Role of Leptin in Japanese Quail Development

Máãajová M., D. Lamo‰ová, M. Zeman: Role of Leptin in Japanese Quail Development. Acta Vet Brno 2002, 71: 473-479. Leptin, a hormone produced mainly by adipose tissue, controls energy balance and can also influence the embryonic development and growth. In our study we investigated the effects of leptin administration during the embryonic development on hatching rate, posthatching growth and development of Japanese quail (Coturnix japonica). At embryonic day 5, 0.1 μg or 1 μg of recombinant mice leptin in 50 μl of phosphate buffered saline was injected into the albumen of incubating eggs. Birds from each group were weighed daily and killed by decapitation at day 0, 2, 5, 7, 14, 21, 28, 35, 42, 49 and 56 of postembryonic life. Plasma concentrations of triiodothyronine (T3), testosterone and total lipids in plasma and in the liver were measured. Quail treated with leptin hatched earlier and expressed a higher body weight during the whole experimental period than the control group (p < 0.05). Significant changes in T3 concentrations, total lipids and body weight were seen immediately after hatching and shortly before sexual maturity. Testosterone levels and eggs laying were higher in leptin groups in comparison to controls. We suppose that the endocrine milieu of the developing avian embryo is influenced by leptin and the administration of this hormone during embryonic life can accelerate embryonic development of birds. birds, embryogenesis, growth, reproduction, triiodothyronine, testosterone, total lipids Leptin plays an important role as a signal of the body fat content to the brain, where it regulates food intake and energy expenditure (Fr iedman and Halaas 1998). It is important not only for the control of food intake and energy balance, but it functions also as a metabolic and neuroendocrine hormone. Leptin has been implicated as a mediator of diverse physiological features, including reproduction, growth, hematopoiesis, renal function, immune functions and angiogenesis (Ahima et al. 1996). Surprisingly, it is synthesized at a high rate in the placenta and may function as a growth factor for the fetus, signaling the nutritional status from the mother to her fetus (Masuzaki et al. 1997). This hormone is a growth factor for many cell types, including lymphocytes (Lord et al. 1998), cultured tracheal epithelial cells, lung squamous cells (Tsuchiya et al. 1999), pancreatic (Tanabe et al. 1997) and embryonic cells. Moreover, leptin seems to have a key role in the regulation of the reproductive system, at both peripheral and central levels. There are also known interactions between leptin and sex hormones: for example, it is known, that leptin stimulates GnRH, FSH and LH release (Yu et al. 1997). Chicken leptin mRNA appears to be most abundant in the liver, which is the major site of fat synthesis in birds. The presence of leptin mRNA in the liver suggests that birds may have a different mechanism for regulating food intake and energy expenditure than mammals (Ashwel l et al. 1999). Leptin expression was also detected in the chick embryonic liver and yolk sac (Ashwel l et al. 1999), suggesting that leptin may have a role in the chick embryo similar to that hypothesized for leptin in the placenta of mammals. In the developing embryo, leptin expression is detectable as early as 72 hours of incubation (McMurtry et al. 2000). ACTA VET. BRNO 2002, 71: 473–479 Address for correspondence: Mgr. Mariana Máãajová Institute of animal Biochemistry and Genetics, SASci, Moyzesova 61 900 28 Ivanka pri Dunaji, Slovakia Phone: +421-2-45943 232 Fax: +421-2-45943 232 E-mail: mariana.kubovicova@saavba.sk http://www.vfu.cz/acta-vet/actavet.htm It has been found that recombinant murine leptin has a stimulatory effect on the proliferation of cultured embryonic chicken muscle and liver cells (Lamo‰ová and Zeman 2001). Therefore, the purpose of this study was to determine if recombinant murine leptin injected into hatching eggs of Japanese quail during the embryonic period acts as a growth factor and subsequently influences postembryonic growth, food intake and sexual development. Materials and Methods Eggs of Japanese quail from our breeding colony were divided into three groups (93 in each group). The average weight of eggs in every group was equalized before the experiment (9.20 ± 0.25 g). Eggs were incubated in a forced draught incubator with automatic turning every two hours at 37.5 ± 0.2 °C and 50-60 % relative humidity. At embryonic day 5 recombinant murine leptin (Pepro Tech, EC, England) in 50 μl of phosphate buffered saline (PBS) was injected into the albumen of eggs. Two different concentrations of leptin were used: 0.1 μg (L1) and 1 μg (L2). The control group (C) was injected with 50 μl of PBS. After hatching each group of birds was kept in a separate box up to day 35 of postembryonic life (PD 35), after which the animals were placed in individual cages. Continuous lighting at intensity 6080 lux was imposed. Quail were fed ad libitum with feeding mash (21% crude protein and 12 MJ/kg of metabolizable energy). Individual body weight and food intake (per group to PD 35 and then individually) were recorded daily. Six animals from each group were decapitated at PD 0, 2, 5., 7, 14, 21, 28, 35, 42, 49, 56. Blood samples were collected into heparinized tubes and then centrifuged. Plasma was stored at –20 °C until assayed for testosterone (RIA, Immunotech, Slovakia), T3 (RIA, Immunotech, Slovakia) and total lipids in plasma (Bio-la-test, Lachema, Czech Republic). Liver was weighted and taken for determination of the total lipids (Bio-la-test, Lachema, Czech Republic). Data are presented as means ± S.E.M. The results were analysed by two-way analysis of variance (ANOVA) and Student’s t-test.

Leptin plays an important role as a signal of the body fat content to the brain, where it regulates food intake and energy expenditure (Friedman and Halaas 1998).It is important not only for the control of food intake and energy balance, but it functions also as a metabolic and neuroendocrine hormone.Leptin has been implicated as a mediator of diverse physiological features, including reproduction, growth, hematopoiesis, renal function, immune functions and angiogenesis (Ahima et al. 1996).Surprisingly, it is synthesized at a high rate in the placenta and may function as a growth factor for the fetus, signaling the nutritional status from the mother to her fetus (Masuzaki et al. 1997).This hormone is a growth factor for many cell types, including lymphocytes (Lord et al. 1998), cultured tracheal epithelial cells, lung squamous cells (Tsuchiya et al. 1999), pancreatic (Tanabe et al. 1997) and embryonic cells.Moreover, leptin seems to have a key role in the regulation of the reproductive system, at both peripheral and central levels.There are also known interactions between leptin and sex hormones: for example, it is known, that leptin stimulates GnRH, FSH and LH release (Yu et al. 1997).
Chicken leptin mRNA appears to be most abundant in the liver, which is the major site of fat synthesis in birds.The presence of leptin mRNA in the liver suggests that birds may have a different mechanism for regulating food intake and energy expenditure than mammals (Ashwell et al. 1999).Leptin expression was also detected in the chick embryonic liver and yolk sac (Ashwell et al. 1999), suggesting that leptin may have a role in the chick embryo similar to that hypothesized for leptin in the placenta of mammals.In the developing embryo, leptin expression is detectable as early as 72 hours of incubation (McMurtry et al. 2000).
It has been found that recombinant murine leptin has a stimulatory effect on the proliferation of cultured embryonic chicken muscle and liver cells (L a m o ‰ o v á and Z e m a n 2001).Therefore, the purpose of this study was to determine if recombinant murine leptin injected into hatching eggs of Japanese quail during the embryonic period acts as a growth factor and subsequently influences postembryonic growth, food intake and sexual development.

Materials and Methods
Eggs of Japanese quail from our breeding colony were divided into three groups (93 in each group).The average weight of eggs in every group was equalized before the experiment (9.20 ± 0.25 g).Eggs were incubated in a forced draught incubator with automatic turning every two hours at 37.5 ± 0.2 °C and 50-60 % relative humidity.At embryonic day 5 recombinant murine leptin (Pepro Tech, EC, England) in 50 µl of phosphate buffered saline (PBS) was injected into the albumen of eggs.Two different concentrations of leptin were used: 0.1 µg (L1) and 1 µg (L2).The control group (C) was injected with 50 µl of PBS.After hatching each group of birds was kept in a separate box up to day 35 of postembryonic life (PD 35), after which the animals were placed in individual cages.Continuous lighting at intensity 60-80 lux was imposed.Quail were fed ad libitum with feeding mash (21% crude protein and 12 MJ/kg of metabolizable energy).Individual body weight and food intake (per group to PD 35 and then individually) were recorded daily.Six animals from each group were decapitated at PD 0, 2, 5., 7, 14, 21, 28, 35, 42, 49, 56.Blood samples were collected into heparinized tubes and then centrifuged.Plasma was stored at -20 °C until assayed for testosterone (RIA, Immunotech, Slovakia), T3 (RIA, Immunotech, Slovakia) and total lipids in plasma (Bio-la-test, Lachema, Czech Republic).Liver was weighted and taken for determination of the total lipids (Bio-la-test, Lachema, Czech Republic).
Data are presented as means ± S.E.M.The results were analysed by two-way analysis of variance (ANOVA) and Student's t-test.

Results
In all trials quail from groups treated with leptin during the embryonic period hatched earlier (about 5 -24 h) than the control group.Leptin injected into eggs did not evoke developmental malformations in quail and did not change the gross weight of internal organs.The hatchability was similar in all groups (C: 71.2%; L1: 69.5%; L2: 73.6%).The mean body weights of hatched quail were as follows: C group: 6.23 ± 0.07 g; L1 group: 6.69 ± 0.07 g (p < 0.001 vs. C) and L2 group: 6.46 ± 0.07 g (p < 0.05 vs. C).
There was a significant effect of treatment (F2,2960=16.6,p < 0.001) on body weight.Mean body weights across the whole observation period were higher in birds from both groups treated with leptin compared to those in the control group (p < 0.05) (Fig. 1).T3 levels were significantly lower in L1 and L2 groups (p < 0.05) on the day of hatching in comparison with the control (C: 7.44 ± 1.28 nmol/l; L1: 4.86 ± 0.23 nmol/l; L2: 4.26 ± 0.08 nmol/l).In the following days, the concentrations of plasma T3 in treated quail did not differ from control levels.
The highest food intake was observed in the group treated with the lower leptin concentration (L1), but the differences between groups were not significant., where A is the asymptotic size, K the rate constant of the equation and ti is the inflection point of the growth curve (Starck and Ricklefs, 1998).Solid lines -control group, dashed lines -L1 group (0.1µg of leptin) and dotted lines -L2 group (1,0 µg of leptin).
daily body weight gain up to somatic maturity, was also recorded in the L1 group.The feed efficiency was approximately equal in all three groups (Table 1).
Liver weights from all groups did not differ during the period of intensive growth, but was higher in the L1 group during the egg laying period (Fig. 2A).The standard developmental pattern of total lipids in the liver exhibited higher values after hatching, decreased levels during the rapid growth phase and the expected increase afterwards, which paralleled egg laying rate (Fig. 2B).A significant decrease in total lipids in plasma was found on the day of hatching in both leptin groups.Later, during the period of intensive growth and before sexual maturity, the differences among the groups were not significant (Fig. 2C).
An earlier onset of egg laying was observed in females treated with 0.1 µg of leptin (L 1 -2 days) compared with the control group.Females from leptin groups reached 100% eggs laying earlier than the control group.The production of total egg mass (total weight of eggs) per one quail and the egg laying rate were higher in both leptin groups (Fig. 3A,B).The highest average production of egg mass per quail and per day was in the L 1 group (6.15 g, in L 2 6.01 g, and in the control group 5.43 g).

Discussion
Leptin gene homologues have been cloned in several species.The leptin homology among avian and mammalian species is a subject of discussion (Taouis et al. 1998;Friedman-Einat 1999), but recent phylogenetic analysis shows that the mouse leptin gene sequence has a higher similarity with the chicken sequence than with those of other mammals (Doyon et al. 2001).Because of the close similarity in the primary structures of the mammalian and avian leptin, and because it has been found that chick embryonic muscle cells and hepatocytes are sensitive to mouse leptin (Lamo‰ová and Zeman 2001), we used commercially available recombinant murine leptin in our experiments.
Leptin probably plays an important role in embryonic development, as evidenced by production of leptin in placenta and widespread expression of leptin and its receptors in Table 1 The feed consumption converted to body weight gain (g/g) per one quail from day 5 till day 56 of life.
C -control group; L1 -0.1 µg of leptin ; L2 -1 µg of leptin.(Masuzaki et al. 1997).It has been hypothesized that leptin may play a physiological role in the implantation of fertilized eggs and in the subsequent early embryonic development (Gonzalez 2000).The avian embryo, which develops in isolation and is not maternally influenced, seems to be an appropriate model for studying the developmental processes induced by leptin in higher vertebrates.The presence of leptin mRNA was demonstrated in the brain, bursa Fabricii, heart, liver, muscle, and spleen of 5-day-old Leghorn embryo (Ashwell et al. 1999).Moreover, leptin expression was detectable as early as 72 hours in the developing chick embryo (McMurtry et al. 2000).Presence of leptin mRNA in the developing chick embryo, similar to rodent embryos (Hoggard et al. 1997) suggests that leptin has paracrine and endocrine effects on embryogenesis.Leptin expression in the embryonic yolk sac implicates that this hormone may control energy expenditure by mediating nutrient transfer to the developing embryo.Our data suggest that administration of leptin into quail eggs at ED 5 may affect the developing neuro-endocrine axis.In all our experiments quail from groups treated with leptin hatched significantly earlier than those from the control group, and their body weights were higher throughout the whole time period in which we observed them.This means that the higher leptin levels during embryonic development caused the faster development of the embryo.
It is known, that concentrations of serum T3 in Japanese quail are highest during internal pipping and then decrease abruptly to the day of hatching (M cNabb 1987).In our experiments we measured significantly lower concentration of T3 in leptin treated groups in comparison to controls at the day of hatching (< 24 h old).This fact corresponds with the earlier hatching of quail from leptin treated groups.
It has been proposed that leptin is one of the signals controlling sexual maturation and is involved in the regulation of reproductive functions (Reitman et al. 2001).Administration of leptin to infertile ob/ob (leptin deficient) mice stimulates all aspects of their reproductive endocrine system and rescues their fertility (Barash et al. 1996;Chehab et al. 1997).Injection of leptin accelerates sexual maturation in normal weanling mice as well as the timing of the first litter (Ahima et al. 1997).These data suggest that this hormone functions as a trophic factor for the reproductive system in mammals, but there are still very limited data in birds.In our experiments quail females from both leptin treated groups reached sexual maturity earlier than controls (evaluated by the first laid egg and reaching 100% of egg laying).We hypothesize that the higher leptin levels during embryonic development evoked permanent changes in the neuroendocrine system controlling reproductive processes and consequently inducing earlier sexual maturity (higher concentration of testosterone in males and earlier onset of egg laying in females).Higher concentrations of lipids in the liver and higher weights of the liver in the leptin groups are also probably connected with the earlier onset of egg laying.
In summary, our results suggest that murine recombinant leptin administered during the embryonic period acts as a growth factor and accelerate avian development.

Fig. 1 .
Fig.1.Growth curves of Japanese quail after in ovo leptin administration on day 5 of incubation.Body weight data were fitted by Gompertz function: W=A*e e (-K*(t-ti)) , where A is the asymptotic size, K the rate constant of the equation and ti is the inflection point of the growth curve(Starck and Ricklefs, 1998).Solid lines -control group, dashed lines -L1 group (0.1µg of leptin) and dotted lines -L2 group (1,0 µg of leptin).

Fig. 2 :
Fig.2: A-Liver weights, B-amount of total lipids in liver and C-concentrations of total lipids in plasma of Japanese quail after in ovo leptin administration on day 5 of incubation.Open columns designate the control group, left hatched columns L 1 and shaded columns L 2 group.* P < 0.05; ** P < 0.01 vs. control.Data are means from six animals ± S.E.M.
Fig.3: A-Production of total egg mass and B-rate of egg production by one quail after in ovo leptin administration on day 5 of incubation.The legend is the same as in Fig.1.Data are means from 6-8 animals.

Table 2
Weight of testes (g) of Japanese quail after in ovo leptin administration on day 5 of incubation.