Leptin and hypothalamic gene expression in early-and late-maturing Bos indicus Nellore heifers

We investigated wether early-maturing or late-maturing Bos indicus Nellore heifers produced more leptin mRNA in adipose tissues and altered expression of hypothalamic genes related to leptin signaling. Six prepubertal and six pubertal heifers aged about 34 months and weighing 280 kg to 300 kg each were selected from a population of 100 Nellore heifers. Real-time PCR was used to quantify the expression of the leptin gene (LEP) in adipose tissues and the long isoform of the leptin receptor gene (Ob-Rb), the NK2 homeobox 1 hypothalamic marker gene NKX2-1, the suppressor of cytokine signaling 3 gene (SOCS-3), the neuropeptide Y genes (NPY) and the NPY G-protein coupled receptor genes NPY-Y1 and NPY-Y4 in the hypothalamus. Heifers attaining puberty earlier showed significantly greater LEP expression in adipose tissues (p < 0.05) and there was tissue interaction (p < 0.05). Hypothalamic expression of Ob-Rb, NKX2-1, NPY and SOCS-3 did not differ between groups, but in early-maturing heifers there was a tendency for lower expression of NPY-Y1 (8.3-fold less) and NPY-Y4 (14.3-fold less) compared to late-maturing heifers (p = 0.1). These results suggest that a combination of higher LEP expression, lower NPY-Y1 and NPY-Y4 expression could be a factor in regulating puberty in early-maturing B. indicus heifers.


Introduction
Older age at puberty is responsible for lower slaughter rates in cattle production systems based on Bos indicus (Artiodactyla, Bovidae) breeds raised on pastures (Martin et al., 1992) because although B. indicus cattle are better adapted to harsh tropical conditions than Bos taurus breeds, they generally reach puberty when they are older and heavier (Thallman et al., 1999;Rodrigues et al., 2002).Thus, selection of early-maturing B. indicus heifers as well as nutritional protocols to advance puberty have received a great deal of interest from the scientific community (Vargas et al., 1998;Martinez-Velazquez et al., 2003).
The hypothalamic maturation process and the metabolic signal involved in regulation of puberty are not well understood (Kinder et al., 1995).Leptin, a 16 kDa protein hormone coded for by the LEP gene (also called the obese (ob) gene), has a key role in regulating energy intake and expenditure and has been proposed as an indicator of body adiposity and, in rodents, has a permissive role on puberty (Smith et al., 2002).Leptin acts in the hypothalamus through the long isoform of the leptin receptor (Ob-Rb).However, the molecular mechanism by which leptin signaling in the hypothalamus might be involved in initiation of puberty has not been elucidated.One possible mechanism is through neuropeptide Y (NPY) signaling, this peptide increases dramatically in cerebrospinal fluid during undernutrition and negatively modulates secretion of luteinizing hormone (LH) when centrally infused into cattle (Gazal et al., 1998).Given the inhibitory role of NPY on sexual maturation, leptin-mediated suppression of NPY expression in the arcuate nucleus is probably important in controlling pubertal development (Pedrazzini et al., 2003).
The action of NPY in the hypothalamus is modulated by a family of G-protein coupled receptors, consisting of at least five distinct members (Y1, Y2, Y4, Y5 and Y6) (Blomqvist and Herzog, 1997).The products of NPY-Y1 and NPY-Y4 mediate the detrimental effects of NPY on the gonadotrope axis through knockout models (Sainsbury et al., 2002;Pedrazzini, 2004).Our hypothesis was that latematuring B. indicus heifers have less LEP expression in adipose tissue and/or less sensitivity to leptin signaling in the hypothalamus.It was our objective to test whether earlymaturing B. indicus heifers had greater amounts of leptin mRNA in adipose tissues, and altered expression of hypothalamic genes related to leptin signaling.

Animals and treatments
In November 2003, 100 pastured Bos indicus heifers were selected based on breed attributes (Nellore), month of birth (November-2001), and body weight (between 280 kg and 300 kg), from a population of 500 heifers in a herd at the Hildergard Georgina Von Pritzelwitz experimental station managed by the University of São Paulo in Londrina, Paraná, Brazil.These 100 heifers were submitted to rectal palpation and scored as prepubertal or postpubertal according to the presence or absence of a palpable corpus luteum (CL) and ovary size.After scoring, 15 prepubertal heifers and 15 postpubertal heifers, born in the same month with similar body weight and body condition score, were selected and given a prostaglandin (PGF 2 α) injection to induce estrus (Ciosin, Coopers, Brazil).The occurrence of estrus was visually monitored in these 30 heifers on the third and fourth day after the PGF 2 α injection, and on the fourth day all 15 heifers that were previously scored as prepubertal and showed no sign of estrus after PGF 2 α injection were weighed and submitted to rectal palpation to confirm an absence of CL.All animal procedures were conducted in accordance with the Guidelines for the Care and Use of Agricultural Animals in Agricultural Research and Teaching (FASS, 1999).
Six heifers that had palpable CL and showed estrus after PGF 2 α injection (early-maturing), and six heifers that had no CL or signs of estrus (late-maturing) were selected for the experiment and slaughtered on the fifth day after PGF 2 α injection.The prepubertal or postpubertal conditions of the heifers were confirmed at slaughter by dissection of the ovaries.At slaughter, samples from the subcutaneous, omental and perirenal adipose tissues, and hypothalamus were collected, frozen in liquid nitrogen, and stored at -80 °C for subsequent analysis.The optical chiasm and mammilary bodies were used as anatomical markers for collecting the hypothalamus.For statistical purposes, the treatments were defined as the two heifer groups, early-maturing or late-maturing heifers.
Total RNA from tissue samples was isolated using TRIzol ® Reagent (Invitrogen, USA) according to Chom-czynski and Saachi (1987).The quality of isolated RNA was determined by measuring the absorbance at 260 and 280 nm and its integrity was verified as mainly 18S and 28S rRNA by electrophoresis in 1% (w/v) agarose gel.

Reverse Transcription (RT) PCR
We used 5 μg of total RNA from each tissue sample for cDNA synthesis.After denaturing at 70 °C for 10 min, half of the sample (2.5 μg) was reverse-transcribed into cDNA with 0.5 μg of oligo thymidine and 200 units of Superscript II reverse transcriptase (RT) (Invitrogen) in a final volume of 20 μL, for 60 min at 42 °C.The other half was incubated without reverse transcriptase and used as a negative control in polymerase chain reactions (PCR) to confirm the absence of residual genomic DNA contamination.
Oligonucleotide primer pairs specific for LEP, Ob-Rb, suppressor of cytokine signaling 3 (SOCS-3), NPY, NPY-Y1 and NPY-Y4 were designed based on bovine Gen-Bank sequences (Table 1).One μL of each RT reaction was used as template for PCR reactions in a final volume of 50 μL with 1.5 mM MgCl 2 , 0.4 mM of each deoxynucleotide triphosphate (Invitrogen), 0.25 μM of each primer (Invitrogen), 4 units of Taq polymerase (Invitrogen), and 1× PCR buffer (Invitrogen).The following amplification conditions were used: 95 °C for 5 min followed by 40 cycles at 95 °C for 45 s, 55 °C for 60 s and 72 °C for 1 min.After the last cycle, the reactions were held at 72 °C for 10 min.
Primers specific for the ribosomal protein gene RP-L19 (also known as L19) were used as positive controls for all samples to verify the success of the RT reactions, and primers specific for the NK2 homeobox 1 hypothalamic marker gene NKX2-1 (also known as TITF1) were used to verify possible sampling variations when dissecting the hypothalamus.In addition to incubating the RNA without reverse transcriptase, negative control reactions were performed similarly without addition of a template from the RT reaction.Amplified cDNA were visualized by agarose gel electrophoresis and staining with ethidium bromide.

Quantification of gene expression
Quantification of gene expression was performed using the LightCycler Real-Time PCR System (Roche Diagnostics; Switzerland) based on the second derivative maximum method.Samples without cDNA were used as a negative control.With this method a second derivative maximum within the exponential phase of the amplification curve is linearly related to the starting concentration of template cDNA molecules.
A master-mix of the reaction components was prepared with 1.1 mM MgCl 2 , 110 nM forward primer, 110 nM reverse primer, 222 μM dNTP, 278 mg/L bovine serum albumin, 0.08 units/μL of Taq Platinum DNA poly-merase (Invitrogen) and 45,450-fold diluted nucleic acid stain Sybr Green I (Invitrogen).LightCycler mastermix (18 μL) was filled in the LightCycler glass capillaries, and 2 μL of cDNA added as PCR template.The capillaries were closed, centrifuged for 5 s at 700 x g and placed into the LightCycler rotor.The following LightCycler experimental run protocol was used: denaturation program (95 °C for 5 min), amplification and quantification program repeated 40 times (95 °C for 10 s, 55 °C for 15 s, 72 °C for 20 s), melting curve program (75-95 °C with a heating rate of 0.1 °C per second and a continuous fluorescence measurement) and a final cooling step to 40 °C.

Statistical analysis
To calculate gene expression, a relative quantification method was used (Yuan et al., 2006): where E is the amplification efficiency of each gene, Ct the threshold cycle and ΔCt the late-maturing (LM) threshold cycle minus the early-maturing threshold cycle (ΔCt = Ct LM -Ct EM ).The relative expression method was used to minimize possible variations due to the efficiency of reverse transcription and quantity of template utilized.A dilution curve with a series of cDNA concentrations was calculated for each gene to obtain the amplification efficiency.The SAS procedure Proc Mixed (SAS, 2000) was used to perform simple linear regression for each group based on the model described by Yuan et al. (2006).The amplification efficiency (E) was calculated as E = 2 (-1/slope) .If the E values were not different than 2, then the gene expression ratio (R) was calculated using the equation R = 2 -ΔΔCt , in which When the E values were different than 2 we adjusted ΔΔCt using the percentage amplification efficiency (PE) to give ΔΔCt_adj = PE RP-L19 * ΔCt RP-L19 -PE target *ΔCt target and the gene expression ratio was calculated R = 2 -ΔΔCt_adj .Data were analyzed using analysis of covariance (ANCOVA) considering the fixed effects of treatment, gene and the treatment × gene interaction, as well as the random effects of the animals (treatment) and gene × treatment (animal).Significance was assumed to be p < 0.05 and tendency was assumed to be p < 0.1.

Results
When testing the efficiency of gene amplification, regression analysis resulted in regression coefficient (R 2 ) values above 0.96, indicating a linear relationship between starting cDNA concentration and Ct.The efficiency of amplification for LEP, RP-L19 and NPY-Y1 indicated a slope significantly different than -1, therefore the ratio of gene expression was calculated considering the calculated efficiency of amplification (Table 2).For the other genes, efficiency was considered to be equal to 2.
Heifers that attained puberty earlier had greater leptin gene expression in adipose tissue (p < 0.05, Table 3), and there was a significant treatment by tissue interaction (p < 0.05, Table 3).After adjusting for differences in the housekeeping gene RP-L19 expression, LEP expression was detected by real-time PCR at 2.1 cycles earlier in the postpubertal heifers than in the prepubertal heifers (ΔΔCT = -2.1,Table 3), which represents an average increase of 4.3-fold in LEP expression among the three adipose tissue depots (Figure 1).Early-maturing heifers had greater LEP expression in omental fat depot (10-fold increase, p = 0.05) and subcutaneous fat depot (6.9-fold increase, p = 0.01), while there was no effect on LEP ex- pression in the perirenal fat depot (1.4-fold increase, p > 0.60) (Figure 1).Hypothalamic expression of the long isoform of leptin receptor (Ob-Rb) was not different between heifer groups (p > 0.50, Table 4).Dissection of the hypothalamus was based on anatomical markers and there should have been no difference between the two groups of heifers in the area of the brain that was sampled.This was checked by analyzing the expression of the NK2 homeobox 1 hypothalamic marker gene NKX2-1 (also known as TITF-1) which is only expressed in the hypothalamus and is not involved in reproductive events (Suzuki et al., 1998).We found that NKX2-1 expression was not affected by treatment (p > 0.90, data not shown), demonstrating that there was no difference between the heifer groups in the area of the brain sampled.There was also no treatment effect on SOCS-3 expression by the hypothalamus (p > 0.80, Table 4).The expression of NPY in the hypothalamus was not statistically different (p > 0.7) between heifer groups (Table 4).However, for heifers that reached puberty earlier there was a tendency (p = 0.1) for less expression of NPY-Y1 (8.3-fold less) and NPY-Y4 (14.3-fold less), as shown in Table 4 and Figure 2. When the data for both NPY receptors, NPY-Y1 and NPY-Y4 were analyzed together, there was a statistically significant (p = 0.03) 11-fold reduction in expression in earlymaturing heifers (data not shown).

Discussion
The increased LEP expression in adipose tissue of early-maturing heifers supports the idea that the circulating concentration of leptin is an important signal for the initiation of pubertal processes (Ahima et al., 1997), and suggests that heifers with greater adipose tissue LEP expression could attain puberty earlier and at lighter body weight.Although well adapted to tropical grazing conditions, B. indicus cattle reach puberty at a much later age than most B. taurus breeds, even when both breeds are raised under similar environmental and nutritional conditions (Rodrigues et al., 2002).Later puberty results in a later age at first calving and reduces the slaughter rate and 660 Vaiciunas et al.   overall efficiency of cow-calf operations using B. indicus breeds in grazing systems.
In both B. indicus and B. taurus heifers, early stage of sexual maturation is regulated by the maturation of the hypothalamus (Evans et al., 1994;Rodrigues et al., 2002).The physiological regulation for maturation of the hypothalamus leading to puberty is not well understood, but clearly both body weight and chronological age influence the onset of puberty (Moran et al., 1989;Kinder et al., 1995).Nutrition is also an important element determining reproductive status in cattle and other mammals.A complex and controversial relationship between body fatness and the control of the reproductive axis was proposed by Frisch et al. (1980).
More recently, it has been postulated that leptin could explain the link between body fat, nutrition and control of the reproductive axis, with plasma concentrations of leptin being permissive to puberty in rodents (Barash et al., 1996).Exogenous administration of leptin in feed restricted beef heifers is able to increase LH concentrations and gonadotropin-releasing hormone (GnRH) stimulated LH secretion by the pituitary (Amstalden et al., 2002;Maciel et al., 2004a;Zieba et al., 2004).However, leptin administration for 40 days in well fed heifers was not able to advance puberty or increase LH secretion (Maciel et al., 2004b;Zieba et al., 2004).These and other studies suggest that acute or chronic feed restriction can sensitize the reproductive axis to leptin (Dyer et al., 1997;Nagatani et al., 2000).Taken together, these results indicate that in B. indicus heifers raised on pasture, with less body fat, averaging 300 kg at age 36 months, as is very common in grazing systems, the circulating concentrations of leptin should be limiting GnRH secretion and the onset of puberty.Therefore, those heifers with greater adipose tissue LEP expression could reach puberty earlier at a lower body weight.
Our results agree with the findings of other workers showing that amount of leptin mRNA is less in subcutaneous tissue than in perirenal adipose tissue (Kim et al., 2000;Yang et al., 2003).The reason for the greater LEP expression in perirenal fat could be explained by a greater adipocyte diameter (Yang et al., 2003).Fasting-induced decrease of LEP expression occurs predominantly in the subcutane-ous fat, while there is no effect in either perirenal or omental fat depots (Kim et al., 2000).In our study, attainment of puberty was related to LEP expression in the subcutaneous and omental fat depots, but not in the perirenal fat depot.
In humans and rodents, leptin signaling in the hypothalamus is essential for sexual maturation and leptin receptor deficient rodents cannot attain puberty (Huszar et al., 1997).This suggests that it is possible that early-maturing heifers could have a greater Ob-Rb expression in the hypothalamus and, therefore, reach puberty with less circulating leptin.However, our results do not support this hypothesis because B. indicus heifers that reached puberty earlier had greater LEP expression but equal Ob-Rb expression in the hypothalamus.In another study with cattle, Ob-Rb expression in the hypothalamus was not different in Holstein and Charolais bulls (both B. taurus) at 18 months of age, although Holstein cattle reached puberty on average  at an earlier age than Charolais cattle (Ren et al., 2002).However, Ob-Rb expression in the hypothalamus of rams was altered by photoperiod, with rams subjected to a long day-length photoperiod being sexually inactive, had similar circulating leptin but greater Ob-Rb expression than rams exposed to a short-day photoperiod (Clarke et al., 2003).
The expression of NPY was also higher in the hypothalamus of rams subjected to a long-day photoperiod (Clarke et al., 2003).Because NPY is a potent inhibitor of GnRH secretion by the hypothalamus, this could explain why rams under a long-day photoperiod were sexually inactive although they had similar plasma leptin concentration and greater Ob-Rb expression than sexually active rams under a short-day photoperiod (Clarke et al., 2003).
In mammalian cell lines, SOCS-3 acts as an inhibitor of leptin signaling and leptin administration is capable of increasing SOCS-3 mRNA in the hypothalamus of ob/ob mouse and other mammals (Bjorbaek et al., 1998;Eyckerman et al., 2000).It is thought that there is a feedback mechanism, by which an increase of leptin induces SOCS-3 expression and directly inhibits leptin receptor signaling, which would be responsible for creating leptin resistance (Bjorbaek et al., 1998).In our study, although there was greater adipose tissue LEP expression in heifers that attained puberty earlier SOCS-3 expression in the hypothalamus was not altered.
The reproductive axis of cattle is very sensitive to leptin when the cattle are malnourished (Maciel et al., 2004a) but leptin fails to stimulate the hypothalamic-adenohypophyseal axis of well-nourished sheep and cattle, suggesting that physiological resistance to leptin may occur in animals that are in neutral or positive energy balance (Amstalden et al., 2005).It was thought that well-fed animals could be resistant to leptin due to increased SOC-3 expression in the hypothalamus and adenohypophysis, however fasting increased and did not decrease the amounts of SOCS-3 mRNA in the adenohypophysis of cows (Amstalden et al., 2005).Taken together, these results suggest that SOCS-3 expression is not related to control of the reproductive axis in cattle.
Neurons from the arcuate hypothalamus nucleus produce NPY, one of the most abundant peptides in the hypothalamus (Friedman and Halaas, 1998;Williams et al., 2000).The various NPY functions are mediated by a family of NPY receptors (Y1, Y2, Y4, Y5 and Y6) (Gehlert, 1999) and NPY signaling in the hypothalamus regulates the effects of leptin on reproductive activity in rodents and primates (Aubert et al., 1998;Sainsbury et al., 2002).In ob/ob rats, a lack of leptin inhibition leads to chronically elevated NPY expression in the hypothalamus, while treatment with leptin reduces NPY and restores fertility (Sainsbury et al., 2002).Also, when centrally administered, NPY greatly inhibits sexual function in rats (Pierroz et al., 1996).Based on these observations it is possible that changes in hypothalamic NPY signaling could be involved in sexual matura-tion of heifers.In our study, although hypothalamic expression of NPY was the same in early-maturing or late-maturing heifers there was a tendency for less expression of the NPY-Y1 and NPY-Y4 receptors in the hypothalamus of early-maturing heifers.
The importance of NPY-Y1 signaling for the continuous maturation of the prepubertal hypothalamus has been demonstrated in knock-out models.Animals deficient in the Y1 receptor have increased pituitary LH and increased seminal vesicle size after 48 h of starvation (Pedrazzini et al., 1998).Daily injections of leptin into juvenile Y1 -/-female mice causes an advancement in puberty compared to wild-type mice, which is accompanied by an increase in uterus weight.An improved function of the gonadotropic axis is also seen in Y1 -/-, ob/ob double knockout mutant mice, which suggests that the permissive role of leptin in attainment of puberty is likely mediated by NPY and NPY-Y1 signaling (Pralong et al., 2002).Ablation of the Y4 receptor in ob/ob mice also restores fertility to 100% in male mice and improves fertility in female double knockout mice by 50% (Sainsbury et al., 2002).
In conclusion, LEP expression was greater in adipose tissues and expression of NPY receptor genes was less in the hypothalamus of early-maturing heifers.These results suggest that, because of the lower expression of NPY receptor genes, the hypothalamus of heifers reaching puberty earlier could be less sensitive to NPY inhibition.So, there are two detected pathways that could be responsible for hastened puberty in these heifers, greater LEP expression by the adipose tissue, and lesser expression of NPY receptor genes in the hypothalamus.
where Ct is the threshold cycle, RP-L19 is the ribosomal protein gene, PE is the percentage amplification efficiency, and ΔCt = Ct LM -Ct EM .† SEM = Standard Error of the Mean.

Figure 1 -
Figure 1 -Ratio of leptin gene expression in the adipose tissue depots of early-maturing and late-maturing Bos indicus Nellore heifers.Lines on bars are standard errors.*Ratio different from 1, p < 0.05.**Ratio different from 1, p < 0.01.

Figure 2 -
Figure 2 -Ratio of hypothalamic gene expression in early-maturing and late-maturing Bos indicus Nellore heifers.Lines on bars are standard errors.Tendencies for treatment differences (p < 0.10) between means are indicated by the symbol, ‡ bellow the appropriate bars.

Table 1 -
Oligonucleotide primer pairs designed for use in polymerase chain reaction (PCR) amplification

Table 2 -
Efficiency of gene amplification using real-time polymerase chain reaction amplification.

Table 3 -
Quantification of gene expression in the adipose tissue depots of early-maturing (EM) and late-maturing (LM) Bos indicus Nellore heifers.

Table 4 -
Quantification of hypothalamic gene expression in early-maturing and late-maturing Bos indicus Nellore heifers.PE RP-L19 × ΔCt RP-L19 -PE target × ΔCt target , where Ct is the threshold cycle, RP-L19 is the ribosomal protein gene, PE is the percentage amplification fficiency, and ΔCt = Ct LM -Ct EM .