General and Comparative Endocrinology 199 (2014) 38–45

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Growth hormone (GH) and GH-releasing hormone (GHRH): Co-localization and action in the chicken testis Carlos G. Martínez-Moreno a,c, Luz M. López-Marín b, Martha Carranza a, Daniel Giterman c, Steve Harvey c, Carlos Arámburo a, Maricela Luna a,⇑ a b c

Departamento de Neurobiología Celular y Molecular, Instituto de Neurobiología, Universidad Nacional Autónoma de México, Campus Juriquilla, Querétaro 76230, Mexico Departamento de Nanotecnología, Centro de Física Aplicada y Tecnología Avanzada, Universidad Nacional Autónoma de México, Campus Juriquilla, Querétaro 76230, Mexico Department of Physiology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada

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Article history: Received 1 June 2013 Revised 21 January 2014 Accepted 26 January 2014 Available online 4 February 2014 Keywords: Chicken growth hormone (cGH) Growth hormone releasing hormone (GHRH) Testes Proliferation Secretion GH isoforms

a b s t r a c t Growth hormone (GH) gene expression is not confined to the pituitary gland and occurs in many extrapituitary tissues, including the chicken testis. The regulation and function of GH in extrapituitary tissues is, however, largely unknown. The possibility that chicken testicular GH might be regulated by GH-releasing hormone (GHRH), as in the avian pituitary gland, was investigated in the present study. GHRH colocalized with GH in the germinal epithelium and in interstitial zones within the chicken testes, particularly in the spermatogonia and spermatocytes. In testicular cell cultures, exogenous human GHRH1–44 induced (at 1, 10 and 100 nM) a dose-related increase in GH release. Western blot analysis showed a heterogeneous pattern in the GH moieties released during GHRH stimulation. 26 kDa monomer GH was the most abundant moiety under basal conditions, but 15 and 17 kDa isoforms were more abundant after GHRH stimulation. GHRH treatment also increased the abundance of PCNA (proliferating cell nuclear antigen) immunoreactivity in the testes. This may have been GH-mediated, since exogenous GH similarly increased the incorporation of (3H)-thymidine into cultured testicular cells and increased their metabolic activity, as determined by increased MTT reduction. Furthermore, GH and GHRH immunoneutralization blocked GHRH-stimulated proliferative activity. In summary, these results indicate that GHRH stimulates testicular GH secretion in an autocrine or paracrine manner. Data also demonstrate proliferative actions of GHRH on testicular cell number and suggest that this action is mediated by local GH production. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction It is now well established that growth hormone (GH) gene expression is not confined to the pituitary gland and occurs widely in extrapituitary tissues (Harvey, 2010), including the testis. In the mature testes, the presence of GH mRNA has been reported in humans (Untergasser et al., 1998), chickens (Harvey et al., 2004) and the Japanese eel (Miura et al., 2011). GH-immunoreactivity (GH-IR) was also found in the chicken testis during growth and sexual maturation (Luna et al., 2004; Martínez-Moreno et al., 2011). In the chicken testis, the localization of GH was mainly within germ cells (spermatogonia, spermatocytes and spermatids) and was largely associated with a submonomeric isoform of 17 kDa, rather than

⇑ Corresponding author. Address: Departamento de Neurobiología Celular y Molecular, Instituto de Neurobiología, Universidad Nacional Autónoma de México, Campus Juriquilla, Querétaro, Qro 76230, Mexico. Fax: +52 (442) 238 1005. E-mail address: [email protected] (M. Luna). http://dx.doi.org/10.1016/j.ygcen.2014.01.013 0016-6480/Ó 2014 Elsevier Inc. All rights reserved.

with the 22 kDa monomer that is most abundant in the pituitary gland (Luna et al., 2004; Martínez-Moreno et al., 2011). The testis is not just a site of GH production, but also a target site of GH action, since the GH receptor (GHR) gene is ubiquitously expressed within testicular cells (Hull and Harvey, 2000), including those of the germ-line, as well as in Sertoli, Leydig and peritubular cells (Martínez-Moreno et al., 2011). These receptors may thus respond in an endocrine way to pituitary GH in circulation and in autocrine or paracrine (or intracrine) ways to GH produced locally in the testis. Within the testes, GH is thought to induce the renewal of spermatogonia (Loir, 1999; Matsushima et al., 1986), to promote sperm motility (Champion et al., 2002), to induce testicular differentiation (Hull and Harvey, 2000) and to increase steroidogenesis (Hull and Harvey, 2000). The regulation of testicular GH secretion is, however, unknown. GH secretion from the pituitary gland is primarily stimulated by GH-releasing hormone (GHRH) (Gahete et al., 2009; Harvey et al., 1985). In extrapituitary tissues, GHRH also stimulates GH release from immune tissues (Clark, 1997), in which it is produced and

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acts locally (Guarcello et al., 1991; Weigent and Block, 1990; Weigent et al., 1991). GH production in retinal ganglion cells (RCGs) is similarly found to be stimulated by GHRH (Harvey et al., 2012), which is also present in RGCs (Harvey et al., 2012) and thus likely to act in autocrine or paracrine ways in GH regulation. As GHRH and its receptor are also found in the chicken testis (Wang et al., 2007), the possibility that it regulates testicular GH secretion was therefore examined, as was the possibility that GHRH has other direct or indirect actions in testicular function. 2. Materials and methods 2.1. Animals and tissues Adult male chickens were kept in a vivarium at our institute with food and water ad libitum. Animals were maintained with a controlled light cycle (12 h light/12 h dark). The roosters were sacrificed by decapitation according to the Institute’s Bioethics Committee regulations and the testes were collected in Hank’s salts solution in the presence of 5% penicillin/streptomycin. The left testicle was collected from all the adult chickens (32–35 week old chickens) at an age when fertility peaks and plasma and testicles GH levels remain constant (Scanes and Harvey, 1981; Luna et al., 2004). The testicles were selected according to weight (8–15 g), size (3.5–5 cm long) and shape (avoiding abnormalities). An additional group of testis from early adult chickens (20 week old) was added for GH secretion studies determination by ELISA. 2.2. Hematoxilin/eosin staining and immunohistochemistry For immunohistochemical analysis, testes were fixed with Bouin-Hollande sublimate for 48 h, dehydrated in ethanol, and embedded in paraffin. Sections (10 lm) were cut with a microtome and mounted on pretreated glass slides (Fisherbrand, Fisher Scientific, Edmonton, Canada). Paraffin was removed from the slides with Citrisolv (Fisher). Sections were rehydrated in absolute alcohol (100%), ethanol (95, 70, 50 and 30%) and distilled water; finally the testicular sections were equilibrated with PBS for 1 h. For the hematoxilin/eosin staining, testicular sections were incubated for 3 min in Harris hematoxylin [0.5% hematoxylin (Merck Darmstadt, Germany)] in a solution containing 5% ethanol, 10% aluminum sulfate (Sigma Aldrich, St. Louis, MO, USA) and 0.25% mercury (II) oxide red (Sigma Aldrich), washed in tab water for 1 min, then immersed 5 times in blueing solution (0.1% NH4OH in deionized water), and finally rinsed with distilled water. Sections were further incubated in eosin Y (Sigma Aldrich; 0.25% in 60% acidified ethanol) for 15 s, rinsed with distilled water. Slides were dehydrated in ethanol (50%, 50%, 96% and 100%) and dipped in xylene (3 times) before being mounted with Entellan (Merk). For immunohistochemistry, tissues were boiled in citrate buffer (10 nM sodium citrate, 0.05% Tween 20; pH 6.0) for 20 min to unmask epitopes (after rehydration). GH-IR was determined with rabbit polyclonal antibody directed to recombinant chicken GH (Bleed 1/9) (Harvey and Scanes, 1977), diluted 1:500 in TPBS with 1% nonfat dry milk (Bio-Rad Laboratories, Inc., Hercules, CA) or with sheep polyclonal antibody against chicken growth hormone (developed by Cheung and Hall (1990) and generously provided by Dr. T.R. Hall, Ciba-Geigy, Switzerland). Co-localization of growth hormone releasing hormone was complemented with a polyclonal antibody directed to chicken GHRH (Harvey et al., 2012) (developed by Washington Biotechnology, Simpsonville, MD, USA) raised in rabbits (at 1:500 in TPBS with 1% non-fat milk). Negative controls were performed by substituting the primary antibodies for normal rabbit or sheep serum or omitting it completely. Also, label specificity was determined by pre-absorbing the primary antibod-

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ies (1 ll) with rcGH (10 lg) and GHRH (10 lg) (Fig. 1). Tissue sections were incubated overnight (4 °C) with primary antibodies. After washing (3  10 min) in TPBS, sections were incubated for 2 h with Alexa Fluor 488 goat anti-rabbit IgG antibody (Invitrogen Molecular Probes, Eugene, Oregon, USA) or Alexa Fluor 647 donkey anti-sheep IgG antibody (Invitrogen), both at concentrations of 1:1000 in TPBS with 1% non-fat dry milk (Bio-Rad). After washing in TPBS (3  10 min), the sections were mounted onto slides using Prolong Gold mounting medium (Invitrogen, No. P36930). 2.3. Confocal microscopy Slides were visualized using a Leica TCS SP5 confocal microscope. Imaging was performed using a Leica HCX plan-apochromatic oil immersion confocal objective lens with a magnification of 40 and a numerical aperture of 1.44. Gamma adjustments were performed using Leica Application Suite Advanced Fluorescence (LASAF) microscope software. Subsequent image processing was performed using ImageJ software (developed by NIH, freeware). 2.4. Primary testicular cell culture The enzymatic dispersion of testicular tissue was performed (Nagano et al., 1998), but using collagenase rather than a collagenase-trypsin mix. Briefly, the tunica albuginea (external gonad layer) was removed, and the tissue was cut into small pieces using sterile scissors and a scalpel. Homogenized tissue was washed and resuspended on DMEM (Gibco, Grand Island, NY, USA). Large aggregates were mechanically disaggregated with a Pasteur pipette followed by 3 washes with DMEM (Gibco). The tissue pieces (approximately 3 g) were transferred to a new 50 ml plastic centrifuge tubes (Corning Inc., NY, USA) and incubated with 0.3% collagenase type 2 (Worthington Biochemical Corp., Lakewood, NJ, USA) in DMEM (Gibco) with moderate stirring for 1 h at 37 °C. The cell suspension was washed with 15 ml DMEM (2 times) and resuspended in 25 ml DMEM for cell quantification. The number of cells and viability was determined by the trypan blue exclusion technique (Kumi-Diaka and Butler, 2000). In these testicular cultures, 19.6% of the cells were identified as spermatogonia, 31.9% were spermatocytes and 24.6% were identified as spermatids (Martínez-Moreno et al., 2011). For GH release determination, 5  106 cells previously counted in a hemocytometer, were incubated for 24 h for at 37 °C; cell culture was performed in 60  15 mm culture dishes in 5 ml Megacell (Sigma–Aldrich Inc, St. Louis, MO, USA, a commercial medium containing 3% fetal bovine serum and amino acid supplements). After the stabilization period (the first 24 h after cell dispersion), culture media were discarded and replaced with 5 ml M199 (Gibco) with treatments, described below, for 2 h at 37 °C, and then collected. The longer-term cell proliferation assays utilized 5  105 cells in 200 ll Megacell (Sigma–Aldrich) in 24-well plates for the determination of PCNA (proliferating cell nuclear antigen) levels and for assessment of 3H-thymidine incorporation. Cultures (5  105 cells) for the assessment of metabolic activity as determined by the MTT assay, where suspended in 100 ll Megacell in 96-well plates. All cultures for proliferation were stabilized in Megacell media (Sigma–Aldrich) with 1% penicillin/streptomycin for 24 h and then the cells were incubated at 37 °C in a water-saturated atmosphere of 95% air and 5% CO2 for 24 h in the presence of treatments. 2.5. Treatments The GH releasing activity of GHRH (Sigma–Aldrich, human synthetic 1–44 peptide, G8895) was first examined, at concentrations of 1, 10 and 100 nM, since this is an established secretagogue for pituitary GH release in chickens (Harvey, 1999). These treatments

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Fig. 1. Co-localization of growth hormone (GH) and growth hormone releasing hormone (GHRH) immunoreactivity in the testis of a 35-week-old chicken. Chicken testicular sections were stained with Harris hematoxylin/eosin (H&E) staining (A–C): spermatogonias (SPG arrows), spermatocytes (arrowheads), and elongated spermatids (within squares). Confocal images of double stained sections of adult testis (D–I) show: GH-IR cells (red, 1st Ab: polyclonal sheep anti-chicken GH at 1:500; 2nd Ab: donkey antisheep IgG-Alexa Fluor 647 at 1:1000) and GHRH (green, 1st Ab: a polyclonal rabbit anti-chicken GHRH at 1:500; 2nd Ab: goat anti-rabbit IgG-Alexa Fluor 488 at 1:500). Colocalization of GH and GHRH in germ cells is shown in yellow after merging images (F and I). Arrows show immune-positive SPG’s; arrowheads show spermatocytes; (⁄) shows an interstitial cell; inserted squares show elongated spermatids; dotted line shows basal membrane (b.m). i.z., interstitial zone; t.a., tunica albuginea. Representative of at least 4 testes. The lower panels show negative controls and the absence of specific staining by the pre-absorption of the antibodies with excess (10 lg) human GHRH (J) and excess (10 lg) recombinant chicken GH (K), respectively. In both cases the primary antibodies were incubated with the exogenous antigen for 16 h at 4 °C. The lower panel also shows an unstained section (L) incubated in the absence of either primary antibodies and in the presence of secondary antibodies Alexa Fluor 488 and 647. Scale bars, 50 lm (A), 10 lm (B, D–F, J–L), 5 lm (G–I).

were applied during 2 h in serum-free M199 medium. Control incubations were in the absence of GHRH as well as in the presence of anti-cGHRH antibody (1:100 dilution) (Harvey et al., 2012) and anti-rcGH antibody (CAP1; 1:100 dilution) (Arámburo et al., 2001) for immunoneutralization. In the cell proliferation assays, (PCNA, MTT and 3H-thymidine incorporation), recombinant chicken GH (rcGH, American Cyanamid, Princeton, NJ, USA Lot-100) treatments (1, 10 and 100 nM) were applied to cultures in Megacell medium for 24 h. 3H-thymidine and GH were added to the respectively cultures simultaneously. 2.6. Preparation of culture media and cell extracts for ELISA and Western Blot Cell culture media (5 ml) were collected from culture dishes using a Pasteur pipette, and transferred to 15 ml plastic tubes and stored at 80 °C until concentration and dialysis. Centriprep

centrifugal filter units with Ultracel-10 membranes (10,000 nominal molecular weight limit) (Merck Millipore Ltd. Tullagreen, Carrigtwohill, Co. Cork, IRL) were used for concentration and dialysis. Concentration was performed adding 5 ml of media culture to the centriprep unit and centrifuged (at 2500g) into 1 ml of concentrated culture media. Dialysis was performed adding 10 ml of distilled water to final volume (1 ml) of the concentrated culture media in the same centrifugal filter unit (10 K NMWL), and re-filtered by centrifugation into 1 ml of concentrated and dialyzed sample. The sample was dried on a vacuum centrifugal lyophilizer, and resuspended in distilled water (55 ll) for ELISA and in sample buffer (35 ll) for Western blot analysis. Cells were harvested with a cell scraper in the presence of an EDTA-free protease inhibitor cocktail (Mini-complete, Roche Diagnostics, Indianapolis, IN, USA) containing 1 mM PMSF, pH 9.0. The cells were centrifuged at 16,000g at 4 °C for 5 min, and the pellet was homogenized using a pellet pestle rod (3 times). The superna-

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tants were collected, and the protein content of each sample was determined by the Bradford micro-method (Bio-Rad). 2.7. Elisa The presence of GH-immunoreactivity in the extracts was investigated using an enzyme-linked immunosorbant assay (ELISA) (Martínez-Coria et al., 2002). Briefly, 96-well microtiter plates (Immulon 2HB, Chantully, VA, USA) were coated overnight at 4 °C, with 12 ng rcGH in 100 ll 1 M carbonate buffer, pH 10.3. The plates were extensively washed with TPBS (0.01 M sodium phosphate, 0.15 mM NaCl, 0.05% v/v Tween 20, pH 7) using an automatic microplate immunowasher (Bio-Rad). This procedure was performed after each incubation step. Dialyzed and concentrated culture media, cell extracts or serial dilutions of rcGH (1250– 0.5 ng/ml) in TPBS were then incubated for 16 h with 100 ll of primary antibody diluted 1:100,000 with TPBS and 1% w/v nonfat dried milk. The antibody was raised in rabbits against purified pituitary derived chicken GH (Martínez-Moreno et al., 2011) and is specific for GH and has no cross-reactivity (