1.  INTRODUCTION

       The profound effects of systemic hormones on all aspects of pre- and postnatal development, differentiation and secretory activity of mammary gland are well documented. The cyclic events of mammary growth and differentiation that occur during pregnancy and lactation are controlled by various steroid and peptide hormones. At the cellular and tissue level the action of many of these hormones is mediated by growth factors in an intracrine, autocrine, juxtacrine or paracrine manner. In the present communication we report on the expression of various growth factors (IGF-I, IGF-II, FGF- 1, FGF-2, TGF-α) in the bovine mammary gland during well defined stages of mammogenesis, lactation and involution. Additionally the expression pattern of GHR during these stages will be discussed.

2.  MATERIAL AND METHODS

2.1  Animals, tissue sampling and preparation.

     Mammary tissue was obtained from 27 German Brown Swiss heifers and cows at distinct periods of mammary gland development immediately after slaughter. The material studied comprised mammary tissue from non-pregnant and primigravid heifers, cows during lactogenesis, early and late lactation and dry, non pregnant cows. The following classification of animals was established:I       Mammogenesis
I. 1 Mammogenesis-ductal growth (non pregnant heifers)
I.2 Mammogenesis-lobuloalveolar development during first pregnancyI.2.1: days 194-213 of pregnancy I.2.2: days 255-272 of pregnancyII            Lactogenesis: days 5-11 post partumIII          Galactopoiesis 111.1  peak lactation111.2  late lactationIV  Involution (3-4 weeks dried off, non pregnant)
         For histology and immunohistochemistry, tissue samples (approximately15 mm long and 5 mm thick) were fixed in Bouin's solution or in methanol/glacial acid (ratio 1:1, w/v) for 24 h, dehydrated in a graded series of ethanol, cleared in xylene and embedded in paraffin. Serial sections (5 µm) were cut on a Leitz microtome and mounted on glass slides. Following deparaffinization, the presence of growth factors (IGF-I, IGF-II, TGF-α, FGF-1, FGF-2, and receptors (FGFR and GHR) was demonstrated immunohistochemically by the streptavidin-biotin horseradish peroxidase complex (ABC) technique. Controls were performed by a) omission of the primary antibody; b) replacing the antiserum against the growth factors or FGFR and GHR by normal mouse serum (Sigma, Munich) in differentconcentrations (dilution 1:5; 1:10; 1:100). c) Preabsorption of the primary antibody. It was performed by preincubation of the primary antibody for 24 h at 40°C with the respective recombinant growth factors in a siliconized polypyrene tube before incubation on the slides.

2.2  RNA extraction

      Total RNA was isolated from bovine tissues using a specifically adapted guanidium thiocyanate/phenol procedure. The RNA pellet was washed in 75% ethanol and diluted in distilled, DEPC treated water. Concentration of RNA was measured by photometry. For RT-PCR of GHR, total RNA was prepared from snap frozen bovine mammary gland tissue using Tripure Reagent (Sigma, Deisenhofen, Germany) according to the manufacturer's instructions.

2.3  Reverse transcription polymerase chain reaction  (RT-PCR)      

       For RT-PCR, total cellular RNA was denatured at 65°C for 10 min, quick-cooled on ice and reverse transcribed in a final volume of 20 ml. When RNA had been prepared using snap-frozen tissue and Tripure Reagent, 5 mg RNA were used for the RT reaction. The RT reaction mix included 1x ExpandTM Reverse Transcriptase buffer (50 mM Tris-HCl, 40 mM MgCl2, 0.5% Tween 20 (v/v), pH 8.3), 10 mM DTT, 1.6 mg oligo dT, 0.75 mM dNTP, 3 mM MgCl2, 30 units RNase inhibitor and 50 units ExpandTM reverse transcriptase. All reagents were purchased from Boehringer Mannheim, Germany. The reaction mix contained 1x reaction buffer (10 mM Tris, 50 mM KCI, 1.5 mM MgCl2, pH 8.3), 0.4 mM dNTP, 4 units Taq polymerase (Boehringer, Mannheim, Germany) and 100 pmol of each primer. Controls were performed by using RNA instead of cDNA in the PCR reaction. The primers used for RT-PCR for the different growth factors and for GHR as well as the specific conditions of amplification are described previously.

2.4   RNase protection assay

    The total RNA (30 mg) was introduced into a commercial RNase protection assay (RPA) (Ambion, Texas, USA) and performed as previously described4. Precast 10% polyacrylamide gels supplied with 7 M urea (Cleangels, Pharmacia, Freiburg, Germany) were used. Sense and 32P-antisense   riboprobes    were    generated   by   subcloning   of   the    desired homologues gene products according to the manufacturer's instructions (PCR-Cloning and RNA-Transcription Kit; Stratagene, La Jolla, CA, USA). To quantify the mRNA of the growth factors by RPA, increasing amounts of in vivo synthesized sense RNA were hybridised with the respective P-antisense riboprobes and compared with the mammary RNA sample using densitometry. There was a linear increase in the abundance of RNase-protected sense/antisense fragments between 0.25 and 5 pg. Individual samples were analysed to verify RT-PCR results and afterwards pooled RNA was examined to obtain the expression pattern throughout the different stages of mammary gland development.

 3.  RESULTS AND DISCUSSION