JCS02704 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. The tFGFR-1flag construct and the methodology used to genotype FGFR-1 transgenic mice. (A) The tFGFR-1 protein coding region was fused to the mouse Prm1 promoter, while a flag-tag sequence was incorporated at the 3¢ end for transgene detection. A Sv40 polyadenylation sequence, Sv40 poly (A), was incorporated to enhance stability of the transgenic transcript and prevent translational delay. (B) The initial genotyping of FGFR-1 transgenic mice was achieved using a three-pronged PCR screening strategy. Mice that were positive for all three PCR screens were considered transgenic, i.e. numbers: 4, 6, 8-10, 13, 14 and 16. (C) Southern blot screening for the identification of DH4/9 mice, using a 630 bp cDNA probe designed to the 3¢ end of the FGFR-1 transgene. Digested gDNA from wild-type, single heterozygous mice were used as controls. The blot shown demonstrated that two of the mice screened were DH4/9 as they showed a combination of unique bands from the two single heterozygous lines (arrows).

• Supplemental Figure 2 -

  Fig. S2. The developmental expression of Snt-2 mRNA during rat spermatogenesis and FGFR-1 protein in rat and mouse epididymal sperm tails. (A) Northern blot analysis of total rat testis RNA showed Snt-2 was up-regulated during days 25-90 of testis development in accordance with the appearance of round through to elongated spermatids in the testis. (B) In order to determine whether FGFR-1 protein was expressed in mature sperm, sperm and sperm tails were isolated from both rat and mouse cauda epididymides and Western blot analysis was performed using a rabbit polyclonal FGFR-1 antiserum. The image shows that both the long (FGFR-1L) and short (FGFR-1S) isoforms of FGFR-1, at approximately 150 kDa and 110 kDa, were present in rat and mouse testis and sperm (Bernard et al., 1991). (C and D) The immunofluorescent localization of FGFR-1 protein on caudal epididymal sperm. Arrows indicate sperm tail staining. The lack of immunofluorescent staining when the FGFR-1 serum was replaced by an irrelevant Ig indicates the specificity of FGFR-1 staining.

• Supplemental Figure 3 -

  Fig. S3. The expression of flag-tagged protein in tFGFR-1 mouse testes. Immunohistochemistry was employed to determine the localization of the FGFR-1 transgenic protein within the mouse testes. FGFR-1 transgenic protein was localized to the elongating and elongated spermatids (arrows). Numbers in the top right hand corner indicate the line from which tissue was obtained. Scale bars, 50 mm. wt: wild type mice.

• Supplemental Figure 4 -

  Fig. S4. Serum hormone levels in wild type and tFGFR-1 mice. The serum hormone levels of FSH (A), activin A (B) and inhibin (C) were all unchanged compared to wild-type animals in all of the transgenic FGFR-1 mouse lines.