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An intense anti-androgen binding protein (ABP) reaction was noted over the cytoplasm of Sertoli cells at all stages of the cycle of the seminiferous epithelium of wild-type mice; the reaction extended from the base of the epithelium to the lumen (Fig. 4, a, c, and e). No staining was observed over germ cells. In FORKO mice, the staining over Sertoli cells at all stages of the cycle appeared weak and in some areas completely missing (Fig. 4, b, d, and f). A nonspecific staining of Leydig cells was evident in the interstitial spaces inherent to the anti-ABP protein construct (see Materials and Methods), and the fact that Leydig cells express GSTs. Control sections showed no staining over the epithelium (Fig. 4a, inset). Electron Microscopic Appearance of the Testis of FORKO Mice While the nucleus of the affected Sertoli cells in FORKO mice was intact, showing a pale-stained uncondensed chromatin pattern and conspicuous nucleolus and satellite bodies as seen in wild-type mice (Fig. 5a), their cytoplasm was grossly disrupted; this was especially evident in the mid and apical areas of their cytoplasm (Figs. 5b, 6, 7b, and 8). In wild-type mice, the organelles in the expansive Sertoli cell cytoplasm were embedded in a compact, finely floc-culent ground substance, with adjacent Sertoli cells being tethered together by a conspicuous Sertoli-Sertoli blood testis barrier (Fig. 5a). FIG. 4. Light micrographs of seminiferous tubules of wild-type (a, c, e) and FORKO (b, d, f) mice at 3 mo of age immuno-stained with an anti-ABP antibody. In wild-type mice at early (a, stage II-III), mid (c, stage VII), and late (e, stage XII) stages of the cycle, a reaction is evident in Sertoli cells, either basally or as distinct bands radiating across the width of the epithelium (arrows). This is in contrast to the weak staining or absence of a reaction over Sertoli cells of FORKO mice (arrows), seen at stages I (b), VII (d), and XII (f). The reaction over Leydig cells in the interstitial space (IS) is nonspecific. SE, Seminiferous epithelium; L, lumen; N, nucleus of Sertoli cell. a-f) Original magnification X390; inset in a, original magnification X250. FIG. 5. Electron micrographs of the base of the seminiferous epithelium of wild-type (a) and FORKO mice at 3 mo of age (b). In a, organelles in the Sertoli cell cytoplasm, such as Golgi apparatus (G), lyso-somes (Ly), mitochondria (M), and endoplasmic reticulum (ER), are embedded in a finely flocculent ground substance (star). The Sertoli-Sertoli blood testis barrier (open arrows) is intact and composed of bundles of filaments (small arrows) overlaid by flattened cisternae of endoplasmic reticulum (arrowheads). The Sertoli cell nucleus (N) is pale stained and shows a prominent nucleolus (n) and closely associated satellite body (s) in this plane of section. In b, the Sertoli cell cytoplasm is dilated in the midarea of the seminiferous epithelium (stars), where large membranous whorls are evident (curved arrows). Nearer to the base of the epithelium, the Sertoli cytoplasm shows small territories enclosing spherical mitochondria (M), ly-sosomes (Ly), and a pale stained nucleus (N), all embedded in a finely flocculent ground substance (asterisks). The large dilated Sertoli cytoplasm surrounds spherical germ cells (Gc) that do not show any structural abnormalities. a) Original magnification X14 000; (b) original magnification X6600. FIGS. 6 and 7. FIG. 6. Electron micrograph of the base and midregion of the seminiferous epithelium of a 6-mo-old FORKO mouse. Areas of Sertoli cell cytoplasm (asterisks) located basally are in contact with the basement membrane (Bm) and contain mitochondria (M), lyso-somes (Ly), and cisternae of endoplasmic reticulum (ER), embedded in a finely floc-culent ground substance. In the midregion of the epithelium, a large dilation of Sertoli cell cytoplasm (star), containing large membranous profiles (curved arrows), is separated from the basal cytoplasm of Sertoli cell cytoplasm presumably due to the plane of section. In the upper right hand corner of this dilation, mitochondria (M), a Golgi apparatus (G), and lysosomes (Ly) are evident, with two mitochondria (M) appearing to be floating freely in the interior of this dilation. Another large dilated space (long dashed arrow) extending toward the lumen appears to be confluent with the basal area of Sertoli cell cytoplasm containing intact organelles and ground substance. As both dilated spaces contain organelles and are delimited by a plasma membrane (arrows), they are considered to be dilations of the Sertoli cell cytoplasm and not extensions of the lumen. The Sertoli-Sertoli blood testis barrier at the base of the epithelium is evident and appears intact (open arrows). Germ cells (Gc) do not show any apparent structural abnormalities of their cytoplasm, nucleus, or organelles, and the organelles of the Sertoli cell themselves appear intact. Original magnification X8600. FIG. 7. Heads of elongating spermatids enveloped by Sertoli cell processes in wild-type (a) and FORKO (b) mice at 3 mo of age. In a, the cytoplasm of the Sertoli cell processes (S) enveloping the spermatid head contains mitochondria (M), lysosomes (Ly), and ER, embedded in a finely flocculent ground substance. The ectoplasmic specializations, comprised of thick bundles of filaments (small arrows) and overlying flattened ER cisternae (arrowheads), are closely applied to the spermatid head. In b, the Sertoli cell process enveloping the spermatid head is greatly dilated and appears predominately organelle free (star). The ectoplasmic specialization adhering to the spermatid head appears to be less extensive, showing fewer bundles of filaments (small arrows) and at times dilated ER cisternae (arrowheads). However, the plasma membrane of the Sertoli cell process is closely apposed to the spermatid head (long arrows) and to that applied to the elongating germ cell (Gc) cytoplasm (short arrows). A, Acrosomes of spermatid head; N, nucleus of spermatid heads. a) Original magnification X16100; (b) original magnification X16 800.