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Ultimobranchial gland respond in a different way in male and female fresh water teleost Mastacembelus armatus (Lacepede) during reproductive cycle
Animal Reproduction Science 156 (2015) 111–117
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Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
Ultimobranchial gland respond in a different way in male and female fresh water teleost Mastacembelus armatus (Lacepede) during reproductive cycle Sushant Kumar Verma ∗ , Abdul Alim Co-operative College (Ranchi University), Jharkhand, India
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Article history: Received 12 December 2014 Received in revised form 27 February 2015 Accepted 11 March 2015 Available online 19 March 2015 Keywords: Mastacembelus armatus Ultimobranchial gland 17 -estradiol 17 ␣-methyltestosterone Testicular cycle Ovarian cycle
a b s t r a c t The present study was carried out to analyze the differences in the activity of ultimobranchial gland (UBG) between male and female fresh water teleost Mastacembelus armatus during reproductive cycle. Considerable variations in the nuclear diameter of UBG cells and plasma calcitonin (CT) levels during different reproductive phases of testicular and ovarian cycle suggested that the activity of the UBG depends upon the sexual maturity of fishes. A positive correlation was observed between plasma CT and sex steroid levels and the gonadosomatic index in both sexes which further confirmed the involvement of UBG in the processes related to gonadal development in fishes irrespective of the sex. Sudden increase in the level of plasma CT and nuclear diameter of UBG cells after administration of 17 ␣-methyltestosterone in males and 17 -estradiol in females during resting phase of the reproductive cycle clearly showed that UBG becomes hyperactive with increases in the level of sex steroids. Plasma calcium level was also found to be positively correlated with gonadal maturation in females. However no such change in plasma calcium level in relation to testicular cycle was observed. Thus it can be concluded that UBG becomes hyperactive during gonadal maturation but its role differs between male and female fishes. In females it may involved in both gonadal maturation and plasma calcium regulation while in males its involvement in calcium regulation was not justified. Variations in the level of CT during various phases of testicular cycle evidenced its involvement in gonadal maturation only. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The ultimobranchial gland (UBG) in teleosts is located in an area ventral to the oesophagus and is involved in the synthesis and storage of a polypeptide called calcitonin (CT). In mammals CT is considered as one of the important calcium regulating hormones produced mainly by the
∗ Corresponding author. Tel.: +91 7587300084. E-mail address: [email protected] (S.K. Verma). http://dx.doi.org/10.1016/j.anireprosci.2015.03.007 0378-4320/© 2015 Elsevier B.V. All rights reserved.
parafollicular ‘C’ cells of the thyroid gland but in fishes its role on calcium homeostasis has long been controversial. Although several authors have been able to show the hypocalcemic action of CT in fish (Srivastav et al., 1998; Suzuki et al., 1999; Mukherjee et al., 2004), conflicting results are often reported (Wendelaar-Bonga and Pang, 1991; Singh and Srivastav, 1993). The hypocalcemic effect of UBG extracts of various fishes in rats has been documented several times (Sasayama et al., 1993), but at the same time no such effect was observed on allogenic injection of UBG extract in various species of fishes (Srivastav, 1989). Hypocalcemic or hypercalcemic role of CT in fishes
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S.K. Verma, A. Alim / Animal Reproduction Science 156 (2015) 111–117
may depend upon the dose or species (Fouchereau-Peron et al., 1987). From the very beginning, the effect of CT on the regulation of reproduction in fishes has been proposed by many workers. UBG becomes hyperactive during gonadal maturation (Ahmad and Swarup, 1990) which may be shown by hypertrophy and hyperplasia of cells (Oguri, 1973) as well as increased calcitonin level, especially in females (Fouchereau-Peron et al., 1990). Administration of 17 estradiol induces UBG to release CT (Suzuki et al., 2004). Salmon calcitonin was found to stimulate the secretion of 17 -estradiol in vitellogenic ovarian follicles of carp Cyprinus carpio (Paul et al., 2008). Plasma calcium rises during gonadal maturation in fishes (Guerreiro et al., 2002) which is more pronounced in females (Srivastava and Srivastava, 1994, 1998). This increase in plasma calcium during ovarian maturation may be due to increased secretion of oestrogen from the ovary (Guerreiro et al., 2001; Gillespie and de Peyster, 2004) as administration of estradiol was found to induce hypercalcemia (Guerreiro et al., 2002; Gillespie and de Peyster, 2004). Estradiol increases the level of plasma calcium by acting directly on gills (Filby and Tyler, 2005) and intestine (Wang et al., 2005) or indirectly via some endocrine factor like PTHrP or a related factor responsive to estradiol (Fuentes et al., 2007). The role of calcitonin in female Mastacembelus armatus has been established as hypocalcemic factor by us in our earlier work (Verma and Alim, 2013), but till date no report exists regarding its role in male fishes or gender specific evaluation of its activity during gonadal maturation in this species which is one among the economically important species in rural parts of India (Verma and Murmu, 2010). Therefore the present study was undertaken with the hypothesis that a positive correlation between plasma calcium and CT level during gonadal maturation will indicate the role of UBG in calcium homeostasis while variation in plasma CT level during different phases of gonadal cycle without any significant change in the level of calcium will indicate towards its possible role in processes related to gonadal maturation. 2. Materials and methods 2.1. Experiment 1 (reproductive cycle experiment) The experimental design for this work was similar to that of our previous work on this species which was related to gender specific differential activity of stanniocalcin (Verma and Alim, 2014). Ten healthy and adult specimens of fish M. armatus consisting of five males and five females were collected every month throughout the year from local ponds with the help of fishermen. After collection fishes were brought to the laboratory for acclimatization to laboratory conditions for 15 days in plastic pool tanks having size of 90 cm diameter and 60 cm height. During this period fish were fed with live earthworms and boiled eggs obtained from local fish feed suppliers. Water was replaced every 24 h to maintain suitable environment for fishes with sufficient oxygen. The tank water was maintained with following
composition: temperature = 28 ± 1.0 ◦ C, salinity = 0.6 ± 0.02 ppt, total hardness = 28 ± 0.05 mg/l, pH = 6.5 ± 0.2 units, dissolved oxygen = 6.6 ± 0.01 mg/l. The photoperiod maintained through the entire experiment was 12:12 h. The average (±SD) body weight and length of male (n = 60) and female (n = 60) fishes were 300 ± 12.50 g, 26 ± 1.86 cm and 350 ± 12.86 g, 31 ± 1.95 cm respectively. After anesthetized with phenoxyethanol the tail was severed and the blood samples were collected from the caudal vessels using a heparinized syringe for estimation of plasma calcium, 17 -estradiol, testosterone and CT level. The fishes were sacrificed, gonads were taken out after which they were weighed (gm) for the determination of gonadosomatic index and fixed in Bouin’s solution for 12–16 h (depending upon the size of the tissue) heart and oesophagus along with UBG were also taken out and fixed in Bouin’s solution for 24 h. 2.2. Histology Paraffin blocks of fixed tissues were prepared and sections of 5–7 m were made using a microtome. The sections were stained in haematoxylin and counterstained in eosin. Histological changes in gonads were observed for classification of gonadal cycle in to different phases. Nuclear diameter of UBG cells (m) was measured by image analyzer microscope (Metavis image analyzing system with Meltmage Lx Software) for which 50 nuclei were randomly selected from every fifth section of the gland. 2.3. Plasma calcium, calcitonin, testosterone and 17ˇ-estradiol estimation After centrifugation in cooling centrifuge (maintained at 4 ◦ C, 4000 rpm for 5 min) plasma was analyzed for total plasma calcium according to the methods of Trinder (1960). A competitive ELISA technique based on competition between free CT in standard or plasma samples and CT immobilized on microtiter plates for the CT antibodies was used for determination of plasma CT level (Sasayama et al., 1996). Plasma testosterone and 17 -estradiol levels were determined by radioimmuno assay method (Istria et al., 1974; Polzonetti et al., 1983) following Guerriero et al. (1988). The sensitivity of testosterone was 7 pg/ml (intraassay, 6%; interassay, 12%), and that of 17 -estradiol was 5 pg/ml (intraassay, 8%; interassay, 12%). The antibody used for testosterone determinations cross-reacted with dihydrotestosterone, and therefore the data are reported as androgens (Guerriero et al., 2005). 2.4. Experiment 2 (steroid injection experiment) Twenty-four live (12 male and 12 female) adult and healthy specimens of M. armatus were collected from local fishermen, during the month of December which is the resting phase of reproduction. They were maintained in laboratory with the conditions as described above in the experimental design segment. The average (±SD) body weight and length of male and female fishes were 286 ± 11.20 g, 23 ± 1.31 cm and 315 ± 11.91 g, 30 ± 2.02 cm respectively. After 15 days the fishes were divided in four
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groups; two groups consisting of 6 female each and other two groups with 6 males each and kept in four separate aquaria of 100 L capacity. Out of the two groups of females one group was injected with 0.1 ml of vehicle (peanut oil) and the other group was administrated with 100 g of 17 -estradiol (sigma) in 0.1 ml of vehicle. One group of males was injected with 0.1 ml of vehicle (peanut oil) while other group of males was injected with 100 g of 17 ␣methyltestosterone (sigma) in 0.1 ml of vehicle. The fishes were injected intraperitonially on alternate days and injections were given at the same time of the day to avoid diurnal variations. The blood samples were analyzed for plasma calcium, 17 -estradiol, testosterone and CT level estimation after 15 days with the methods already described above. 2.5. Statistical analysis Distribution parameters are presented as means and Standard Deviation (SD). To test for differences in the mean values, analysis of variance (ANOVA) was carried out. Differences between means were evaluated by post hoc test. Assignment of data correlation was done by Pearson tests, and the relationships between total plasma calcium and CT were evaluated by linear regression. The accepted statistical significance level was P < 0.05.
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between plasma calcium and CT level (R = 0.9112, y = 27.17x − 27.11) and between plasma calcium and 17 -estradiol (R = 0.8991, y = 4.87 + 2.12x) in female M. armatus during different phases of reproductive cycle (Fig. 3). 17 -estradiol ranged from 1.76 to 5.06 ng/ml and CT ranged from 167 to 368 ng/ml. In both sexes the gonadosomatic index increases from preparatory phase to pre-spawning phase after which it further decreases after spawning in the post spawning phase. 3.3. Nuclear diameter of UBG cells In both male and female fishes nuclear diameter of UBG cells showed a large variation during various reproductive phases (Fig. 4). Maximum nuclear diameter of UBG cells was observed during pre-spawning and spawning phase in both sexes. 3.4. Effects of synthetic steroid administration
3. Results
Very little increase in the plasma calcium level was observed in male fishes after 17 ␣-methyltestosterone administration (Fig. 5). But plasma CT and nuclear diameter of UBG cells was significantly increased. In females administration of 17 -estradiol resulted in an increase in plasma calcium level, nuclear diameter of UBG cells and plasma CT levels (Fig. 6).
3.1. Gonadal cycle
4. Discussion
Five different phases of reproductive cycle were identified in both male and female fishes of M. armatus with the same morphological and histological features as described by us in our previous work on the same species (Verma and Alim, 2014). These stages are – Phase 1 or Resting phase (December–February), Phase II or Preparatory Phase (March–May), Phase III or Pre spawning phase (June–early July), Phase IV or Spawning phase (Late July–September) and Phase V or Post spawning phase (October–November).
Calcitonin (CT) is secreted by ultimobranchial gland in fishes whose definite functions have not been established yet (Miller, 2006; Nag et al., 2007). But many workers claimed its role towards calcium homeostasis (WendelaarBonga and Pang, 1991; Suzuki et al., 2000) especially during reproductive period (Suzuki et al., 2004). It has been proposed that the hypocalcemic role of CT in fishes is more pronounced in female. Although the hypocalcemic role of CT in female M. armatus has been established by us in our previous work (Verma and Alim, 2014), but no such report exists in this regard evaluating the gender specific differential activity of CT during gonadal maturation in this species. Therefore the present study was carried to confer its role in both sexes of M. armatus during gonadal cycle. On the basis of gonadosomatic index and various histological parameters the annual sex cycle of M. armatus has been divided mainly into five phases (Verma and Alim, 2014). The seasonal changes in the activity of UBG, as indicated by increase in the nuclear diameter of UBG cells and plasma CT level was found to increases in parallel with the advancement of the both testicular and ovarian cycle. These concomitant changes occurring in the nuclear diameter of CT cells and CT level with that of testis and ovary growth suggest a role of CT in gonadal maturation in both sexes. Measured level of testosterone in males and 17--estradiol in females during various reproductive phases showed similar pattern in changes as that of GSI. High level of testosterone from August to September indicates milt production in males. On the other hand high values of 17--estradiol during July to September indicate that females would likely
3.2. Plasma calcium, sex steroid and CT level Little variation in plasma calcium level during different phases of testicular cycle was detected. On the other hand a considerable variation in plasma calcium level along with various phases of ovarian cycle was observed (Fig. 1). In females increase in the plasma calcium level was observed during preparatory phase reaching the peak during pre spawning and spawning phase. Afterwards gradually decreases with spawning and reduced to minimum at resting phase. Plasma testosterone and calcitonin level varies significantly during various reproductive phases in male fishes (Fig. 2). Testosterone ranged from 0.61 to 1.87 ng/ml and CT ranged from 153 to 350 ng/ml. There exists very weak correlation between plasma CT and calcium level (R = 0.2298, y = 592.78x − 1891.22), however plasma testosterone and calcium level showed somewhat moderate positive correlation (R = 0.5498, y = 3.24 + 0.03x) during various phases of testicular cycle. In females a strong correlation was established
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Fig. 1. Variations in the level of Plasma calcium in M. armatus during various phases of gonadal cycle. Each value is mean ± SD (n = 5). Means with different letters are significantly different (P ≤ 0.05).
to spawn during these months as increased level of 17-estradiol initiates vitellogenesis (Lee and Yang, 2002). Variation in plasma calcium level with various reproductive phases of ovarian cycle was observed in M. armatus, similar to the results obtained by some workers in other fishes (Srivastava and Srivastava, 1998; Bjornsson and Haux, 1985). Level of 17- estradiol also changes with the ovarian cycle and was found to be maximum during pre-spawning and spawning period, as reported earlier (Woodhead, 1968). Increased level of 17- estradiol during these phases of ovarian cycle initiates vitellogenesis as a result of which protein-bound fraction of plasma calcium level rises (Swarup et al., 1986; Balbontin et al., 1978).
Thus maximum plasma calcium level was observed during pre spawning phase and spawning phase due to increased oestrogen secretion by ovary (Urasa and Wendelaar Bonga, 1985). Similar increase in the level of plasma calcium was also observed after 17--estradiol administration in female fishes. Plasma CT varies in the same manner as that of calcium during ovarian cycle. Thus enhanced activity of UBG as indicated by plasma level of CT and increased nuclear diameter of UBG cells during pre-spawning and spawning phase may be due to an increase in serum calcium level which in turn is the effect of increased secretion of estradiol. This was also evidenced by administration of 17--estradiol
Fig. 2. Variations in plasma CT and testosterone level along with gonadosomatic index in male M. armatus during different phases of testicular cycle. Each value is mean ± SD (n = 5).
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Fig. 3. Variations in plasma CT and 17 -estradiol level along with gonadosomatic index in female M. armatus during different phases of ovarian cycle. Each value is mean ± SD (n = 5).
Fig. 4. Variations in nuclear diameter of UBG cells during different phases of gonadal cycle in male and female M. armatus. Each value is mean ± SD (n = 5). Means with different letters are significantly different (P ≤ 0.05).
Fig. 5. Effect of 17 ␣-methyltestosterone administration on plasma calcium, plasma calcitonin and nuclear diameter of UBG cells of male M. armatus. Each value is mean ± SD (n = 5). Means with different letters/number are significantly different (P ≤ 0.05).
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Fig. 6. Effect of 17 -estradiol administration on plasma calcium, calcitonin and nuclear diameter of UBG cells of female M. armatus. Each value is mean ± SD (n = 5). Means with different letters/number are significantly different (P ≤ 0.05).
which induced hypercalcemia which in turn stimulated UBG to produce its anti hypercalcemic hormone CT. On the other hand obtained data indicated that in M. armatus there exist no correlation between plasma calcium level and testicular maturation, which is similar to the result obtained by other workers in different fishes (Singh and Srivastav, 1990; Bedjargi et al., 2014). Plasma calcium was found to remain constant throughout the testicular cycle. Also no significant increase in plasma calcium level was observed after 17 ␣-Methyltestosterone administration in males during resting phase of testicular cycle. However a significant increase in the level of CT was observed. This indicates the involvement of CT in some other process of gonadal development rather than calcium homeostasis in males. Our present study clearly indicated that UBG act differentially in male and female fishes of M. armatus. In females it is involved in calcium regulation during ovarian cycle but in males it may involve in some activity related to testicular maturation in spite of calcium homeostasis. Conflict of interest None. Acknowledgement The authors would like to thank the staff at the Co-operative college (Ranchi University) Jamshedpur for allowing the use of all facilities during this study. References Ahmad, N., Swarup, K., 1990. Seasonal changes in structure and changes in serum calcium level and the reproductive cycle of a freshwater female catfish, Mystus vittatus (Bloch). Euro. Arch. Biol. (Bruxelles) 101, 285–294.
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Contents lists available at ScienceDirect
Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
Ultimobranchial gland respond in a different way in male and female fresh water teleost Mastacembelus armatus (Lacepede) during reproductive cycle Sushant Kumar Verma ∗ , Abdul Alim Co-operative College (Ranchi University), Jharkhand, India
a r t i c l e
i n f o
Article history: Received 12 December 2014 Received in revised form 27 February 2015 Accepted 11 March 2015 Available online 19 March 2015 Keywords: Mastacembelus armatus Ultimobranchial gland 17 -estradiol 17 ␣-methyltestosterone Testicular cycle Ovarian cycle
a b s t r a c t The present study was carried out to analyze the differences in the activity of ultimobranchial gland (UBG) between male and female fresh water teleost Mastacembelus armatus during reproductive cycle. Considerable variations in the nuclear diameter of UBG cells and plasma calcitonin (CT) levels during different reproductive phases of testicular and ovarian cycle suggested that the activity of the UBG depends upon the sexual maturity of fishes. A positive correlation was observed between plasma CT and sex steroid levels and the gonadosomatic index in both sexes which further confirmed the involvement of UBG in the processes related to gonadal development in fishes irrespective of the sex. Sudden increase in the level of plasma CT and nuclear diameter of UBG cells after administration of 17 ␣-methyltestosterone in males and 17 -estradiol in females during resting phase of the reproductive cycle clearly showed that UBG becomes hyperactive with increases in the level of sex steroids. Plasma calcium level was also found to be positively correlated with gonadal maturation in females. However no such change in plasma calcium level in relation to testicular cycle was observed. Thus it can be concluded that UBG becomes hyperactive during gonadal maturation but its role differs between male and female fishes. In females it may involved in both gonadal maturation and plasma calcium regulation while in males its involvement in calcium regulation was not justified. Variations in the level of CT during various phases of testicular cycle evidenced its involvement in gonadal maturation only. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The ultimobranchial gland (UBG) in teleosts is located in an area ventral to the oesophagus and is involved in the synthesis and storage of a polypeptide called calcitonin (CT). In mammals CT is considered as one of the important calcium regulating hormones produced mainly by the
∗ Corresponding author. Tel.: +91 7587300084. E-mail address: [email protected] (S.K. Verma). http://dx.doi.org/10.1016/j.anireprosci.2015.03.007 0378-4320/© 2015 Elsevier B.V. All rights reserved.
parafollicular ‘C’ cells of the thyroid gland but in fishes its role on calcium homeostasis has long been controversial. Although several authors have been able to show the hypocalcemic action of CT in fish (Srivastav et al., 1998; Suzuki et al., 1999; Mukherjee et al., 2004), conflicting results are often reported (Wendelaar-Bonga and Pang, 1991; Singh and Srivastav, 1993). The hypocalcemic effect of UBG extracts of various fishes in rats has been documented several times (Sasayama et al., 1993), but at the same time no such effect was observed on allogenic injection of UBG extract in various species of fishes (Srivastav, 1989). Hypocalcemic or hypercalcemic role of CT in fishes
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may depend upon the dose or species (Fouchereau-Peron et al., 1987). From the very beginning, the effect of CT on the regulation of reproduction in fishes has been proposed by many workers. UBG becomes hyperactive during gonadal maturation (Ahmad and Swarup, 1990) which may be shown by hypertrophy and hyperplasia of cells (Oguri, 1973) as well as increased calcitonin level, especially in females (Fouchereau-Peron et al., 1990). Administration of 17 estradiol induces UBG to release CT (Suzuki et al., 2004). Salmon calcitonin was found to stimulate the secretion of 17 -estradiol in vitellogenic ovarian follicles of carp Cyprinus carpio (Paul et al., 2008). Plasma calcium rises during gonadal maturation in fishes (Guerreiro et al., 2002) which is more pronounced in females (Srivastava and Srivastava, 1994, 1998). This increase in plasma calcium during ovarian maturation may be due to increased secretion of oestrogen from the ovary (Guerreiro et al., 2001; Gillespie and de Peyster, 2004) as administration of estradiol was found to induce hypercalcemia (Guerreiro et al., 2002; Gillespie and de Peyster, 2004). Estradiol increases the level of plasma calcium by acting directly on gills (Filby and Tyler, 2005) and intestine (Wang et al., 2005) or indirectly via some endocrine factor like PTHrP or a related factor responsive to estradiol (Fuentes et al., 2007). The role of calcitonin in female Mastacembelus armatus has been established as hypocalcemic factor by us in our earlier work (Verma and Alim, 2013), but till date no report exists regarding its role in male fishes or gender specific evaluation of its activity during gonadal maturation in this species which is one among the economically important species in rural parts of India (Verma and Murmu, 2010). Therefore the present study was undertaken with the hypothesis that a positive correlation between plasma calcium and CT level during gonadal maturation will indicate the role of UBG in calcium homeostasis while variation in plasma CT level during different phases of gonadal cycle without any significant change in the level of calcium will indicate towards its possible role in processes related to gonadal maturation. 2. Materials and methods 2.1. Experiment 1 (reproductive cycle experiment) The experimental design for this work was similar to that of our previous work on this species which was related to gender specific differential activity of stanniocalcin (Verma and Alim, 2014). Ten healthy and adult specimens of fish M. armatus consisting of five males and five females were collected every month throughout the year from local ponds with the help of fishermen. After collection fishes were brought to the laboratory for acclimatization to laboratory conditions for 15 days in plastic pool tanks having size of 90 cm diameter and 60 cm height. During this period fish were fed with live earthworms and boiled eggs obtained from local fish feed suppliers. Water was replaced every 24 h to maintain suitable environment for fishes with sufficient oxygen. The tank water was maintained with following
composition: temperature = 28 ± 1.0 ◦ C, salinity = 0.6 ± 0.02 ppt, total hardness = 28 ± 0.05 mg/l, pH = 6.5 ± 0.2 units, dissolved oxygen = 6.6 ± 0.01 mg/l. The photoperiod maintained through the entire experiment was 12:12 h. The average (±SD) body weight and length of male (n = 60) and female (n = 60) fishes were 300 ± 12.50 g, 26 ± 1.86 cm and 350 ± 12.86 g, 31 ± 1.95 cm respectively. After anesthetized with phenoxyethanol the tail was severed and the blood samples were collected from the caudal vessels using a heparinized syringe for estimation of plasma calcium, 17 -estradiol, testosterone and CT level. The fishes were sacrificed, gonads were taken out after which they were weighed (gm) for the determination of gonadosomatic index and fixed in Bouin’s solution for 12–16 h (depending upon the size of the tissue) heart and oesophagus along with UBG were also taken out and fixed in Bouin’s solution for 24 h. 2.2. Histology Paraffin blocks of fixed tissues were prepared and sections of 5–7 m were made using a microtome. The sections were stained in haematoxylin and counterstained in eosin. Histological changes in gonads were observed for classification of gonadal cycle in to different phases. Nuclear diameter of UBG cells (m) was measured by image analyzer microscope (Metavis image analyzing system with Meltmage Lx Software) for which 50 nuclei were randomly selected from every fifth section of the gland. 2.3. Plasma calcium, calcitonin, testosterone and 17ˇ-estradiol estimation After centrifugation in cooling centrifuge (maintained at 4 ◦ C, 4000 rpm for 5 min) plasma was analyzed for total plasma calcium according to the methods of Trinder (1960). A competitive ELISA technique based on competition between free CT in standard or plasma samples and CT immobilized on microtiter plates for the CT antibodies was used for determination of plasma CT level (Sasayama et al., 1996). Plasma testosterone and 17 -estradiol levels were determined by radioimmuno assay method (Istria et al., 1974; Polzonetti et al., 1983) following Guerriero et al. (1988). The sensitivity of testosterone was 7 pg/ml (intraassay, 6%; interassay, 12%), and that of 17 -estradiol was 5 pg/ml (intraassay, 8%; interassay, 12%). The antibody used for testosterone determinations cross-reacted with dihydrotestosterone, and therefore the data are reported as androgens (Guerriero et al., 2005). 2.4. Experiment 2 (steroid injection experiment) Twenty-four live (12 male and 12 female) adult and healthy specimens of M. armatus were collected from local fishermen, during the month of December which is the resting phase of reproduction. They were maintained in laboratory with the conditions as described above in the experimental design segment. The average (±SD) body weight and length of male and female fishes were 286 ± 11.20 g, 23 ± 1.31 cm and 315 ± 11.91 g, 30 ± 2.02 cm respectively. After 15 days the fishes were divided in four
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groups; two groups consisting of 6 female each and other two groups with 6 males each and kept in four separate aquaria of 100 L capacity. Out of the two groups of females one group was injected with 0.1 ml of vehicle (peanut oil) and the other group was administrated with 100 g of 17 -estradiol (sigma) in 0.1 ml of vehicle. One group of males was injected with 0.1 ml of vehicle (peanut oil) while other group of males was injected with 100 g of 17 ␣methyltestosterone (sigma) in 0.1 ml of vehicle. The fishes were injected intraperitonially on alternate days and injections were given at the same time of the day to avoid diurnal variations. The blood samples were analyzed for plasma calcium, 17 -estradiol, testosterone and CT level estimation after 15 days with the methods already described above. 2.5. Statistical analysis Distribution parameters are presented as means and Standard Deviation (SD). To test for differences in the mean values, analysis of variance (ANOVA) was carried out. Differences between means were evaluated by post hoc test. Assignment of data correlation was done by Pearson tests, and the relationships between total plasma calcium and CT were evaluated by linear regression. The accepted statistical significance level was P < 0.05.
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between plasma calcium and CT level (R = 0.9112, y = 27.17x − 27.11) and between plasma calcium and 17 -estradiol (R = 0.8991, y = 4.87 + 2.12x) in female M. armatus during different phases of reproductive cycle (Fig. 3). 17 -estradiol ranged from 1.76 to 5.06 ng/ml and CT ranged from 167 to 368 ng/ml. In both sexes the gonadosomatic index increases from preparatory phase to pre-spawning phase after which it further decreases after spawning in the post spawning phase. 3.3. Nuclear diameter of UBG cells In both male and female fishes nuclear diameter of UBG cells showed a large variation during various reproductive phases (Fig. 4). Maximum nuclear diameter of UBG cells was observed during pre-spawning and spawning phase in both sexes. 3.4. Effects of synthetic steroid administration
3. Results
Very little increase in the plasma calcium level was observed in male fishes after 17 ␣-methyltestosterone administration (Fig. 5). But plasma CT and nuclear diameter of UBG cells was significantly increased. In females administration of 17 -estradiol resulted in an increase in plasma calcium level, nuclear diameter of UBG cells and plasma CT levels (Fig. 6).
3.1. Gonadal cycle
4. Discussion
Five different phases of reproductive cycle were identified in both male and female fishes of M. armatus with the same morphological and histological features as described by us in our previous work on the same species (Verma and Alim, 2014). These stages are – Phase 1 or Resting phase (December–February), Phase II or Preparatory Phase (March–May), Phase III or Pre spawning phase (June–early July), Phase IV or Spawning phase (Late July–September) and Phase V or Post spawning phase (October–November).
Calcitonin (CT) is secreted by ultimobranchial gland in fishes whose definite functions have not been established yet (Miller, 2006; Nag et al., 2007). But many workers claimed its role towards calcium homeostasis (WendelaarBonga and Pang, 1991; Suzuki et al., 2000) especially during reproductive period (Suzuki et al., 2004). It has been proposed that the hypocalcemic role of CT in fishes is more pronounced in female. Although the hypocalcemic role of CT in female M. armatus has been established by us in our previous work (Verma and Alim, 2014), but no such report exists in this regard evaluating the gender specific differential activity of CT during gonadal maturation in this species. Therefore the present study was carried to confer its role in both sexes of M. armatus during gonadal cycle. On the basis of gonadosomatic index and various histological parameters the annual sex cycle of M. armatus has been divided mainly into five phases (Verma and Alim, 2014). The seasonal changes in the activity of UBG, as indicated by increase in the nuclear diameter of UBG cells and plasma CT level was found to increases in parallel with the advancement of the both testicular and ovarian cycle. These concomitant changes occurring in the nuclear diameter of CT cells and CT level with that of testis and ovary growth suggest a role of CT in gonadal maturation in both sexes. Measured level of testosterone in males and 17--estradiol in females during various reproductive phases showed similar pattern in changes as that of GSI. High level of testosterone from August to September indicates milt production in males. On the other hand high values of 17--estradiol during July to September indicate that females would likely
3.2. Plasma calcium, sex steroid and CT level Little variation in plasma calcium level during different phases of testicular cycle was detected. On the other hand a considerable variation in plasma calcium level along with various phases of ovarian cycle was observed (Fig. 1). In females increase in the plasma calcium level was observed during preparatory phase reaching the peak during pre spawning and spawning phase. Afterwards gradually decreases with spawning and reduced to minimum at resting phase. Plasma testosterone and calcitonin level varies significantly during various reproductive phases in male fishes (Fig. 2). Testosterone ranged from 0.61 to 1.87 ng/ml and CT ranged from 153 to 350 ng/ml. There exists very weak correlation between plasma CT and calcium level (R = 0.2298, y = 592.78x − 1891.22), however plasma testosterone and calcium level showed somewhat moderate positive correlation (R = 0.5498, y = 3.24 + 0.03x) during various phases of testicular cycle. In females a strong correlation was established
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Fig. 1. Variations in the level of Plasma calcium in M. armatus during various phases of gonadal cycle. Each value is mean ± SD (n = 5). Means with different letters are significantly different (P ≤ 0.05).
to spawn during these months as increased level of 17-estradiol initiates vitellogenesis (Lee and Yang, 2002). Variation in plasma calcium level with various reproductive phases of ovarian cycle was observed in M. armatus, similar to the results obtained by some workers in other fishes (Srivastava and Srivastava, 1998; Bjornsson and Haux, 1985). Level of 17- estradiol also changes with the ovarian cycle and was found to be maximum during pre-spawning and spawning period, as reported earlier (Woodhead, 1968). Increased level of 17- estradiol during these phases of ovarian cycle initiates vitellogenesis as a result of which protein-bound fraction of plasma calcium level rises (Swarup et al., 1986; Balbontin et al., 1978).
Thus maximum plasma calcium level was observed during pre spawning phase and spawning phase due to increased oestrogen secretion by ovary (Urasa and Wendelaar Bonga, 1985). Similar increase in the level of plasma calcium was also observed after 17--estradiol administration in female fishes. Plasma CT varies in the same manner as that of calcium during ovarian cycle. Thus enhanced activity of UBG as indicated by plasma level of CT and increased nuclear diameter of UBG cells during pre-spawning and spawning phase may be due to an increase in serum calcium level which in turn is the effect of increased secretion of estradiol. This was also evidenced by administration of 17--estradiol
Fig. 2. Variations in plasma CT and testosterone level along with gonadosomatic index in male M. armatus during different phases of testicular cycle. Each value is mean ± SD (n = 5).
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Fig. 3. Variations in plasma CT and 17 -estradiol level along with gonadosomatic index in female M. armatus during different phases of ovarian cycle. Each value is mean ± SD (n = 5).
Fig. 4. Variations in nuclear diameter of UBG cells during different phases of gonadal cycle in male and female M. armatus. Each value is mean ± SD (n = 5). Means with different letters are significantly different (P ≤ 0.05).
Fig. 5. Effect of 17 ␣-methyltestosterone administration on plasma calcium, plasma calcitonin and nuclear diameter of UBG cells of male M. armatus. Each value is mean ± SD (n = 5). Means with different letters/number are significantly different (P ≤ 0.05).
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Fig. 6. Effect of 17 -estradiol administration on plasma calcium, calcitonin and nuclear diameter of UBG cells of female M. armatus. Each value is mean ± SD (n = 5). Means with different letters/number are significantly different (P ≤ 0.05).
which induced hypercalcemia which in turn stimulated UBG to produce its anti hypercalcemic hormone CT. On the other hand obtained data indicated that in M. armatus there exist no correlation between plasma calcium level and testicular maturation, which is similar to the result obtained by other workers in different fishes (Singh and Srivastav, 1990; Bedjargi et al., 2014). Plasma calcium was found to remain constant throughout the testicular cycle. Also no significant increase in plasma calcium level was observed after 17 ␣-Methyltestosterone administration in males during resting phase of testicular cycle. However a significant increase in the level of CT was observed. This indicates the involvement of CT in some other process of gonadal development rather than calcium homeostasis in males. Our present study clearly indicated that UBG act differentially in male and female fishes of M. armatus. In females it is involved in calcium regulation during ovarian cycle but in males it may involve in some activity related to testicular maturation in spite of calcium homeostasis. Conflict of interest None. Acknowledgement The authors would like to thank the staff at the Co-operative college (Ranchi University) Jamshedpur for allowing the use of all facilities during this study. References Ahmad, N., Swarup, K., 1990. Seasonal changes in structure and changes in serum calcium level and the reproductive cycle of a freshwater female catfish, Mystus vittatus (Bloch). Euro. Arch. Biol. (Bruxelles) 101, 285–294.
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