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
The expression of eight different growth factors in roe deer testis was detected using a RT-PCR approach. Primers were synthesised according to the corresponding bovine or
human growth factors. Primer sequences, expected fragment sizes and amplified regions within the targeted growth factor are described in Table 1. VEGF primers were designed
to detect all four VEGF isoforms Houck et al., 1991 although VEGF
165
is reported to be the most common form Houck et al., 1992. In case of bFGF the three possible isoforms
differ at their N-terminus, therefore, a single PCR to detect all isoforms was not possible. After optimisation, PCRs targeting IGF-II, TGF-a, TGF-b
1
and TGF-b
3
generated single fragments that were directly sequenced. Thus, their identity as growth factor sequences
was confirmed. In the case of aFGF, bFGF, IGF-I and VEGF the bovine primers generated multiple PCR products which were all cloned and subsequently sequenced. In each case a
growth factor derived fragment could be identified. Sequence information from the cloned fragments was used for the design of roe deer specific primer pairs. Since the PCRs for
IGF-II, TGF-a and TGF-b
3
with either bovine or cervine primers were not optimal, roe deer specific primers were derived from the sequenced fragment.
All PCR products generated with bovine primers are listed in Table 2 together with their nucleotide and amino acid homologies in comparison to the respective bovine and human
growth factor cDNA and protein sequences. In the case of the vascular endothelial growth factor two fragments were generated, corresponding to two of the four known isoforms
Houck et al., 1991. Sequence comparison showed that the shorter VEGF fragment corre- sponded to the VEGF
121
isoform whereas the longer one corresponded to VEGF
165
Houck et al., 1991. Fig. 1 shows the PCR fragments obtained from different roe deer testicular
growth factors. According to our knowledge this is the first study reporting the detection of aFGF, bFGF, IGF-I, IGF-II, TGF-a, TGF-b
1
, TGF-b
3
and VEGF gene expression in roe deer testes. The consensus sequences obtained from six animals for roe deer aFGF,
bFGF, IGF-I and IGF-II, TGF-a, TGF-b
1
, TGF-b
3
and VEGF were submitted to Genbank and have been assigned accession numbers AF152586–AF152594, respectively Table 2.
Homology percentage data are based on the fragments that were originally obtained with bovine derived primers. All roe deer growth factor fragments showed a higher degree of
similarity to their bovine homologs than to the corresponding human growth factors.
Compared to their bovine homologs, two roe deer growth factor fragments TGF-b
1
, bFGF had no amino acid aa exchanges, three differed in one aa IGF-I: S118A, TGF-b
3
: A354S, VEGF: G113S and one had two aa exchanges TGF-a: L63V, T106S, compared to
sheep. A larger number of exchanges were seen in IGF-II five positions: D104A, V105L, Q115R, I118T, A136V and in the aFGF fragment eight positions: S32R, Y36H, C62S,
L104I, I113T, H117Y, H121N, R131S.
The standardised GF expression data from three sampling months always separated by a period of 4 intermittent months clearly indicate differing regulation patterns for the GF’s
investigated. The sampling months represent the pre-rutting April, rutting August and
A. Wagener et al. Animal Reproduction Science 64 2000 65–75 69
70 A. Wagener et al. Animal Reproduction Science 64 2000 65–75
Table 2 Homologies between DNA and protein sequences of roe deer, bovine and human growth factors
Growth factor
GF Total
fragment length bp
Length of roe deer specific sequence
wo primers bp Homology between
roe deer and bovine sequences nucleotides
amino acids Homology between
roe deer and human sequences nucleotides
amino acids Genbank
accession number
aFGF 364
320 96,392
91,391 AF152586
bFGF 360
325 97,2100
94,598 AF152587
IGF-I 239
199 96,598
9098 AF152588
IGF-II 379
340 96,895
86,785 AF152589
TGF-a 258
223 96,497
a
94,197
a
AF152590 TGF-b
1
200 152
96,7100 90,2100
AF152591 TGF-b
3
280 244
n.e. 94,398
AF152592 VEGF
121
278 236
98,398 93,694
AF152593 VEGF
165
411 369
98,999 95,996
AF152594
a
Homology to ovine nucleotide and protein sequences; n.e.: no entry no Genbank entry available regarding bovine or ovine TGF-b
3
sequences.
post-rutting December season. Fig. 2 shows the approximate course of the gene expression from six representative growth factors. Expression of IGF-I and TGF-b
1
seem to reach their peak during rutting season with TGF-b
1
having a prolonged high expression. The expression of IGF-II and VEGF, however, appear to be down regulated during rutting season. The
expression of bFGF and TGF-a seem to remain relatively uninfluenced throughout the year although a timespan of 4 month between the sample yielding might obscure actual changes.
Since only two animals were available for each month, statistical analysis was not performed.
Fig. 1. Gelelectrophoresis of roe deer testis growth factor fragments, obtained by RT-PCR; lanes; M: molecular weight marker; 1: aFGF; 2: bFGF; 3: IGF-I; 4: IGF-II; 5: TGF-a; 6: TGF-b
1
; 7: TGF-b
3
; 8: VEGF.
A. Wagener et al. Animal Reproduction Science 64 2000 65–75 71
Fig. 2. Relative expression levels of different growth factors during pre-rutting April, rutting August and post-rutting December season. Values arbitrary units represent the ratio of mean GFmean GAPDH expression,
measured video densitometrically.
4. Discussion
