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Fig. 2. Western blots of whole ovary extracts. Proteins were separated
on a 10% SDS/polyacrylamide gel. Lane a, immunodetection
with the anti-Xfin2 antibody, raised against a non finger region of
Xfin. Lane b, staining with the anti-Xfm antibody, as in Fig. la. The
same 120 kDa antigen is stained by the two different antisera
(arrow). Lane c, staining with the preimmune serum. Lane d, staining
with the anti-Xf/n serum which has been preincubated with the
fusion protein immobilized on nitrocellulose filters.
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Fig. 3. Western blot analysis of the expression of )(fin protein in
oogenesis. Staged oocyte (I-VI) extracts, prepared as explained in
Experimental Procedures, were separated on a 7.5% SDS/polyacrylamide
gel and probed with the anti-Xfin antibodies. The same
amount of total proteins (180 ~g) was loaded on each lane. The
arrow points to the 120 kDa protein species.
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Fig. 4. Western blot analysis of the developmental expression of the Xfin gene product in embryogenesis. Staged embryo extracts and unfertilized
eggs were separated on SDS-PAGE and stained as above (see Fig. 3). The same amount of total proteins (180 ~g) was loaded on each lane. ec,
early cleavage; Ic, late cleavage; m, morula; b, blastula; g, gastrula; n, neurula; or, ovary; e, eggs; t, tail bud; mr, muscular response; h, hatching.
The arrow points to the 120 kDa protein species.
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Fig. 5. In situ hybridization of in vitro synthesized X fin RNA to
previtellogenic (a) and vitellogenic (b) oocytes. In either case, no
preferential distribution of Xfin mRNA was found. In (b), notice the
dilution effect of the Xfin mRNA level between previtellogenic and
vitellogenic oocytes, nu, nucleus; fc, follicle cells; yp, yolk platelets.
The exposure times were 5 days (a) and 13 days (b). Giemsa stain.
Scale bars = 10/zm.
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Fig. 6. Immunostaining of oocyte sections with the anti-Xfin antibodies.
Notice the dilution effect between vitellogenic and previtellogenic
oocytes. Scale bar = 10/xm.
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Fig. 7. Expression of Xfm protein in adult tissues. 180 /~g of total proteins were loaded on each lane. The same extracts were stained with
anti-Xfm antibodies (a) or with anti-enolase antibody (b) to monitor antigen recovery. The numbers refers to the M r in kDa of the two respective
antigens, indicated by arrows, br, brain; iu, lung; k, kidney; te, testis; g, gut; m, skeletal muscle; h, heart; ov, ovary.
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Fig. 8. Immunostaining of testis (a-d) and kidney (e-f) sections with the anti-Xfin antibody. (a) Spermatocysts at various stages of maturation.
The staining is localized in the cytoplasm of primary spermatocytes at a late pachytene stage of the first meiotic prophase; spermatocysts of
secondary spermatogonia (sg) and early pachytene (pa) primary spermatocytes are unstained. (b) Spermatocyst of round spermatids. Notice the
staining over the cytoplasmic bridges. The arrow points to a Sertoli cell. (c) Spermatocyst of spermatids at the beginning of spermiogenesis,
showing the maturing germ cells aligned on the cyst wall. The nuclei are unstained. Mature spermatozoa (sp) are indicated. (d) Spermatocyst of
elongated spermatids which do not retain staining, that is instead concentrated over the residual bodies (arrow). (e), (f) Kidney sections showing
only one specific portion of the nephric tubule stained by the antibody. Notice that nuclei are unstained. Some cells of the unstained portion of
the tubule are engaged in mitosis (arrow). Glomeruli (gl) are unstained. Scale bars = 10 g.m; a, b, c, d, same magnification as f.
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Fig. 9. In situ hybridization with Xfin anti-sense probe on testis sections. (a) A labelled spermatocyst of primary spermatocytes at late pachytene
stage (arrow) and a spermatocyst of round spermatids showing a decrement in the labelling (arrowhead). (b), (c) Labelled spermatocysts of
primary spermatocytes at diplotene stage (arrows). In (c) a spermatocyst of primary spermatocytes at zygo-pachytene stage showing no labelling is indicated (arrowhead). (d) A control section hybridized with an Xfin sense probe. The exposure time was 10 days. Giemsa stain. Scale bars = 10um; a, same magnification as d; b, same magnification as c.
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