Expression levels of THRr gene in G5 fish
Changes in external environmental temperature was received upon entering pre-adulthood, thus the activation of the fish's sensor system in response to the temperature showed significant effect. Thermal receptors found in the pineal organ are an important system involved in the activation of fish reproductive pathways (Pankhurst 2016). Environmental temperature fluctuations influence the pineal thermal receptor gene (thyroid hormone receptor gene, THRr) in response to environmental temperature modulation and the information is transmitted to the hypothalamic neuroendocrinal system. THRr expression can further influence the GnRH hormone associated with fish reproduction, including the GnRH receptor (GnRHr) (Bhat et al. 2015; Crisanti et al. 2001; Kazeto et al. 2005).
Analysis of THRr gene expression in temperature treatments showed that 26°C had the highest expression effect compared to 22°C and 30°C among transgenic fish, but did not have a significant effect at both temperatures in both transgenic and non-transgenic fish (Figs. 7A and 7B). In this research, C. gariepinus treated at 25°C showed the highest level of core clock gene expression in the pineal organ compared to temperatures of 15°C and 35°C (Saha et al. 2018). The similarity in the expression levels of the THRr and core clock genes in the pineal indicates that the response of thermal receptor cells to temperature is in the range of 25–26°C for the catfish group. At high temperatures (30°C) and 35°C there was a decrease in the expression level of the THRr and core clock genes, while at relatively low temperatures (22°C and 15°C), the expression levels were lower than at 30°C and 35°C. Similar results were shown in the development of European eel (Anguilla anguilla) larvae which decreased when treated with a temperature of 22°C (Politis et al. 2017). This indication clearly shows that the expression level of the THRr gene is influenced by low temperature or high temperature, where high temperature tends to reduce the expression level, while low temperature causes a decrease in the expression level. The optimal temperature that provides an increase in high expression levels of the THRr gene for transgenic G5 mutiara catfish (average 3.40) and non-transgenic (average 2.30) is at 26°C. The higher expression level of the THRr gene in transgenic catfish compared to non-transgenic fish was thought to be related to GH overexpression in transgenic fish. Thyroid hormone collaborates with growth hormone in the growth and reproduction process of fish (Lema et al. 2022; Ojima & Iwata 2020), thus it can be explained that the level of THR gene expression (including THRr) was found in the highest level when treated at a temperature of 26°C, whereas this condition did not occur in non-transgenic fish. Transgenic G5 catfish containing CgGH inserts is stably transmitted from its transgenic mutiara catfish broodstock while transgenic genes in fish were known to be repeated in a head-to-tail fashion proved in transgenic salmon (containing PRL-lacZ) (Uzbekova et al., 2003) and transgenic coho salmon (Oncorhynchus kisutch) (containing a salmon growth hormone OnMTGH1) (Uh et al., 2006).
Expression levels of GnRHr gene in G5 fish
Thyroid hormone receptors (THRr) are found in the ovarian and testicular tissue of channel catfish (Kazeto et al. 2005), indicating that THRr is indirectly involved in the fish reproductive pathway. THRr gene expression in the ovary increases to its highest peak when oocyte growth reaches the vitellogenic phase, this indication shows that THRr plays an important role in vitellogenesis and gonadal maturation. Thyroid hormones work together with other hormones along the hypothalamic-pituitary-gonad pathway which are involved in reproductive aspects, including the GnRHr hormone. So, TRHr is involved in the growth and development of the fish reproductive system (Habibi et al. 2012). Indirectly, THRr (pineal) gene expression can influence the level of GnRHr (hypothalamic) gene expression.
The GnRHr expression level treated at 26°C (average 4.23) in G5 transgenic mutiara catfish was higher than in other treatments (Figs. 8A and 8B). The increase in GnRHr expression levels at 26°C is relatively similar to the THRr gene expression levels at that temperature. This indication shows that the increase in THRr expression is indirectly followed by an increase in GnRHr gene expression. This consistency suggests an influence of THRr on GnRHr expression. In contrast, temperatures of 22°C (average 2.69) and 30°C (average 2.76) tended to reduce the level of GnRHr gene expression, and there was no significant difference between transgenic and non-transgenic fish at 30°C. These results indicated that GH-transgenesis did not induce GnRHr expression levels at 30°C and showed similar effects in non-transgenic fish. The expression level of GnRHr at 22°C was lower than at 26°C in transgenic catfish, indicating that lower temperature decreased the expression level, whereas warm temperatures increased the expression level of GnRHr to a greater extent. Meanwhile, high temperature (30 °) had an effect on reducing GnRHr expression levels. Effect of high temperature (34°C) was known to reduce the expression level of the GnRH gene in Amargosa pupfish (Cyprinodon nevadensis amargosae), while a temperature of 24°C is the optimal temperature to stimulate ovarian growth (Lema et al. 2022). These results are relevant to the expression level of the GnRHr gene found in G5 transgenic mutiara catfish, that the optimum temperature that induces the highest level of GnRHr expression was 26°C.
Expression levels of FSHr gene in G5 fish
Temperature is one of the environmental factors involved in regulating fish reproduction, especially during the periods of gonad growth, vitellogenesis, spawning and egg hatching (Bromage et al. 2001; Levy et al. 2011). Oocyte growth is influenced by fluctuations in environmental temperature which can modulate the expression levels of genes involved in the secretion of gonadotrophin hormones which are important in stimulating oocyte development and growth. Two genes involved in regulating the secretion of fish gonadotropin hormones are follicle stimulating hormone (FSH) and luteinizing hormone (LH) which are found in the pituitary (David & Degani 2011; Mateos et al. 2002; Yu et al. 2022). Pituitary secretion of FSH and LH hormones is regulated by the hypothalamic GnRH hormone, where the expression of the GnRH gene (also including the GnRHr gene) plays a role in regulating the expression of the FSH and LH genes for the production of these two gonadotrophin hormones. The FSH hormone receptor (FSHr) is located in the gonad, thus this receptor is involved in the gonadal maturation process and can be used as a representation of increased FSH gene expression (Anderson et al. 2019). Induction of appropriate temperatures can stimulate increased expression of genes involved in steroidogenesis which leads to spawning of broodstock. During oocyte growth, the expression level of FSH genes (including FSHr) is influenced by environmental temperature fluctuations.
Results of FSHr gene expression analysis of G5 transgenic mutiara catfish at a temperature of 26°C was higher than other treatments and significantly different from non-transgenic fish (Figs. 9A and 9B). The expression level of the FSHr gene in transgenic catfish at 22°C (average 4.84) was higher than in non-transgenic fish, but lower than at 26°C (average 7.25) in transgenic fish. Likewise, at 30°C, the FSHr expression level was not much different from that at 22°C. This result is similar to research on juvenile hybrid sturgeon (Acipenser baerii ♀× Acipenser schrenckii ♂), which the optimal temperature that induces high expression of the FSH gene occurs at 27°C, when compared to 21°C and 30°C (Chen et al. 2022). It was further stated that this optimum temperature is needed to regulate the development of fish gonads, especially FSH is needed to increase the size of the oocyte follicle during the development stage of the oogonia into primary oocytes. At 26°C treatment, the increase in FSHr gene expression is inseparable from the influence of hypothalamic GnRHr gene expression where the expression level of the pineal THRr gene equally increase, while at 30°C shows an effect of decreased expression. At the temperature of 22°C, expression levels of the THRr, GnRHr, FSHr genes were found lower compared to the temperature of 26°C treatment (Figs. 7B, 8B, 9B). Optimum temperatures (around 26°C) are needed for gonad maturation and encourage the spawning process of many cyprinids and other warmwater fishes (Chen et al. 2022). Low temperatures (22°C) or high temperatures (30°C) do not have much influence on accelerating gonad maturation and stimulating spawning of brood fish. It is therefore understandable that the expression levels of genes involved in the pineal (THRr)-hypothalamic (GnRHr)-pituitary (FSHr) pathway were consistently at high levels when exposed to 26°C water temperature compared to 22°C and 30°C.
GH levels and gonadal growth of G5
Serum GH levels of G5 transgenic mutiara catfish were significantly higher than non-transgenic fish at temperatures of 22°C, 26°C, 30°C and serum GH of transgenic catfish kept at 26°C was higher than other treatments (Fig. 7A). The results of measuring GH levels show that temperature treatment (especially in 26°C) induced higher GH levels (average 6.13) than temperatures of 22°C and 30°C, both in transgenic and non-transgenic catfish. Pituitary GH secretion involves stimulation of hypothalamic GnRH, because GnRH is a factor that stimulates GH, LH or FSH secretion (Li et al. 2002). The level of GnRH expression can be represented by the GnRH gene receptor (GnRHr). High temperature induction (30°C) tends to reduce GH levels in transgenic and non-transgenic catfish, as does low temperature (22°C). The decrease in GH levels at high and low temperatures is closely related to the decrease in GnRHr expression levels, indicating that GH secretion levels are indirectly regulated by GnRHr expression levels. The increase in GnRHr expression levels at warm temperatures (26°C) was accompanied by increased secretion of GH levels in both transgenic and non-transgenic fish (Figs. 5B and 7A), indicating a close relationship between GnRHr expression and GH levels which is influenced by temperature.
The consistency of GH levels in different temperature treatments was also followed by an increase in the ovarian weight of G5 fish, whereas the temperature of 26°C gave a higher increase in the ovarian weight of transgenic and non-transgenic catfish than temperatures of 22°C and 30°C (Fig. 7B). The decrease in GH levels at temperatures of 22°C and 30°C affected the lower growth of ovarian weight in G5 fish. This was in line with previous studies where an optimum temperature (26°C) induced a higher increase in ovarian weight of tilapia (Orechromis niloticus) compared to low temperatures (16°C) (Gopal et al. 2014), showing similar results in G5 catfish where increased levels of GH in stimulated the gonad growth of G5 female catfish. Other research shows that a temperature of 27°C can significantly induce increased expression of genes involved in reproduction and growth in the brain and pituitary pathways of female and male blue gouramis fish (Trichogaster tricopterus) than temperatures of 23°C and 31°C. The expression level of the FSH gene in the pituitary significantly decreased at 31°C compared to 23°C and 27°C, and was implicated in reducing the GSI value of fish (David & Degani 2011). Overall, both GH levels and ovarian weight gain in G5 transgenic mutiara catfish were higher than non-transgenic fish, with an increase in GH levels of 2.92 times at a temperature of 26°C, 2.48 times at a temperature of 22°C and 1.83 times at a temperature of 30°C. The high levels of GH are thought to be closely related to the overexpression of GH in transgenic catfish at a temperature of 26°C, whereas at temperatures of 22°C and 30°C, it causes a decrease in GH levels which is followed by a decrease in ovary weight in both transgenic and non-transgenic fish.
Estradiol levels gonadal development of G5 fish
The increased FSHr gene expression pituitary leads to increased GH pituitary and E2 levels in the gonad, inducing the enzyme activity 17α-hydroxylase/C17,20-lyase and p450 aromatase a (cyp19a1a) for oocyte maturity, and ovarian weight; as a consequence, female gonad development increases in female coho salmon when treated with optimum temperature (28°C) (Anderson et al. 2019; Sua-Cespedes et al. 2021). Low temperature (23°C) reduces gene expression levels in the brain (including GnRHr) which in turn causes a decrease in pituitary GH and E2 levels in the ovaries. E2 plays an important role in carrying out vitellogenesis processes, which involve yolk protein accumulation, causing oocyte growth, and ovary enlargement, as represented by ovaries weight (Levy et al. 2011; Melamed et al. 1998). So here, it is shown that temperature fluctuations affect the expression of genes involved in the hypothalamic-pituitary-gonadal pathway that induces GH and E2 levels that lead to oocyte maturation.
In the warm temperature treatment on G5 transgenic mutiara catfish, the increase average weight of ovaries was associated with the stimulation of E2 levels under 26°C (Figs. 7B and 8A) which was higher than non-transgenic fish and other temperature treatments. This increase in GH and E2 levels leads to oocyte growth and as a consequence the development of female gonads leading to the ovarian ripening stage. Similar result was also found in the 27°C temperature treatment in the female blue gourami (Levy et al. 2011). The gonad development rate of female transgenic and non-transgenic G5 mutiara catfish was reduced when treated with temperatures of 22°C and 30°C (Fig. 8B). However, ovary development in transgenic catfish was faster than non-transgenic catfish at a temperature of 30°C, indicating that transgenesis-GH could maintain oocyte growth in transgenic fish. GH can induce the IGF-1 gene in liver and gonads (stimulating E2 production), indicating that GH is involved in yolk protein synthesis during vitellogenesis and steroid production to initiate oocyte growth during gonadal maturation (Buwono et al. 2019b; Gomez et al. 1999; Swanson et al. 2003; Wong et al. 2006). Rearing female fish broodstock requires an optimum temperature range to initiate the reproductive cycle and sustain oocyte growth and maturation (Uchida et al. 2003).
This indicates that the oocyte development stage of G5 transgenic mutiara catfish can reach early-late vitellogenic stages when treated with temperatures of 22°C, 26°C and 30°C, whereas in non-transgenic fish slow down oocyte growth (Fig. 8B), where at 22°C and 30°C, the oocyte development was in 'immature' status in G5 non-transgenic fish. Meanwhile, at a temperature of 30°C, ovarian development reaches the pre-vitellogenic stage. In treatment A (transgenic 22°C and C* (non-transgenic 30°C) an oogonium containing a nucleus was in the middle of an oocyte follicle, and some oogonium continued to develop to form cortical alveoli (Fig. 8B), showing the growth from primary oocytes to secondary oocytes to pre-vitellogenic. This oocyte development is similar to the results of research on female blue gourami fish which were reared at a temperature of 23°C for 9 days showing the pre-vitellogenic stage, while at a temperature of 31°C they were at the early vitellogenic stage (Levy et al. 2011). This slow oocyte growth is closely related to a decrease in E2 levels and GH levels which stimulate the formation of vitellogenin during the early stages of oocyte maturation (Hermelink et al. 2011). Consistent slow development of oocytes (secondary oocyte development stage) was found in non-transgenic G5 catfish at temperatures of 22°C and 26°C as a consequence of reduced levels of GH and E2. In contrast, in G5 transgenic catfish at temperatures of 22°C, 26°C and 30°C, oocyte development reached the late-vitellogenic stage to the ootid stage, indicating that GH-transgenesis increased oocyte development higher than in non-transgenic fish. The oocytes in non-transgenic fish (treatment A* temperature 22°C and B* temperature 26°C) were in the primary growth stage, the oogonium stage, characterized by oogonia proliferation, which is a common feature found during early oocyte growth. In treatment B, the oocyte was in late-vitellogenic stage, marked with the formation of yolk granule (YG) as a stage of yolk accumulation early in the ovary ripening period (Fig. 8B). Mature oocytes developed, which were characterized by the formation of theca cells and granulosa cells, which play a role in the aromatization of androgens to E2 to induce vitellogenin production during early to mature oocytes. YG formation increases, causing an enlargement in oocyte size and migration of germinal vesicle toward the periphery known as Germinal Vesicle Break Down (GVBD), indicating that the oocyte is in the mature stage. During vitellogenesis before the oocyte matures, induction at the hypothalamic level is required, especially GnRH levels. This includes an increase in GnRHr expression to stimulate an increase in LH and FSH levels (including FSHr) as a signal to start the formation of maturing induction hormone in the development of secondary oocytes into ootids (Ohta et al. 2002; Yaron & Levavi-Sivan 2006).
CgGH expression and GSI Levels in G5 fish
The following information explains the role of temperature in regulating CgGH expression in transgenic fish in relation to the GSI level. Figure 12A explains that the GSI value observed from gonad growth of female transgenic fish which was influenced by temperature. This temperature determines the expression level of CgGH at the temperature of 26°C, the average growth of GSI was higher compared to 22°C and 30°C treatment. The consistency was shown from the expression levels in Fig. 12B where significantly higher expression levels in transgenic fish was regulated at the temperature of 26°C. This proves that the growth of transgenic female catfish is influenced by the expression level of the CgGH insert and this highest expression value is directly supported by a temperature of 26°C. The GSI value at a temperature of 30°C decreased and suppressed the expression level in transgenic fish, while at a low temperature of 22°C the expression level was not maximally induced and was not the ideal temperature for maximum expression of CgGH. The GSI value and average gonad growth in transgenic catfish were also lower than at 26°C. The presence of CgGH is advantageous compared to transgenic fish because the average GSI value in all treatments is higher than the GSI value of transgenic fish except in 26°C. Catfish highly responds to temperature during rearing catfish outside its temperature preferences may cause stress. GSI was used previously as a successful indicator of temperature treatments in catfish (Al-Deghayem et al. 2017). In addition to the proven synergism between GH and ovarian development, temperature was also a critical factor affecting catfish (Singh and Lal, 2008). Since water rearing temperature affects the fish pineal organ of catfish within the complexity of a receptor rhythm and its infliction upon reproductive cycle (Saha et al. 2020), preferred temperature and the presence of CgGH over expression proved to cause accelerated gonad maturation in transgenic female fish compared to non-transgenic counterparts.