Atypical development of Sertoli cells and impairment of spermatogenesis in the hypogonadal (hpg) mouse

Abstract

Testes of hypogonadal (hpg) mice show arrested postnatal development due to congenital deficiencies of gonadotrophin-releasing hormone (GnRH) and gonadotrophin synthesis and secretion. Follicle-stimulating hormone (FSH), androgen or oestrogen treatment restore qualitatively normal spermatogenesis in hpg testes. Understanding the cellular and molecular changes accompanying hormone-induced spermatogenesis in hpg mice requires detailed morphological analyses of the germ cells and Sertoli cells in the untreated hpg testis. We compared seminiferous epithelial cytology in adult hpg, immature and adult wild-type mice using unbiased optical disector-based stereology, immunolocalization of Sertoli cell microtubules (MT), espin (a component of the blood-testis barrier), markers of Sertoli cell maturity (p27(kip1) and WT-1), and electron microscopy. Hpg testes had marked reductions in weight, seminiferous cord volume and length, and severe spermatogenic impairment with germ cells per testis < 1% of adult wild-type testes. Sertoli cell nuclei expressed WT-1 in hpg testes, but often were centrally located, similar to 9-14-day-old wild-type testes, and they expressed p27(kip1), indicating that hpg Sertoli cells were post-mitotic. Hpg testes had significantly (P < 0.05) reduced Sertoli cells per testis (0.56 million) compared with 10-day wild-type (1.15 million) and adult wild-type testes (2.06 million). Immunofluorescence labelling of normal adult Sertoli cells showed supranuclear MT columns and basally located espin, but these features were absent in 10-day-old and hpg Sertoli cells. Hpg Sertoli cells showed pleomorphic nuclear ultrastructure with mature-type nucleoli, similar to normal adult-type Sertoli cells, but hpg Sertoli cells exhibited incomplete tight junctions that lacked ectoplasmic specializations. We conclude that in hpg mice, chronic gonadotrophin insufficiency restrains Sertoli cell proliferation and maturation, forming pseudo-adult-type Sertoli cells that are incapable of supporting germ cell proliferation and maturation.

Fig. 1

Histology of seminiferous epithelium in adult wild-type (A–C), 10-day-old wild-type (D–F) and adult hpg testis (G–I). (A) Adult wild-type seminiferous epithelium at stage IX of spermatogenic cycle showing nuclei of Sertoli cells (S), spermatogonium (SG), pachytene primary spermatocytes (P) and early elongating spermatids (SD). (B) Detail of a mature Sertoli cell nucleus with a tripartite nucleolus. (C) Highmagnification of A showing leptotene (L) and pachytene (P) primary spermatocytes between which is a thin curvilinear structure (arrow) representing inter-Sertoli cell junctional complex. (D) Ten-day wild-type testis showing basal and central Sertoli cell nuclei (S), spermatogonia (SG) and occasional spermatocytes (arrow). (E) sDetail of Sertoli cell nuclei showing ovoid and angular shapes with small nucleoli (arrowheads) and heterochromatin patches (arrows). (F) Most advanced germ cell type is occasional primary spermatocytes showing condensed chromatin (arrows). (G) hpg testis cord showing basal and central Sertoli cell nuclei (S), spermatogonia (SG) and central confluence of Sertoli cell cytoplasm (asterisks). Degenerative cellular debris (arrows) and hydropic-type germ cells are indicated (arrowhead). (H) Detail of Sertoli cell nuclei showing large and double-nucleoli (arrows). (I) Most advanced germ cells are zygotene primary spermatocytes with thickened chromatin profiles. Scale bars in A, D, G, = 20 µm; all others = 10 µm.

Fig. 2

Expression of WT-1 (upper panels) and p27 (lower panels) immunoreactivity in adult hpg mouse testis (far left column: hpg) or wild-type mouse testes on postnatal day 9 (second left column: +/+d9), day 14 (second to right column: +/+d14) or adult (far right column: +/+adult). For each antigen, the upper row are micrographs obtained with a × 20 objective (scale bars = 30 µm), the lower row obtained with a × 50 objective (scale bars = 10 µm).

Fig. 3

Immunofluorescence demonstration of microtubules (â-tubulin) indicated by green fluorescence and espin (red fluorescence). Nucleic acids are shown by blue fluorescence. A,D are adult wild-type testes; B,E are 10-day wild-type testes, and C,F are hpg testes. (A) Normal adult seminiferous tubule showing tall columns of microtubules in Sertoli cells and surrounding the basal spermatogonia with circular cell nuclei. (B) Ten-day seminiferous cord showing positive tubulin label among circular/irregularly shaped basal cells and weak labelling more centrally where negative profiles of immature Sertoli cells are noted (arrows). (C) hpg testis showing positive tubulin label of circular, basal germ cells and absence of tubulin in the centre of the cord. Nucleic acids (blue fluorescence) label more centrally located cells, the identity of which cannot be confirmed. (D) Normal adult testis showing arching and curvilinear segments of the espin component of inter-Sertoli cell tight junctions (arrows). Espin in ectoplasmic specializations of spermatid bundles is indicated (circle). Nucleic acids in nuclei of spermatocytes and spermatids are shown by blue fluorescence. (E) Ten-day testis showing lack of or very weak labelling for espin; blue fluorescence indicates nucleic acids. (F) hpg testis showing lack of positive label for espin in the seminiferous cords; blue fluorescence indicates germ and Sertoli cell nuclei. Scale bars, 20 µm.

Fig. 4

Electron micrographs of adult wild-type (A–C), 10-day wild-type (D–F) and adult hpg testes (G–K). (A) Typical normal adult Sertoli cell with nucleolus and annular chromatin (arrowhead) and curved inter-Sertoli cell junctions (arrows). (B) Sertoli cell tripartite nucleolar complex showing central nucleolus and flanking chromatin. (C) Inter-Sertoli cell junctional complex between apposing Sertoli cells. (D) Ten-day testis with compacted immature Sertoli cell nuclei (S). (E) Immature Sertoli cell nucleus with patches of heterochromatin associated with nuclear membrane (arrows). (F) Unspecialized plasma membranes (arrow) between adjacent Sertoli cell nuclei (S). (G) hpg testis showing basal and more central Sertoli cell nuclei (S). (H) Sertoli cell tripartite nucleolar complex with central chromatin and peripheral nucleoli. (I) hpg Sertoli cell nucleolar complex showing granular nucleolus and annular chromatin. (J) Plasma membranes between hpg Sertoli cell nuclei (S) showing segments of close apposition of membrane. (K) Plasma membranes between adjacent hpg Sertoli cells showing individual cytoplasmic densities. Scale bars in A,D,G = 10 µm; E = 3 µm; B,I = 2 µm; C,F,H = 1 µm; J,K = 0.5 µm.