Neuropeptidome of the Hypothalamus and Pituitary Gland of Indicine

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Neuropeptidome of the hypothalamus and pituitary gland of indicine x taurine heifers: evidence of differential neuropeptide processing in the pituitary gland before and after puberty Kasey L. DeAtley, Michelle L Colgrave, Angela Canovas, Gene Wijffels, Ryan L. Ashley, Gail A Silver, Gonzalo Rincon, Juan F. Medrano, Alma Islas-Trejo, Marina R. S. Fortes, Antonio Reverter, Laercio Porto-Neto, Sigrid A. Lehnert, and Milton G. Thomas J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.7b00875 • Publication Date (Web): 07 Mar 2018 Downloaded from http://pubs.acs.org on March 7, 2018

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Journal of Proteome Research

Neuropeptidome of the hypothalamus and pituitary gland of indicine x taurine heifers: evidence of differential neuropeptide processing in the pituitary gland before and after puberty

Kasey L. DeAtley†Φ*, Michelle L. Colgrave‡, Angela Canovas††, Gene Wijffels‡, Ryan L. Ashley†, Gail A. Silver†, Gonzalo Rincon§, Juan F. Medranoψ, Alma Islas-Trejoψ, Marina R. S. FortesΠ, Antonio Reverter‡, Laercio Porto-Neto‡, Sigrid A. Lehnert‡ and Milton G. Thomas∫

†

Department of Animal and Range Sciences, New Mexico State University, Las Cruces, 88003,

USA; ‡ CSIRO, Agriculture and Food, 306 Carmody Road, St. Lucia, QLD 4067, AU; ††

Department of Animal Biosciences, University of Guelph, ON N1G 2W1, CA; ψDepartment of

Animal Science, University of California, Davis, CA 95616, USA; §Zoetis Animal Health, Kalamazoo, MI, USA; ΠSchool of Chemistry and Molecular Biosciences, University of Queensland, Brisbane St Lucia, QLD, 4042 and the Queensland Alliance for Agriculture and Food Innovation, Australia; ∫Department of Animal Science, Colorado State University, Fort Collins, CO 80523, USA

1 ACS Paragon Plus Environment

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ABSTRACT: Puberty in cattle is regulated by an endocrine axis which includes a complex milieu of neuropeptides in the hypothalamus and pituitary gland. The neuropeptidome of hypothalamic-pituitary tissue of pre- (PRE) and post-pubertal (POST) Bos indicus-influenced heifers was characterized, followed by quantitative analysis of 51 fertility-related neuropeptides in these tissues. Comparison of peptide abundances with gene expression levels allowed assessment of post-transcriptional peptide processing. Based on classical cleavage, 124 mature neuropeptides from 35 precursor proteins were detected in hypothalamus and pituitary gland tissues of three PRE and three POST Brangus heifers. An additional 19 peptides (cerebellins, PEN peptides) previously reported as neuropeptides that do not follow classical cleavage were also identified. In the pre-pubertal hypothalamus a greater diversity of neuropeptides (25.8%) were identified relative to post-pubertal heifers, while in the pituitary gland 38.6%, more neuropeptides were detected in the post-pubertal heifers. Neuro-tissues of PRE and POST heifers revealed abundance differences (p < 0.05) in peptides from protein precursors involved in packaging and processing (e.g. the granin family and ProSAAS) or neuron stimulation (PENK, CART, POMC, cerebellins). On its own, the transcriptome data of the precursor genes could not predict the neuropeptide profile in the exact same tissues in several cases. This provides further evidence of the importance of differential processing of the neuropeptide precursors in the pituitary before and after puberty.

Key words: cattle, heifer, hypothalamus, neuropeptide, neuropeptidome, peptide processing, pituitary gland, puberty, transcriptome, transcript.

INTRODUCTION


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Reproduction in mammals involves hormones from the hypothalamus, pituitary gland, and gonads, known as the hypothalamo-pituitary-gonadal endocrine axis.1,2 The transition from pre- to post-puberty in females is characterized by a biphasic pituitary gland response and the secretion of gonadotropins, luteinizing hormone (LH) and follicle stimulating hormone (FSH). Most important is the coupled pulsatile secretion of the hypothalamic-decapeptide, gonadotropin-releasing hormone (GnRH) by the hypothalamus and LH by the pituitary.3-5 Although our understanding of the molecular and physiological mechanisms driving puberty in humans and model species is already relatively detailed, investigation of the genes coding for elements of the central control of reproduction in livestock species is a worthwhile goal. Not only do such studies provide comparative information for vertebrate reproductive biology more generally, but they have the potential to lead to the development of tools for genetic selection and reproductive management in agricultural animals such as cattle. Genes encoding the precursors of regulatory neuropeptides are obvious candidates, and indeed SNP associated with genes coding for peptides, such as PENK, PROP1 and NPY, have been implicated in regulation of puberty in cattle.6-9 Neuropeptides are important regulators of many physiological processes such as reproduction, circadian rhythm, energy metabolism, appetite, and body temperature homeostasis.10-12 Hypothalamic neuropeptides are products of neuronal and glial cells, which make up the majority of the cellular population, and are secreted into the hypothalamohypophyseal portal system to stimulate or inhibit synthesis and secretion of pituitary gland hormones.13-14 Most studies of neuropeptides, puberty and reproduction of livestock have been constrained to investigating a few mature neuropeptides due to the limitations of reagents and the sensitivity of the technology to detect mature neuropeptides and their variants.



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Mass spectrometry (MS) is an effective technology for study of neuropeptides because it enables simultaneous detection of multiple peptides (i.e., the neuropeptidome) with high sensitivity.15-18 Peptidome studies, informed by well annotated genome sequences, identify the numerous endogenous peptides within a tissue and allow detection of post-translational modifications that cannot be predicted. These studies greatly expand our knowledge of genetic and physiologic regulation of fertility beyond genome-wide association study (GWAS) and transcriptome (RNA-Seq).19,6 Neuropeptidome reports, and the workflow from mass spectrometry analyses to database matching, were initiated in studies of rodents.20-23 Colgrave et al.15 expanded these efforts by using a combination of thermal stabilization and MS technologies to identify 140 neuropeptides in hypothalamic tissue of two mature Bos indicus beef cows. These results stimulated us to also analyze these types of tissues using multiple reaction monitoring (MRM) techniques to detect differential peptide expression in bovine reproductive models. The initial objective of this study was to describe the neuropeptidome of the hypothalamus and pituitary gland of pre- and post-pubertal Brangus heifers, which are Bos indicus-influenced composite cattle used in beef production systems in warm and hot climates. Subsequent objectives included quantitative assessment of neuropeptides known to affect reproduction by employing MRM-MS studies and to evaluate the strength of association of the quantitative peptide data and their gene expression values measured with RNA-Seq in a previous study.6 This effort highlighted the prospect of differential peptide processing where transcription was seen to be similar amongst tissues, which contained different levels of individual peptide products. This is the first comprehensive analysis of the pre- and post-puberty neuropeptidome of the pituitary and hypothalamus in any mammalian species. The data and subsequent analyses


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present evidence of differential processing of the neuropeptide precursors in the pituitary before and after puberty (and to a lesser extent in the hypothalamus). It should be noted that the transcriptome data for precursor genes in these very same tissues, were unable to predict the neuropeptide profile in several cases.

EXPERIMENTAL PROCEDURES Heifer puberty phenotypes and tissues The animals used in this study, their management and assessment of puberty has been described by Canovas et al.6 In brief, pre-pubertal (PRE; n = 4) heifers were 352.8 days of age and weighed 272.6 kg when tissues were harvested, whereas post-pubertal (POST; n = 4) were 381.5 days of age at tissue harvest and weighed 354.7 kg. Puberty was defined as when two consecutive serum progesterone values >1 ng/mL were observed 3 to 4 days apart. The animals were managed according to a protocol approved by the Institutional Animal Care and Use Committee of New Mexico State University (protocol #2010-013). Additional detail on the animal experiment is provided in Methods S1. The pubertal (POST) heifers had experienced increased levels of serum leptin and IGF-I and had completed an estrous cycle prior to tissues being harvested in the mid-luteal phase of the estrous cycle (i.e., 10 days post-estrous). Hypothalamus and the anterior and posterior pituitary gland tissues were obtained approximately 15 min post-mortem and immediately snap-frozen in liquid nitrogen. The hypothalamus was a 1 cm3 of tissue that included pre-optic and arcuate nuclei regions as well as any portion of the median eminence adjacent to the third cerebroventricle. The pituitary gland was the entire gland including both the anterior and posterior glands. During tissue sampling and laboratory processing, we failed to collect or



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process the hypothalamus of two heifers and the pituitary gland of one heifer. Therefore, 13 tissue samples (PRE hypothalamus: n = 3; PRE pituitary: n = 4; POST hypothalamus: n = 3; POST pituitary: n = 3) were available for LC-MS/MS analysis and 12 tissue samples (PRE hypothalamus: n = 3; PRE pituitary: n = 3; POST hypothalamus: n = 3; POST pituitary: n = 3) were available for RNA-seq and multiple reaction monitoring (MRM) mass spectrometry analyses. Statistical analyses in this study were conducted within tissues, so presentation of results, (i.e., titles of tables and figures) clarify the n of each analysis.

Transcriptomics data Hypothalamus and pituitary RNA-Seq data were obtained from Canovas et al6 (Table S2 and S3). Procedures of RNA extraction, sequencing and determining reads per kilobase of exon per million reads (RPKM) for each gene have been described in detail by Canovas et al.6

Thermal denaturation and peptide extraction Whole, frozen hypothalamus and pituitary gland samples were dissected to 5 mm diameter and treated with the Stabilizor T1 (Denator, Gӧteborg, Sweden) as described previously 15

to stabilize tissue peptide content for extraction and MS analyses. Briefly, 5 mm diameter

tissue samples were placed in a Maintainor card consisting of a Teflon-lined chamber. The chamber was evacuated (5-10 mbar) before the card entered the instrument for the peptide stabilization process. The time for sample treatment was determined automatically by a Class 1 laser measurement. The sample was then rapidly heated to 90˚C (not exceeding 95˚C) by the aluminum heating blocks. Subsequently, tissues were stored at -80˚C until peptide extraction. Stathmin, a highly conserved-ubiquitous protein, was used as a marker of sample integrity.


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Frozen and thermally denatured hypothalamus and pituitary gland samples were further dissected into smaller fragments and randomized to ensure each technical replicate was representative of the entire tissue. Hypothalamus samples (4 x 35 mg) and pituitary gland samples (2 x 35 mg) were processed in small portions to replicate previous studies. Individual portions were suspended in a cold acetic acid extraction solution (11 µL 0.25% acetic acid/mg tissue) and homogenized using microtip sonication (60 Sonic Dismembrator, Fisher Scientific, Pittsburgh, PA) at power level 3 for 10 s. The solutions were then centrifuged at 20,000 x g for 30 min. Supernatant was collected and applied to a Nanosep 10 kDa Omega filter (Pall Corporation, Ann Arbor, MI). Samples were centrifuged at 20,000 x g for 90 min. This initial filtrate was collected and put aside. Remaining tissue from the first centrifugation was re-suspended in 200 µL 0.25% acetic acid wash and re-centrifuged for 30 min; supernatant was collected and applied to the 10 kDa filter then centrifuged for 90 min at 20,000 x g (i.e., first wash). An additional 100 µL 0.25% acetic acid was added to filter as a second wash and centrifuged for 30 min. In total, ~300 µL of filtrate (~200 µL from first wash and 100 µL from second wash) were collected and pooled with the initial filtrate. The hypothalamic extract were pooled to yield neuropeptides derived from 70 mg of tissue (two technical replicates), whereas the pituitary gland extract was kept separate to yield neuropeptides derived from 35 mg of tissue (two technical replicates). The pooled filtrates were concentrated with a SpeedVac concentrator (Thermo Scientific, Pittsburgh, PA) to 20 µL volume and stored at -20˚C until MS analyses.

LC-MS/MS analysis



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Hypothalamus and pituitary gland extracts derived from 70 and 35 mg of tissue were reconstituted in 70 and 35 µL of 1% formic acid, respectively; thus, diluted to the equivalence of 1 mg tissue/µL. Samples (20 µL) were analyzed with a TripleTOF 5600 mass spectrometer (SCIEX, Redwood City, CA) for identification of neuropeptides. Peptides were chromatographically resolved on a nano HPLC (Shimadzu Scientific, Rydalmere, Australia) using an Agilent Zorbax (Agilent Technologies, Santa Clara, CA) MS C18 300 Å, column (150 mm x 75 µm) with a particle size of 3.5 µm. The mobile phases consisted of solvent A (0.1% formic acid in H2O) and solvent B (0.1% formic acid, 9.9% H2O and 90% acetonitrile). Peptides were loaded onto a C18 trap column and washed for 4 min at 30 μL/min to remove salts and other interfering reagents. The valve was switched such that the trap was in line with the column and the peptides were eluted at 300 nL/min using a linear gradient of 2-40% B over 30 min. The HPLC eluant was directed into a nanoelectrospray ionization source operated in a positive ion electrospray mode with an ionspray voltage of 2,500 V and an interface heater temperature of 150˚C. Analyses were performed in Information Dependent Acquisition (IDA) mode using Analyst TF 1.5 software (SCIEX). For IDA, survey scans were acquired over the mass range m/z 300-1800 for 0.5 s. The 20 most intense ions detected in the survey scan with charge state 2+ to 5+ were selected for tandem mass spectrometry (MS/MS). Tandem mass spectra were acquired over the mass range m/z 80-1800 with a 50 ms accumulation time using rolling collision energy settings. Precursor ions were selected twice and then excluded for 8 s.

Peptide Identification


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Data were processed with the Paragon algorithm of ProteinPilot 4.0 software (SCIEX). Mass spectra were searched against bovine precursor (171 entries) and bovine peptide (357 entries) sequence collections, previously reported by Colgrave et al.15 and the Uniprot bovine sequence database (version 2012/05). Taxonomy was restricted to bovine and other classifications included: 1) no cysteine alkylation and 2) no digestion enzyme. The false discovery rate (FDR) was calculated using the PSPEP algorithm embedded within ProteinPilot. Proteins (precursor protein sequences) were considered to be identified using an FDR set to 1%, while peptides were considered identified if they were above 95% confidence. Peptides that showed high quality MS/MS spectra with scores below these thresholds were manually verified or discarded. Peptides were classified as degradation products if N- and C-terminal residues did not follow classic cleavage patterns (i.e., cleavage at non-dibasic amino acid motifs and Cterminal fragments) and discarded.

Relative quantitation of peptides Multiple reaction monitoring (MRM) mass spectrometry was used for quantitation of selected neuropeptides. The peptides were selected on three criteria: (1) they were reported in the study of Colgrave et al. 15; (2) they were observed in the qualitative analyses; or (3) they were described in literature as having a physiological role in mammalian puberty. Peptides were analyzed on a 4000 QTRAP mass spectrometer (SCIEX) equipped with a nanospray II ionization source operated in positive ion mode. Samples were chromatographically separated on an Eksigent Nano 1D Plus system (Dublin, CA) using a Proteopep II column (150 mm x 75 μm) with a particle size of 5 μm and a linear gradient of 5- 40% acetonitrile over 40 min with a flow rate of 300 nL/min. The eluent for the HPLC was coupled directly to the mass spectrometer.



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Data were acquired and processed using Analyst v1.5.2 software (SCIEX). The scan type was set to MRM. The source conditions were: ionspray voltage 2,300 V, curtain gas 20, GS1 25, GS2 0, interface temperature 150°C. The resolution was set to low for both Q1 and Q3. MRM transitions were built using spectral information obtained on the TripleTOF 5600 MS. The collision energy was set according to the mass and charge of each precursor ion. The MRM transitions were scheduled based on their expected retention time using a 4 min window with a 1.5 s cycle time. Neuropeptide quantification was accomplished by integrating the peak area of the most intense MRM transition for each peptide using MultiQuant software (SCIEX). The average peak area was determined by taking the mean of three replicate injections (i.e., technical replicates).

Statistics Qualitative mass spectrometry data, generated via the TripleTOF 5600 MS, were summarized using an Excel (Microsoft Inc., Seattle, WA) spreadsheet based on the inputs of pubertal status, tissue, peptide sequence detected for individual heifer and m/z of each peptide. The number of peptides in a tissue (i.e., hypothalamus and pituitary gland) and pubertal group (i.e., PRE and POST) were compared with a one-way ANOVA and the PROC GLM procedures of SAS (version 9.2, SAS Inst., Inc., Cary, NC). Note: these statistical analyses were conducted within tissues, so presentation of results, (i.e., titles of tables and figures) clarify the n of each analysis. Also, Neuropeptide determinations were based upon manual validation of classic cleavage patterns (i.e., dibasic cleavage, C-terminal amidation, N-terminally acetylation, etc.).2426

Peptides not exhibiting classic cleavage patterns were determined to be degradation

products.27,15 Frequencies of differential and commonly expressed potential neuropeptides


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between pubertal group (i.e., PRE or POST), tissues and individual heifers were conducted with the FREQUENCY function of the Excel (Microsoft Inc., Seattle, WA) spreadsheet. Venn diagrams were generated using Venn Diagram Plotter (http://omics.pnl.gov/software/VennDiagram Plotter.php). Pearson’s correlations were estimated with the PROC CORR procedure of SAS as to evaluate associations of hormone levels at slaughter as well as gene expression level (RNA-RPKM) with peptide abundance.

RESULTS The neuropeptide of the hypothalamus and pituitary gland In total, 863 peptides were observed in the hypothalamus and pituitary gland of the preand post-pubertal heifers (Table S1); however, 83.4% were designated as degradation products. The remaining 16.6% (n = 143; Table S1) were determined to be neuropeptides based on classic cleavage or literature reports. Stathmin, a highly conserved-ubiquitous protein, was used as a proxy for sample integrity. Evidence of its degradation product stathmin [2-14] was only found in two pituitary samples (i.e., heifers 36 and 105; Table S1), demonstrating that our tissue sampling and thermal denaturation protocols had been largely effective in stabilizing peptides prior to extraction.15 Across all the tissues, 143 processed neuropeptides, derived from 35 precursors were identified, 4.3 with a range of 1-37 peptides (Table S1). Most of the peptides observed to be differentially expressed in this study are annotated with peptide packaging and processing functions (i.e., chromogranin-secretogranin family, PCSK1N-derived peptides) as well as neuron stimulatory factors such as PENKB (prodynorphin), CART, POMC and cerebellin. Only 11 peptides with post-translational chemical modifications (amidation, acetylation, etc.) were detected.



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The overall complexity of the peptidome of the two tissues studied underwent marked changes between the pre- and post-pubertal states. Before puberty, the hypothalamus contained a greater number of individual peptides than after, while 38.6% more individual peptides were identified in the pituitary after puberty, compared with the pre-pubertal state. Our findings on differential and commonly expressed neuropeptides in hypothalamus and pituitary gland from PRE and POST heifers are summarized in Figure 1.

Relative quantification of neuropeptides - Evidence for differential processing in the preand post-puberty pituitary gland Out of the 143 identified neuropeptides, 51 were selected for quantitative analysis via MRM mass spectrometry. The selected neuropeptides were detected in most of the tissue extracts in the untargeted analyses and are acknowledged products of processing from various precursors in the literature and public databases. The quantitative data for each peptide in each tissue are presented in Tables 1 and 2. In the hypothalamus, only three peptides differed (p ≤ 0.05) among PRE- and POSTheifers (Table 1; secretogranin-1 [146-159], cerebellin-4 [66-80] and proenkephalin-B [186208]). Table 2 lists the 20 peptides that differed (p < 0.05) in the pituitary gland between PRE and POST heifers. Transcriptomics (RNASeq) data was available for the same tissues studied here. 6 When the peptidomics data was compared with transcriptomics data, it became evident that levels of precursor transcripts were not highly correlated with the neuropeptide products detected in the same tissues. For example, the levels of the proprotein convertase subtilisin/kexin type 1 inhibitor (PCSK1N) transcript were not significantly different between any PRE and POST


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tissues and the levels of each peptide derived from the PCSK1N transcript were not different between the PRE and POST hypothalamus samples (Figure 2A). However, when the levels of nine individual neuropeptide products of this transcript in the pituitary were examined (Tables 12), a more dynamic picture emerges. Four neuropeptides, GAV, PEN, PEN-21 and PEN-20 were significantly more abundant in the POST pituitary gland relative to the PRE pituitary gland. The POST pituitary gland contents were 3.9-, 4.1-, 2.6- and 14.7-fold higher in the POST pituitary respectively. Big SAAS and Little SAAS [3-18] were found at significantly higher levels in the PRE pituitary. The POST pituitary gland content of GAV, PEN-21 and PEN-20 was significantly elevated relative to the PRE and POST hypothalamus sample content (ranging 3.2- to 8.4-fold higher). In contrast, Big-LEN and Little SAAS [3-18] were more abundant in the hypothalamus relative to pituitary gland of either pre- or post-pubertal heifers (Figure 2A). Mature Little SAAS was at least 3-fold more abundant in the PRE hypothalamus relative to the PRE pituitary (Figure 2A). Proenkephalin B presents an example of dynamic hypothalamic transcription which translates into higher levels of its peptide product in the pituitary (Figure 3A). The apparent higher levels of prodynorphin gene expression in the post-pubertal hypothalamus was not significant (p = 0.08) in transcription in the PRE and POST hypothalami, but in contrast a 2.7fold lower (p < 0.05) level of the peptide product of this gene, proenkephalin B [186-208] was observed in the POST hypothalamus. Prodynorphin transcription was detected at a very low level in the pituitary glands, however the resulting PENKB [186-208] peptide was detected at high levels in the POST pituitary. Similarly, the transcription level of the somatostatin gene was very low in the pituitary glands relative to the hypothalamus in both pre-and post-pubertal heifers (Figure 3B).



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Somatostatin transcription was similar in the PRE hypothalamus relative to POST, and the amount of the hypothalamic somatostatin-28 [89-100] peptide was similar in both states. While the somatostatin mRNA was transcribed at very low levels in the pituitary, its peptide product was detected in this tissue and changed significantly following puberty: the PRE pituitary gland had 2.6-fold more somatostatin-28 [89-100] (p = 0.002) than the POST pituitary gland (Figure 3B). Figure 3C-3D present two examples of high levels of transcription in the PRE and POST pituitary glands and very different levels of different peptide end products in the same tissues. Specifically, transcription of proopiomelanocortin (POMC) was not significantly different in the PRE and POST pituitary glands, however, the PRE pituitary contained over 30-fold higher (p = 0.013) levels of the POMC Joining Peptide [109-129] than its POST counterpart. Conversely, CLIP [146-167], also derived from POMC, was 10-folder higher (p = 0.001) in the POST pituitary gland. Secretogranin-2 transcription was similar in PRE and POST pituitary glands; however, the post-puberty pituitary gland contained more than 25-fold more (p = 0.025) secretoneurin [114] (Figure 3D). In contrast, secretogranin-1 transcript and neuropeptide levels observed in the pituitary gland exhibited the expected scenario of relatively higher levels of transcription in the PRE pituitary gland resulting in increased neuropeptide content for most of the secretogranin-1 neuropeptide products that were quantified by MRM (Figure S2). Chromogranin A (CHGA) (Figure 3E) transcription levels in the PRE and POST hypothalami were overall similar. In both of these tissues, similar levels of the WE-14 peptide were detected, but the GV-19 peptide was not detectable in either hypothalamic state. However, in the pituitary glands, similar levels of transcription of the CHGA gene were detected, but the


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POST pituitary gland had higher (p < 0.05) content of the both GV-19 and WE-14 (7.5- and 3.8fold respectively) than the PRE pituitary.

Correlation of neuropeptide content with transcription The results presented in Figures 2 and 3 revealed some disparities between transcription levels (RKPM) and neuropeptide yield. Correlations among these metrics are presented in Tables S2 and S3. Few significant correlations were detected between RNA-Seq and pituitary peptide peak areas among the PRE and POST heifers. These results prompted an examination of the peptide yield for the level of transcription. The ratio of peptide peak area divided by the RPKM for its precursor transcript was calculated for each peptide/precursor for each tissue and pubertal state to allow a comparison of peptide yield against transcription in each specific case (Table 3). We observed that the ratio of specific neuropeptide/precursor RPKM differed between the PRE and POST pituitary glands in a number of examples (Table 3). While seven of the nine PCSK1N-derived neuropeptides showed a higher content in the post-pubertal state, three of the seven were deemed not significant because of the variation noted within the biological groups which was likely exacerbated by the low number of animals available (n = 3). The peptide/transcription expression ratios for the four PSCK1N products: GAV, PEN, PEN-21 and PEN-20, were higher (p < 0.05) than the PRE pituitary ratio. The fold change in the ratios between PRE and POST for these peptides and their precursor transcription was 2.6- to 14.7 (Table 3). Of the five secretogranin-1 neuropeptides, four revealed different peptide/transcript expression ratios when comparing the post- vs. pre-puberty tissue (Table 3). BAM-1745 and secrotogranin-1 [487-506] gave approximately 2-fold higher (p < 0.03) peptide-to-transcript



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ratios in the POST pituitary. Conversely, the POST pituitary ratios for secretogranin-1 [411-427] and secretogranin-1 [557-564] were 5 to 8-fold lower (p < 0.05) than those of the PRE pituitary. The peptide-to-transcript ratio was 2-fold higher (p < 0.05) for cerebellin-1 [58-71] in the PRE pituitary also, and the related peptides cerebellin-1 [57-71] and cerebellin-1 [57-72] trended in the same direction. In an extreme example, there was a 38-fold increase (p < 0.05) in peptide/transcript expression ratio for secretoneurin [1-14] in the POST pituitary gland relative to the PRE pituitary gland. The POMC Joining Peptide [109-129] peptide yield offers an example of a 20-fold change in the opposite direction, wherein the PRE pituitary gland tended to yield more (p < 0.05) peptide per POMC transcript, however this difference was not statistically significant. The hypothalamic tissues provided only one example of a significant change in peptide-to-transcript with secretogranin-1 [276-306] showing close to 2-fold higher values in the PRE pituitary tissue (Table 3).

DISCUSSION The hypothalamic-pituitary-gonadal endocrine axis, especially as it relates to puberty, has been well described in ruminants with an emphasis placed on nutritional signaling.2,4,28 The animal model (PRE vs POST) described herein was designed from the body morphometric measures and endocrine profiles previously described in studies of Bos taurus x Bos indicus heifers.29-31 The transcriptomes of the reproductive and metabolic tissues (i.e., ~ 17,832 genes expressed in eight tissues, which included hypothalamus and pituitary gland) of the PRE and POST heifers of our study were described by Canovas et al.6 The objective of the current study was to describe the hypothalamic-pituitary gland neuropeptidome of PRE and POST heifers,


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based on the well-documented role of neuropeptides as autocrine, paracrine and endocrine regulators of mammalian reproduction at the level of the hypothalamus and pituitary gland.16,32-34 This effort included the identification of 143 peptides

Neuropeptidome of the Hypothalamus and Pituitary Gland of Indicine

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