Materials and methods Directory UMM :Data Elmu:jurnal:A:Animal Reproduction Science:Vol64.Issue1-2.Dec2000:

66 A. Wagener et al. Animal Reproduction Science 64 2000 65–75 recrudescence during the transitions between breeding and non-breeding periods. There- fore, they require mechanisms for the stimulation of cell proliferation and spermatogenesis during testis growth, and also for cell apoptosis during testis involution. These mechanisms require participation of hormones such as testosterone. The production of testosterone by Leydig cells seems to ensure the survival of germ cells Tapanainen et al., 1993; Thompson, 1994. The hormonal regulation of the changes in testis growth is mediated by paracrine and autocrine effects of different growth factors. Growth factors are key regulator molecules which affect cell proliferation, meiosis and differentiation. Several growth factors such as FGFs, IGFs, TGFs have been isolated from testis tissue and evidence suggests that these factors play a role in the control of spermatogenesis for review: Lamb, 1993; Spiteri-Grech and Nieschlag, 1993; Kierszenbaum, 1994; Smith and Conti, 1996. These investigations were mostly carried out with non-seasonal laboratory animals or in vitro studies. How- ever, very little is known about the regulation of spermatogenesis in seasonally breeding ruminants. In deer species, so far only involvement of endocrine IGF-I in seasonal growth processes has been shown; for example in red deer by a correlation of the IGF-I secretion to the annual rhythm of antler development Suttie et al., 1989. As a prerequisite to test the hypothesis that the above mentioned growth factors are in- volved in processes such as spermatogenesis, cell proliferation and apoptosis in roe deer testes, the expression of the corresponding genes needs to be investigated and was there- fore analysed using the reverse transcriptase polymerase chain reaction RT-PCR with the subsequent comparison of results of the periods before, during and after the rut.

2. Materials and methods

2.1. Animals and tissues Roe deer testis tissue was obtained following castration from six free ranging animals immediately after capture. The animals were immobilised i.m., using a blow pipe with xylazin 2 mgkg body mass bm in combination with ketamin 4 mgkg bm. Anaesthesia was achieved with 1.5–2 isofluran. After the procedure the anaesthesia was antagonised with atipamezol 0.0875 mgmg xylazin. After its removal the testis was dissected into small pieces and immediately frozen in liquid nitrogen. 2.2. Reverse transcription RT Total RNA was prepared from 100 mg testis tissue with TRIzol reagent GIBCO Life Technologies. Isolated RNA was quantified spectrophotometrically at 260 nm. The first cDNA strand was generated from 1 mg of total RNA in a 50 ml RT-reaction. RNA was denaturated together with 0.5 mg random hexamer primers Pharmacia for 5 min at 70 ◦ C. RT was performed with 200 units U moloney Mouse leukaemia virus reverse transcriptase M-MLV RT, Promega, Madison, WI, USA. The reaction mixture also contained 25 U ribonuclease inhibitor Promega, 50 mM Tris–HCl, pH 8.3, 3 mM MgCl, 10 mM DTT, 75 mM KCl and 0.5 mM of each deoxynucleosidetriphosphate dNTP. RNA was reversely transcribed at 37 ◦ C 30 min, 42 ◦ C 30 min, followed by a 5 min enzyme inactivation period at 99 ◦ C. The reaction mixture was then chilled to 4 ◦ C. A. Wagener et al. Animal Reproduction Science 64 2000 65–75 67 2.3. PCR-reaction All RNA samples tested negative for the presence of contaminating DNA in a PCR with 80 ng total RNA that had not been reversely transcribed using glyceraldehyde-3-phosphate dehydrogenase GAPDH specific primers for 36 cycles 94 ◦ C 30 s, 50 ◦ C 30 s, 72 ◦ C 45 s. Primer design for aFGF, bFGF, IGF-I and IGF-II was based on sequences of bovine andor human aFGF bovine: Halley et al., 1988, human: Chiu et al., 1990, bFGF bovine: Abraham et al., 1986, human: Prats et al., 1989, IGF-I bovine: Fotsis et al., 1990, human: Steenbergh et al., 1991, IGF-II bovine: Brown et al., 1990, human: LeRoith and Roberts, 1993, TGF-b 3 human: ten Dijke et al., 1988 and VEGF bovine: Leung et al., 1989, human: Houck et al., 1991. Bovine VEGF reverse primers were from Garrido et al. 1993 and cervine TGF-a and TGF-b 1 primers from Francis and Suttie 1998. Single PCR fragments were directly sequenced; in case of multiple PCR fragments all products were cloned into pGemT vector Promega according to the manufacturer’s instructions. All inserts were sequenced in order to identify growth factor specific fragments. In the case of aFGF, bFGF, IGF-I, VEGF Wagener et al., 1999, TGF-a and TGF-b 3 PCR fragments were first obtained using bovine primers. Sequencing of these fragments allowed the design of roe deer specific primers. All primers were synthesised by BioTeZ Ltd. Berlin, Germany. Either 2.5 ml of the RT reaction containing 50 ng of cDNA or 2.5 ml of water as negative control, were used in a 50 ml PCR reaction. Synthesis of a GAPDH gene fragment served as RT and PCR control. PCRs were carried out as follows: GAPDH: 1.5 U Taq-Polymerase PAN Systems, 0.2 mM of each specific primer, 2 mM MgCl 2 , 0.2 mM dNTPs; aFGF, bFGF, IGF-I, IGF-II, TGF-a, TGF-b 1 , TGF-b 3 , VEGF: 1.5 U Taq-Polymerase, 0.2 mM of each specific primer, 1.5 mM MgCl 2 , 0.2 mM dNTPs. All PCRs were initially denaturated for 3 min at 94 ◦ C and had a final extension phase of 10 min at 72 ◦ C. Individual amplification programmes were applied for GAPDH 27 cycles at 94 ◦ C 30 s, 50 ◦ C 30 s, 72 ◦ C 45 s, aFGF 30 cycles at 94 ◦ C 30 s, 50 ◦ C 30 s 72 ◦ C 45 s, bFGF 31 cycles at 94 ◦ C 30 s, 55 ◦ C 30 s, 72 ◦ C 45 s, IGF-I, 29 cycles at 94 ◦ C 30 s, 61 ◦ C 30 s, 72 ◦ C 45 s, IGF-II 30 cycles at 94 ◦ C 30 s, 62 ◦ C 30 s, 72 ◦ C 45 s, TGF-a 35 cycles at 94 ◦ C 30 s, 62 ◦ C 30 s, 72 ◦ C 45 s, TGF-b 1 30 cycles at 94 ◦ C 30 s, 61 ◦ C 30 s, 72 ◦ C 45 s, TGF-b 3 31 cycles at 94 ◦ C 30 s, 63 ◦ C 30 s, 72 ◦ C 45 s and VEGF 34 cycles at 94 ◦ C 30 s, 61 ◦ C 30 s, 72 ◦ C 45 s. One set of PCR fragments for all 8 GF’s and GAPDH was always generated from the same cDNA. Primers are listed in Table 1 together with the localisation of the amplified fragments within the corresponding growth factor. Fragments were separated on a 1.5 agarose gel and visualised on an ultraviolet transilluminator by ethidium bromide staining. The optical density of detected fragments was determined using the Video Densitometer with a Cybertech Image Capture Computer and WinCam 2.2. Image Software Cybertech. The levels of the respective growth factors relative to the level of GAPDH present, were expressed as the appropriate growth factorGAPDH ratio. 2.4. Sequencing PCR products were purified using a PCR purification kit Qiagen, Germany with a subsequent precipitation step with sodium acetateethanol. The sequencing PCR was carried out with BigDyeTerminator TM mix Applied Biosystems according to the dideoxymethod 68 A. Wagener et al. Animal Reproduction Science 64 2000 65–75 Sanger et al., 1977. The PCR products were sequenced bidirectionally using an A310 DNA Sequence Analyzer Applied Biosystems, USA.

3. Results