MATERIALS AND METHODS

RNA isolation and reverse transcription: Total RNA was purified with Trizol reagent (Invitrogen) and treated with RQ1 RNase free DNase (Promega) to remove genomic DNA contamination. One microgram of total RNA was used for reverse transcription reaction with ReverTraAce (Toyobo) and random primer (Invitrogen), according to the manufacturer’s instructions. Quantitative PCR was performed with Taqman RT-PCR using Premix Ex Taq (Takara) and analyzed with the 7300 real-time PCR system (Applied Biosystems).

Probe sequences used in quantitative PCR: FAM, 6-carboxyfluorescin; TAMRA, 6-carboxytetramethylrhodamine, β-actin.

Forward, 5’-TGCCATGTATGTGGCCATCC-3’; reverse, 5’-CGCTCGGCTGTGGTGGTAA-3’; probe, 5’-FAM-ACTCTGGGGACGGGGTCA-TAMRA-3’, bcOBP.

Forward, 5’-GCACATGCCAGTTTTTCTCA-3’; reverse, 5’-GGAATCCCCCTCTCATTGTT-3’; probe, 5’-FAM-AAGGTTCCGCCAAAGGAACCA-TAMRA-3’.

Tissue collection and preparation: We used sick cows in lactation stage. The tissues without suspicion of pathological features were used. The tissues were fixed with 4% paraformaldehyde in PBS and embedded in paraffin for in situ hybridization. Serial sections were cut at 5 μm thickness and then placed onto 3-aminopropyltriethoxysilane-coated glass slides.

Probes and labeling: Oligo-DNA complementary to a part of bcOBP mRNA was synthesized together with additional three ATT repeats at the 3’-end and 5’-end and haptenized with DIG Oligonucleotide Tailing Kit (Roche), according to the manufacturer’s instructions.

In situ hybridization: Paraffin sections were deparaffinized and rehydrated with a toluene-ethanol series. The slides were treated with PBS for 10 min and proteinase K (10 μg mL^-1, 37°C, for 15 min), successively. After postfixation with 4% paraformaldehyde (PFA) in Phosphate Buffered Saline (PBS) (5 min), the sections were immersed in 2 mg mL^-1 glycine in PBS (30 min) and kept in 40% deionized formamide in 4×SSC until used for hybridization. Hybridization was carried out at 42°C for 12-16 h with Dig-labeling oligo-cDNA for bcOBP dissolved in the hybridization medium. After repeated washings, the slides were incubated with POD-conjugated ovine anti-DIG Fab fragment (Roche). The sites of peroxidase activity were visualized with DAB substrate (Roche) according to the manufacturer’s instructions.

RESULTS

Quantification of the expression of bcOBP mRNA: To quantitate the expression of bcOBP mRNA, we isolated mRNA from bovine organs. TaqMan quantitative PCR (qPCR) was performed for those samples. qPCR analysis revealed that the expression was found in the principal organs such as heart, liver, kidney, node and mammary gland (Fig. 1). In addition, the expression level of bcOBP mRNA in heart is as much as in kidney among principal organs (Fig. 1).

Localization of bcOBP mRNA in mammary gland: To investigate the origin of bcOBP, we detected bcOBP mRNA in mammary gland with in situ Hybridization (ISH). In mammary glands, the higher expression in the mammary alveolus than in supporting tissues of mammary glands has been observed (Fig. 2).

Fig. 1:	Quantitative PCR analysis of bcOBP transcription in several tissues. Transcript levels were normalized to β-actin. Shown are the averages and Standard Derivations (SD) in three independent experiments

Fig. 2(a-b):	Tissue section in situ detection of bcOBP mRNAs. (a) DIG-POD-DAB stained (×100) mammary gland sections were hybridized with digoxigenin-labelled bcOBP antisense probes and (b) Control (×100). No signal was obtained with sense probes used as negative control

DISCUSSION

In the present study, we discovered that bcOBP mRNA were expressed in principal organs, indicating that bcOBP might be involved in various physiological functions. Importantly, the expression level of bcOBP mRNA in heart is as much as in kidney. This fact exhibits a possibility that bcOBP has important roles for maintenance of homeostasis. In human, two novel protein genes (hOBP a and hOBP b) were found (Lacazette et al., 2000). hOBP a had an oral sphere expression profile and hOBP b was expressed in genital sphere organs (Lacazette et al., 2000). They hypothesized that two hOBP had critical roles for maintenance of homeostasis in human body (Lacazette et al., 2000). However, the functional roles of bcOBP and two kinds of hOBP remain unknown. Our findings provide a fundamental concept for further study such as their biological functions.

bcOBP was found in bovine colostrums (Fukuda et al., 2009). No tight in the hypothesis and findings on the origin of bcOBP. There was a hypothesis that bcOBP found in bovine colostrums were secreted from mammary gland. Mammary gland, especially mammary alveolus, might secrete bcOBP into colostrums because higher expression of bcOBP mRNA in mammary alveolus than in supporting tissues (Fig. 2). This result supported the possibility that a bcOBP protein found in bovine colostrums was secreted from mammary alveolus in mammary gland. There is also a possibility that other organs which express bcOBP mRNA secret bcOBP protein to blood and then bcOBP-blood is converted to colostrums. Alpha-1-acid glycoprotein (AGP), found in human blood (Piafsky et al., 1978), is also a lipocalin in bovine milk (D'Amato et al., 2009). AGP in human blood is secreted from liver (Fournier et al., 2000). AGP in bovine milk is considered to be synthesized in mammary tissues and in somatic cells (Ceciliani et al., 2007). With this condition, no finding was determined.

ACKNOWLEDGMENTS

We are grateful to Dr. Yoshiyasu Kobayashi, Hisashi Inokuma, Hidefumi Furuoka and their colleagues for technical supports for tissue samplings. This study is financially supported by the Global Center of Excellence "Frontier Program for Animal Global Health and Hygiene", Ministry of Education, Culture, Sports, Science and Technology, Japan and Japan Society for the Promotion of Science.