TMCnet News
Establishment and characterization of dairy cow growth hormone secreting anterior pituitary cell model [In Vitro Cellular & Developmental Biology](In Vitro Cellular & Developmental Biology Via Acquire Media NewsEdge) Received: 17 April 2013 / Accepted: 7 July 2013 / Published online: 20 September 2013 / Editor: T. Okamoto © The Society for In Vitro Biology 2013 Abstract A dairy cow anterior pituitary cell (DCAPC) model was established in vitro for the study of growth hormone (GH) synthesis and secretion in the anterior pituitary gland of the dairy cow. Pituitary glands were obtained from Holstein dairy cows' heads cut by electric saw, and the posterior pituitary glands were removed to obtain integrated anterior pituitary glands. Immunohistochemistry assay of GH in the anterior pituitary glands showed that most somatotrophs were located within the lateral wings of the anterior pituitary. Tissues of the lateral wings of the anterior pituitary were dispersed and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. The DCAPCs displayed a monolayer, cobblestone, epithelial-like morphology which are the typical characteristics of the anterior pituitary cells. The DCAPCs were subcultured continuously over ten passages. GH immunoreactivity was present in DCAPCs at passage 10. The transcription of the bovine GH mRNA in DCAPCs at passage 10 was decreased to below 50% compared with the lateral wings of the anterior pituitary tissues. Thus, our DCAPCs model is effective for the in vitro examination of GH synthesis and secretion in the dairy cow anterior pituitary gland. The effects of transforming growth factor beta 1 (TGF-ß1) and interferon-? (IFN-?) on the expression of GH mRNA in DCAPCs at passage 3 were also investigated. There were no obvious changes in transcription of the GH gene after treatment with TGF-ß1 for 24 h, while IFN-? increased transcription of the GH gene in a dose-dependent manner. Keywords Dairy cow. Anterior pituitary gland. Growth hormone. TGF-ß1. IFN-? Introduction The pituitary gland is an important component of the endocrine system, which forms an important link between the central nervous system and other endocrine glands. Growth hormone (GH) is a polypeptide hormone synthesized and secreted by the anterior pituitary gland, which plays a key role in regulating ruminant mammary gland development and lactation (Akers 2006). In lactating cows, bovine GH induced proliferation of mammary parenchyma and growth of epithelial cells and also increased the cell renewal in the mammary gland (Capuco et al. 2001). GH increases milk protein gene expression in bovine mammary explants and mammary epithelial cells (Yang et al. 2005; Yonekura et al. 2006). GH treatment can activate intracellular pathways in mammary epithelial cells involved in the regulation of milk protein synthesis via the Janus kinase and signal transducers and activators of transcription signaling (Bionaz and Loor 2011). Considering its irreplaceable regulatory role in lactation, research on endocrinology of lactation has always focused on the factors and mechanisms affecting GH synthesis and release. Factors affecting GH synthesis and secretion in the dairy cow include hypothalamic factors, peripheral hormones, cytokines, and dietary nutrients (Katoh et al. 2007). These conclusions are mostly based on the research in vivo, and the mechanisms involved remain poorly understood. Cell culture is one of the most convenient tools for understanding the mechanisms of hormone synthesis and secretion. A number of immortal somatotropic lines of rat have been established, such as GH1, GH3, GH4, GH4C1, and MtTcelllines (Ooietal.2004).These immortalizedcelllines have contributed much to the understanding of pituitary physiology and have provided useful models to understand the mechanisms of pituitary hormone synthesis and release. Ingram et al. (1988) described the morphological characteristics of bovine lactotrophs separated by Percoll gradient centrifugation. Nagai et al. (2008) established a bovine anterior pituitary-derived cell line that expresses interleukin (IL)-18 and IL-18 receptor. The establishment of pure bovine somatotropic cell lines is useful for studying the mechanisms of bovine GH synthesis and secretion. Thus, the primary objective of this investigation was to establish a dairy cow anterior pituitary cell (DCAPCs) line that expresses GH in vitro to study the molecular and cellular mechanisms of GH synthesis and secretion regulated by hypothalamic factors, peripheral hormones, cytokines, or dietary nutrients. Transforming growth factor beta 1 (TGF-ß1) and Interferon-? (IFN-?)regulate pituitary function in mouse, rat, bovine, and human endocrine systems (Vankelecom et al. 1990; Mueller and Kudlow 1991; de Hofland et al. 1999;Oomizuetal.2000). However, the effect and detailed mechanisms by which TGF-ß1andIFN-? mediate GH gene transcription remain unclear. Therefore, the GH mRNA level of DCAPCs affected by TGF-ß1andIFN-? was analyzed individually. Materials and Methods Immunohistochemistry assay of GH in the anterior pituitary gland. Pituitary glands were extracted from the heads of ten Holstein cows cut by electric saw at a local slaughterhouse and transported to the laboratory in Hanks' balanced salt solution (HBSS) solution without calcium and magnesium (CMFHBSS) to which was added 50 µg streptomycin/ml, and 100 IU penicillin/ml at 5°C within 2 h. The posterior pituitary was removed with precision medical scissors and tweezers to obtain integrated anterior pituitary gland. Three integrated anterior pituitary glands were obtained in this step. Then the anterior pituitary glands were fixed with 4% formalin solution for 48 h, dehydrated in ethanol, and embedded in paraffin wax. The anterior pituitary glands were cut into several sections (2 mm thickness) for paraffin blocks. Then, paraffin blocks were cut into several sections (5 µm thickness) using a microtome and mounted onto poly-L-lysine-coated slides. The sections were then rehydrated with xylene, 100%, 95%, 90%, 80%, and 70% alcohol, and distilled water. Then the sections were treated with 3% H2O2 for 30 min to quench endogenous peroxides, blocked with goat serum (Maixin-Bio, Fujian, China) for 30 min, incubated overnight at 4°C with rabbit anti-GH antibody at 1:1,500 dilution (Chemicon International, Millipore, Billerica, MA). The primary antibody of the negative control was replaced with PBS. The secondary antibody was biotin-goat anti-rabbit IgG (Maixin-Bio). The staining was visualized with an 3,3?-diaminobenzidine tetrahydrochloride (Maixin-Bio) kit. The nuclear counterstaining was followed by hematoxylin staining (MP Biomedicals, Shanghai, China). Cytoplasms that were yellow to brown were considered as positive cells. Isolation and culture of DCAPCs. Tissues where somatotrophs were located were diced into small pieces at less than 1 mm3 and incubated in CMF-HBSS containing 0.3% I type collagenase (Sigma, Shanghai, China), 0.1% hyaluronidase (Sigma), and 0.1 0/00 Dnase (Sigma) at 37°C for 2 h. The dispersed cells were washed three times with HBSS, resuspended in Dulbecco's modified Eagle's medium (DMEM; Gibco, Carlsbad, CA) supplemented with 10% fetal bovine serum(FBS; Gibco) at seeding density of 1x103 cells/ml. Then, the cells were seeded into a 75-cm2 culture flask (Corning, Tewksbury, MA) and incubated at 37°C in a humidified atmosphere containing 5% CO2. Cell morphology was observed under inverted phase contrast microscope. Growth characteristics of passaging DCAPCs. After 6 d in culture, the cells were treated with 0.5% trypsin (Sigma) and 0.02% EDTA in CMF-PBS and then collected in centrifuge tubes (Corning). The supernatant was removed and the cell pellet was resuspended in fresh medium at a concentration of 1x104 cells/ml, and cells were seeded into 6-cm cell culture dishes (Corning) for subculturing. Cell growth curves were obtained by a hemocytometer counting at passages 1, 5, and 10. Cells were stained with trypan blue for viability detection. The cell morphology was observed under inverted phase contrast microscope. Fibroblast-like shape cells were observed after the 7th passage of DCAPCs. Further separation of fibroblasts from epithelial-like cells was necessary to establish near-fibroblast free cultures. Thus, pituitary epithelial-like cells and fibroblasts were separated according to their differing sensitivity to trypsin using 0.5% trypsin and 0.15% trypsin plus 0.02% EDTA as previously described (Tong et al. 2012). Protocol for freezing and thawing DCAPCs was established. The composition of freezing liquid (10 ml) was: DMEM medium, 6 ml; dimethylsulfoxide (Sigma), 1 ml; and FBS, 3 ml. Cells were stored in liquid nitrogen by slow freeze-method until thawing. DCAPCs were thawed by routine method and were seeded into 6-cm cell culture dishes at a concentration of 1x104 cells/ml. Trypan blue staining was used to cells and determine survival rates. Immunocytochemistry assay of GH in DCAPCs. DCAPCs at the 2nd, 5th, and 10th passages were incubated onto poly-Llysine-coated cover slips at seeding density of 5x103cells/well in a 24-well cell culture plate. Cover slips were washed three times with PBS for 5 min, and fixed for 10 min in 80% cold acetone, washed again, and then incubatedfor30minin0.1%TritonX-100atambient temperature. The cover slips were treated by the same method. Determination of GH, PRL, and Pit-1 gene expression. Total RNA of the DCAPCs was isolated using Trizol reagent (Sigma). The first strand of cDNA was performed with a reverse transcription kit (Takara). Bovine glyceraldehyde-3phosphate dehydrogenase (GAPDH), GH, prolactin (PRL) and pituitary-specific transcription factor 1 (Pit-1) mRNA expression in the cells was identified by RT-PCR with crossintron primers as previously described (Laporta et al. 2011). The primer pairs for GAPDH were:5?-TGCCCAGAATATC ATCCC-3? and 5?-AGGTCAGATCCACAACAG-3?, which yielded a 134-bp fragment. The primer pairs for GH were: 5?AGATCCTCAAGCAGACCTA-3? and 5?-AGGTACGTC T CCGTCTTA-3?, which yielded a 121-bp fragment. The primer pairs for PRL were: 5?-TATGAAAGGAGCCCCAG ATG-3?and 5?-CACACAGGGTAGGGC TCAGT-3?, which yielded a 137-bp fragment. The primer pairs for Pit-1 were: 5?-TTCTGCAACTCTGCCTCTGA-3?and 5?-CCATAGGT CGATGACTGGT-3?, which yield a 148-bp fragment. The GH and PRL gene transcription level in the anterior pituitary gland and DCAPCs at the 1st, 4th, 7th, and 10th passages was also detected by quantitative RT-PCR(Bio-RAD, California, USA) analysis using the SYBR Green QuantiTect RTPCR Kit (Roche, Basel, Switzerland), and performed in triplicate for each sample. The qRT-PCR experiment was set up according to the MIQE guidelines (Bustin et al. 2009). The relative expression level for GH and PRL were calculated relative to GAPDH (the normalizer) using the comparative cycle threshold method (Lisowski et al. 2008). Cytokine treatment of DCAPCs. DCAPCs in logarithmic growth phase at passage 2 were plated at seeding density of 1x104 cells/ml in each dish of a 6-well cell culture plate. After 24 h, the medium was replaced with DMEM medium supplemented with 0.1% FBS and 0.5% bovine serum albumin (MP Biomedicals) and incubatedfor4hforserumstarvation. TGF-ß1 (cell culture grade, Sigma) or IFN-? (cell culture grade, Sigma) solution were added to the culture medium of each well, and the final concentrations of TGF-ß1orIFN-? were 0, 5.0, 10.0, 20.0, 40.0, and 80.0 ng/ml. After further culture for 24 h, the cells were collected to assess the change of GH mRNA synthesis. Each treatment concentration was replicated 9 times. GH expression was evaluated using the same method. Results Immunohistochemistry. No immunoreactivity for GH was observed in control sections when the primary antibody was replaced with PBS (Fig. 1b). The GH positive cells were widespread within the anterior pituitary gland except the pars tuberalis (Fig. 1a-c). Most somatotrophs were located within the lateral wings of the anterior pituitary with strong GH immunoreactivity (Fig. 1a (5), g). A few somatotrophs were scattered in the median wedge (Fig. 1a (4), f). Low GH immunoreactive cells were observed in the anterolateral (Fig. 1a (2), d) and pars distalis (Fig. 1a (6), h) of the anterior pituitary gland. Several somatotrophs with moderately GH immunoreactivity clustered in the central wedge adjacent to pars tuberalis (Fig. 1a (3), e). Cell morphology. Based on the above results, tissues of the lateral wings of the anterior pituitary were dispersed and cultured in DMEM medium supplemented with 10% FBS. The primary cells adhered to the plate within 12 h after plating. Cell islands were observed at 3 d after plating (Fig. 2a). These colonies expanded to cover large areas after 5 d in culture (Fig. 2b). Epithelial-like colonies with characteristic closely opposed cobblestone morphology were typically observed at 6dafterplating(Fig.2c).Theanteriorpituitarycellsatthe4th, 7th, and 10th passages showed a flattened morphology (Fig. 2d-f). Low confluence DCAPCs at passage 3 presented short-spindle shape and the volume of single cell became bigger than primary cells (Fig. 2d). Fibroblast-like cells were observed at the 7th passage (Fig. 2e) and began to predominate at the 10th passage (Fig. 2f). Near-fibroblast-free cultures of DCAPCs were established and found to be more resistant than fibroblast cells to treatment with trypsin and EDTA when the cells were detached from their substratum. The morphology of fibroblast cells was shown as long spindles (Fig. 3a). Pure cultures of DCAPCs formed a monolayer and aggregated with the characteristic cobblestone morphology of epithelial-like cells (Fig. 3b). Growth characteristics of passaging DCAPCs. DCAPCs at passages 1, 5, and 10 grew slowly within the first day (lag phase), from the second to fifth day when the cells appeared to rapidly proliferate (log phase), and after the sixth day when the proliferation of the cells slowed down (stationary phase). The cell growth curves appeared as "S" shaped (Fig. 4). This growth met the requirements for in vitro studies sufficiently although the cell proliferation rate of DCAPCs at passages 5 and 10 was lower than that of passage 1. Most cells attached to plastic substrate after 10 h in culture. The survival rate of freezing DCAPCs was 83% after being stored in liquid nitrogen for 3 mo. Immunocytochemistry. Very low immunoreactivity was observed in the negative control slide (Fig. 5a). The positive immunocytochemistry staining for cell GH was observed in DCAPCs at the 2nd, 5th, and 10th passages (Fig. 5b-d). As shown in Fig. 5, the GH immunoreactivity of DCAPCs at the 2nd passage was strong. The GH immunoreactivity of DCAPCs at the 5th and 10th passages was moderate. All pituitary cells (in any passage) obtained from the lateral wings of the anterior pituitary were deemed GH immunoreactive. However, not all cells were observed GH immunoreactive in pituitary immunohistochemistry results. This result suggests that the method of isolation of DCAPCs from the lateral wings of the anterior pituitary is fit for establishment of pure bovine somatotropic cell lines. GH and Pit-1 gene expression. The mRNA fragments of GAPDH, GH, PRL, and Pit-1 were amplified (Fig. 6a), and the derived PCR products were sequenced with 100% homology with the published sequences of bovine GAPDH, GH, PRL, and Pit-1. The expression levels of the GH gene were constant in DCAPCs at the 4th, 7th, and 10th passages (Fig. 6b). There was no obvious change in the transcription of bovine GH gene in DCAPCs at passage 10 and the lateral wings of the anterior pituitary tissues (Fig. 6b, p > 0.05). Transcription of the bovine PRL gene was significantly lower in DCAPCs at passages 7 and 10 than in the lateral wings of the anterior pituitary tissues (Fig. 6b, p < 0.05). Cytokine effects on GH gene expression of DCAPCs. Treatment with IFN-? for24hresultedinadose-dependentmanner increase in the transcription of the GH gene in DCAPCs (Fig. 7a). The GH gene expression was significantly upregulated at 10.0 (p<0.05), 20.0 (p<0.05), 40.0 (p<0.01), and 80.0 ng/ml (p<0.01). There was no obvious change in transcription of the GH gene after 24 h treatment with TGF-ß1at5.0, 10.0, 20.0, 40.0, and 80.0 ng/ml (Fig. 7b, p>0.05). Discussion The bovine pituitary gland is an orbicular-ovate structure located at the base of the brain in a bony cavity known as the sella turcica. Despite its small size, the pituitary plays a critical role in the ruminant and regulates a broad range of physiological processes involved in growth, reproduction, lactation, and stress. A steer pituitary gland can produce 107 cells using collagenase I treatment (Ingram et al. 1988). The pituitary gland is composed of two distinct parts: the posterior lobe and anterior lobe. The mature bovine anterior pituitary is composed of six specialized hormone secreting cells: somatotrophs, lactotrophs, corticotrophs, thyrotrophs, mammosomatotrophs, and gonadotrophs. Results obtained from the studies that used cells from the integrated anterior pituitary gland are not precise. Gradient centrifugation is the main technique for separating and isolating pure samples of cells, organelles, viruses, and other subcellular particles based on the difference of density or size. However, a major disadvantage of this technique is producing a low final yield of surviving cells. It seems, therefore, that existing in vitro approaches are not effective for the investigation of GH synthesis and secretion in the dairy cow anterior pituitary gland. Serial dilution cloning of adherent mammalian cells is commonly used for establishment of near-fibroblast free cultures for reliable results (Aso et al. 1995;ChaturvediandSarkar 2006; Nagai et al. 2008; Ding etal. 2001). Previously, we have tried to establish fibroblast-free cultures by the limiting dilution method as described by Nagai et al. (2008). About 3 wk later, several clones were grown separately. However, it was difficult to obtain the expected results as the cells with a low proliferation activity could not form subclones. The low proliferation activity of the cells which were derived from a single cell clone may be associated with the very low seeding density or the source of pituitary gland. Here, we described the establishment and characterization of a DCAPC model. The total viable yield was in the range 3-6x105 cells per lateral wing of the anterior pituitary gland and viability was normally greater than 95%. Serial subcultures were performed to obtain a sufficient number of dairy cow GH secreting anterior pituitary cells for analysis. However, during subculturing the use of culture medium containing FBS may lead to a reduced expression of GH, and a change in morphology to a fibroblast-like shape (Rattner 1997). A method of purification of DCAPCs was developed by using differential cell adhesion properties and was fit for the establishment of bovine fibroblast-free somatotropic cell lines. Here, the DCAPCs were subcultured continuously over 10 passages. GH immunoreactivity was present in all DCAPCs at passages 10. Transcription of the bovine GH gene in DCAPCs at passage 10 were decreased less than 40% compared with the lateral wings of the anterior pituitary tissues. Thus, our DCAPC model can be effectively used in vitro for the study of GH synthesis and secretion in dairy cow anterior pituitary gland. Long-term passage capacity of the cells will facilitate the study of GH synthesis and secretion. Next, we will check the passaging capacity of calf pituitary cells in later stages so as to establish DCPACs with extended usage. TGF-ß is a protein that controls proliferation, cellular differentiation, and other functions in most cells (Hentges and Sarkar 2001; Moustakas et al. 1995). TGF-ß is a secreted protein that exists in at least three isoforms called TGF-ß1, TGF-ß2, and TGF-ß3. TGF-ß1 belongs to a group of intramammary auto/paracrine inhibitors of bovine mammary gland epithelial cell growth and inducers of apoptosis (Zarzynska et al. 2005). Exogenous TGF-ß1 inhibits basal secretion of PRL and stimulated basal secretion of GH in a rat pituitary monolayer culture system (Murata and Ying 1991). This inhibitory effect of TGF-ß1 on PRL production is mainly through transcriptional regulation of the PRL gene. TGF-ß1 may act through a complex signaling pathway that involves multiple DNA elements within the PRL promoter (Farrow and Gutierrez-Hartmann 1999). Here, we found that there was no obvious change in transcription of the GH gene after 24 h treatment with TGF-ß1. IFN-? coordinates a diverse array of cellular programs through transcriptional regulation of immunologically relevant genes. Exogenous IFN-? inhibits stimulated PRL and GH secretion in normal rat anterior pituitary cell cultures (Vankelecom et al. 1990). IFN-? can increase the activity of human GH gene promoter in rat pituitary GH3 cells. The stimulatory action of IFN-? appears to require the intracellular mitogen-activated protein kinase-dependent signaling pathway (Gong et al. 2003). We found that IFN-? could increase the transcription of the GH gene in DCAPCs in a dosedependent manner. The mechanisms of this action need to be researched more deeply. In summary, we established a dairy cow GH secreting anterior pituitary cell model that was well characterized morphologically and biochemically and easily maintained in vitro. The model can be used successfully for the study of the mechanisms of hypothalamic factors, peripheral hormones, cytokines, or dietary nutrients regulating GH synthesis and release. Acknowledgments This work was founded by the National Key Basic Research Program of China (project no. 2011CB100805), Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT, no. IRT1248), and Jilin Scientific and Technological Development Program (project no. 20130206036NY). References Akers R. M. Major advances associated with hormone and growth factor regulation of mammary growth and lactation in dairy cows. J. Dairy Sci. 89: 1222-1234; 2006. Aso H.; Abe H.; Nakajima I.; Ozutsumi K.; Yamaguchi T.; Takamori Y.; Kodama A.; Hoshino F. B.; Takano S. A preadipocyte clonal line from bovine intramuscular adipose tissue: nonexpression of GLUT-4 protein during adipocyte differentiation. Biochem. Biophys. Res. Commun. 213(2): 369-375; 1995. Bionaz M.; Loor J. J. Gene networks driving bovine mammary protein synthesis during the lactation cycle. Bioinform. Biol. Insights 5: 83-98; 2011. Bustin S. A.; Benes V.; Garson J. A.; Hellemans J.; Huggett J.; Kubista M.; Mueller R.; Nolan T.; Pfaffl M. W.; Shipley G. L.; Vandesompele J.; Wittwer C. T. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin. Chem. 55: 4611-622; 2009. Capuco A. V.; Wood D. L.; Baldwin R.; McLeod K.; Paape M. J. Mammary cell number, proliferation, and apoptosis during a bovine lactation: relation to milk production and effect of bST. J. Dairy Sci. 84: 2177-2187; 2001. Chaturvedi K.; Sarkar D. K. Isolation and characterization of rat pituitary endothelial cells. Neuroendocrinology 83(5-6): 387-393; 2006. de Hofland L. J.; Herder W. W.; Waaijers M.; Zuijderwijk J.; Uitterlinden P.; van Koetsveld P. M.; Lamberts S. W. Interferon-alpha-2a is a potent inhibitor of hormone secretion by cultured human pituitary adenomas. J. Clin. Endocrinol. Metab. 84(9): 3336-3343; 1999. DingW.J.;TangY.;SongY.L.;SuZ.D.;LiC.;LiuA.T.;HuX.;JiangH. Isolation of murine muscle-derived stem cells with preplate technique combined with limited dilution technique. Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 15(36): 6797-6801; 2011. Farrow K. N.; Gutierrez-Hartmann A. Transforming growth factorbeta1 inhibits rat prolactin promoter activity in GH4 neuroendocrine cells. DNA Cell Biol. 18: 863-873; 1999. Gong F. Y.; Deng J. Y.; Shi Y. F. IFN-? increases the hGH gene promoter activity in rat GH3 cells. Horm. Res. 60: 14-20; 2003. Hentges S. T.; Sarkar D. K. Transforming growth factor-beta regulation of estradiol induced prolactinomas. Front. Neuroendocrinol. 22: 340-363; 2001. Ingram C. D.; Keefe P. D.; Wooding F. B. P.; Bicknell R. J. Morphological characterisation of lactotrophs separated from the bovine pituitary by a rapid enrichment technique. Cell Tissue Res. 252: 655-659; 1988. Katoh K.; Takahashi T.; Kobayashi Y.; Obara Y. Somatotropic axis and nutrition in young ruminants around weaning time. Asian-Aust. J. Anim. Sci. 20: 1156-1168; 2007. Laporta J.; Driver A.; Khatib H. Short communication: expression and alternative splicing of POU1F1 pathway genes in preimplantation bovine embryos. J. Dairy Sci. 94(8): 4220-4223; 2011. Lisowski P.; Pierzcha M.; Gooecik J. Evaluation of reference genes for studies of gene expression in the bovine liver, kidney, pituitary, and thyroid. J. Appl. Genet. 49(4): 367-372; 2008. Moustakas A.; Takumi T.; Lin H. Y.; Lodish H. F. GH3 pituitary tumor cells contain heteromeric type I and type II receptor complexes for transforming growth factor ß and activin-A. J. Biol. Chem. 270: 765-769; 1995. Mueller S. G.; Kudlow J. E. Transforming growth factor-beta (TGF beta) inhibits TGF alpha expression in bovine anterior pituitaryderived cells. Mol. Endocrinol. 5(10): 1439-1446; 1991. Murata T.; Ying S. Y. Transforming growth factor-beta and activin inhibit basal secretion of prolactin in a pituitary monolayer culture system. Proc. Soc. Exp. Biol. Med. 198: 599-605; 1991. Nagai Y.; Ogasawara H.; Taketa Y.; Aso H.; Tanaka S.; Kanaya T.; Watanabe K.; Ohwada S.; Muneta Y.; Yamaguchi T. Bovine anterior pituitary progenitor cell line expresses interleukin (IL)-18 and IL-18 receptor. J. Neuroendocrinol. 20: 1233-1241; 2008. Ooi G. T.; Tawadros N.; Escalona R. M. Pituitary cell lines and their endocrine applications. Mol. Cell. Endocrinol. 228: 1-21; 2004. Oomizu S.; Honda J.; Takeuchi S.; Kakeya T.; Masui T.; Takahashi S. Transforming growth factor-alpha stimulates proliferation of mammotrophs and corticotrophs in the mouse pituitary. J. Endocrinol. 165(2): 493-501; 2000. Rattner A. Characterization of human osteoblastic cells: influence of the culture conditions. In Vitro Cell. Dev. Biol. Anim. 33: 757-762; 1997. Tong H. L.; Li Q. Z.; Gao X. J.; Yin D. Y. Establishment and characterization of a lactating dairy goat mammary gland epithelial cell line. In Vitro Cell. Dev. Biol. Anim. 48: 149-155; 2012. Vankelecom H.; Carmeliet P.; Heremans H.; Van Damme J.; Dijkmans R.; Billiau A.; Denef C. Interferon-gamma inhibits stimulated adrenocorticotropin, prolactin, and growth hormone secretion in normal rat anterior pituitary cell cultures. Endocrinology 126: 2919-2926; 1990. Yang J.; Zhao B.; Baracos V. E.; Kennelly J. J. Effects of bovine somatotropin on beta-casein mrna levels in mammary tissue of lactating cows. J. Dairy Sci. 88: 2806-2812; 2005. Yonekura S.; Sakamoto K.; Komatsu T.; Hagino A.; Katoh K.; Obara Y. Growth hormone and lactogenic hormones can reduce the leptin mrna expression in bovine mammary epithelial cells. Domest. Anim. Endocrinol. 31: 88-96; 2006. Zarzynska J.; Gajewska M.; Motyl T. Effects of hormones and growth factors on TGF-ß1 expression in bovine mammary epithelial cells. J. Dairy Res. 72: 39-48; 2005. Jian-Fa Wang, Shou-Peng Fu, and Su-Nan Li contributed equally to this work. J.-F * Wang S.-P.Fu * S.-N.Li * Z.-Q.Yang * W.-J. Xue * Z.-Q. Li * W. Wang * J.-X. Liu() College of Veterinary Medicine, Jilin University, Changchun 130062, People's Republic of China e-mail: [email protected] W. Wang e-mail: [email protected] (c) 2014 Society for In Vitro Biology |