 Mishra A, Joy KP (2006) Effects of gonadotrophin in vivo and 2-hydroxyestradiol-17b in vitro on follicular steroid hormone profile associated with oocyte maturation in the catfish Heteropneustes fossilis. J Endocrinol 189:341–353 Munro AD (1990) Tropical freshwater fish. In: Munro AD, Scott AP, Lam TJ (eds) Reproductive seasonality in teleosts: environmental influences. CRC Press, Boca Raton, pp 145–239 Norris OD, Carr AJ (1997) Endocrine disruption: biological bases for health effects in wildlife and humans. Oxford University Press, Oxford, pp 1–477 O’Brien CS, Bourdo R, Bradshaw WE, Holzapfel CM, Cresko WA (2012) Conservation of the photoperiodic neuroendocrine axis among vertebrates: evidence from the teleost fish, Gasterosteus aculeatus. Gen Comp Endocrinol 178:19–27 Ota Y, Ando H, Ueda H, Urano A (1999a) Differences in seasonal expression of neurohypophysial hormone genes in ordinary and precocious male masu salmon. Gen Comp Endocrinol 116:40–48 Ota Y, Ando H, Ueda H, Urano A (1999b) Seasonal changes in expression of neurohypophysial hormone genes in the preoptic nucleus of immature female masu salmon. Gen Comp Endocrinol 116:31–39 Rao NV, Haider S (1992) Oocyte maturation in Clarias batrachus: in vitro effect of various gonadotropins and homologous pituitary homogenate. Indian J Exp Biol 30:235–237 Rodrıguez L, Zanuy S, Carrillo M (2001) Influence of day length on the age at first maturity and somatic growth in male sea bass (Dicentrarchus labrax L.). Aquaculture 196:159–175 Rodrı´guez-Illamola A, Lo´pez Patin˜o MA, Soengas JL, Ceinos RM, Mı´guez JM (2011) Diurnal rhythms in hypothalamic/ pituitary AVT synthesis and secretion in rainbow trout: evidence for a circadian regulation. Gen Comp Endocrinol 170:541–549 Senthilkumaran B, Joy KP (1995) Changes in hypothalamic catecholamines, dopamine-b-hydroxylase, and phenylethanolamine-N-methyltransferase in the catfish Heteropneustes fossilis in relation to season, raised photoperiod and temperature, ovariectomy, and estradiol-17b replacement. Gen Comp Endocrinol 97:121–134 Singh V, Joy KP (2008) Immunocytochemical localization, HPLC characterization, and seasonal dynamics of vasotocin in the brain, blood plasma and gonads of the catfish Heteropneustes fossilis. Gen Comp Endocrinol 159:214–225
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1186 Singh V, Joy KP (2009a) Relative in vitro seasonal effects of vasotocin and isotocin on ovarian steroid hormone levels in the catfish Heteropneustes fossilis. Gen Comp Endocrinol 162:257–264 Singh V, Joy KP (2009b) Effects of hCG and ovarian steroid hormones on vasotocin levels in the female catfish Heteropneustes fossilis. Gen Comp Endocrinol 162:172–178 Singh V, Joy KP (2010) An involvement of vasotocin in oocyte hydration in the catfish Heteropneustes fossilis: a comparison with effects of isotocin and hCG. Gen Comp Endocrinol 166:504–512 Singh V, Joy KP (2011) Vasotocin induces final oocyte maturation and ovulation through the production of a maturation-inducing steroid in the catfish Heteropneustes fossilis. Gen Comp Endocrinol 174:15–21 Singh RK, Chaube R, Joy KP (2013) Differential and reproductive stage-dependent regulation of vasotocin secretion
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Fish Physiol Biochem (2015) 41:1173–1186 by catecholamines in the catfish Heteropneustes fossilis. Comp Biochem Physiol A 166:619–626 Sundararaj BI (1981) Reproductive physiology of teleost fishes: a review of present knowledge and needs for future research, Aquaculture Development and Coordination Programme, Food and Agriculture Organization of the United Nations, Rome. ADCP/REP/81/16, pp 1–82 Tyler CR, Sumpter JP (1996) Oocyte growth and development in teleosts. Rev Fish Biol Fisher 6:287–318 Urano A, Kubokawa K, Hiraoka S (1994) Expression of the vasotocin and isotocin gene family in fish. In: Sherwood NM, Hew CL (eds) Fish physiology, vol XIII. Academic Press, New York, pp 101–132 Wallace RA (1985) Vitellogenesis and oocyte growth in nonmammalian vertebrates. In: Browder LW (ed) Developmental biology, Vol. 1, oogenesis. Plenum Press, New York, pp 127–177
Changes in vasotocin levels in relation to ovarian development in the catfish Heteropneustes fossilis exposed to altered photoperiod and temperature Radha Chaube . Rahul Kumar Singh . Keerikattil P. Joy
Received: 27 September 2014 / Accepted: 16 May 2015 / Published online: 31 May 2015 Ó Springer Science+Business Media Dordrecht 2015
Abstract Photoperiod and temperature are the major proximate factors that activate the brain–pituitary– gonadal–endocrine axis stimulating gonadal recrudescence. Vasotocin (VT), the basic nonapeptide hormone, is secreted by the nucleus preopticus in the hypothalamus and released from the pituitary into circulation as a neurohormone for physiological actions. Additionally, VT is secreted de novo in the ovary of the catfish and has been implicated in ovarian functions. In the present study, we evaluated the changes in VT secretion during altered photoperiod and temperature exposure. The ovarian changes were monitored over gonadosomatic index (GSI) and plasma steroid hormone levels. Exposure of the catfish to long photoperiod (LP, 16L:08D) daily, alone or in combination with high temperature (HT, 28 ± 2 °C), for 14 or 28 days resulted in a decrease in brain– pituitary VT level with a concomitant increase in plasma and ovarian VT levels. The changes were greater in the LP ? HT group on day 28. Concurrently, the treatments stimulated the GSI and plasma estradiol-17b (E2), testosterone (T) and progesterone
(P4) levels with higher more responses in the LP ? HT group. Exposure of the catfish to short photoperiod (SP, 08L:16D) daily or total darkness (TD, 24L:00D) daily, with or without changing the ambient temperature, for 14 or 28 days produced a depressing effect on VT, GSI and steroid hormone levels, the range of the response varied with the temperature. The brain VT level was low except in the TD ? NT group. Plasma and ovarian VT levels decreased more in the SP and TD groups under ambient temperature than in the groups at the raised temperature. The GSI and plasma steroid hormones (E2, T and P4) responded in a similar manner. Plasma cortisol level registered a significant increase in all the groups compared to the initial control groups, and the increase was significantly higher on day 28. The simultaneous activation of VT secretion and ovarian recrudescence by photoperiod and temperature suggests the peptide’s involvement in the hormonal control of gametogenesis. Keywords Catfish Photoperiod Temperature Steroid hormones Vasotocin
R. Chaube R. K. Singh K. P. Joy (&) Department of Zoology, Centre of Advanced Study, Banaras Hindu University, Varanasi 221 005, India e-mail: [email protected]; [email protected]
Introduction
R. Chaube (&) Zoology Department, Mahila Mahavidyalaya, Banaras Hindu University, Varanasi 221 005, India e-mail: [email protected]
Photoperiod and temperature are the main proximate factors that control the annual or seasonal reproductive cycle. The seasonally changing day length
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(photoperiod) is considered responsible for cueing and timing (environmental zeitgeber) of reproduction in the majority of fishes (Lam 1983; Bromage et al. 2001; O’Brien et al. 2012; Borg 2010). The zeitgeber (s) influence the endogenous rhythms resulting in synchronous spawning within each population at approximately the same time of the year. The changes in photoperiod and temperature activate the brain– pituitary–gonad (BPG)–endocrine axis signalling initiation, maintenance and culmination of breeding activity. Many fishes rely on the increasing photoperiod–temperature or declining photoperiod–temperature as the cue for starting gonadal recrudescence (Sundararaj 1981; Lam 1983). Spawning is induced by warm or cool temperature conditions. Gonad growth and maturation in many carps, catfish and other cyprinids in tropical and subtropical waters are influenced by temperature (Sundararaj 1981; Munro 1990; Borg 2010; O’Brien et al. 2012). The manipulation of these factors can accelerate or decelerate gametogenic and growth activities, and the strategy has acquired aquaculture importance (Tyler and Sumpter 1996; Davies et al. 1999; Rodriguez et al. 2001; Gines et al. 2003). Heteropneustes fossilis is one of the most extensively studied fish related to environmental control of reproduction (Sundararaj1981). Joy and Senthilkumaran (1995) have demonstrated that altered photoperiod and temperature influence the BPG-endocrine axis in the catfish. Exposure to a long photoperiod regime (16L:8D) for 30 days, without altering the ambient temperature, stimulated plasma gonadotropin-II (LH), estradiol-17b (E2) and testosterone levels, and gonadosomatic index (GSI) in female catfish. Vasotocin is the basic nonapeptide hormone in teleosts, synthesized in the hypothalamic magnocellular neurosecretory neurons of the nucleus preopticus (NPO) and axons project to the neurohypophysis where the hormone is stored and/or released (Urano et al. 1994; Acher 1996). In the catfish, the distribution of VT in all components of the BPG axis: hypothalamus, pituitary and gonads, was demonstrated previously (Singh and Joy 2008). VT has been known to play a predominant regulatory/modulatory role in osmoregulation, cardiovascular activity, metabolism, reproduction and behavior (Balment et al. 2006; Goodson and Thompson 2010). In the central nervous system, VT may act as a neurotransmitter and/or neuromodulator controlling reproductive functions,
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social hierarchy and behavior (Foran and Bass 1999; Chaube et al. 2012; Singh et al. 2013). Daily or seasonal activity in VT neuron morphology and number, peptide level, or pro-VT and receptor gene expression has been reported in some fish species (Hontela and Lederis 1985; Gilchriest et al. 1998; Kulczykowska 1999; Ota et al. 1999a, b; Gozdowska et al. 2006; Marsuka et al. 2007; Rodriguez-Illamola et al. 2011). The functional significance of these rhythms is not clearly elucidated but may reflect, and influenced by, subtle changes in environmental factors like photoperiod and temperature. In the catfish, the reproductive role of VT was extensively investigated and the evidence indicates that VT is a reproductive hormone, similar to gonadotropin. VT showed seasonal changes in immunoreactivity in the neurons of the NPO and axonal terminals in the posterior neurohypophysis, and in its concentration in the brain– pituitary, plasma and ovary/testis, correlated with the annual reproductive cycle of the catfish (Singh and Joy 2008). Further, in vitro follicular culture studies have demonstrated a direct physiological role of VT in ovarian steroidogenesis, final oocyte maturation, oocyte hydration, prostaglandin secretion and ovulation (Singh and Joy 2009a, 2010, 2011; Joy and Singh 2013). VT secretion is regulated by E2 (Singh and Joy 2009b; Chaube et al. 2012; Singh et al. 2013). In view of its direct reproductive involvement in the catfish, it is hypothesized that the secretion of VT would be modulated by environmental photoperiod and temperature, similar to pituitary gonadotropin (s) and ovarian steroid hormones. This assumption was tested by increasing or decreasing the environmental conditions (photoperiod and temperature) to stimulate or inhibit ovarian development in the catfish during early ovarian recrudescence (preparatory phase). Under these conditions, the changes in VT levels were compared with those in the GSI and steroid hormones to draw functional parallelisms.
Materials and methods Collection and acclimatization Adult female H. fossilis (40–50 g) were collected from local fish markets in Varanasi in the first week of March, which is the preparatory phase of the annual reproductive cycle (gonadosomatic index, GSI-
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0.62 %, early vitellogenic stage). The fish were maintained in 60-L experimental plastic tanks (1 9 1 9 0.2 m) under natural photoperiod and temperature (12.5L:11.5D, 18 ± 2 °C) for at least 48 h prior to the start of the experiment, to overcome stress due to transportation, and fed daily with goat liver ad libitum. All experiments were performed in accordance with the guidelines of the Animal Ethical Committee, Banaras Hindu University, Varanasi. Chemicals (Arg8)-Vasotocin enzyme immunoassay kit (Catalogue No. S-1239, EIAH 8121) was purchased from Bachem Peninsula Laboratories, California, USA. Estradiol-17b (No.E-2031), testosterone (No. AA E-1300), progesterone (No. FR E-2500) and cortisol (No. MS E-5000) ELISA kits, Labor Diagnostika Nord GmbH & Co. KG, Germany, were purchased locally. Solid-phase extraction (SPE) C18 cartridge was purchased from Ranbaxy Fine Chemicals Ltd. Ghaziabad, India. Other chemicals used in the study were of HPLC grade and purchased from E. Merck, Mumbai, India. Degassed and filtered nanopure water (Barnstead International, Dubuque, IO, USA) was used throughout for ELISA. Experimental study The experiments were carried out in the preparatory phase of the gonad from March 5 to April 5, 2012. The acclimated fish were divided into nine groups of 15 fish each and exposed to the altered photoperiod and temperature conditions for 14 and 28 days, as described below. Group 1 (NP ? NT) Control, maintained under normal photoperiod and normal temperature (12.5L:11.5D, 18 ± 2 °C), sampled with other groups after 14 or 28 days. Group 2 (NP ? HT) Normal photoperiod (12.5L:11.5D) and high temperature (28 ± 2 °C) group. The water temperature was elevated with a thermostatically controlled aquarium heater.
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Group 3 (LP ? NT) Long photoperiod (16L:08D) and normal temperature (18 ± 2 °C) group. Light was on from 06 to 22 h. The light source was a 23-W compact fluorescent lamp fixed to the ceiling of the animal house. Group 4 (LP ? HT) Long photoperiod (16L:08D) and high temperature (28 ± 2 °C) group. Light was on from 06 to 22 h.
Group 5 (SP ? NT) Short photoperiod (08L:16D). Light was on from 10 to 18 h and normal temperature (18 ± 2 °C). Group 6 (SP ? HT) Short photoperiod (08L:16D). Light was on from 10 to 18 h and high temperature (28 ± 2 °C) group. Group 7 (TD ? NT) Total darkness (24D:00L). The fish were maintained in a dark chamber for 14 or 28 days under normal temperature (18 ± 2 °C). Group 8 (TD ? HT) Total darkness (24D:00L). The fish were maintained in a dark chamber for 14 or 28 days under high temperature (28 ± 2 °C). During the experiment, the fish were fed with goat liver twice daily ad libitum. At the end of the experiments, the fish were weighed and killed by decapitation between 0900 and 1100 hours. The fish maintained in total darkness were killed under red light. Blood was collected in heparinized tubes. Plasma was separated by centrifugation at 2800g for 15 min at 4 °C and stored at -80 °C till assayed for VT and steroid hormones. Brain and ovary were removed and collected in dry acetone and stored at -80 °C for VT assay. The GSI (%) was calculated as weight of ovary/weight of fish 9 100.
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VT extraction and assay Acetone-dried brains, ovarian pieces and plasma were processed for VT extraction, as described earlier in our laboratory (Singh and Joy 2008; Chaube et al. 2012). The tissue supernatant and medium were extracted through a solid-phase extraction column: first the incubation medium followed by the supernatant of the tissue of the same incubation. The samples were airdried and stored at -20 °C (3–4 days) for the EIA. The samples were reconstituted with acidified ethanol prior to the assay. The assay was performed according to the instruction manual (Bachem Peninsula Laboratories, California, USA, Catalogue No S-1239EIAH8121). The detection range was 0.01–10 ng/mL. The cross-reactivity of the kit for other peptides was VT—100 %, isotocin—0 %, oxytocin—0 %. The cross-reactivity with vasopressin and lysine vasopressin, according to the manual, was 100 and 0 %, respectively, which we did not check in the assay. The intra- and inter-assay variations were 7.4 and 6 %, respectively. Steroid extraction and assay The tissues were homogenized separately in 4 vol of cold PBS (0.02 M, phosphate-buffered saline, pH 7.4) with an ultrasonic homogenizer (XL-2000 Microson, Misonix, USA) at 0 °C for 5–10 s. The homogenate was centrifuged at 500g for 20 min at 4 °C and extracted with 3 vol of diethyl ether, three times. The ether phase was collected and pooled group-wise evaporated and dried under N2 gas and stored at -20 °C till estimation. Plasma and incubation medium were directly extracted with diethyl ether, as described above. The ether phase was collected and pooled group-wise, evaporated, dried under gas and stored at -20 °C till the assay.
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Validation of E2, testosterone, progesterone (P4) and cortisol assays The response–concentration curves for E2, testosterone, P4 and cortisol were linear over 20–3200 pg/mL, 0.1–20 ng/mL, 0.1–60 ng/mL and 0.5–60 lg/dL range, respectively. The sensitivity of the E2, testosterone, P4 and cortisol assay was 10 pg/mL, 0.022 ng/mL, 0.1 ng/mL and 0.4 lg/dL, respectively. Cross-reactivity of the E2 antibody was E2—100 %, estriol—1.6 %, estrone— 1.3 %, progesterone—0.1 %, cortisol—0.1 %, testosterone—0 %, deoxycorticosterone—0 % and 17, 20bDP—0 %. Cross-reactivity of the testosterone antibody was testosterone—100 %, 5a-DHT—5.2 %, androstenedione—1.4 %, androstanediol—0.8 %, P4—0.5 %, androsterone—0.1 %, E2—0.001 % and 17, 20b-DP—0 %. Cross-reactivity of the P4 antibody was P4—100 %, 11-OHP—100 %, 17-OHP—0.4 %, deoxycorticosterone—1.7 %, 17,20b-DP—4.6 %, E2—0 %, testosterone—0.0004 %, corticosterone— 0.3 %, pregnenolone—0.2 %, 5a-Androstan-3b, 17bdiol—0.3 % and cortisol—0 %. Cross-reactivity of the cortisol antibody was cortisol—100 %, prednisolone —13.6 %, corticosterone—7.6 %, deoxycorticosterone—7.2 %, P4—7.2 %, cortisone—6.2 %, deoxycortisol—5.6 %, prednisone—5.6 %, dexamethasone—1.6 %, 17-OHP—0 %, 17,20b-DP—7.6 %, E2—0 %, testosterone—0 % and pregnenolone— 0 %. Known concentrations of E2, testosterone, P4 and cortisol were processed in the same manner as samples to calculate percentage recovery. The percentage recovery was E2 (77.417 pg/mL added)— 98.2 ± 1 % (n = 5), testosterone (1.1 ng/mL added) —94 ± 3 % (n = 5), P4—97 ± 2 % (n = 3) and cortisol—93.8 ± 1.6 % (15.3 lg/dL added). The coefficients of inter and intra-assay variations were 9.2 and 9.9 % for E2, 7.7 and 7.3 % for testosterone, 10.4 and 11.4 % for P4, and 5.6 and 5.76 % for cortisol, respectively.
Steroid hormone assays E2, testosterone, progesterone and cortisol were assayed by specific ELISA kits (Labor Diagnostika Nord GmbH & Co. KG, Germany). Standards and samples were processed according to the manufacturer’s instructions. Absorbance was taken at 450 nM using a Multiscan microplate reader (Thermo Electron Corporation, USA). The details of the steroid assay were already described (Chaube et al. 2014).
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Statistical analysis Data were expressed as mean ± SEM and checked for homogeneity and normality. Levene’s test of equality of error variances found that the error variances of the dependent variables are equal across groups (P [ 0.05) and the data were homogeneous. The data followed normal distribution in Kolmogorov–
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Effects of altered photoperiod and temperature on brain, plasma and ovarian VT levels
significantly but the magnitude of decrease was low. The altered photoperiod (P) and temperature (T) showed significant overall effects on the brain VT levels (two-way ANOVA, P \ 0.001; FP = 108.61, FT = 112.60, and the interaction between the two, FP 9 T = 86.24 for 14 days; FP = 146.72, FT = 123.62, and the interaction between the two, FP 9 T = 48.61 for 28 days). The highest brain VT level was observed in the TD ? NT groups, followed by the SP ? NT group, and the lowest in the LP ? HT group.
Brain VT
Plasma VT
The treatments produced an overall significant effect on brain VT level (Fig. 1; two-way ANOVA, P \ 0.001; FTreatment = 9.74, FDuration = 8.8, and the interaction between the treatment and duration, FT 9 D = 6.16). The VT level decreased significantly compared to the control group, and the scale of reduction was in the order LP ? HT (Group 4), NP ? HT (Group 2) and LP ? NT (Group 3) groups (P \ 0.05). The reduction was the highest on day 28 in the LP ? HT (Group 4) group. In the TD ? NT (Group 7) group, the VT level increased over the control group. In other groups, the level decreased
The treatments produced an overall significant effect on plasma VT level (Fig. 2; two-way ANOVA, P \ 0.001; FTreatment = 23.8, FDuration = 16.2, and the interaction of the two, FT 9 D = 11.12). The VT level increased maximally in the LP ? HT groups, followed by NP ? HT groups at both times (P \ 0.05). In the LP ? HT (Group 4) group, the increase was higher more in the 28-day group. The VT level increased in the LP ? NT (Group 3) and SP ? HT (Group 6) groups at both times compared to the control group. In the TD groups, a significant decrease was noticed in the TD ? NT (Group 7)
Smirnov test. The data were analyzed by a two-way ANOVA (P \ 0.001), taking treatment and duration, and photoperiod and temperature, as independent variables. Multiple group comparisons were done by the Newman–Keuls test (P \ 0.05).
Results
Fig. 1 Effects of photoperiod and temperature treatments on brain VT level in the female catfish H. fossilis. Data were expressed as mean ± SEM and analyzed by two-way ANOVA (P \ 0.001), followed by Newman–Keuls test (P \ 0.05). The groups with different letters are significantly different, and those with the same letters are not significant. NP normal photoperiod, LP long photoperiod, SP short photoperiod, TD total darkness, NT normal (ambient) temperature, HT high temperature
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Fig. 2 Effects of photoperiod and temperature treatments on plasma VT level in the female catfish H. fossilis. Data were expressed as mean ± SEM and analyzed by two-way ANOVA (P \ 0.001), followed by Newman–Keuls test (P \ 0.05). The groups with different letters are significantly different, and those with the same letters are not significant. Other abbreviations as in Fig. 1
groups at both times; however, on day 28 the VT level increased significantly in the TD ? HT (Group 8) group. The altered photoperiod and temperature showed significant overall effects on the plasma VT levels (two-way ANOVA, P \ 0.001; FP = 46.82, FT = 52.13, FP 9 T = 98.10 for 14 days; FP = 73.12, FT = 68.12, FP 9 T = 115.32 for 28 days). The highest plasma VT level was noticed in the LP ? HT group and the lowest in the TD ? NT group.
was decreased in the SP ? NT (Group 5) group but increased in the SP ? HT (Group 6) group. In the TD ? HT (Group 8) group, a significant increase was noticed on day 28. The ovarian VT levels showed significant interaction with the altered photoperiod and temperature treatments (two-way ANOVA, P \ 0.001; FP = 102.02, FTe = 115.38, FP 9 T = 128.17 for 14 days and FP = 118.60, FT = 134.12, FP 9 T = 85.11 for 28 days). The ovarian VT levels were the highest in the LP ? HT group and lowest in the TD ? NT group.
Ovarian VT The treatments produced an overall significant effect on the ovarian VT level (Fig. 3; two-way ANOVA, P \ 0.001; FTreatment = 33.4, FDuration = 23.35, and the interaction of the two, FT 9 D = 9.04). Ovarian VT level showed a significant increase in the NP ? HT (Group 2) and LP ? HT (Group 4) groups at both times with the maximal increase in the LP ? HT (Group 4) group in comparison with the control (P \ 0.05). A significant increase was also noticed in the LP ? NT (Group 3) group on day 28. The VT level was inhibited in the TD ? NT (Group 7) groups strongly at both times, followed by the SP ? NT (Group 5) group. In the SP groups, the inhibition was seen on day 14 in both temperature groups. On day 28, however, the VT level
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Effects of altered photoperiod and temperature on GSI The treatments produced an overall significant effect on the GSI (Fig. 4; two-way ANOVA, P \ 0.001; FTreatment = 442.67; Fduration = 151.25, and the interaction between the two, FT 9 D = 36.20). The GSI did not vary between the NP ? NT (Group 1) groups on day 14 and day 28, and between the NP ? NT (Group 1) group (control), and SP ? NT (Group 5) and SP ? HT (Group 6) groups on day 14. In the NP ? HT (Group 2), LP ? NT (Group 3), LP ? HT (Group 4) and TD ? HT (Group 8) groups, a significant increase was noticed in comparison with the control groups at both times. The increase was
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Fig. 3 Effects of photoperiod and temperature treatments on ovarian VT level in the female catfish H. fossilis. Data were expressed as mean ± SEM and analyzed by two-way ANOVA (P \ 0.001), followed by Newman–Keuls test (P \ 0.05). The groups with different letters are significantly different, and those with the same letters are not significant. Other abbreviations as in Fig. 1
Fig. 4 Effects of photoperiod and temperature treatments on gonadosomatic index (GSI) of the female catfish H. fossilis. Data were expressed as mean ± SEM and analyzed by two-way ANOVA (P \ 0.001), followed by Newman–Keuls test (P \ 0.05). The groups with different letters are significantly different, and those with the same letters are not significant. Other abbreviations as in Fig. 1
maximal in the LP ? HT (Group 4) group, followed by the NP ? HT (Group 2) and LP ? NT (Group 3) groups. The TD ? NT group showed a decrease in the GSI at both times. In the short photoperiod exposure,
no significant effect was seen on day 14; however, on day 28, a decrease was noticed in the SP ? NT (Group 5) group and an increase in the SP ? HT (Group 6) group (P \ 0.05). The GSI level showed significant
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interaction between the altered photoperiod and temperature treatments (two-way ANOVA, P \ 0.001; FP = 15.84, FT = 12.17, FP 9 T = 28.17 for 14 days and FP = 21.63, FT = 18.09, FP 9 T = 21.66 for 28 days). The LP ? HT group showed the highest GSI increase, and the TD ? NT group showed the least increase. Effects of altered photoperiod and temperature on plasma steroid hormone levels
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(Group 7) groups at both intervals, and also in the TD ?HT (Group 8) group on day 14. The plasma E2 levels showed significant interaction between the altered photoperiod and temperature treatments (two-way ANOVA, P \ 0.001; FP = 110.08, FT = 86.12, FP 9 T = 96.18 for 14 days and FP = 115.02, FT = 96.17, FP 9 T = 103.26 for 28 days). The E2 level was the highest in the LP ? HT group and lowest in the SP ? NT and TD ? NT groups. Plasma T
Plasma E2 The treatments produced an overall significant effect on plasma E2 (Fig. 5; two-way ANOVA, P \ 0.001; FTreatment = 103.27, FDuration = 73.32, and the interaction between the two, FT 9 D = 101.12). The highest increase was noticed in the LP ? NT (Group 3) and LP ? HT (Group 4) groups at both intervals compared to the control group. A significant increase was also noticed on day 28 in the NP ? HT (Group 2) and SP ? HT (Group 6) groups. The highest reduction was noticed in the SP ? NT (Group 5) and TD ? NT
Fig. 5 Effects of photoperiod and temperature treatments on plasma E2 level in the female catfish H. fossilis. Data were expressed as mean ± SEM and analyzed by two-way ANOVA (P \ 0.001), followed by Newman–Keuls test (P \ 0.05). The
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The treatments produced an overall significant effect on plasma testosterone (Fig. 6; two-way ANOVA, P \ 0.001; FTreatment = 93.14, FDuration = 69.13, and the interaction between the two, FT 9 D = 58.14). Plasma T level increased in the control group and increased further in the LP ? HT (Group 4) at both intervals (P \ 0.05). A significant increase was also noticed on day 28 in the LP ? NT (Group 3) and NP ? HT (Group 2) groups. The highest reduction was noticed in the TD ? NT (Group 7) group, followed by SP ? NT (Group 5), TD ? HT (Group 8)
groups with different letters are significantly different, and those with the same letters are not significant. Other abbreviations as in Fig. 1
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Fig. 6 Effects of photoperiod and temperature treatments on plasma testosterone level in the female catfish H. fossilis. Data were expressed as mean ± SEM and analyzed by two-way ANOVA (P \ 0.001), followed by Newman–Keuls test (P \ 0.05). The groups with different letters are significantly different, and those with the same letters are not significant. Other abbreviations as in Fig. 1
and SP ? HT (Group 6) groups when comparisons were made with the control group. The plasma T level showed significant interaction between the altered photoperiod and temperature treatments (two-way ANOVA, P \ 0.001; FP = 83.41, FT = 62.17, FP 9 T = 52.73 for 14 days and FP = 80.32, FT = 58.08, FP 9 T = 48.17 for 28 days). The T level was the highest in the LP ? HT group and the lowest in the TD ? NT group.
photoperiod and temperature treatments (two-way ANOVA, P \ 0.001; FP = 102.17, FT = 74.32, FP 9 T = 63.24 for 14 days and FP = 107.24, FT = 68.92, FP 9 T = 58.90 for 28 days). The P4 level was showed the highest increase in the LP ? HT group and the least increase in the TD ? NT and TD ? HT groups.
Plasma cortisol Plasma P4 The treatments produced an overall significant effect on plasma P4 (Fig. 7; two-way ANOVA, P \ 0.001; FTreatment = 104.28, FDuration = 81.48, and the interaction between the two, FT 9 D = 43.24). Plasma P4 increased significantly in the LP ? NT (Group 3) and LP ? HT (Group 4) groups, compared to the control group. The increase was maximal in the LP ? HT (Group 4) group. In the NP ? HT (Group 2) group, the increase was noticed on day 28. In the SP ? NT (Group 5), TD ? NT (Group 7), SP ? HT (Group 6) and TD ? HT (Group 8) groups, the P4 level decreased significantly from the control group. The P4 level registered the lowest value in the TD ? NT (Group 7) group at both intervals. The plasma P4 level showed significant interaction between the altered
Plasma cortisol level showed an overall significant effect following the treatments (Fig. 8; twoway ANOVA, P \ 0.001; FTreatment = 104.18, FDuration = 81.2, and the interaction between the two, FT 9 D = 79.28). The cortisol level increased in all groups compared to the control groups, and the increase was higher on 28. The highest increase was noticed in the LP ? HT (Group 4) and TD ? HT (Group 8) groups. The plasma cortisol level showed significant interaction between the altered photoperiod and temperature treatments (two-way ANOVA, P \ 0.001; FP = 108.26, FT = 83.12, FP 9 T = 76.24 for 14 days and FP = 110.35, FT = 86.42, FP 9 T = 63.08 for 28 days). The cortisol level was the highest in the TD ? HT group, followed by LP ? HT group, and lowest in the SP ? HT group.
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Fig. 7 Effects of photoperiod and temperature treatments on plasma progesterone level in the female catfish H. fossilis. Data were expressed as mean ± SEM and analyzed by two-way ANOVA (P \ 0.001), followed by Newman–Keuls test (P \ 0.05). The groups with different letters are significantly different, and those with the same letters are not significant. Other abbreviations as in Fig. 1
Fig. 8 Effects of photoperiod and temperature treatments on plasma cortisol level in the female catfish H. fossilis. Data were expressed as mean ± SEM and analyzed by two-way ANOVA (P \ 0.001), followed by Newman–Keuls test (P \ 0.05). The groups with different letters are significantly different, and those with the same letters are not significant. Other abbreviations as in Fig. 1
Discussion To our knowledge, this is the first report describing effects of altered photoperiod and temperature on VT
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secretion in the BPG axis. The preparatory phase (early March) heralds the initiation of gonadal recrudescence in the catfish when both photoperiod and temperature start increasing after the winter/
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spring transition, and this period is ideal for studying the effects of photothermal manipulations on gonadal activity. The catfish were exposed to a summer-like condition (long photoperiod (16L) with or without raising the temperature or a winter-like condition (short photoperiod (8L) or totally deprived of light (24 h darkness) with or without raising the temperature. The high temperature chosen falls within the range of temperatures experienced by the catfish during summer months (May–June), and 8-h light regime used is shorter by 2 h what the fish experiences in the nature. The exposure of the catfish to long photoperiod, with or without changing the ambient temperature, stimulated gonadal activity as evident from the increase in the GSI and plasma levels of ovarian steroid hormones compared to the control groups. The exposure of the catfish to short photoperiod and total darkness, with or without changing the ambient temperature, had a decelerating effect on the BPG axis. The GSI was significantly low in the 14-day TD ? NT group (Group 7), and 28-day SP ? NT (Group 5) and TD ? NT groups. In the 14-day TD ? HT group (Group 8), a marginal but significant increase was noticed compared to the control group. The steroid hormone (E2, T and P4) levels except cortisol (see below) were significantly low or not changed compared to the control group. These results are similar to that reported by earlier workers in the catfish (Sundararaj 1981; Joy and Senthilkumaran 1995; Senthilkumaran and Joy 1995). A highly significant stimulatory effect on the GSI was seen in the LP groups and NP ? HT groups compared to the control groups. This shows that light and temperature can independently stimulate gonadal activity, but the combination produced the maximal response, as in the nature. Given alone, temperature had a greater effect on the GSI than photoperiod, as has been also reported previously in this species (Sundararaj 1981). The SP and TD had a depressing effect on the GSI, regardless of the temperature conditions. Taken together, an optimal photoperiod is important for gonadal recrudescence to start upon which temperature exerts the stimulatory actions. E2 is the functionally active estrogen in teleosts and stimulates vitellogenesis and oocyte growth (Fostier et al. 1983; Wallace 1985; Tyler and Sumpter 1996). P4 and T are intermediates in the biosynthesis of E2. Hence, we investigated the dynamics of the steroids after exposure to various treatments. Plasma E2 level
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increased significantly in the LP groups at both intervals and on day 28 in the NP ? HT group (Group 2). There is no significant variation between the LP alone and LP ? HT groups (Group 3 and 4). It appears that, like the GSI, light and temperature may act independently on E2 production but the photoperiod has an edge over the temperature. Like the GSI, the exposure to SP and TD inhibited the steroid level below the control group. However, the high temperature could maintain the E2 level (14-day group) or elevate it (28-day group) compared to the control groups. The changes in the GSI and E2 level are parallel under the altered conditions. The increase in the E2 level and GSI could be due to increased secretion of pituitary gonadotropin, as reported by Joy and Senthilkumaran (1995) in LP ? NT-maintained catfish. Photoperiod and temperature act at the level of the hypothalamo-hypophyseal system to modulate gonadotropin secretion (Joy and Senthilkumaran 1995; Senthilkumaran and Joy 1995). The environmental factors modulate monoaminergic activity differentially. Testosterone is the immediate precursor of E2, and its level did not vary greatly on exposure to the altered photoperiod and temperature perhaps due its conversion. Even then, a general trend can be seen with its level increased under normal or LP conditions with or without changing the temperature. Under SP or TD conditions, the T level was significantly low under ambient conditions or was maintained (SP ? HT Group 6 and 14-day TD ? HT Group 8). The P4 level showed a pattern of changes similar to E2 and was significantly high in the LP ? NT (Group 3), LP ? HT (Group 4) and NP ? HT (Group 2—day 28) groups. In the SP and TD groups, the steroid level was low or maintained compared to the parallel control group. The brain–pituitary–adrenal axis is crucial for vertebrates to cope up with various forms of stress. In teleosts, cortisol is the main corticosteroid secreted by the interrenal tissue (homologue of adrenal cortex) and functions as both glucocorticoid and mineralocorticoid (Norris and Carr 1997). The teleost ovary is also a site of corticosteroid synthesis (Mishra and Joy 2006), and corticoids like cortisol and deoxycorticosterone are implicated in final oocyte maturation (Sundararaj 1981; Rao and Haider 1992). The apparent increase in the cortisol level in all the treatment groups may be primarily a stress response, increasing carbohydrate
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metabolism related to yolk synthesis or general energy demand. The exposure to SP, TD and high temperature conditions may be more stressful than the LP conditions, judging from the higher increase in cortisol level. Parallel with the changes in ovarian recrudescence, the altered photothermal treatments modulated VT secretion in the BPG axis. In general, the exposure to high temperature with or without altering the photoperiod stimulated a greater secretion of VT (release). However, plasma and ovarian VT levels recorded the highest levels in the LP ? HT Group 4. There may be other factors regulating the hormone dynamics in the circulation. It is not clear whether the increase in ovarian VT level was due to a direct action of photoperiod and temperature on the ovary. It is likely that the increased secretion of gonadotropins may increase the ovarian level of VT (Singh and Joy 2009b). Under the SP and TD conditions, brain VT levels did not change from the control group levels or increased, and plasma and ovarian VT level decreased with the lowest level in the TD ? NT Group 7. The changes can be attributed to an inhibition of the hormone release from the brain. However, the high temperature exposure lowered the inhibitory effect of the SP and TD conditions. It is inferred that both photoperiod and temperature act synergistically to modulate the VT secretion under a threshold photoperiod. The results explain the seasonal changes in VT secretion in the BPG axis during the reproductive cycle of the catfish, reported by us earlier (Singh and Joy 2008). With the rise in photoperiod and temperature, plasma and ovarian VT level increased to the peak in the spawning phase (July) and declined thereafter. The brain VT level increased to the peak early in the preparatory phase (March–April), decreased subsequently due to increased release into the circulation. Though VT is involved in multiple functions (see Introduction), the increased secretion of VT can be attributed to its physiological role in ovarian functions, as demonstrated in this species. Under in vitro conditions, VT stimulates steroidogenesis, stimulating E2 in a biphasic manner and P4, 17-hydroxyprogesterone and 17, 20b-dihydroxy-4-pregnen-3-one, 17, 20b-DP (Singh and Joy 2009a). Further, it stimulates final oocyte maturation, oocyte hydration, prostaglandin secretion and ovulation (Singh and Joy 2010, 2011; Joy and Singh 2013). A functional interaction between VT and ovarian steroids has been demonstrated, implying feedback control of each other’s secretory
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activity. E2 modulated follicular VT secretion biphasically, while progestin steroids (P4 and 17,20b-DP) stimulated VT secretion positively (Singh and Joy 2009b). Ovariectomy decreased brain VT levels duration-dependently, and E2 replacement reversed the ovariectomy effects dose-dependently (Singh and Joy 2009b; Chaube et al. 2012). Working further on the mechanism of E2 regulation of VT, it was shown that the E2 action was mediated through the catecholaminergic system, dopamine (DA) inhibits, and noradrenaline (NA), and adrenaline stimulates brain VT (Chaube et al. 2012; Singh et al. 2013). Senthilkumaran and Joy (1995) have shown that photoperiod and temperature act differentially on hypothalamic catecholamines. Exposure of the catfish to LP and HT inhibited DA activity but stimulated NA and adrenaline activities. Exposure to SP and TD conditions had opposite effects on the catecholamines. Taken together, photoperiod and temperature act on the hypothalamic catecholaminergic system to regulate VT differentially. Estrogen-negative feedback acts on the catecholaminergic system to fine-tune the regulation of VT secretion. Since photoperiod and temperature-induced changes on the catecholaminergic system also regulate gonadotropin secretion and E2 exerts negative feedback effect on it through the catecholaminergic system (Senthilkumaran and Joy 1995), it is very likely that the external and internal signals converge on a common mechanism to regulate both VT and gonadotropin secretion. Such a regulatory coupling can bring functional coordination of the two systems on gonad functions over time. In conclusion, the present study demonstrated photoperiod and temperature modulation of VT secretion in the BPG axis. The simultaneous activation of VT secretion and ovarian recrudescence by long photoperiod and high temperature points to the peptide’s participation in the hormonal control of gametogenesis. Acknowledgments R.K.S. is a recipient of Senior Research Fellowship (CAS-Merged R-A/C Grant) of Department of Zoology, Banaras Hindu University. This work was partially supported by a UGC Research grant (SR/34-436/2008) to R.C, New Delhi.
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