Abstract
Pituitary adenylate cyclase-activating polypeptide (PACAP), a pleiotropic neuropeptide, has diverse functions in mammals. However, studies of the expression and function of PACAP and its receptor in fish, particularly in the reproductive system, are still limited. In this report, semi-quantitative RT-PCR and immunohistochemical staining were performed to identify expression domains of commercially important tilapia (Oreochromis mossambicus). PACAP (tpacap38) and its type I receptor (tpac1-r). Transcripts were detected in the brain, gallbladder, gill, heart, intestine, kidney, muscles, pancreas, spleen, stomach, testes, and ovaries, but not in the liver. Expression of tpacap38 and tpac1-r mRNA in brain tissue was significantly higher in both sexes compared with other tissues. Addition of exogenous ovine PACAP38 (0.25–5 nM), cAMP analog (dibutyryl-cAMP, 0.25–1.5 mM) or forskolin (adenylate cyclase activator, 1–10 μM) significantly upregulated tpacap38 in the gonads via a dose- and time-dependent fashion. This effect reached a maximal level at 2 h after induction, and then decreased with prolonged culture for up to 4 or 8 h. Additionally, the expression levels of tpac1-r were not significantly affected by ovine PACAP38 or dibutyryl-cAMP in either sex. Forskolin had a slightly inductive effect and its function could be suppressed with the addition of protein kinase A (PKA) inhibitor, H89 (10 μM), indicating involvement of the cAMP-PKA signaling pathway in the regulation of tpacap38. Expression of tpacap38 and tpac1-r in the gonads of tilapia suggests that PACAP may mediate gonadotropin action via paracrine/autocrine mechanisms in this bony fish.
Introduction
Pituitary adenylate cyclase-activating polypeptide (PACAP) is a multifunctional neuropeptide that is widely dispersed in the CN and peripheral tissues including the intestine, lung, adrenal gland, testis, and ovary in mammals (Arimura et al. 1991, Köves et al. 1995, Rawlings & Hezareh 1996, Arimura 1998, Sherwood et al. 2000, Wang et al. 2003, Maruyama et al. 2006). PACAP plays a regulatory role in the endocrine system and in the stress response, in the nervous system as a neurotransmitter or vasodilator, in the immune system as a neuromodulator, in carbohydrate and protein metabolism, and as a neurotrophic, neuroprotective or proliferative factor in the brain (Arimura 1998, Maruyama et al. 2006, Wu et al. 2006 and references therein). This peptide has been characterized as part of the glucagons/vasoactive intestinal peptide (VIP)/secretin/GHRH superfamily, based on its high amino acid sequence homology with VIP. PACAP exists in two biologically active forms: one with 38 amino acids and one with 27, designated PACAP38 and PACAP27 respectively (Miyata et al. 1989, 1990). GHRH is encoded in the same gene with PACAP in protochordates, fish, amphibians, and birds (Lo et al. 2007 and references therein), whereas they are encoded by two separate genes in mammals (Sherwood et al. 2000, Wang et al. 2003). At some point before or during the emergence of mammals, a divergence resulted in separate genes encoding PACAP and GHRH (Fradinger & Sherwood 2000, Sherwood et al. 2000, Wong et al. 2000, Lee et al. 2007).
Three PACAP receptors (PAC1-R, VPAC1-R, and VPAC2-R) have been identified. All are G protein-coupled receptors with a classical structure of seven-transmembrane domains (Harmar et al. 1998). PACAP type I receptors (PAC1-R) have a high binding affinity for PACAP38 and PACAP27, but a much lower affinity toward VIP. PACAP type II receptors (VPAC1-R and VPAC2-R) have similar binding affinities for PACAP27, PACAP38, and VIP (Ishihara et al. 1992, Hashimoto et al. 1993, Lutz et al. 1993). Signal transduction of all the three receptors stimulates adenylate cyclase (AC) activity that results in increased levels of intracellular cAMP (Schomerus et al. 1994, Delporte et al. 1995, Pisegna & Wank 1996). It has been recently demonstrated that PACAP can induce somatolactin (SL) α and SLβ gene expression in the pituitary cells of grass carp (Ctenopharyngodon idella) by first binding to PAC-I receptors. This activates the AC/cAMP/protein kinase A (PKA) and phospholipase C (PLC)/inositol 1, 4, 5-trisphosphate (IP3)/protein kinase C pathways and involved in the Ca2+/calmodulin (CaM)/CaMK-II cascades (Jiang et al. 2008a, 2008b).
The presence of PACAP and its receptors in the testis and ovary suggest that they might play important roles in the vertebrate hypothalamo–pituitary–gonadal axis to regulate gonadal activities (Wang et al. 2003, Sherwood et al. 2007). In the rat ovary, the inductive effect of gonadotropins on the expression of PACAP and it receptors in granulosa cells in vivo and in vitro has been elucidated (Gräs et al. 1996, Ko et al. 1999, Lee et al. 1999, Ko & Park-Sarge 2000, Koh et al. 2000, Park et al. 2000). PACAP stimulates cAMP accumulation and steroidogenesis in a dose-dependent manner from cultured granulosa cells (Zhong & Kasson 1994, Heindel et al. 1996). In rat testes, neither PACAP nor PACAP receptors have been found to be expressed in rat Sertoli or Leydig cells (Shioda et al. 1994, Romanelli et al. 1997). However, it has been suggested that PACAP can stimulate cAMP accumulation and secretion of lactate, estradiol, and inhibin in Sertoli cells (Heindel et al. 1992), as well as induce cAMP accumulation and testosterone secretion in Leydig cells in a dose-dependent manner (Romanelli et al. 1997, Rossato et al. 1997, El-Gehani et al. 1998, Vaudry et al. 2000). Additionally, PACAP expression in the rat testis may be positively regulated, at least in part, by FSH released from the pituitary (Shioda et al. 1994, Romanelli et al. 1997, Sherwood et al. 2000).
It has also been shown that PACAP can activate protein synthesis in spermatocytes or inhibit synthesis in spermatids in vitro (West et al. 1995). In fish, transcript expression of pacap and its receptors has been detected in the brain, pituitary, spinal cord, gastrointestinal tract, ovary, testis, kidney, liver, heart, gill (Wong et al. 2000), and eye (Fradinger & Sherwood 2000). Concurrent with the wide range of tissue distribution in mammals, Pacap is considered to be pleiotropic (Arimura 1998, Wong et al. 2000, Small & Nonneman 2001). PACAP 1) effectively stimulates the secretion of GH (Parker et al. 1997, Montero et al. 1998, Wong et al. 1998, 2000, Wirachowsky et al. 2000, Rousseau et al. 2001) and gonadotropin (Chang et al. 2001); 2) it functions in the gut by affecting contractions of the intestine (Matsuda et al. 2000, Olsson & Holmgren 2000); 3) it can trigger catecholamine secretion from chromaffin cells (Montpetit & Perry 2000); 4) it controls the differentiation and proliferation of cells during zebrafish ontogeny (Mathieu et al. 2004); 5) it is essential for brain development in zebrafish (Sherwood & Wu 2005, Sherwood et al. 2007); and 6) it enhances the maturation rate of rat and zebrafish oocytes (Apa et al. 1997, Wang et al. 2003).
The above data suggest that PACAP is an important paracrine/autocrine regulator in the gonads of both mammals and fish (Zhou et al. 1997, Vaudry et al. 2000, Wang et al. 2003). Recently, it has been reported that the growth rates of catfish (Clarias gariepinus), tilapia (Oreochromis niloticus), and carp (Cyprinus carpio) were enhanced by the addition of both catfish PACAP-related peptide (PRP) and PACAP recombinant peptides (Lugo et al. 2008). However, recombinant grass carp peptide specifically inhibited T98G human glioblastoma cell proliferation (Wang et al. 2008). These results suggest that the growth-promoting effect of PACAP may be species-specific.
Several studies have provided strong evidence that PACAP and its receptors act as an intragonadal regulator in mammals, however, information regarding the roles of these peptides in fish gonads is lacking (Wang et al. 2003 and references herein). In this report, transcript expression and protein localization of tpacap38 and tpac1-r in the tissues of commercially important tilapia (O. mossambicus) were investigated by RT-PCR and immunohistochemical staining. The expression of tpacap38 and tpac1-r was measured in response to forskolin (an activator of AC), H89 (a specific inhibitor of cAMP-dependent PKA), and increasing intracellular cAMP levels.
Results
Alignment of the amino acid sequences of the tilapia PACAP38 (tpacap38) and its type I receptor (tpac1-r) classes of proteins
The predicted sequence of tilapia PACAP38 has 97.4% similarity and 89.5% identity with its mammalian orthologue (human, cattle, sheep, dog, rat, and mouse; Table 1). It has identities of 100% for stargazer, 94.7% for channel catfish and Thai catfish, 92.1% for zebrafish-2, goldfish-a, and sockeye salmon, 89.5% for goldfish-b, grass carp, and rainbow trout, 84.2% for zebrafish-1, and 81.5% for stingray.
Comparison of amino acid sequence of pituitary adenylate cyclase-activating polypeptide (PACAP38, tpacap38) gene from tilapia (Oreochromis mossambicus) with other animals.
| Species | Common name | Similarity (%) | Identity (%) | Accession number or reference |
|---|---|---|---|---|
| Oreochromis mossambicus | Mozambique tilapia | / | / | AY522580 |
| Uranoscopus japonicus | Stargazer | 100 | 100 | P81039 |
| Ictalurus punctatus | Channel catfish | 94.7 | 94.7 | AF321243 |
| Clarias macrocephalus | Thai catfish | 94.7 | 94.7 | CAA55684 |
| Clarias gariepinus | African catfish | 94.7 | 94.7 | Lugo et al. (2008) |
| Danio rerio | Zebrafish-1 | 89.5 | 84.2 | NP999880 |
| Danio rerio | Zebrafish-2 | 94.7 | 92.1 | AF329730 |
| Carassius auratus | Goldfish-a | 94.7 | 92.1 | Kwok et al. (2006) |
| Carassius auratus | Goldfish-b | 94.7 | 89.5 | Kwok et al. (2006) |
| Oncorhynchus nerka | Sockeye salmon | 94.7 | 92.1 | P41585 |
| Ctenopharyngodon idella | Grass carp | 94.7 | 89.5 | EF592488 |
| Oncorhynchus mykiss | Rainbow trout | 92.1 | 89.5 | AAK28557 |
| Dasyatis akajei | Stingray | 92.1 | 81.5 | Matsuda et al. (1998) |
| Homo sapiens | Human | 97.4 | 89.5 | AAI01804 |
| Bos taurus | Cattle | 97.4 | 89.5 | AAY16443 |
| Ovis aries | Sheep | 97.4 | 89.5 | NM001009776 |
| Canis lupus familiaris | Dog | 97.4 | 89.5 | XP849191 |
| Rattus norvegicus | Norway rat | 97.4 | 89.5 | NM016989 |
| Mus musculus | House mouse | 97.4 | 89.5 | NP033755 |
| Gallus gallus | Chicken | 94.7 | 86.8 | AAB52100 |
| Xenopus laevis | African clawed frog | 97.4 | 86.8 | NP001081947 |
The predicted sequence of tilapia Pac1-r has identities of 93.5, 93.9, and 85.9% with goldfish, zebrafish, and grass carp respectively (Table 2). The similarity with mammals (human, dog, rat, and mouse) is 90.4% and the identity is 84.8%.
Comparison of amino acid sequence of PAC1-R (tpac1-r) gene from tilapia (Oreochromis mossambicus) with other animals.
| Species | Common name | Similarity (%) | Identity (%) | Accession number or reference |
|---|---|---|---|---|
| Oreochromis mossambicus | Mozambique tilapia | / | / | AY830121 |
| Carassius auratus | Goldfish | 96.5 | 93.5 | AF048820 |
| Takifugu rubripes | Fugu rubripes 1A | 97.1 | 96.5 | AJ490804 |
| Takifugu rubripes | Fugu rubripes 1B | 91.7 | 84.0 | AJ494861 |
| Danio rerio | Zebrafish | 96.5 | 93.9 | NP001013462 |
| Ctenopharyngodon idella | Grass carp | 91.8 | 85.9 | EU305549 |
| Homo sapiens | Human | 90.4 | 84.8 | NP001109 |
| Canis lupus familiaris | Dog | 90.4 | 84.8 | XP539503 |
| Rattus norvegicus | Norway rat | 90.4 | 84.8 | NP598195 |
| Mus musculus | House mouse | 90.4 | 84.8 | P70205 |
| Gallus gallus | Chicken | 94.7 | 91.8 | ABQ63080 |
| Xenopus laevis | African clawed frog | 90.0 | 85.9 | AAF16939 |
Tissue distribution of tpacap38 and tpac1-r detected by semi-quantitative RT-PCR
In order to detect the expression of tpacap38 and tpac1-r transcript in various tissues including the brain, gall bladder, gill, heart, intestine, kidney, liver, muscle, pancreas, spleen, stomach, and gonads, semi-quantitative RT-PCR was performed with cDNA from both males (Fig 1A and C) and females (Fig. 1B and D). The brain was chosen as the standard control because it has the highest level of PACAP expression in mammals (Vaudry et al. 2000). The results showed that tpacap38 and tpac1-r were expressed at various levels in all samples tested except the liver. The RT-PCR products of tpacap38 and tpac1-r from tilapia tissues had the predicted size of 199 and 276 bp respectively. The RT-PCR product of the β-actin control had the predicted size of 675 bp. PCR products were normalized to the corresponding β-actin levels. The tpacap38 and tpac1-r levels in both female and male brain were significantly higher than those in other tissues (P<0.05). The levels of tpacap38 were significantly lower in gonads and tpac1-r levels were significantly lower in muscle of both sexes (P<0.05).
Semi-quantitative RT-PCR analysis of male (A and C) and female (B and D) tilapia tissues using specific primers for tpacap38 (A and B) and pacap type I receptor (tpac1-r; C and D). (Representative pictures are shown). B, brain; Ga, gallbladder; Gi, gill; H, heart; I, intestine; K, kidney; L, liver; Mu, muscle; P, pancreas; Sp, spleen; St, stomach; O, ovary; T, testis. a,b,c,d,e,fDifferent superscripts among groups indicate statistically significant differences (P<0.05).
Citation: REPRODUCTION 137, 3; 10.1530/REP-08-0422
Immunohistochemical localization of tpacap38 and tpac1-r
Sections of brain, gallbladder, gill, heart, intestines, kidney, liver, muscle, pancreas, spleen, stomach, and gonads (ovary or testis) after immunohistochemical staining for PACAP and PAC1-r are shown in Fig. 2. The data reveal that PACAP and PAC1-r proteins are expressed in the cytoplasm of cells of the brain cortex (Fig. 2A1-2) and white matter (Fig. 2A3-4), gall bladder (Fig. 2C1–4), muscle filaments in heart (Fig. 2D1–4), and skeletal muscle (Fig. 2H1–4), in centroacinar cells of the pancreas (Fig. 2I1–4), red pulps of the spleen (Fig. 2J1–4), in the epithelium of the gill (Fig. 2C1–4), intestine (Fig. 2E1–4), renal tubules (Fig. 2F1–4), and submucosa cells of the stomach (Fig. 2K1–4). In the gonads, both proteins are expressed in the cytoplasm of granulosa cells, but not theca cells around oocytes (Fig. 2L1–4), and in the interstitial cells, but not sperm of the testis (Fig. 2M1–4). However, no PACAP or PAC1-r was detected in hepatocytes (Fig. 2G). The same results were observed for both sexes. Similar results were observed in the 1-month-old juveniles. The expression of PACAP protein (Fig. 2N) seemed more extensive than Pac1-r protein (Fig. 2O) in the brain (Fig. 2N1 and O1) and other internal organs (Fig. 2N2 and O2).
Immunohistochemical staining of PACAP and PACAP type I receptor in brain (A), gallbladder (B), gill (C), heart (D), intestine (E), kidney (F), liver (G), muscle (H), pancreas (I), spleen (J), stomach (K), ovary (L) testis (M), and 1-month-old whole juveniles of tilapia. Control groups of PACAP are shown. The blue arrowheads indicate PACAP and PACAP type I receptor proteins. BrC, brain cortex; BrW, brain white matter; CaC, centroacinar cells; CaM, cardic muscle; Du, duct; GiE, epithelial of gill; Gra, granulosa cells; Hep, hepatocytes; InE,epithelial of intestines; InC, interstitial cells; LP, lamina propira; MuF, muscular filament; O, oocyte; PT, portal tract; RP, red pulp; RT, renal tubules; Sp, sperm; The, theca cells; SM, submucosa cells; StE, epithelial of stomach. A2–M2 and A4–M4 are the insets from A1–M1 and A3–M3 respectively. A–M, A1–M1 and A3–M3: Scale bar=100 μm; A2–M2 and A4–M4: Scale bar=20 μm; N and O: Scale bar=1 mm; N1, N2, O1 and O2: Scale bar=20 μm.
Citation: REPRODUCTION 137, 3; 10.1530/REP-08-0422
No PACAP or PAC1-r protein was found in the control groups of the tissues tested here (data for Pac1-r protein were not shown) of either sex. The immunohistochemical results supported the observations from RT-PCR.
Endogenous expression of tpacap38 and tpac1-r in tilapia gonads
In order to detect the endogenous expression levels of tpacap38 and tpac1-r transcripts in testes and follicles before supplement treatments, an 8 h in vitro culture without supplements was conducted. Figure 3 shows the tpacap38 (A and B) and tpac1-r (C and D) expression levels in the testes (A and C) and follicles (B and D) normalized to corresponding levels of β-actin. The levels of tpacap38 and tpac1-r mRNA expressed in fresh gonads (0 h) of both sexes were significantly higher than those at other time points (P<0.05). Expression levels decreased within 1 h after culture (P<0.05) and continued to decrease over a 4-hour culture period. There were no significant differences among the levels after 5–8 h of culture (P>0.05), except of tpacap38 in the testis at 8 h (P<0.05). However, lower levels of tpacap38 and tpac1-r were observed at 8 h in both sexes. Subsequent experiments were designed based on these results.
Semi-quantitative RT-PCR analysis of the endogenous expression of tpacap38 (A and B) and tpac1-r (C and D) in the gonads of male (A and C) and female (B and D) tilapia after cultured for different periods (0–8 h) without supplement. Results for the 0 h (control group) are set as 1. (Representative pictures are shown). a,b,c,d,e,fDifferent superscripts among groups indicate statistically significant differences (P<0.05).
Citation: REPRODUCTION 137, 3; 10.1530/REP-08-0422
Ovine PACAP38 regulates the expression of tpacap38 and tpac1-r in tilapia gonads
Dose-dependent stimulation of tpacap38 was observed in both sexes (Fig. 4). The levels of tpacap38 mRNA expressed in both sexes were higher than the levels in the fresh and control groups at concentrations of 1.50 and 5.00 nM (P<0.05; Fig. 4A and B). However, the expression levels in the gonads after 0.25 nM stimulation were similar to those in the fresh and control groups (P>0.05). This suggests that an ovine PACAP38 range over 1.50 nM is the optimal physiological concentration for inducing its effect on tpacap38 expression and ovine PACAP38 had a similar stimulating effect in both sexes. Expression of tpacap38 transcript increased at 2 h and decreased by 4 h after stimulation with 5.00 nM ovine PACAP38 in both sexes (P<0.05; Fig. 4C and D), and reached levels similar to those of fresh and control groups by 8 h (P>0.05).
Effects of ovine PACAP38 on the expression of tpacap38 in male (A and C) and female (B and D) gonads using semi-quantitative RT-PCR analysis. In the dose-response experiment (A and B), gonads were treated for 2 h with different doses of ovine PACAP38 (0.25, 1.50, and 5.00 nM) after an 8-hour preculture, and a control group was treated with DMEM only. For the time course experiments (C and D), 5 nM ovine PACAP38 was applied to the cultured gonads for 2, 4, or 8 h after the preculture and before RNA extraction, and the gonads after 8 h preculture (0 h) were considered to be as control groups. Each value represents the mean±s.e.m. of three independent RT-PCR replicates, and the electrophoretic image is shown at the top of each graph. The expression levels were normalized to β-actin and expressed as the fold-change to the respective control. Results for the control groups are set as 1. a,b,c,dDifferent superscripts among groups indicate statistically significant differences (P<0.05).
Citation: REPRODUCTION 137, 3; 10.1530/REP-08-0422
By contrast to the response of tpacap38, the expression levels of tpac1-r were constant in both sexes, with little response to ovine PACAP38 at any concentration (P>0.05; Fig. 5A and B). Still, these levels were higher than in the control groups (P<0.05). The same results were observed in the time-course experiment. No significant differences were observed between the expression levels at each time point and fresh groups (P>0.05; Fig. 5C and D); instead, significant difference was only observed between fresh and control groups (P<0.05).
Effects of ovine PACAP38 on the expression of tpac1-r in male (A and C) and female (B and D) gonads using semi-quantitative RT-PCR analysis. In the dose-response experiment (A and B), the gonads were treated for 2 h with different doses of ovine PACAP38 (0.25, 1.50, and 5.00 nM) after an 8-hour preculture, and a control group was treated only with DMEM. For the time course experiments (C and D), 5 nM of ovine PACAP38 was applied to the cultured gonads for 2, 4 or 8 h after the preculture and before RNA extraction, and the gonads after 8 h preculture (0 h) were considered to be as control groups. Each value represents the mean±s.e.m. of three independent RT-PCR replicates and the electrophoretic image is shown at the top of each graph. The expression levels were normalized to β-actin and expressed as the fold-change to the respective control. Results for the control groups are set as 1. a,b,c,dDifferent superscripts among groups indicate statistically significant differences (P<0.05).
Citation: REPRODUCTION 137, 3; 10.1530/REP-08-0422
The data indicated that ovine PACAP38 has more inductive effect on the expression of tpacap38 than on tpac1-r.
db-cAMP regulates the expression of tpacap38 and tpac1-r in tilapia gonads
Dose-dependent stimulation of tpacap38 mRNA expression by db-cAMP was observed in both sexes (Fig. 6) with the maximal response reached at 1.50 mM. The data shown in Fig. 6A and C clearly indicate the presence of a plateau phase for cAMP stimulation of pacap mRNA expression. The disappearance of the inductive effects at high concentration of db-cAMP could be due to the upper-response limit of the assay (Wang et al. 2003). The levels of tpacap38 mRNA expressed in both sexes were higher than the levels in the fresh and control groups at the concentrations of 1.50 mM and 5.00 mM (P<0.05; Fig. 6A and B). The expression levels after 0.25 nM stimulation, however, were similar with those in the fresh and control groups (P>0.05). This suggests that the db-cAMP range at 1.50 nM is the optimal physiological concentration for inducing its effect on tpacap38 expression. db-cAMP had a similar stimulatory effect in both sexes. In the time-course experiment, expression of tpacap38 mRNA increased at 2 h and decreased by 4 h in both sexes after incubation with 1.50 nM db-cAMP (P<0.05; Fig. 6C and D). The level in males at 8 h was similar to those in the fresh and control groups (P>0.05; Fig. 6C), but reached similar levels of fresh and 4 h groups in the female set (P>0.05; Fig. 6D).
Effects of db-cAMP on the expression of tpacap38 in male (A and C) and female (B and D) gonads using semi-quantitative RT-PCR analysis. In the dose-response experiment (A and B), the gonads were treated for 2 h with different doses of db-cAMP (0.25, 1.50, and 5.00 mM) after an 8-hour preculture, and a control group was treated only with DMEM. For the time course experiments (C and D), 1.5 mM of db-cAMP was applied to the cultured gonads for 2, 4, or 8 h after the preculture and before RNA extraction, and the gonads after 8 h preculture (0 h) were considered to be as control groups. Each value represents the mean±s.e.m. of three independent RT-PCR replicates and the electrophoretic image is shown at the top of each graph. The expression levels were normalized to β-actin and expressed as the fold-change to the respective control. Results for the control groups are set as 1. a,b,cDifferent superscripts among groups indicate statistically significant differences (P<0.05).
Citation: REPRODUCTION 137, 3; 10.1530/REP-08-0422
Similar to the response of tpacap38, the expression levels of tpac1-r were increased (female) or similar (male) to those of fresh groups after 1.50 nM of db-cAMP stimulation in both sexes, and slightly decreased at 5.00 nM (Fig. 7A and B). The levels of the fresh and treatment groups were higher than in the control groups (P<0.05). In the time-course experiment, 1.50 nM of db-cAMP had an inductive effect on the expression of tpac1-r at 2 h (P<0.05; Fig. 7C and D), the effect diminished with increasing culture periods in both sexes, and the expression levels were relatively lower than those of tpacap38. The lowest expression levels were observed in control groups (P<0.05).
Effects of db-cAMP on the expression of tpac1-r in male (A and C) and female (B and D) gonads using semi-quantitative RT-PCR analysis. In the dose-response experiment (A and B), the gonads were treated for 2 h with different doses of db-cAMP (0.25, 1.50, and 5.00 mM) after an 8-hour preculture, and a control group was treated only with DMEM. For the time course experiments (C and D), 1.5 mM of db-cAMP was applied to the cultured gonads for 2, 4 or 8 h after the preculture and before RNA extraction, and the gonads after 8 h preculture (0 h) were considered to be as control groups. Each value represents the mean±s.e.m. of three independent RT-PCR replicates and the electrophoretic image is shown at the top of each graph. The expression levels were normalized to β-actin and expressed as the fold-change to the respective control. Results for the control groups are set as 1. a,b,cDifferent superscripts among groups indicate statistically significant differences (P<0.05).
Citation: REPRODUCTION 137, 3; 10.1530/REP-08-0422
The above data indicate that db-cAMP has a more inductive effect on the expression of tpacap38 than on tpac1-r.
Effects of forskolin and H89 on the expression of tpacap38 and tpac1-r in tilapia gonads
Treatment of cultured tilapia gonads with forskolin for 2 h significantly increased the expression of tpacap38 in a dose-dependent manner (P<0.05), with the maximal response reached at 10 nM in both sexes (Fig. 8A and B). The same results were observed in those groups treated with ovine PACAP38 or cAMP analogs (Figs 4 and 6). When forskolin (10 μM) was applied together with H89 (10 μM), the expression of tpacap38 was repressed (P>0.05; Fig. 8C and D), and no inhibitory effect of H89 was observed as compared with the control groups (P>0.05). H89 (10 μM) completely blocked forskolin (10 μM)-stimulated expression of tpacap38 almost down to basal levels in both sexes (P>0.05).
Effects of forskolin on the expression of tpacap38 in male (A and C) and female (B and D) gonads in the absence or presence of H89 using semi-quantitative RT-PCR analysis. The gonads were treated for 2 h with different doses of forskolin (1, 5, and 10 μM) (A and B) or 10 μM of H89 (C and D) after an 8-hour preculture and before RNA extraction, and a control group was treated only with DMEM. Each value represents the mean±s.e.m. of three independent RT-PCR replicates and the electrophoretic image is shown at the top of each graph. The expression levels were normalized to β-actin and expressed as the fold-change to the respective control. Results for the control groups are set as 1. a,b,cDifferent superscripts among groups indicate statistically significant differences (P<0.05).
Citation: REPRODUCTION 137, 3; 10.1530/REP-08-0422
The inductive effect of forskolin on the expression of tpac1-r in male tilapia gonads was not significant at the concentrations between 1 and 5 μM (P>0.05; Fig. 9A). The inductive effect became significant at 10 μM (P<0.05) when the expression levels were still not as high as those of tpacap38. Forskolin had a better dose-dependent inductive effect on the expression of tpac1-r at the tested concentrations in the females (P<0.05; Fig. 9B), and the expression reached a plateau at 5 and 10 μM.
Effects of forskolin on the expression of tpac1-r in male (A and C) and female (B and D) gonads in the absence or presence of H89 using semi-quantitative RT-PCR analysis. The gonads were treated for 2 h with different doses of forskolin (1, 5, and 10 μM) (A and B) or 10 μM of H89 (C and D) after an 8-hour preculture and before RNA extraction, and a control group was treated only with DMEM. Each value represents the mean±s.e.m. of three independent RT-PCR replicates and the electrophoretic image is shown at the top of each graph. The expression levels were normalized to β-actin and expressed as the fold-change to the respective control. Results for the control groups are set as 1. a,b,cDifferent superscripts among groups indicate statistically significant differences (P<0.05).
Citation: REPRODUCTION 137, 3; 10.1530/REP-08-0422
The expression of tpac1-r was repressed by H89 (10 μM) with forskolin (10 μM) in males (P<0.05; Fig. 9C), though the level was not as low as that of tpacap38 (Fig. 8C). A similar result was observed in females; H89 blocked the forskolin-stimulated expression of tpac1-r (P<0.05; Fig. 9D).
Forskolin (10 μM) was shown to increase tpac1-r mRNA in follicular & testicular cultures to levels significantly higher than that of the fresh control (Fig. 9A and B). However, similar results could not be repeated in the experiments with H89 treatment (Fig. 9C and D). These data only indicate that the PACAP38 of ovine or piscine origin can stimulate the expression of the genes encoding both tpacap38 and its tpac1-r and that its effect is conveyed by the cAMP transduction pathway.
Discussion
The present study reveals the expression domains of PACAP38 and PACAP type I receptor (Pac1-r) in the tissues of tilapia, a euryhaline commercially important teleost. Presumably, these two genes could be involved in regulating gonadal function via a paracrine/autocrine mechanism. The alignment of the amino acid sequences of tilapia PACAP38 and PAC1-r show a high degree of evolutionary conservation with mammals, birds, other fish, and amphibians. Vip and PACAP type I receptor family members (90–100% similarities and 82–100% identities; Tables 1 and 2) show similar results and are reported in previous studies on carp, catfish, fugu, goldfish, rainbow trout, salmon, tilapia, and zebrafish (McRory et al. 1995, Parker et al. 1997, Wong et al. 1998, 2000, Fradinger & Sherwood 2000, Small & Nonneman 2001, Montpetit et al. 2003, Wang et al. 2003, Cardoso et al. 2004, Kwok et al. 2006, Lo et al. 2007, Sze et al. 2007). This indicates a close relationship between teleosts and other vertebrates. Fish pacap consists of five exons and four introns, and the coding region is located in exon 5, whereas the bulk of fish GHRH sequence is encoded in exon 4 (Parker et al. 1997, Wong et al. 2000). It has been suggested that the ancestral gene of PACAP may have originated from the glucagon gene family via exon duplication followed by gene duplication (Sherwood et al. 2000, Wong et al. 2000). Numerous studies have also shown that two populations of pacap mRNAs, a short form coding only for pacap and a long form coding for both PACAP and GHRH have been identified in various tissues of bony fish (Wong et al. 2000, Wang et al. 2003, Wu et al. 2006), and in our previous tilapia study (Lo et al. 2007). This duplication event may have occurred at some point close to the divergence of the piscine lineages (Fradinger & Sherwood 2000, Sherwood et al. 2000, Wong et al. 2000, Lee et al. 2007). This evidence raises the possibility that pacap and GHRH expression in fish could be differentially regulated (Wong et al. 2000, Wang et al. 2003, Wu et al. 2006). The biological actions of PACAP are mediated through 3 different types of G protein-coupled receptors, named Pac1-r, Vpac1-r, and Vpac2-r. It is still unkown whether, Vpac1-r and Vpac2-r are expressed in tilapia and, if so, in what tissues.
Our RT-PCR and immunohistochemical studies showed that PACAP38 and PACAP receptor Pac1-r expression in tilapia seem to be ubiquitous, in contrast to other fish species (Tables 3 and 4). Tilapia pacap38 and tpac1-r mRNA and protein can be detected in different tissues including brain, gallbladder, gill, heart, intestine, kidney, liver, muscle, pancreas, spleen, stomach, testis, and ovary, excluding the liver. Consistent with our results, the absence of pacap38 in liver has also been reported in the studies of carp (Kwok et al. 2006), catfish (McRory et al. 1995, Small & Nonneman 2001), rainbow trout (Krueckl & Sherwood 2001), sockeye salmon (Parker et al. 1997), and zebrafish (Wang et al. 2003). It is expressed, however, in the liver of goldfish, though at a low level (Wong et al. 2000). Like pacap38, lack of pac1-r expression in the liver has also been reported in studies of goldfish (Wong et al. 1998, 2000, Kwok et al. 2006) and fugu (Cardoso et al. 2004). Although tpacap38 and tpac1-r are present in other organs of the same embryonic endoderm-mesoderm origin (e.g., stomach, intestine or pancreas or gall bladder), none of them are present in liver cells of O. mossambicus in our present results. Similar results were also reported in human liver cells (Wei & Mojsov 1996, Cassiman et al. 2007). The tissue-specific expression pattern described for different fish species is not the same and this could be due to difficulties in detecting low levels of expression in some tissues (Wong et al. 2000).
Gene expression of pituitary adenylate cyclase-activating polypeptide (pacap38 in tissues of different fish.
| Species/organs | Brain | Astrocyte | Eyeball | Gall bladder | Gill | Heart | Intestine | Kidney | Liver | Muscle | Pancreas | Spleen | Stomach | Pituitary | Ovary | Testis | References |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Grass carp (Ctenopharyngo- don idella) | + | ND | ND | ND | − | − | − | − | − | − | ND | − | ND | − | ND | ND | Sze et al. (2007) |
| Goldfish (Carassius auratus) | + | ND | ND | ND | + | + | + | + | + | ND | ND | ND | ND | + | + | + | Kwok et al. (2006) |
| Zebrafish (Danio rerio) | + | ND | + | ND | − | − | + | − | − | ± | ND | ND | ND | ND | + | + | Fradinger & Sherwood (2000) and Wang et al. (2003) |
| Sockeye salmon (Oncorhynchus nerka) | + | ND | ND | ND | ND | − | + | ND | − | ND | ND | ND | ND | ND | ND | ND | Parker et al. (1997) |
| Stargazer (Uranoscopus japonicus) | + | ND | ND | ND | ND | ND | ND | ND | ND | ND | ND | ND | ND | ND | ND | ND | Matsuda et al. (2000) |
| Thai catfish (Clarias mucrocephalus) | + | ND | ND | ND | ND | ND | ND | ND | − | − | − | ND | + | − | + | + | McRory et al. (1995) |
| Channel catfish (Ictalurus punctatus) | ND | ND | ND | ND | − | − | + | − | − | + | − | − | ND | ND | + | + | Small & Nonneman (2001) |
| Tilapia (Oreochromis mossambicus) | + | + | ND | ND | ND | ND | ND | ND | ND | ND | ND | ND | ND | + | ND | ND | Lo et al. (2007) |
| Tilapia (Oreochromis mossambicus) | + | ND | ND | + | + | + | + | + | − | + | + | + | + | ND | + | + | This work |
+, detectable; −, not expressed; ND, not determined.
Gene expression of pituitary adenylate cyclase-activating polypeptide (pacap38) type I receptor in tissues of different fish.
| Species/organs | Brain | Astrocyte | Eyeball | Gall bladder | Gill | Heart | Intestine | Kidney | Liver | Muscle | Pancreas | Spleen | Stomach | Pituitary | Ovary | Testis | References |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Goldfish (Carassius auratus) | + | ND | ND | − | ± | + | ± | − | − | − | ND | − | ND | + | − | − | Wong et al. (1998) and Kwok et al. (2006) |
| Rainbow trout (Oncorhynchus mykiss) (VPAC1) | + | ND | ND | ND | + | + | + | + | + | ND | ND | + | ND | ND | ND | ND | Montpetit et al. (2003) |
| Rainbow trout (Oncorhynchus mykiss) (VPAC2) | + | ND | ND | ND | − | + | + | − | + | ND | ND | + | ND | ND | ND | ND | Montpetit et al. (2003) |
| Fugu rubripes | + | ND | ND | ND | + | − | − | − | − | ND | ND | − | ND | + | + | + | Sze et al. (2007) |
| Zebrafish (Danio rerio) | + | ND | + | ND | + | − | + | ND | ND | + | ND | ND | ND | ND | + | + | Fradinger et al. (2005) |
| Sea bream (Sparus auratus) | + | ND | ND | ND | − | + | + | + | − | ND | ND | ND | ND | + | + | + | Cardoso et al. (2007) |
| Tilapia (Oreochromis mossambicus) | + | + | ND | ND | ND | ND | ND | ND | ND | ND | ND | ND | ND | + | ND | ND | Lo et al. (2007) |
| Tilapia (Oreochromis mossambicus) | + | ND | ND | + | + | + | + | + | − | + | + | + | + | ND | + | + | This work |
+, detectable; −, not expressed; ND, not determined.
Such a wide range of tissue distribution of tpacap38 and tpac1-r in tilapia is comparable with the situation in mammals and may indicate that these peptides are involved in a wide array of biological functions in fish (Wong et al. 2000). The expression of tpacap38 and tpac1-r in the brain and other tissues indicates that they act not only as a hypophysiotropic hormone, but also as an autocrine and paracrine factor in various organs as well (Vaudry et al. 2000). The follicular granulosa cells and interstitial cells in testes are likely the major targets for pacap38 and pac1-r based on our current data. The expression levels of these two peptides in tilapia gonads suggest a local paracrine/autocrine role of these peptides in the regulation of gonad. Vpac2-r is the most abundantly expressed in cultured zebrafish follicle cells (Wang et al. 2003). In mammals, RT-PCR analysis and in situ hybridization have demonstrated the expressions of all 3 types of PACAP receptors in the ovary (Pesce et al. 1996, Scaldaferri et al. 1996, Gräs et al. 2000, Ko & Park-Sarge 2000, Park et al. 2000, Wang et al. 2003). The evidence for these expression patterns in tilapia have yet to be determined.
The development and function of vertebrate gonads are mainly controlled by pituitary gonadotropins. The activities of gonadotropins are modulated by many intraovarian paracrine/autocrine growth factors, such as activin, insulin-like growth factors, epidermal growth factor, and others including PACAP (Danforth 1995, Gräs et al. 1996, Ko et al. 1999, Lee et al. 1999, Koh et al. 2000, Ge 2000, Wang et al. 2003). Our previous study has shown that exogenous ovine PACAP38 (1 nM) can stimulate the expression of tpacap38 in cultured tilapia astrocytes (Lo et al. 2007). Here, we demonstrate that ovine PACAP38 (0.25–5 nM), a cAMP analog (db-cAMP; 0.25–1.5 mM), and forskolin (1–10 μM) can significantly upregulate tpacap38 expression in tilapia gonads in a dose- and time-dependent manner. This effect reached its maximum at 2 h after induction and decreased with prolonged culture for 4–8 h. Similar results with the expression pattern of vpac2-r are also reported for zebrafish ovarian follicle cells after induction by hCG (Wang et al. 2003).
These data are consistent with the postulation that PACAP acts as a paracrine factor, and gonadotropin significantly increases the production of intracellular cAMP and expression level of zfpacap38 mRNA in cultured gonads (Wang et al. 2003). However, the inductive effect with increasing concentration is not yet known. In this report, the expression level of tpacap38 in cultured tilapia follicles was higher than that in cultured testis after induction by exogenous ovine PACAP38, cAMP analog (db-cAMP) or forskolin in the dose- or time-dependent experiment described. This result is consistent with the well known concept that PACAP is transiently induced after LH surge in vivo or gonadotropin supplement in vitro in the granulose cells of mice, rats, and human (Wang et al. 2003, Morelli et al. 2008 and references herein). As shown in Fig. 5A and B (for PACAP treatment) and Fig. 7A and B (for db-cAMP treatment), tpac1-r mRNA levels were significantly higher after drug treatment compared with the respective control groups. Even though forskolin only had a slightly inductive effect on the expression of tpac1-r in the present results, the significantly stimulatory effect of exogenous ovine PACAP38 shown here and the stimulatory effect of gonadotropins on PACAP and/or PACAP receptors expression in the gonads of both fish and mammals shown in other reports indicate that the gonadal PACAP-mediated paracrine system and its regulation are well conserved, and this system could play fundamental roles in the development and function of gonads in vertebrates (Wang et al. 2003, Barberi et al. 2007 and references herein).
The stimulation of tpacap38 in astrocytes (Lo et al. 2007) and gonads in response to treatment with db-cAMP, together with the effects of forskolin completely blocked by H89, as described in zebrafish ovarian follicle cells (Wang et al. 2003), indicate that activation of the cAMP-PKA signaling pathway mediates upregulation of tpacap38 expression with exogenous ovine PACAP38 (Lo et al. 2007). As described by Wang et al. (2003) in zebrafish follicle cells, the enhanced upregulation of tpacap38 by the addition of a cAMP analog suggests that PACAP in tilapia gonads could serve either as a downstream factor to relay the actions of gonadotropins from one cell type to another in a paracrine manner, or as part of a closed-loop positive feedback mechanism to amplify the actions of gonadotropins in an autocrine manner. The functional role of tpacap38 could be related to the gonadal activin/follistatin system as observed in zebrafish (Wang et al. 2003). Other studies have demonstrated the presence of 2 cAMP response elements in the promoter of rat and mouse PACAP genes (Yamamoto et al. 1998, Park et al. 2000, Lo et al. 2007). Additionally, PACAP-induced differentiation is mediated by the production of cAMP in neuroblastoma cells, pheochromocytoma cells, cerebellar granule cells, and embryonic stem cells (Lo et al. 2007 and references herein). In other experiments, the expression levels of tpacap38 and tpac1-r in tilapia gonads were not affected by supplements of β-estradiol and hydrocortisone (data not shown). Although the exogenous effect of hCG and db-cAMP on steroidogenesis in the ovary of tilapia have been investigated and published as early as 1984Bogomolnaya & Yaron 1984, the exogenous PACAP and forskolin effect on steroidogenesis in tilapia gonads still remains to be investigated.
In conclusion, the upregulation of tpacap38, stimulated by the addition of exogenous ovine PACAP38, db-cAMP or forskolin, reveals that PACAP in gonads acts either as a paracrine mediator between interstitial/granulosa cells (PACAP) and other interstitial/granulosa cells (Pac1-r) or as an autocrine system (PACAP and PAC1-r colocalization), thus regulating the function of vertebrate gonads.
Materials and Methods
Animals
Sexually-mature male and female adult tilapia (O. mossambicus) were maintained in the laboratory at the Department of Molecular Biotechnology, Da-Yeh University, where they were kept in an aerated tank at room temperature (RT, 25–28 °C) for a 14h light:10h darkness photoperiod. Principles of animal care published by the National Laboratory Animal Center, National Science Council, Taiwan, were followed (Huang et al. 2007).
Reverse transcription PCR (RT-PCR)
For detection of tilapia PACAP38 (tpacap38) and PACAP type I receptor (tpac1-r) transcript in different tissues, mature individuals (♀, n=6, 102.7±16.4 g, gonadosomatic index (GSI)=3.0±0.6; ♂, n=6, 159.5±53.6 g, GSI =0.7±0.2, spawning season) were killed by cold shock, after which the brain, gallbladder, gill, heart, intestine, kidney, liver, muscle, pancreas, spleen, stomach, and gonads (ovary with follicle diameters ≥2 mm or testis) were rapidly excised. Total RNA was extracted from the organs with RNazol (Tel-Test, Friendswood, TX, USA) following the instructions from the manufacturer. cDNA was synthesized using an RT-PCR system (SuperScript II Reverse Transcriptase, Invitrogen) as described previously (Huang et al. 2001, 2007). Gene-specific primers for the coding region of tpacap38 (GenBank accession no. AY522580), tpac1-r (GenBank accession no. ), and β-actin (GenBank accession no. ) used in the PCR were as described previously (Huang et al. 2001, 2007, Lo et al. 2007). β-actin expression served as an internal control for normalization.
One microgram of cDNA was used as template for PCR that was carried out in the presence of 10 mM Tris–HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl2, 50 μM dNTPs, 2 pmol of β-actin primers, 8 pmol of the other primer sets, and 1 μl Taq DNA polymerase (5 U/μl, Platinum, Invitrogen). The amplification conditions were as follows: 1 cycle at 94 °C for 5 min (denaturation); various cycles (30 cycles for tpacap38, 35 cycles for tpac1-r, and 27 cycles for β-actin) at 94 °C for 30 s (denaturation), 55 °C for 30 s (annealing), and 72 °C for 30 s (extension); 1 cycle at 72 °C for 10 min (final extension). The PCR products were analyzed on a 1.2% agarose gel and were sequenced directly. Nucleic acid and putative amino acid sequences were compared with all published sequences on the genetic computer program (Huang et al. 2001, 2007).
Immunohistochemical localization of PACAP38 and PACAP type I receptor
Along with RNA extractions, samples of brain, gill, heart, intestine, stomach, kidney, gallbladder, muscle, liver, pancreas, spleen, testis, and ovary from mature individuals were fixed with 4% buffered paraformaldehyde (P-6148, Sigma) and 2% glutaraldehyde (Merck) or 4% buffered formaldehyde (Merck) for over 6 h at 4 °C. The samples were subsequently dehydrated in an ascending series of ethanol and then embedded in paraplast (Merck) as described previously (Huang et al. 2007). Tissue sections were cut at 5 μm and mounted on glass slides. Sections were pretreated with 3% H2O2 (100% methanol and 3% H2O2 mixed 1:1) in methanol for inactivation of endogenous peroxidase, and the sections were incubated with anti-PACAP and PACAP type I receptor (PAC1-R) polyclonal antibodies (0.2 μg in 50 μl PBS, sc-25439 and sc-30018, rabbit anti human origin; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) for 1 h at RT or overnight at 4 °C and then washed three times in PBS. The section slides were incubated with secondary antibodies from a detection kit (N-Histofine, Simple stain MAX PO-R; Nichirei, Tokyo, Japan) for 30 min at RT and washed three times in PBS. Finally, stained signals of specimens were developed using the Liquid DAB (3, 3’-diaminobezidine tetrahydrochloride) Substrate Kit (Zymed Laboratories, Inc., San Francisco, CA, USA). Counterstaining was performed with eosin Y (Pacific CSA International Inc., Walnut, CA, USA). Control groups were incubated as described above without anti-PACAP or PAC1-R antibodies according to the protocol provided by the manufacture in the detection kit. Photomicrographs were taken with an Olympus microscope (BX 51, Olympus, Tokyo, Japan) by a CCD camera (DXM-1200, Nikon, Tokyo, Japan) (Huang et al. 2007).
Effects of chemicals on the in vitro expression of tpacap38 and tpac1-r in gonads
Chemicals
Ovine PACAP38 (A1439), dibutyryl cyclic AMP (db-cAMP; D0260), forskolin (F6886), and H89 (dihydrochloride hydrate; B1427) were purchased from Sigma. The preparation of these chemicals has been described previously (Lo et al. 2007).
Primary cultures and treatments
Mature individual tilapia (♀, n=12, 68.2±4.9 g, GSI=4.3±0.3; ♂, n=12, 84.9±4.0 g, GSI=0.5±0.1; spawning season) were killed following cold shock on ice anesthetic for 10 min. The testes and ovarian follicles (diameter≥2 mm) were immediately excised from the control group for RNA extraction (testes and follicles without any treatment were defined as the fresh control groups) or for subsequent treatments (experimental groups).
For assaying the endogenous expression of tpacap38 and tpac1-r transcripts in the follicles and testes, we employed a modified version of a previously published method (Huang et al. 2007). Follicles (≥2 mm diameter, 30 follicles/well) and testes (cut into 2×2 mm pieces, 10 pieces/well) were first cultured for 8 h in a six-well plate (15×10 mm/well, Cat. No. 140675, Nunc, Roskilde, Denmark) in DMEM solution (D-8900, Sigma), supplemented with 100 IU/ml Penicillin and 100 μg/ml Streptomycin (Penstrep, Gibco) at 28 °C in 5% CO2 with saturated humidity. RNA was extracted at each time point.
After the 8 h preculture treatment, follicles and testes were washed with fresh DMEM and separated into two parts. The first part was used for experiments lasting 2 h (dose dependent) with varying concentrations of ovine PACAP38 (0.25, 1.5, and 5 nM) and db-cAMP (0.25, 1.5, and 5 mM), and a control group was treated with only DMEM. The second part was used for experiments (time course) incubating the tissue in DMEM with 1.5 mM db-cAMP or 5 nM ovine PACAP38 for varying culture periods (2, 4, and 8 h), and the gonads after 8 h preculture (0 h) were considered to be as control groups. Another treatment was conducted with various concentrations of forskolin (1, 5, and 10 μM) or 10 μM of forskolin in the presence or absence of 10 μM of H89 (a specific inhibitor of cAMP-dependent (PKA)) for an additional 2 h culture after the 8 h preculture, and the control groups were as those in dose-dependent experiments described.
After incubation, the total RNA of tested gonads was isolated and RT-PCR was used to detect tpacap38 and tpac1-r expression patterns. Each treatment was repeated independently in triplicate.
Semi-quantification of PCR products
Quantification of the level of PCR products after electrophoresis was conducted using a densitometer (Personal Densitometer SI, Molecular Dynamics, Sunnyvale, CA, USA) with software provided by the manufacturer. The signal of β-actin was used as an internal control and normalized with every band in the same treatment for calculation and comparison with other treatments. The relative expression level of tpacap38 and tpac1-r in each experiment was expressed as the ratio of tpacap38 and tpac1-r mRNA to β-actin mRNA (Huang et al. 2001, 2007). The results of control groups (0 h in endogenous expression experiment, and testes or ovarian follicles cultured for 8 h without supplement in the following time course experiments or for additional 2 h without supplement after 8 h preculture in the following dose dependent experiments) were set as 1. Results from three independent experiments were expressed as the mean ±s.e.m. In all experiments, significance values (P<0.05) were compared by a Duncan multiple range test after one-way ANOVA.
Declaration of interest
There is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work was supported by the Council of Agriculture, grants 96AS-1.2.1-ST-a2(36) (ROC) and 95AS-6.2.1-ST-a1(37) (ROC) to Dr Wei-Tung Huang.
Acknowledgements
Tilapia used in this work were kindly provided by Dr Jen-Leih Wu of Academia Sinica, Taiwan.
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