Characterization of gender-specific bovine serum
Kim, Jihoe; Kim, Minsoo; Nahm, Sang-Soep; Lee, Dong-Mok; Pokharel, Smritee; Choi, Inho
2011-06-01 00:00:00
Animal Cells and Systems Vol. 15, No. 2, June 2011, 147154 Characterization of gender-specific bovine serum a b b a a a Jihoe Kim , Minsoo Kim , Sang-Soep Nahm , Dong-Mok Lee , Smritee Pokharel and Inho Choi * a b School of Biotechnology, Yeungnam University, Gyeongsan, Korea; College of Veterinary Medicine, Konkuk University, 1 Hwayangdong, Gwangjingu, Seoul, 143-701, Korea (Received 6 December 2010; received in revised form 27 January 2011; accepted 1 February 2011) Animal cell cultures generally require a nutrient-rich medium supplemented with animal serum. Adult bovine serum contains a variety of nutrients including inorganic minerals, vitamins, salts, proteins and lipids as well as growth factors that promote animal cell growth. To evaluate the potential use of gender-specific bovine serum (GSBS) for cell culture, the biochemical properties of male serum (MS), female serum (FS) and castrated-male serum (CMS) were investigated. Overall, the chemical profile of GSBS was similar to that of bovine references except for glucose, creatine kinase, lactate dehydrogenase and potassium. FS showed elevated total protein and sodium concentrations compared to MS and CMS. Proteins present in MS, FS and CMS but absent in fetal bovine serum (FBS) were selected by two-dimensional gel electrophoresis and identified by peptide mass fingerprinting. Some of the identified proteins are known to be involved in immune responses and the others have unknown physiological roles. Moreover, it was found that some proteins such as alpha-2-macroglobulin appeared to be gender-specific with higher contents in FS. Insulin and testosterone was significantly higher in MS, and 17b-estradiol and estrone were higher in FS, as compared to the other sera. Taken together, the results indicate that each GSBS has a different ratio of components. Differences in serum constituents may affect cell cultures in a different manner and could be beneficial, depending on the specific aim of cell cultures. Keywords: adult bovine serum; serum proteins; cell culture Introduction using charcoal-dextran has been used to study the cellular effect of steroids. However, the charcoal- Use of animal serum in culture media to reproduce in dextran process used to eliminate certain steroid vivo conditions started with the development of cell components in the serum also removes some desir- culture technique in 1900s. Serum provides an able components such as fatty acids, vitamins, optimum cell culture environment by providing electrolytes and metabolites essential for cell growth, albumin, steroids, cytokines and growth factors. In and imparts additional variables to the experiment addition, serum helps to maintain pH, osmotic (Cao et al. 2009). Thus, gender-specific sera with high pressure and electrolytes that play critical roles in or low contents of testosterone and estrogen can cell growth and morphology (Pilili et al. 2010). As provide an in situ environment for certain specific cell examples, albumin helps in transport and acts as an cultures. antioxidant (Francis 2010), and vitronectin/fibronec- In Korea, up to 650,000 cattle are slaughtered every tin aids in cell spreading and heparin and collagen year and around 25 million liters of blood waste is binding activity (Hayman et al. 1985; Aday et al. produced (Roc ¸a 2002). Only a small amount of bovine 2011). However, substitution of serum in cell culture blood from slaughter-houses is used currently for the media has not been successful due to the presence of production of plasma proteins (Belhocine et al. 1998), ambiguous factors. probiotics (Chang and Shin 1998) and food additives. Endogenous hormones in animal sera, which vary Steroids in blood are potent environmental contami- according to gender, have a greater effect in vitro nants and continuous exposure to them can lead to (Milo et al. 1976). Serum that is rich in testosterone testicular, breast and prostate cancer, endometriosis, enhances the myoblast formation of satellite and polycystic ovarian syndrome and decreased semen C3H10T1/2 cells (Sinha-Hikim et al. 2003). Estrogen quality (Foster 2008). Therefore, recycling bovine and estrone enhance growth of breast cancer cells blood in the preparation of serum for cell culture (Lee et al. 2009) and higher insulin content enhances could help decrease the environmental burden. In the growth of vascular endothelial cells and leuko- addition, we have previously reported the advantage cytes (Hirata et al. 2005). Steroid-free serum prepared of using gender-specific bovine serum (GSBS) as *Corresponding author. Email: inhochoi@ynu.ac.kr ISSN 1976-8354 print/ISSN 2151-2485 online # 2011 Korean Society for Integrative Biology DOI: 10.1080/19768354.2011.577584 http://www.informaworld.com 148 J. Kim et al. compared with fetal bovine serum (FBS), at least in Hormone analysis certain type of cell cultures (Lee et al. 2009). In the Hormone level was analyzed according to Lee et al. present work, we characterized the sera from male, (2010). Enzyme-linked immunosorbent assay (ELISA) female and castrated-male cattle. kits for testosterone, estrone and 17b-estradiol (DRG Instruments GmbH, Margurg, Germany) were used for hormonal analysis. Standard controls of known con- centration, or test serum and enzyme conjugate, were Materials and methods added in a specific-antibody-coated microtiter well and Reagents and equipment incubated for 12 h at room temperature. The mixture All chemicals used in this study were of analytical was removed and unbound samples were washed thrice grade and were purchased from Sigma-Aldrich Korea with washing solution. After adding substrate solution (Seoul, Korea) unless otherwise mentioned. Pharma- for 1530 min, the enzymatic reaction was terminated lyte (pH 3.510) was from GE Healthcare Korea by adding stop solution. The absorbance at 450 nm was (Seoul, Korea) and IPG Dry Strips (pH 410 NL, 24 measured within 10 min using an ELISA plate reader cm long) were from Genomine (Pohang, Korea). (Microplate autoreader; Bio-Rad, Hercules, CA, USA). Modified porcine trypsin (sequencing grade) was A bovine insulin ELISA kit from ALPCO Diagnostics from Promega Korea (Seoul, Korea). Analytical re- (Salem, NH, USA) was used for quantitative analysis agents and equipments used for serum analyses were of insulin following manufacture’s protocol. BS-400 from Mindray (Shenzhen, China), Hemavet from CDC Technologies (Oxford, CT, USA) and EasyLyte from Medica (Bedford, MA, USA). Preparation of serum protein samples for two dimensional-polyacrylamide gel electrophoresis (2D-PAGE) Serum isolation Pooled serum samples of castrated (n14), male (n18) and female (n15) cattle were prepared by Blood samples from 47 bovine individuals (34-year- mixing equal volumes of individual serum in each old females, n15; 2-year-old males, n18; 2-year- group. The pooled serum samples were dissolved in old castrated males, n 14) were collected in sterile 7 M urea, 2 M thiourea containing 4% (w/v) 3-[(3- bottles from a local slaughter-house at Yeongcheon, cholamidopropyl)dimethylammonio]-1-propanesulfo- Gyeongsanbuk-do, Republic of Korea. The blood nate (CHAPS), 1% (w/v) dithiothreitol (DTT) and 2% samples were quickly stored at 48C and serum was (v/v) pharmalyte and 1 mM benzamidine. Proteins were isolated according to Lee et al. (2009). Collected extracted for 1 h at room temperature with vortexing. blood samples were quickly stored at 48C for 45h. After centrifugation at 15,000 g for 1 h at 158C, The separated serum was collected in a sterile bottle insoluble material was discarded and the soluble and stored at 208C for 24 h after centrifugation at fraction was used for 2D-PAGE. Protein loading was 5000 rpm at 48C for 20 min. The frozen serum was normalized as previously described (Bradford 1976). thawed and centrifuged at 7000 rpm for 20 min, and the supernatant was collected prior to inactivation at 568C for 30 min. Finally, the serum was filtered 2D-PAGE and quantitative analysis of image through a 0.22 mm pore size filter for bacterial IPG dry strips were equilibrated for 1216 h with 7 M eradication and stored at 208C until further use. urea, 2 M thiourea containing 2% CHAPS, 1% DTT, 1% pharmalyte, and loaded with 1 mg of the protein samples prepared as above. Isoelectric focusing (IEF) Chemical analysis was performed at 208C using a Multiphor II electro- All equipment used for serum chemical analyses was phoresis unit and EPS 3500 XL power supply (Amer- in routine use and was calibrated daily. The choices sham Biosciences, Piscataway, NJ, USA) following the of analytical methods were based on the general manufacturer’s instructions. For IEF, the voltage was veterinary clinical pathology practice (Table 1; linearly increased from 150 to 3500 V during 3 h for Stockham and Scott 2008). All biochemical data sample entry followed by a constant 3500 V, with were analyzed using SPSS version 12.0 (SPSS, focusing complete after 96 kVh. Prior to electrophor- Chicago, IL, USA). Statistical differences were com- esis in the second dimension, strips were incubated for pared by one-way ANOVA test followed by Bonfer- 10 min in equilibration buffer (50 mM Tris-Cl, pH 6.8, roni tests for multiple comparisons. Differences with containing 6 M urea, 2% sodium dodecyl sulfate (SDS) a P valueB0.05 were considered as significant. and 30% glycerol), first with 1% DTT and second with Animal Cells and Systems 149 Table 1. Reagents and equipment used for blood analysis. Test Analytical method Reagent Equipment 1 2 Total protein Biuret Cormary BS-400 Albumin Bromocresol green colorimetric ELItech BS-400 Aspartate aminotransferase Kinetic ELItech BS-400 Alanine transaminase Kinetic ELItech BS-400 Alkaline phosphatase Kinetic ELItech BS-400 Total cholesterol Enzymatic ELItech BS-400 Total bilirubin Modified Malloy-Evelyn ELItech BS-400 Glucose Enzymatic ELItech BS-400 Blood urea nitrogen Enzymatic ELItech BS-400 Creatinine Jaffe’s Cormary BS-400 Creatine kinase Kinetic ELItech BS-400 Hemoglobin Electrical impedence Reagent for Hemavet 850FS Hemavet 850FS 5 6 Sodium Ion selective electrode Easy electrolytes EasyLyte Potassium Ion selective electrode Easy electrolytes EasyLyte Chloride Ion selective electrode Easy electrolytes EasyLyte Calcium o-Cresolphthalein complexone assay SIEMENS BS-400 Phosphorus U.V. end point ELItech BS-400 1 2 3 4 5 6 Poland; MINDRAY, CHINA; France; CDC Tech., USA; Medica, USA; USA. 2.5% iodoacetamide. Equilibrated strips were inserted 50% acetonitrile/0.1% TFA, and subjected to matrix- onto SDS-PAGE gels (2024 cm, 1016%). SDS- assisted laser desorption/ionization-time of flight PAGE was performed using Hoefer DALT 2D system (MALDI-TOF) analysis using an the Ettan MALDI- (Amersham Biosciences) following the manufacturer’s TOF Pro apparatus (Amersham Biosciences, Piscat- instructions. All 2D gels were run at 208C for 1700 Vh, away, NJ, USA) as previously described (Fernandez and Coomassie G250 stained as previously described et al. 1998). Spectra were collected from 350 shots per (Anderson et al. 1991). Quantitative analysis of digi- spectrum over an m/z range of 6003000 and calibrated tized images was done using the PDQuest version 7.0 by two-point internal calibration using trypsin auto- software (BioRad), according to the protocols provided digestion peaks (m/z 842.5099, 2211.1046). The peak list by the manufacturer. The quantity of each spot was was generated using the Ettan MALDI-TOF Pro normalized by total valid spot intensity. Evaluation Module version 2.0.16. The threshold used for peak-picking was 5000 for minimum resolution of monoisotopic mass and 2.5 for S/N. The search program MASCOT (http://www.matrixscience.com/) was used In-gel protein digestion for protein identification by PMF. The following para- Selected protein spots were digested in-gel using meters were used for the database search: trypsin as the modified porcine trypsin in a manner similar to that cleaving enzyme, a maximum of one missed cleavage, previously described (Shevchenko et al. 1996). Gel iodoacetamide (Cys) as a complete modification, oxida- pieces were washed with 50% acetonitrile to remove tion (Met) as a partial modification, monoisotopic SDS, salt and stain, dried to remove solvent and then masses and a mass tolerance of 9 0.1 Da. The PMF rehydrated with trypsin (810 ng/ml) and incubated acceptance criterion was probability scoring. 810 h at 378C. The proteolytic reaction was termi- nated by addition of 0.5% trifluoroacetic acid (TFA). Peptides were recovered by combining the aqueous Results phase from several extractions of gel pieces with 50% aqueous acetonitrile. After concentration the peptide Biochemical profile mixture was desalted using C ZipTips (Millipore, Table 2 presents chemical profiles of male serum (MS), Billerica, MA, USA), and peptides were eluted in female serum (FS) and castrated-male serum (CMS). 15 ml of acetonitrile. Most of the serum constituent levels were within the bovine reference range. However, glucose, creatine kinase, lactate dehydrogenase (LDH) and potassium Peptide mass fingerprinting (PMF) showed higher average values in all three groups An aliquot of peptides prepared as above was mixed with compared with the reference values. Although all three an equal volume of a-cyano-4-hydroxycinnamic acid in groups were within the reference ranges, total protein 150 J. Kim et al. Table 2. Summary of blood chemical profiles in male, female and castrated male cattle. Reference range* (unit) Male (range) Female (range) Castrated (range) a, b a b 7.4 7.6 7.0 Total protein 5.78.1 (g/dl) (6.28.7) (6.89.6) (6.57.6) 3.7 3.6 3.6 Albumin 3.04.3 (g/dl) (3.34.0) (3.44.0) (3.43.8) 92.5 79.3 74.8 Aspartate aminotransferase 78132 (u/l) (73.0127.0) (48.0103.0) (52.097.0) 23.4 20.9 20.7 Alanine transaminase 1140 (u/l) (13.028.0) (13.027.0) (12.028.0) 109.7 60.4 89.3 Alkaline phosphatase 0500 (u/l) (49.0212.0) (32.0101.0) (60.0184.0) 177.2 162.6 150.7 Cholesterol 65220 (mg/dl) (119.0272.0) (89.0237.0) (84.0240.0) 0.2 0.2 0.2 Bilirubin 0.010.5 (mg/dl) (0.10.3) (0.10.5) (0.00.3) 164.0 153.7 179.3 Glucose 4575 (mg/dl) (101.0248.0) (54322) (118320) 14.2 14.4 14.0 Blood urea nitroge 6.027.0 (mg/dl) (8.622.1) (11.023.3) (10.416.7) 1.4 1.4 1.3 Creatinine 1.02.0 (mg/dl) (1.11.8) (0.11.9) (1.01.5) 572.9 507.9 471.4 Creatine kinase 105.0409.0 (u/l) (340.0854.0) (193.0878.0) (192.01056.0) 4370.2 4438.5 4279.3 Lactate dehydrogenase 692.01445.0 (u/l) (3179.05506.0) (3006.05847.0) (2770.05637.0) 13.8 13.0 13.5 Hemoglobin 8.015.0 (g/dl) (10.118.6) (11.115.5) (10.318.2) a, b a b 144.6 146.4 144.3 Sodium 132152 (mmol/l) (140.9149.5) (141.2152.4) (140.2148.1) 8.0 7.9 8.3 Potassium 3.95.8 (mmol/l) (5.810.0) (4.710.0) (6.410.0) 100.1 101.3 100.3 Chloride 95110 (mmol/l) (96.8102.5) (96.7105.4) (97.2103.5) 9.4 9.3 9.5 Calcium 9.712.4 (mg/dl) (8.710.2) (8.310.7) (8.710.6) 6.0 5.9 6.4 Phosphorus 5.66.5 (mg/dl) (4.48.1) (3.07.5) (4.39.8) Data from Radostits et al. (2000). Concentrations in GSBS are significantly higher than the bovine reference range. Different superscript designates significant difference (PB0.05). and sodium concentrations in FS were slightly higher 3). Most of the identified proteins (CM-S1, CM-M-S3, than in CMS (Table 2). CM-M-S5, CM-M-S6 and F-S1) were likely to be involved in immune responses. The protein CM-M-S2 was detected at a higher level in CMS and MS (2.9- and 3.8-fold, respectively, compared to the content in FS); Identification of adult bovine-specific serum proteins the protein was identified as alpha-1B-glycoprotein. The proteins in pooled MS, FS and CMS were Protein CM-M-S7 was identified as a hemopexin-like separated by 2D-PAGE (Figure 1). Comparing the protein, and elevated 3.2-fold and 2-fold in CMS and 2D gel images for the samples with the image obtained MS, respectively, compared to FS. Protein CM-F-S1 for a commercial FBS, 70 protein spots were found to was identified as alpha-2-macroglobulin, which binds be specific for MS, FS and CMS. By quantitative to and modulates growth factors, cytokines and comparison of each protein spot, 22 protein spots that hormones (Feige et al. 1996). The protein was exhibited over a 2-fold variation were selected and classified in five gender-specific groups (Figure 2). expressed 2.7-fold and 2.0-fold more in CMS and FS, PMF for the 22 proteins identified eight proteins (Table respectively, than in MS. Animal Cells and Systems 151 Figure 1. 2D-PAGE analysis of female serum. Serum protein samples were prepared and separated according to their isoelectric point (pI) and molecular mass (MW). Protein spots identified by PMF are indicated with circles and numbers, and summarized in Table 3. Hormonal analysis with stressful situations (Cockram and Corley, 1991). Thus, elevated glucose and creatine kinase levels in Testosterone was significantly higher in MS than in FS GSBS may reflect the extreme anxiety in cattle under- and CMS (PB0.01), whereas estrogen levels (both 17b- going slaughter. estradiol and estrone) were highest in FS and lowest in FS showed higher total protein and sodium con- CMS. The insulin level was significantly higher in MS centrations than MS and CMS. Unfortunately, we were compared to FS and CMS (Table 4). unable to find any previous observations that explain sex-related differences in total protein and sodium Discussion concentration in serum. Potential reasons could be pregnancy, since the majority of slaughtered cattle used In order to test the potential use of GSBS for cell in the study were 34 years of age, and may have culture, biochemical constituents, proteins and hor- already experienced several pregnancies. Thus, higher mone in FS, MS, and CMS were investigated in this serum total protein might be related to the necessity for study. In general, the blood chemical profile of GSBS milk production (McAdam and O’Dell 1982). It has was compatible with other bovine strains. In GSBS, been shown that solutes in culture media significantly glucose, creatine kinase, LDH and potassium levels affect cell growth, with the major effect being repro- were higher than the reference ranges. It is most likely duced by simply altering sodium concentration (Rubin that elevated LDH and potassium concentrations were and Chu 1984). Moreover, serum protein is a ready due to hemolysis, which is known to cause leakage of source of unknown growth factors that are likely to LDH and potassium from erythrocytes (Mather and exert diverse effects on cells in culture. Therefore, Mackie 1960; Leung and Henderson 1981). Although elevated total protein and sodium concentrations we took great care while collecting blood samples should be taken into consideration when using female during the slaughter process, hemolysis was unavoid- GSBS. able since the samples were collected from bleeding Although the currently identified proteins were carcasses. The overall values for glucose and creatine selected to be gender-specific, they are more likely to kinase were higher than the reference range in all three be adult bovine-specific (and so not detected in FBS). sera. Traumatic injuries or surgical handling of viscera In fact, proteomic analysis of blood sera is challenging evoke neuroendocrine responses that resulted in hyper- due to the abundant proteins, such as albumin (up to glycemia (Reis et al. 1998). Moreover, marked elevation of serum creatine kinase concentration is associated 60% of total serum proteins) and immunoglobulin in 152 J. Kim et al. Figure 2. Quantitative analysis of putative gender-specific serum proteins. Seventy serum protein spots initially screened as specific for GSBS were quantitatively analyzed. Twenty-two proteins that exhibited variable deviation over 2-fold in quantity between each group of CMS ( ), MS (I) and FS ( ) were selected for further identification and grouped as castrated male- specific (CM-S, A), female-specific (F-S, B), male and female-specific (M-F-S, C), castrated male and male-specific (CM-M-S, D) and castrated male and female-specific (CM-F-S, E). The relative quantity was obtained by comparing spot intensity, and the number on the x-axis indicates the spot number in each group. serum. Hence, the enrichment of minor proteins after CMS) for the screening of gender-specific serum removing abundant serum proteins would enhance the proteins. From the initial screening, some proteins identification of more gender-specific proteins. An- were detected to deviate more significantly in quantity other problem might be the low cut-off value (2-fold between each serum group and were more likely to be in quantity comparing each protein in MS, FS and gender-specific. But the PMF identification was not Table 3. Identification of proteins by PMF analysis. Spot # Protein ID Proteins Sequence coverage (%) Score MW (Da) CM-S1 NP_001035559 Complement C3 12 151 188652 CM-M-S2 NP_001039708 Alpha-1B-glycoprotein precursor 21 108 54091 CM-M-S3 AAB62251 IgM H-chain constant region 32 122 48512 CM-M-S5 NP_001137569 C-reactive protein precursor 22 89 25504 CM-M-S6 CAJ31249 Prepro complement component C3 7 90 188715 CM-M-S7 NP_001029784 Hemopexin precursor 16 104 52974 F-S1 DAA21469 Complement factor H precursor 13 93 99830 CM-F-S1 NP_001103265 Alpha-2-macroglobulin 28 325 168953 PMF was performed as described in the Materials and methods for 22 protein spots selected in Figure 2. Using the MASCOT search program (http://www.matrixscience.com/), eight proteins were identified and summarized. Animal Cells and Systems 153 Table 4. Analysis of hormones in different genders of adult bovine sera. T (ng/ml) E2 (pg/ml) E1 (pg/ml) Insulin (ng/ml) Female 1.16 (0.312.07) 100 (24279) 420 (1051468) 0.45 (0.052.46) a a Male 10.76 (4.7818.62) 69 (13255) 320 (64671) 0.74 (0.022.48) Castrated 1.49 (0.313.21) 27 (1472) 215 (72688) 0.30 (0.050.62) Hormone analysis was done as described in the Materials and methods by ELISA for testosterone (T), estrone (E ), 17b-estradiol (E2) and insulin. B 0.01. successful, probably due to the low amount of proteins ovarian cyclic variation (Dobson et al. 1974). Again, in sera. the increased proliferation in breast cancer cells may be Nevertheless, our proteomic analysis identified due to high estrogen level in female serum compared to adult bovine-specific and putative gender-specific pro- FBS (Lee et al. 2009). In contrast, all testosterone, 17b- teins. More than half of the identified proteins (Table 3, estradiol and estrone were lowest in castrated male components of complement, immunoglobulin) were cattle compared to both the male and female cattle. shown to be directly involved in immune responses of It has been well-documented that muscle develop- adult bovines against potent pathogens encountered ment during and after puberty in animals is closely during their lifetimes. The protein alpha-1B-glycopro- associated with increased circulating sex steroids in tein was grouped in castrated male and male-specific blood secreted from the gonads. Increased amounts of proteins (CM-M-S in Figure 2 and Table 3). Although sex steroids, directly or indirectly via the stimulation of its physiological role has not been clearly understood, other factors affecting muscle cell growth and differ- the protein contains conserved immunoglobulin do- entiation, will eventually lead to muscle development in mains (Ishioka et al. 1986) and may be a marker for animals (Sinha-Hikim et al. 2003). In addition, respon- mycobacterial infections of cattle (Seth et al. 2009). siveness of certain cell types is greatly affected by Another castrated male and male-specific protein, CM- gender-specific sex hormones. For example, estrogens M-S7, was identified as a hemopexin-like protein that are required for breast and uterine cancer cell growth, has 73% amino acid homology with hyaluronidase while androgens are required for prostate cancer cell from porcine liver (Zhu et al. 1994), which catalyzes the growth. In this respect, our previous result that hydrolysis of hyaluronic acid in extracellular matrix demonstrated enhanced cell proliferation and differ- (Stern 2008). The most interesting protein identified in entiation of myogenic satellite cells in MS as compared this report was alpha-2-macroglobulin, which was with FS, CMS as well as FBS highlights the importance specific for proteinase inhibitors and binds growth of optimal serum for cell cultures. The data from this factors and hormones as a carrier (Feige et al. 1996). study suggest the possible application of GSBS as a The protein has autocrine or paracrine roles in substitute for FBS. increasing 17b-estradiol production by granulosa cells (Ireland et al. 2004). The amount of 17b-estradiol in FS was determined to be 1.5-fold that in MS (Table 4), Acknowledgements which was coincident with the relative amount of The gender-specific adult bovine sera were used from the alpha-2-macroglobulin. However, the amount of the Bovine Genome Resource Bank, Yeungnam University, same hormone in CMS did not agree with the alpha-2- Korea. This work was carried out with the support of the macroglobulin analysis. ‘Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ007472)’, ‘Rural Hormone analysis revealed variation in hormonal Development Administration, Republic of Korea. level which is gender-specific. The level of testosterone observed in MS was significantly higher than in FS and CMS (PB0.01). Gender variation in the level of References testosterone may be due to the difference in nutritional Aday S, Hasirci N, Gurhan D. 2011. A ‘cost effective and status (Brito et al. 2007) and age; testosterone level simple culture method for primary hepatocytes. Anim increases each month up until puberty (Barth et al. Cells Syst. 15:1927. 2008). Testosterone in MS may be one of the causes of Anderson NL, Esquer-Blasco R, Hofmann JP, Anderson NG. 1991. 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Insulin enhances increase in satellite cell number in healthy, young men. leukocyte-endothelial cell adhesion in the retinal micro- Am J Physiol Endocrinol Metab. 285:E197E205. circulation through surface expression of intercellular Stern R. 2008. Hyaluronidases in cancer biology. Semin adhesion molecule-1. Microvasc Res. 69:13541. Cancer Biol. 18:275280. Ishioka N, Takahashi N, Putnam FW. 1986. Amino acid Stockham SL, Scott MA. 2008. Fundamentals of veterinary sequence of human plasma alpha 1B-glycoprotein: clinical pathology. 2nd ed. Cambridge: Blackwell. p. 369 homology to the immunoglobulin supergene family. Proc Natl Acad Sci USA. 83:23632367. Zhu L, Hope TJ, Hall J, Davies A, Stern M, Muller-Eberhard Ireland JL, Jimenez-Krassel F, Winn ME, Burns DS, Ireland U, Stern R, Parslow TG. 1994. Molecular cloning of a JJ. 2004. Evidence for autocrine or paracrine roles of mammalian hyaluronidase reveals identity with hemo- alpha 2-macroglobulin in regulation of estradiol produc- pexin, a serum heme-binding protein. J Biol Chem. tion by granulosa cells and development of dominant follicles. Endocrinology. 145:27842794. 269:3209232097.
http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.pngAnimal Cells and SystemsTaylor & Francishttp://www.deepdyve.com/lp/taylor-francis/characterization-of-gender-specific-bovine-serum-DKHVyWCMTY
Animal cell cultures generally require a nutrient-rich medium supplemented with animal serum. Adult bovine serum contains a variety of nutrients including inorganic minerals, vitamins, salts, proteins and lipids as well as growth factors that promote animal cell growth. To evaluate the potential use of gender-specific bovine serum (GSBS) for cell culture, the biochemical properties of male serum (MS), female serum (FS) and castrated-male serum (CMS) were investigated. Overall, the chemical...
Animal Cells and Systems Vol. 15, No. 2, June 2011, 147154 Characterization of gender-specific bovine serum a b b a a a Jihoe Kim , Minsoo Kim , Sang-Soep Nahm , Dong-Mok Lee , Smritee Pokharel and Inho Choi * a b School of Biotechnology, Yeungnam University, Gyeongsan, Korea; College of Veterinary Medicine, Konkuk University, 1 Hwayangdong, Gwangjingu, Seoul, 143-701, Korea (Received 6 December 2010; received in revised form 27 January 2011; accepted 1 February 2011) Animal cell cultures generally require a nutrient-rich medium supplemented with animal serum. Adult bovine serum contains a variety of nutrients including inorganic minerals, vitamins, salts, proteins and lipids as well as growth factors that promote animal cell growth. To evaluate the potential use of gender-specific bovine serum (GSBS) for cell culture, the biochemical properties of male serum (MS), female serum (FS) and castrated-male serum (CMS) were investigated. Overall, the chemical profile of GSBS was similar to that of bovine references except for glucose, creatine kinase, lactate dehydrogenase and potassium. FS showed elevated total protein and sodium concentrations compared to MS and CMS. Proteins present in MS, FS and CMS but absent in fetal bovine serum (FBS) were selected by two-dimensional gel electrophoresis and identified by peptide mass fingerprinting. Some of the identified proteins are known to be involved in immune responses and the others have unknown physiological roles. Moreover, it was found that some proteins such as alpha-2-macroglobulin appeared to be gender-specific with higher contents in FS. Insulin and testosterone was significantly higher in MS, and 17b-estradiol and estrone were higher in FS, as compared to the other sera. Taken together, the results indicate that each GSBS has a different ratio of components. Differences in serum constituents may affect cell cultures in a different manner and could be beneficial, depending on the specific aim of cell cultures. Keywords: adult bovine serum; serum proteins; cell culture Introduction using charcoal-dextran has been used to study the cellular effect of steroids. However, the charcoal- Use of animal serum in culture media to reproduce in dextran process used to eliminate certain steroid vivo conditions started with the development of cell components in the serum also removes some desir- culture technique in 1900s. Serum provides an able components such as fatty acids, vitamins, optimum cell culture environment by providing electrolytes and metabolites essential for cell growth, albumin, steroids, cytokines and growth factors. In and imparts additional variables to the experiment addition, serum helps to maintain pH, osmotic (Cao et al. 2009). Thus, gender-specific sera with high pressure and electrolytes that play critical roles in or low contents of testosterone and estrogen can cell growth and morphology (Pilili et al. 2010). As provide an in situ environment for certain specific cell examples, albumin helps in transport and acts as an cultures. antioxidant (Francis 2010), and vitronectin/fibronec- In Korea, up to 650,000 cattle are slaughtered every tin aids in cell spreading and heparin and collagen year and around 25 million liters of blood waste is binding activity (Hayman et al. 1985; Aday et al. produced (Roc ¸a 2002). Only a small amount of bovine 2011). However, substitution of serum in cell culture blood from slaughter-houses is used currently for the media has not been successful due to the presence of production of plasma proteins (Belhocine et al. 1998), ambiguous factors. probiotics (Chang and Shin 1998) and food additives. Endogenous hormones in animal sera, which vary Steroids in blood are potent environmental contami- according to gender, have a greater effect in vitro nants and continuous exposure to them can lead to (Milo et al. 1976). Serum that is rich in testosterone testicular, breast and prostate cancer, endometriosis, enhances the myoblast formation of satellite and polycystic ovarian syndrome and decreased semen C3H10T1/2 cells (Sinha-Hikim et al. 2003). Estrogen quality (Foster 2008). Therefore, recycling bovine and estrone enhance growth of breast cancer cells blood in the preparation of serum for cell culture (Lee et al. 2009) and higher insulin content enhances could help decrease the environmental burden. In the growth of vascular endothelial cells and leuko- addition, we have previously reported the advantage cytes (Hirata et al. 2005). Steroid-free serum prepared of using gender-specific bovine serum (GSBS) as *Corresponding author. Email: inhochoi@ynu.ac.kr ISSN 1976-8354 print/ISSN 2151-2485 online # 2011 Korean Society for Integrative Biology DOI: 10.1080/19768354.2011.577584 http://www.informaworld.com 148 J. Kim et al. compared with fetal bovine serum (FBS), at least in Hormone analysis certain type of cell cultures (Lee et al. 2009). In the Hormone level was analyzed according to Lee et al. present work, we characterized the sera from male, (2010). Enzyme-linked immunosorbent assay (ELISA) female and castrated-male cattle. kits for testosterone, estrone and 17b-estradiol (DRG Instruments GmbH, Margurg, Germany) were used for hormonal analysis. Standard controls of known con- centration, or test serum and enzyme conjugate, were Materials and methods added in a specific-antibody-coated microtiter well and Reagents and equipment incubated for 12 h at room temperature. The mixture All chemicals used in this study were of analytical was removed and unbound samples were washed thrice grade and were purchased from Sigma-Aldrich Korea with washing solution. After adding substrate solution (Seoul, Korea) unless otherwise mentioned. Pharma- for 1530 min, the enzymatic reaction was terminated lyte (pH 3.510) was from GE Healthcare Korea by adding stop solution. The absorbance at 450 nm was (Seoul, Korea) and IPG Dry Strips (pH 410 NL, 24 measured within 10 min using an ELISA plate reader cm long) were from Genomine (Pohang, Korea). (Microplate autoreader; Bio-Rad, Hercules, CA, USA). Modified porcine trypsin (sequencing grade) was A bovine insulin ELISA kit from ALPCO Diagnostics from Promega Korea (Seoul, Korea). Analytical re- (Salem, NH, USA) was used for quantitative analysis agents and equipments used for serum analyses were of insulin following manufacture’s protocol. BS-400 from Mindray (Shenzhen, China), Hemavet from CDC Technologies (Oxford, CT, USA) and EasyLyte from Medica (Bedford, MA, USA). Preparation of serum protein samples for two dimensional-polyacrylamide gel electrophoresis (2D-PAGE) Serum isolation Pooled serum samples of castrated (n14), male (n18) and female (n15) cattle were prepared by Blood samples from 47 bovine individuals (34-year- mixing equal volumes of individual serum in each old females, n15; 2-year-old males, n18; 2-year- group. The pooled serum samples were dissolved in old castrated males, n 14) were collected in sterile 7 M urea, 2 M thiourea containing 4% (w/v) 3-[(3- bottles from a local slaughter-house at Yeongcheon, cholamidopropyl)dimethylammonio]-1-propanesulfo- Gyeongsanbuk-do, Republic of Korea. The blood nate (CHAPS), 1% (w/v) dithiothreitol (DTT) and 2% samples were quickly stored at 48C and serum was (v/v) pharmalyte and 1 mM benzamidine. Proteins were isolated according to Lee et al. (2009). Collected extracted for 1 h at room temperature with vortexing. blood samples were quickly stored at 48C for 45h. After centrifugation at 15,000 g for 1 h at 158C, The separated serum was collected in a sterile bottle insoluble material was discarded and the soluble and stored at 208C for 24 h after centrifugation at fraction was used for 2D-PAGE. Protein loading was 5000 rpm at 48C for 20 min. The frozen serum was normalized as previously described (Bradford 1976). thawed and centrifuged at 7000 rpm for 20 min, and the supernatant was collected prior to inactivation at 568C for 30 min. Finally, the serum was filtered 2D-PAGE and quantitative analysis of image through a 0.22 mm pore size filter for bacterial IPG dry strips were equilibrated for 1216 h with 7 M eradication and stored at 208C until further use. urea, 2 M thiourea containing 2% CHAPS, 1% DTT, 1% pharmalyte, and loaded with 1 mg of the protein samples prepared as above. Isoelectric focusing (IEF) Chemical analysis was performed at 208C using a Multiphor II electro- All equipment used for serum chemical analyses was phoresis unit and EPS 3500 XL power supply (Amer- in routine use and was calibrated daily. The choices sham Biosciences, Piscataway, NJ, USA) following the of analytical methods were based on the general manufacturer’s instructions. For IEF, the voltage was veterinary clinical pathology practice (Table 1; linearly increased from 150 to 3500 V during 3 h for Stockham and Scott 2008). All biochemical data sample entry followed by a constant 3500 V, with were analyzed using SPSS version 12.0 (SPSS, focusing complete after 96 kVh. Prior to electrophor- Chicago, IL, USA). Statistical differences were com- esis in the second dimension, strips were incubated for pared by one-way ANOVA test followed by Bonfer- 10 min in equilibration buffer (50 mM Tris-Cl, pH 6.8, roni tests for multiple comparisons. Differences with containing 6 M urea, 2% sodium dodecyl sulfate (SDS) a P valueB0.05 were considered as significant. and 30% glycerol), first with 1% DTT and second with Animal Cells and Systems 149 Table 1. Reagents and equipment used for blood analysis. Test Analytical method Reagent Equipment 1 2 Total protein Biuret Cormary BS-400 Albumin Bromocresol green colorimetric ELItech BS-400 Aspartate aminotransferase Kinetic ELItech BS-400 Alanine transaminase Kinetic ELItech BS-400 Alkaline phosphatase Kinetic ELItech BS-400 Total cholesterol Enzymatic ELItech BS-400 Total bilirubin Modified Malloy-Evelyn ELItech BS-400 Glucose Enzymatic ELItech BS-400 Blood urea nitrogen Enzymatic ELItech BS-400 Creatinine Jaffe’s Cormary BS-400 Creatine kinase Kinetic ELItech BS-400 Hemoglobin Electrical impedence Reagent for Hemavet 850FS Hemavet 850FS 5 6 Sodium Ion selective electrode Easy electrolytes EasyLyte Potassium Ion selective electrode Easy electrolytes EasyLyte Chloride Ion selective electrode Easy electrolytes EasyLyte Calcium o-Cresolphthalein complexone assay SIEMENS BS-400 Phosphorus U.V. end point ELItech BS-400 1 2 3 4 5 6 Poland; MINDRAY, CHINA; France; CDC Tech., USA; Medica, USA; USA. 2.5% iodoacetamide. Equilibrated strips were inserted 50% acetonitrile/0.1% TFA, and subjected to matrix- onto SDS-PAGE gels (2024 cm, 1016%). SDS- assisted laser desorption/ionization-time of flight PAGE was performed using Hoefer DALT 2D system (MALDI-TOF) analysis using an the Ettan MALDI- (Amersham Biosciences) following the manufacturer’s TOF Pro apparatus (Amersham Biosciences, Piscat- instructions. All 2D gels were run at 208C for 1700 Vh, away, NJ, USA) as previously described (Fernandez and Coomassie G250 stained as previously described et al. 1998). Spectra were collected from 350 shots per (Anderson et al. 1991). Quantitative analysis of digi- spectrum over an m/z range of 6003000 and calibrated tized images was done using the PDQuest version 7.0 by two-point internal calibration using trypsin auto- software (BioRad), according to the protocols provided digestion peaks (m/z 842.5099, 2211.1046). The peak list by the manufacturer. The quantity of each spot was was generated using the Ettan MALDI-TOF Pro normalized by total valid spot intensity. Evaluation Module version 2.0.16. The threshold used for peak-picking was 5000 for minimum resolution of monoisotopic mass and 2.5 for S/N. The search program MASCOT (http://www.matrixscience.com/) was used In-gel protein digestion for protein identification by PMF. The following para- Selected protein spots were digested in-gel using meters were used for the database search: trypsin as the modified porcine trypsin in a manner similar to that cleaving enzyme, a maximum of one missed cleavage, previously described (Shevchenko et al. 1996). Gel iodoacetamide (Cys) as a complete modification, oxida- pieces were washed with 50% acetonitrile to remove tion (Met) as a partial modification, monoisotopic SDS, salt and stain, dried to remove solvent and then masses and a mass tolerance of 9 0.1 Da. The PMF rehydrated with trypsin (810 ng/ml) and incubated acceptance criterion was probability scoring. 810 h at 378C. The proteolytic reaction was termi- nated by addition of 0.5% trifluoroacetic acid (TFA). Peptides were recovered by combining the aqueous Results phase from several extractions of gel pieces with 50% aqueous acetonitrile. After concentration the peptide Biochemical profile mixture was desalted using C ZipTips (Millipore, Table 2 presents chemical profiles of male serum (MS), Billerica, MA, USA), and peptides were eluted in female serum (FS) and castrated-male serum (CMS). 15 ml of acetonitrile. Most of the serum constituent levels were within the bovine reference range. However, glucose, creatine kinase, lactate dehydrogenase (LDH) and potassium Peptide mass fingerprinting (PMF) showed higher average values in all three groups An aliquot of peptides prepared as above was mixed with compared with the reference values. Although all three an equal volume of a-cyano-4-hydroxycinnamic acid in groups were within the reference ranges, total protein 150 J. Kim et al. Table 2. Summary of blood chemical profiles in male, female and castrated male cattle. Reference range* (unit) Male (range) Female (range) Castrated (range) a, b a b 7.4 7.6 7.0 Total protein 5.78.1 (g/dl) (6.28.7) (6.89.6) (6.57.6) 3.7 3.6 3.6 Albumin 3.04.3 (g/dl) (3.34.0) (3.44.0) (3.43.8) 92.5 79.3 74.8 Aspartate aminotransferase 78132 (u/l) (73.0127.0) (48.0103.0) (52.097.0) 23.4 20.9 20.7 Alanine transaminase 1140 (u/l) (13.028.0) (13.027.0) (12.028.0) 109.7 60.4 89.3 Alkaline phosphatase 0500 (u/l) (49.0212.0) (32.0101.0) (60.0184.0) 177.2 162.6 150.7 Cholesterol 65220 (mg/dl) (119.0272.0) (89.0237.0) (84.0240.0) 0.2 0.2 0.2 Bilirubin 0.010.5 (mg/dl) (0.10.3) (0.10.5) (0.00.3) 164.0 153.7 179.3 Glucose 4575 (mg/dl) (101.0248.0) (54322) (118320) 14.2 14.4 14.0 Blood urea nitroge 6.027.0 (mg/dl) (8.622.1) (11.023.3) (10.416.7) 1.4 1.4 1.3 Creatinine 1.02.0 (mg/dl) (1.11.8) (0.11.9) (1.01.5) 572.9 507.9 471.4 Creatine kinase 105.0409.0 (u/l) (340.0854.0) (193.0878.0) (192.01056.0) 4370.2 4438.5 4279.3 Lactate dehydrogenase 692.01445.0 (u/l) (3179.05506.0) (3006.05847.0) (2770.05637.0) 13.8 13.0 13.5 Hemoglobin 8.015.0 (g/dl) (10.118.6) (11.115.5) (10.318.2) a, b a b 144.6 146.4 144.3 Sodium 132152 (mmol/l) (140.9149.5) (141.2152.4) (140.2148.1) 8.0 7.9 8.3 Potassium 3.95.8 (mmol/l) (5.810.0) (4.710.0) (6.410.0) 100.1 101.3 100.3 Chloride 95110 (mmol/l) (96.8102.5) (96.7105.4) (97.2103.5) 9.4 9.3 9.5 Calcium 9.712.4 (mg/dl) (8.710.2) (8.310.7) (8.710.6) 6.0 5.9 6.4 Phosphorus 5.66.5 (mg/dl) (4.48.1) (3.07.5) (4.39.8) Data from Radostits et al. (2000). Concentrations in GSBS are significantly higher than the bovine reference range. Different superscript designates significant difference (PB0.05). and sodium concentrations in FS were slightly higher 3). Most of the identified proteins (CM-S1, CM-M-S3, than in CMS (Table 2). CM-M-S5, CM-M-S6 and F-S1) were likely to be involved in immune responses. The protein CM-M-S2 was detected at a higher level in CMS and MS (2.9- and 3.8-fold, respectively, compared to the content in FS); Identification of adult bovine-specific serum proteins the protein was identified as alpha-1B-glycoprotein. The proteins in pooled MS, FS and CMS were Protein CM-M-S7 was identified as a hemopexin-like separated by 2D-PAGE (Figure 1). Comparing the protein, and elevated 3.2-fold and 2-fold in CMS and 2D gel images for the samples with the image obtained MS, respectively, compared to FS. Protein CM-F-S1 for a commercial FBS, 70 protein spots were found to was identified as alpha-2-macroglobulin, which binds be specific for MS, FS and CMS. By quantitative to and modulates growth factors, cytokines and comparison of each protein spot, 22 protein spots that hormones (Feige et al. 1996). The protein was exhibited over a 2-fold variation were selected and classified in five gender-specific groups (Figure 2). expressed 2.7-fold and 2.0-fold more in CMS and FS, PMF for the 22 proteins identified eight proteins (Table respectively, than in MS. Animal Cells and Systems 151 Figure 1. 2D-PAGE analysis of female serum. Serum protein samples were prepared and separated according to their isoelectric point (pI) and molecular mass (MW). Protein spots identified by PMF are indicated with circles and numbers, and summarized in Table 3. Hormonal analysis with stressful situations (Cockram and Corley, 1991). Thus, elevated glucose and creatine kinase levels in Testosterone was significantly higher in MS than in FS GSBS may reflect the extreme anxiety in cattle under- and CMS (PB0.01), whereas estrogen levels (both 17b- going slaughter. estradiol and estrone) were highest in FS and lowest in FS showed higher total protein and sodium con- CMS. The insulin level was significantly higher in MS centrations than MS and CMS. Unfortunately, we were compared to FS and CMS (Table 4). unable to find any previous observations that explain sex-related differences in total protein and sodium Discussion concentration in serum. Potential reasons could be pregnancy, since the majority of slaughtered cattle used In order to test the potential use of GSBS for cell in the study were 34 years of age, and may have culture, biochemical constituents, proteins and hor- already experienced several pregnancies. Thus, higher mone in FS, MS, and CMS were investigated in this serum total protein might be related to the necessity for study. In general, the blood chemical profile of GSBS milk production (McAdam and O’Dell 1982). It has was compatible with other bovine strains. In GSBS, been shown that solutes in culture media significantly glucose, creatine kinase, LDH and potassium levels affect cell growth, with the major effect being repro- were higher than the reference ranges. It is most likely duced by simply altering sodium concentration (Rubin that elevated LDH and potassium concentrations were and Chu 1984). Moreover, serum protein is a ready due to hemolysis, which is known to cause leakage of source of unknown growth factors that are likely to LDH and potassium from erythrocytes (Mather and exert diverse effects on cells in culture. Therefore, Mackie 1960; Leung and Henderson 1981). Although elevated total protein and sodium concentrations we took great care while collecting blood samples should be taken into consideration when using female during the slaughter process, hemolysis was unavoid- GSBS. able since the samples were collected from bleeding Although the currently identified proteins were carcasses. The overall values for glucose and creatine selected to be gender-specific, they are more likely to kinase were higher than the reference range in all three be adult bovine-specific (and so not detected in FBS). sera. Traumatic injuries or surgical handling of viscera In fact, proteomic analysis of blood sera is challenging evoke neuroendocrine responses that resulted in hyper- due to the abundant proteins, such as albumin (up to glycemia (Reis et al. 1998). Moreover, marked elevation of serum creatine kinase concentration is associated 60% of total serum proteins) and immunoglobulin in 152 J. Kim et al. Figure 2. Quantitative analysis of putative gender-specific serum proteins. Seventy serum protein spots initially screened as specific for GSBS were quantitatively analyzed. Twenty-two proteins that exhibited variable deviation over 2-fold in quantity between each group of CMS ( ), MS (I) and FS ( ) were selected for further identification and grouped as castrated male- specific (CM-S, A), female-specific (F-S, B), male and female-specific (M-F-S, C), castrated male and male-specific (CM-M-S, D) and castrated male and female-specific (CM-F-S, E). The relative quantity was obtained by comparing spot intensity, and the number on the x-axis indicates the spot number in each group. serum. Hence, the enrichment of minor proteins after CMS) for the screening of gender-specific serum removing abundant serum proteins would enhance the proteins. From the initial screening, some proteins identification of more gender-specific proteins. An- were detected to deviate more significantly in quantity other problem might be the low cut-off value (2-fold between each serum group and were more likely to be in quantity comparing each protein in MS, FS and gender-specific. But the PMF identification was not Table 3. Identification of proteins by PMF analysis. Spot # Protein ID Proteins Sequence coverage (%) Score MW (Da) CM-S1 NP_001035559 Complement C3 12 151 188652 CM-M-S2 NP_001039708 Alpha-1B-glycoprotein precursor 21 108 54091 CM-M-S3 AAB62251 IgM H-chain constant region 32 122 48512 CM-M-S5 NP_001137569 C-reactive protein precursor 22 89 25504 CM-M-S6 CAJ31249 Prepro complement component C3 7 90 188715 CM-M-S7 NP_001029784 Hemopexin precursor 16 104 52974 F-S1 DAA21469 Complement factor H precursor 13 93 99830 CM-F-S1 NP_001103265 Alpha-2-macroglobulin 28 325 168953 PMF was performed as described in the Materials and methods for 22 protein spots selected in Figure 2. Using the MASCOT search program (http://www.matrixscience.com/), eight proteins were identified and summarized. Animal Cells and Systems 153 Table 4. Analysis of hormones in different genders of adult bovine sera. T (ng/ml) E2 (pg/ml) E1 (pg/ml) Insulin (ng/ml) Female 1.16 (0.312.07) 100 (24279) 420 (1051468) 0.45 (0.052.46) a a Male 10.76 (4.7818.62) 69 (13255) 320 (64671) 0.74 (0.022.48) Castrated 1.49 (0.313.21) 27 (1472) 215 (72688) 0.30 (0.050.62) Hormone analysis was done as described in the Materials and methods by ELISA for testosterone (T), estrone (E ), 17b-estradiol (E2) and insulin. B 0.01. successful, probably due to the low amount of proteins ovarian cyclic variation (Dobson et al. 1974). Again, in sera. the increased proliferation in breast cancer cells may be Nevertheless, our proteomic analysis identified due to high estrogen level in female serum compared to adult bovine-specific and putative gender-specific pro- FBS (Lee et al. 2009). In contrast, all testosterone, 17b- teins. More than half of the identified proteins (Table 3, estradiol and estrone were lowest in castrated male components of complement, immunoglobulin) were cattle compared to both the male and female cattle. shown to be directly involved in immune responses of It has been well-documented that muscle develop- adult bovines against potent pathogens encountered ment during and after puberty in animals is closely during their lifetimes. The protein alpha-1B-glycopro- associated with increased circulating sex steroids in tein was grouped in castrated male and male-specific blood secreted from the gonads. Increased amounts of proteins (CM-M-S in Figure 2 and Table 3). Although sex steroids, directly or indirectly via the stimulation of its physiological role has not been clearly understood, other factors affecting muscle cell growth and differ- the protein contains conserved immunoglobulin do- entiation, will eventually lead to muscle development in mains (Ishioka et al. 1986) and may be a marker for animals (Sinha-Hikim et al. 2003). In addition, respon- mycobacterial infections of cattle (Seth et al. 2009). siveness of certain cell types is greatly affected by Another castrated male and male-specific protein, CM- gender-specific sex hormones. For example, estrogens M-S7, was identified as a hemopexin-like protein that are required for breast and uterine cancer cell growth, has 73% amino acid homology with hyaluronidase while androgens are required for prostate cancer cell from porcine liver (Zhu et al. 1994), which catalyzes the growth. In this respect, our previous result that hydrolysis of hyaluronic acid in extracellular matrix demonstrated enhanced cell proliferation and differ- (Stern 2008). The most interesting protein identified in entiation of myogenic satellite cells in MS as compared this report was alpha-2-macroglobulin, which was with FS, CMS as well as FBS highlights the importance specific for proteinase inhibitors and binds growth of optimal serum for cell cultures. The data from this factors and hormones as a carrier (Feige et al. 1996). study suggest the possible application of GSBS as a The protein has autocrine or paracrine roles in substitute for FBS. increasing 17b-estradiol production by granulosa cells (Ireland et al. 2004). The amount of 17b-estradiol in FS was determined to be 1.5-fold that in MS (Table 4), Acknowledgements which was coincident with the relative amount of The gender-specific adult bovine sera were used from the alpha-2-macroglobulin. However, the amount of the Bovine Genome Resource Bank, Yeungnam University, same hormone in CMS did not agree with the alpha-2- Korea. This work was carried out with the support of the macroglobulin analysis. ‘Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ007472)’, ‘Rural Hormone analysis revealed variation in hormonal Development Administration, Republic of Korea. level which is gender-specific. The level of testosterone observed in MS was significantly higher than in FS and CMS (PB0.01). Gender variation in the level of References testosterone may be due to the difference in nutritional Aday S, Hasirci N, Gurhan D. 2011. A ‘cost effective and status (Brito et al. 2007) and age; testosterone level simple culture method for primary hepatocytes. Anim increases each month up until puberty (Barth et al. Cells Syst. 15:1927. 2008). Testosterone in MS may be one of the causes of Anderson NL, Esquer-Blasco R, Hofmann JP, Anderson NG. 1991. 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