Distribution of P12 in Mouse Reproductive Tracts
Hypodermic injection of P12 to rabbits poorly induced the production of P12 antibody. Instead, we immunized the animals by intrasplenic injection of P12. The antiserum prepared from this method showed strong immunoaffinity to P12 as well as to GST-P12 but did not cross-react with GST (cf. lanes 1-3 of Fig. 1). Among the protein components of SVS, which contained a trace amount of P12 (Fig. 1A, lane 4), the antiserum immunoreacted only with P12 (Fig. 1B, lane 4), showing the high specificity of the P12 antibody in the antiserum. Therefore, we used the antiserum for the immunodetection of P12 throughout this study. antibiotic levaquin
We examined the distribution of P12 in the genital tracts of adult mice by Western blot analysis. In the male reproductive tracts, P12 was demonstrated in the homogenates of seminal vesicle, coagulating gland, and prostate, but it was not detectable in the homogenates of other sexual organs such as testis, epididymis, and vas deferens (Fig. 2). This is in agreement with the previous examination of the distribution of P12 mRNA in the male sexual organs. The protein was not found in the female reproductive tracts including ovary, oviduct, uterus, and vagina. The existence of this protein in the three accessory sexual glands of adult male mice was also confirmed in the immunohistochemical staining patterns of P12 in the tissue sections shown in Figure 3.
FIG. 1. Specificity of the P12-induced antiserum. The proteins were resolved by SDS-PAGE on a 15% polyacrylamide gel slab: lane 1, 2.0 ^g of P12; lane 2, 4.0 ^g of GST; lane 3, 4.0 ^g of GST-P12; lane 4, 30 ^g of the saline extract of mouse SVS. A) The proteins were stained with Coomassie brilliant blue dye. B) The proteins in the gel were transferred to nitrocellulose membranes and immunodetected by Western blot with the partially purified P12 antibody in the blocking solution (0.5 ^g/ml) (see text for details).
FIG. 2. Tissue distribution of P12 in sexual organs of adult male mice. Total protein (30 ^g) prepared from the tissue homogenate of mice (12 wk) was run on a 15% polyacrylamide gel by SDS-PAGE, transferred to a nitrocellulose membrane, and immunodetected with the P12 antiserum. Tissues examined are seminal vesicle (lane 1), coagulating gland (lane 2), prostate (lane 3), testis (lane 4), epididymis (lane 5), and vas deferens (lane 6).
FIG. 3. Histochemical staining patterns of P12 on sections of reproductive tracts of adult male mice. Tissue slices of seminal vesicle, coagulating gland, and prostate were histochemically stained for P12 with the P12 antiserum and the anti-rabbit IgG conjugated with alkaline phosphatase. A) P12 was immunolocalized on the epithelium and the cavity of each tissue. B) The specimens were stained as in A except that the P12 antiserum was replaced by normal serum. C) The specimens were stained with hematoxylin and eosin to reveal the morphology. MF, mucosa fold; SM, smooth muscle; MC, mucosa crypt; LF, luminal fluid; GA, glandular alveolus. Bar = 100 ^m.