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Article Type: Original Article

Nutritional Programming of Accelerated Puberty in Heifers: Involvement of POMC Neurones in the Arcuate Nucleus





Rodolfo C. Cardoso,1,2 Bruna R. C. Alves,2 Sarah M. Sharpton,2 Gary L. Williams,1,2 and Marcel Amstalden2*

1

Animal Reproduction Laboratory, Texas A&M AgriLife Research, Beeville, TX, USA

2

Department of Animal Science, Texas A&M University, College Station, TX, USA



*

Both authors contributed equally to this work

Deceased (September, 2014)

Short title: POMC and nutritional acceleration of puberty in heifers Key words: POMC, kisspeptin, arcuate nucleus, puberty, heifers Corresponding author: Gary L. Williams, Ph.D. Texas A&M AgriLife Research 3507 Highway 59E Beeville, TX, 78102 USA

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Tel: +1 (361) 358-4930 Fax: +1 (361) 358-4930 Email: [email protected] ABSTRACT Timing of puberty and subsequent fertility in female mammals are dependent on the integration of metabolic signals by the hypothalamus. Proopiomelanocortin (POMC) neurones in the arcuate nucleus (ARC) comprise a critical metabolic-sensing pathway controlling the reproductive neuroendocrine axis. Melanocyte-stimulating hormone alpha (αMSH), a product of the POMC gene, has excitatory effects on gonadotropin-releasing hormone (GnRH) neurones and fibres containing αMSH project to GnRH and kisspeptin neurones. Because kisspeptin is a potent stimulator of GnRH release, αMSH may also stimulate GnRH secretion indirectly via kisspeptin neurones. Here, we report studies in young female cattle (heifers) to determine whether increased nutrient intake during the juvenile period (4-8 months of age), a strategy previously shown to advance puberty, alters POMC and KISS1 mRNA expression, and αMSH close contacts on GnRH and kisspeptin neurones. In experiment 1, POMC mRNA expression, detected by in situ hybridisation, was greater (P < 0.05) in the ARC in heifers that gained 1 kg/day of body weight (high-gain, HG; n = 6) compared to heifers that gained 0.5 kg/day (low-gain, LG; n = 5). The number of KISS1-expressing cells in the middle ARC was reduced (P < 0.05) in HG compared to LG heifers. In experiment 2, double-immunofluorescence showed limited αMSH-positive close

contacts on GnRH neurones, and the magnitude of these inputs was not influenced by nutritional status. Conversely, a large number of kisspeptin-immunoreactive cells in the ARC were observed in close proximity to αMSH-containing varicosities. Furthermore, HG heifers (n = 5) exhibited a greater (P < 0.05) percentage of kisspeptin neurones in direct apposition to αMSH fibres and an

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increased (P < 0.05) number of αMSH close contacts per kisspeptin cell when compared to LG heifers (n = 6). These results indicate that the POMC-kisspeptin pathway may be important in mediating the nutritional acceleration of puberty in heifers. INTRODUCTION Feeding a high-concentrate diet to young female cattle (heifers) during the juvenile

period, resulting in increased body weight (BW) gain and circulating concentrations of leptin, leads to early maturation of the reproductive neuroendocrine system and earlier puberty (1-4). Importantly, a critical window for the nutritional programming of accelerated puberty occurs around 4 to 6.5 months of age (early juvenile stage), because a period of feed restriction after this time point does not reduce the proportion of heifers that reach early puberty (3). The initiation of a characteristic high-frequency pattern of episodic release of gonadotropin-releasing hormone (GnRH) during pubertal transition is largely dependent upon metabolic cues received during early development. Although it is clear that the adipocyte-derived hormone, leptin, is critically implicated in the pubertal activation of the central reproductive system, the afferent neuronal pathways involved in this process remain unresolved. Proopiomelanocortin (POMC) neurones in the arcuate nucleus (ARC) contain leptin

receptors (5-7), control energy homeostasis (8,9), and have been proposed to play a role in the metabolic control of reproduction in rodents (10). Transgenic mice overexpressing agouti-related peptide (AgRP), an endogenous antagonist of the melanocortin receptors 3 and 4 (MC3R/4R), are obese and infertile (11). POMC neurones have been demonstrated to make direct synaptic contact with GnRH neurones in rats (12, 13) and α-melanocyte-stimulating hormone (αMSH), a product of the POMC gene, activates GnRH neurones in female mice as result of direct postsynaptic activation of MC3R/4R (14). POMC-immunoreactive fibres also project to

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The hypothalamic NPY system also plays an important role in the nutritional regulation of reproduction in females. In prepubertal heifers, elevated nutrient intake during the juvenile period decreased both NPY mRNA expression in the ARC and NPY-immunoreactive inputs to GnRH neurons (21). Because NPY has been demonstrated to inhibit GnRH release in cows (49) and central administration of NPY delays the onset of puberty in rats (50), attenuation of inhibitory NPY tone also appears to be an important event during pubertal transition in heifers. Therefore, collectively, our previous (21) and current findings suggest that functional changes in key metabolic-sensing circuitries, including the NPY and POMC pathways, are likely involved in the nutritional advancement of puberty in heifers. In summary, elevated nutrient intake during the juvenile period increased POMC mRNA

expression in the ARC of prepubertal heifers. Although few GnRH neurones were observed in close apposition with αMSH fibres, kisspeptin neurones in the ARC may constitute a neuronal pathway through which melanocortins mediate the permissive effects of leptin on onset of puberty in heifers.

DECLARATION OF INTEREST The authors of the manuscript have no conflicts of interest to declare.

FUNDING This project was supported by the Agriculture and Food Research Initiative Competitive

Grant no. 2009-65203-05678 from the USDA National Institute of Food and Agriculture.

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AHA) and mediobasal hypothalamus (MBH). (C) Mean (± SEM) percentage of GnRHimmunoreactive neurones in close proximity with αMSH-containing fibres in the POA+AHA and MBH. (D) Mean (± SEM) number of αMSH-containing varicosities in close contact with GnRH-immunoreactive soma and proximal dendrites in the POA+AHA and MBH. Scale bar: (A) 20 μm. LG, low-gain; HG, high-gain. Fig. 5 αMSH close contacts on kisspeptin cell bodies and proximal dendrites in the arcuate nucleus (ARC) of prepubertal heifers. (A) Confocal microscope image depicting a kisspeptinimmunoreactive neurone (red) in close apposition (arrows) with αMSH-containing varicosities (green). (B) Mean (± SEM) number of kisspeptin-immunoreactive cells/animal in the rostral (rARC), middle (mARC), and caudal (cARC) subregions of the ARC. (C) Mean (± SEM) percentage of kisspeptin-immunoreactive neurones in close proximity to αMSH-containing fibres in the ARC. (D) Mean (± SEM) number of αMSH-containing varicosities in close contact with kisspeptin-immunoreactive soma and proximal dendrites in the ARC. Scale bar: (A) 50 μm. LG, low-gain; HG, high-gain. *P < 0.05; **P < 0.01; ***P < 0.001.

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Table 1 Primer sequences for synthesis of DNA template used for in situ hybridisation detection of POMC and KISS1 in bovine preoptic area and hypothalamus.

Gene

Primer sequence (5’- 3’)

5' promoter*

Type of probe

AGAGGACAAGCGTTCTTACT

T7

Sense

TGGCTCTTCTCCGAGGTCA

T3

Antisense

AAGCCCACAGCGGCCGG3

T3

Sense

TCCTTTATTGCTTCGGGACA

T7

Antisense

POMC

KISS1

* Primers were designed including the T3 promoter (5'-AATTAACCCTCACTAAAGGG-3') or T7 promoter (5'-TAATACGACTCACTATAGGG-3') within the 5' end of the sequence.

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detected in these areas was too limited for valid, meaningful comparisons. Images were analysed using the NIS Elements software (Nikon Inc.) after normalisation by the background density of silver grains which was determined from regions adjacent to the area of interest using procedures described previously (23). Anatomical subdivisions of the ARC were determined as described previously in sheep (23). Experiment 2: αMSH immunoreactivity and close contacts on GnRH and kisspeptin neurones in prepubertal heifers Blocks of tissue containing the septum, POA, and hypothalamus obtained from a

different subset of prepubertal heifers (LG, n = 6; HG, n = 5) were cut in approximately 3-mm coronal blocks and placed in a solution of 0.1M phosphate buffer (PB) containing 4% paraformaldehyde for 48 h as described earlier (21). Tissue blocks were then immersed in a PB solution containing 30% sucrose for 5 to 7 days. Tissue blocks were then cut in 50-µm sections using a freezing sliding microtome, placed in cryoprotectant solution, and stored at -20°C until processing. Immunohistochemistry A series of sections 200 μm apart was cut sagittaly in two halves. One series of

hemisections was processed for double-label detection of αMSH and GnRH by immunofluorescence. The contralateral series of hemisections was used for double-label immunofluorescent detection of αMSH and kisspeptin. Procedures were carried out on freefloating sections at room temperature under gentle agitation following procedures similar to those described previously (21, 24). For the αMSH/GnRH double-label immunofluorescence, sections were incubated overnight at room temperature with mouse monoclonal anti-GnRH (1:10,000; Covance, Princeton, USA; cat#SMI-41R), followed by incubation with Alexa 488-

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conjugated goat anti-mouse IgG (1:200; Molecular Probes, Eugene, USA; cat#A-11001). Detection of αMSH was performed by incubation with guinea pig anti-human αMSH antiserum (1:40,000; Biosensis, Adelaide, Australia; cat#GP-030-50) overnight at room temperature, followed by incubation with biotinylated goat anti-guinea pig IgG (1:400; Vector Labs, Burlingame, USA; cat#BA7000) for 1 h. Alexa 555-conjugated streptavidin (1:250, Invitrogen, Carlsbad, USA; cat#S-21381) was used to detect αMSH immunoreactivity. For the αMSH/kisspeptin double-label immunofluorescence, sections were submitted to

heat-induced epitope retrieval by incubation in 10mM Sodium Citrate buffer (pH 6) at 90°C for 18 min to enhance detection of kisspeptin, as described previously (21). Sections were incubated

overnight at room temperature with rabbit anti-kisspeptin10 (1:250,000; graciously provided by Dr. Alain Caraty, INRA Centre de Tours, Nouzilly, France; AC#564), followed by biotinylated goat anti-rabbit IgG (1:400; Vector Labs; cat#BA-1000) and streptavidin-conjugated horseradish peroxidase (1:600; Vectastain Elite ABC, Vector Labs; cat#PK-6100). Alexa 555-conjugated streptavidin (1:250; Invitrogen; cat#S-21381) was used to label kisspeptin immunoreactivity. Detection of αMSH was performed using guinea pig anti-human αMSH antiserum (1:20,000; Biosensis; cat# GP-030-50) followed by incubation with Alexa 488-conjugated goat anti-guinea pig IgG (1:200; Molecular probes; cat#A-11073). After completion of the immunostaining, sections were mounted on glass slides using Gelvatol medium. Controls for the dual-label immunofluorescence procedure included omission of primary

antibodies (anti-GnRH, anti-αMSH, and anti-kisspeptin10) and pre-absorption of primary antibodies with GnRH (100 μM; Sigma-Aldrich, St Louis, USA), αMSH (100 μM; Phoenix

Pharmaceuticals Inc., Burlingame, USA), and ovine kisspeptin (3mM; American Peptide

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Company Inc., Sunnyvale, USA). These procedures eliminated the fluorescent signal for each corresponding antigen. Sections were analysed using an epifluorescent microscope (Nikon eclipse 80i, Nikon

Inc.) and all quantification analyses were performed by an observer blind to the assignment of tissue sections within experimental groups. Close contacts were defined as direct apposition between αMSH-containing varicosities and GnRH- and kisspeptin-immunoreactive soma and proximal dendrites observed in the same focal plane using a 40X objective. To assess the accuracy of this method, images of selected sections containing 10 kisspeptin neurones in the ARC were acquired in Z-stacks of 1-µm optical sections using a Zeiss 510 Meta NLO laserscanning confocal microscope (Zeiss, Heidelberg, Germany). The comparison of direct apposition data obtained by visual counts using the epifluorescent microscope (47 close contacts/10 kisspeptin neurones) and by confocal image analysis (44 close contacts/10 kisspeptin neurones) resulted in 93.6% concordance. In order to determine the number of αMSH- and kisspeptin-immunoreactive cells, and the percentage of ARC kisspeptin cells in close contact to αMSH fibres, 3 comparable sections of the rARC and cARC, and 5 comparable sections of the mARC, were selected from each animal. In our previous study in prepubertal heifers (21), we were not able to reliably detect kisspeptin-immunoreactive cells in the POA due to the limited number of cells observed and high variability between animals. Thus, the number of kisspeptinimmunoreactive cells in the POA and percentage of these cells in direct apposition to αMSH fibres were not determined in the present study. All GnRH neurones visualised in sections containing the POA and MBH were utilised to determine the percentage of GnRH cells in direct apposition to αMSH-containing varicosities. Statistical Analysis

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The JMP software (SAS Inst. Inc., Cary, NC) was used for statistical analyses. The percentage of GnRH and kisspeptin neurones in close proximity to αMSH containing fibres were normalised using the arcsine square root transformation method as described previously (21). Optical density of silver grains per region and per cell, number of KISS1mRNA-expressing cells, number of αMSH- and kisspeptin-immunoreactive cells, mean number of αMSH close contacts to GnRH and kisspeptin neurones, and normalised percentages of GnRH and kisspeptin cells in close proximity to αMSH varicosities were compared between LG and HG groups using the Student’s t-test. Each hypothalamic region was analysed independently. RESULTS Confirmation of prepubertal status at the time of tissue collection No luteal tissue was observed in any of the heifers by post mortem visual inspection of

the ovaries. In agreement, none of the heifers presented circulating concentration of progesterone above 1 ng/ml during the 2 weeks preceding euthanasia. During this period, mean circulating concentrations of progesterone did not differ between LG (0.11 ± 0.01 ng/ml) and HG (0.12 ± 0.01 ng/ml) heifers. Experiment 1: Hypothalamic POMC and KISS1 mRNA expression in prepubertal heifers POMC mRNA expression in the ARC Hybridisation of tissue sections with antisense probe to POMC mRNA labelled an

abundant number of cells in the ARC of prepubertal LG (Fig. 1A) and HG heifers (Fig. 1B). The mean optical density of silver grains in the rARC and mARC was greater (P < 0.05) in HG than LG heifers (Fig. 1C). In the cARC, a trend (P = 0.07) was observed for greater POMC mRNA optical density in HG than LG heifers (Fig. 1C). Furthermore, HG heifers presented greater (P

Nutritional Programming of Accelerated Puberty in Heifers: Involvement of Pro-Opiomelanocortin Neurones in the Arcuate Nucleus.

The timing of puberty and subsequent fertility in female mammals are dependent on the integration of metabolic signals by the hypothalamus. Pro-opiome...
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