Immunolocalization of cytochrome P450 aromatase in rat testis during postnatal development

Aromatization of androgens into estrogens in rat testis is catalyzed by the microsomal enzyme cytochrome P450 aromatase. In this work, aromatase cellular site was investigated in prepuberal, peripuberal and postpuberal testis, from 10-, 21- and 60-day-old rats respectively. Paraffin-embedded testis sections were processed for P450arom immunostaining using a rabbit polyclonal antiserum generated against purified human placental cytochrome P450 aromatase. Next, biotinylated anti-rabbit IgG was applied, followed by ABC/HRP/complex amplification with diaminobenzidine as chromogen. Prepuberal testis sections showed a strong immunoreactivity of aromatase in Sertoli cell cytoplasm while interstitial cells were immunonegative. In peripuberal testis sections, cytoplasmic immunoreaction was weak in Sertoli cells, but it was strong in spermatocytes and sporadic in Leydig cells. Postpuberal testis sections displayed a moderate aromatase immunoexpression in spermatocytes while a strong immunostaining was observed in round and elongated spermatids, as well as in Leydig cells. These results indicate a different age-dependence of aromatase localization in rat testicular cells during gonadal development. In particular, inside the seminiferous tubules, the aromatization site moves from Sertoli cells to late germ cells, suggesting a proliferative role of aromatase in prepuberal testis and its subsequent involvement in meiotic and post-meiotic germ cell maturation.     

The immunolocalization of small nuclear ribonucleoprotein particles in testicular cells during the cycle of the seminiferous epithelium of the adult rat

The objective of this study was to determine the cellular and subcellular distribution of small nuclear ribonucleoprotein particles (snRNPs) in the adult rat testis in relation to the different cell types at the various stages of the cycle of the seminiferous epithelium. The distribution of snRNPs in the nucleus and cytoplasm of germ cells was quantitated in an attempt to correlate RNA processing with morphological and functional changes occurring during the development of these cells. Light-microscopic immunoperoxidase staining of rat testes with polyclonal anti-Sm and monoclonal anti-Y12 antibodies localized spliceosome snRNPs in the nuclei and cytoplasm of germ cells up to step 10 spermatids. Nuclear staining was intense in Sertoli cells, spermatogonia, spermatocytes, and in the early steps of round spermatid development. Although comparatively weaker, cytoplasmic staining for snRNPs was strongest in mid and late pachytene spermatocytes and early round spermatids. Quantitative electron-microscopic immunogold labeling of Lowicryl embedded testicular sections confirmed the light-microscopic observations but additionally showed that the snRNP content peaked in the cytoplasm of midpachytene spermatocytes and in the nuclei of late pachytene spermatocytes. The immunogold label tended to aggregate into distinct loci over the nuclear chromatin. The chromatoid body of spermatids and spermatocytes and the finely granular material in the interstices of mitochondrial aggregates of spermatocytes were found to be additional sites of snRNP localization and were intensely labeled. This colocalization suggests that these dense cytoplasmic structures may be functionally related. Anti-U1 snRNP antibodies applied to frozen sections showed the same LM localization pattern as spliceosome snRNPs. Anti-U3 snRNP antibodies applied to frozen sections stained nucleoli of germ cells where pre-rRNA is spliced.     

Ultrastructure of developing germ cells in the fetal human testis

Electron microscopic studies of the testis were performed on 12 human embryos and fetuses between 9 and 30 weeks post conceptionem. According to their ultrastructural features, the fetal germ cells could be divided into the following three stages of differentiation: 1) gonocytes, 2) intermediate cells, and 3) fetal spermatogonia. Sertoli cells were present among the germ cells in all the testes studied. The gonocytes showed the highest nucleo-cytoplasmic ratio. Their round nucleus contained a centrally located, prominent nucleolus. The cytoplasm displayed a well developed Golgi apparatus, lipid droplets and parallel arrays of short cisternae of the rough surfaced endoplasmic reticulum (rER). Microfilaments were numerous, particularly just beneath the cell membrane. The intermediate cells were found to extend several cytoplasmic processes and to contain a moderate number of long, branched and/or widened rER cisterna which were frequently connected to the perinuclear cisterna. Intermediate cells were often connected to one another by intercellular cytoplasmic bridges. The fetal spermatogonia also displayed cytoplasmic bridges. These cells showed the lowest nucleo-cytoplasmic ratio and more condensed nuclear chromatin. The mitochondria were situated close to the nucleus. Many of them were connected by a cementing substance. Lipid droplets and rER cisternae were rare in these cells. Infoldings of the inner nuclear membrane were often present in the gonocytes and in the intermediate cells, but were rarely observed in the fetal spermatogonia. Glycogen particles, polyribosomes, and chromatoid bodies ("nuage") were present in all the three germ cell types. With the maturation of the fetus, the number of gonocytes was found to decrease, whereas the number of fetal spermatogonia increased. The Sertoli cells also changed their ultrastructure, showing an increase in the number of rER cisternae, as well as of microfilaments, lipid droplets, and secondary lysosomes.     

Sertoli cell behaviors in developing testis cords and postnatal seminiferous tubules of the mouse

Sertoli cells are the primary structural component of the fetal testis cords and postnatal seminiferous tubules. Live imaging technologies facilitate the visualization of cell morphologies and behaviors through developmental processes. A transgenic mouse line was generated using a fragment of the rat Gata4 gene to direct the expression of a dual-color fluorescent protein reporter in fetal and adult Sertoli cells. The reporter encoded a red fluorescent protein, monomeric Cherry (mCherry), fused to histone 2B and enhanced green fluorescent protein (EGFP) fused to a glycosylphosphatidylinositol sequence, with a self-cleaving 2A polypeptide separating the two fusion proteins. After translation, the red and green fluorescent proteins translocated to the nucleus and plasma membrane, respectively, of Sertoli cells. Transgene expression in testes was first detected by fluorescent microscopy around Embryonic Day 12.0. Sertoli cell division and migration were visualized during testis cord formation in organ culture. Initially, the Sertoli cells had mesenchyme-like morphologies and behaviors, but later, the cells migrated to the periphery of the testis cords to become epithelialized. In postnatal seminiferous tubules, Sertoli nuclei were evenly spaced when viewed from the external surface of tubules, and Sertoli cytoplasm and membranes were associated with germ cells basally in a rosette pattern. This mouse line was bred to previously described transgenic mouse lines expressing EGFP in Sertoli cytoplasm or a nuclear cyan fluorescent protein (Cerulean) and mCherry in plasma membranes of germ cells. This revealed the physical relationship between Sertoli and germ cells in developing testis cords and provided a novel perspective on Sertoli cell development.     

Chromatin condensation behaviour of the Y chromosomes in the human testis

The condensation behaviour of the human Y chromosome in germ cells and Sertoli cells of pre- and post-pubertal testes was followed by fluorescence in situ hybridisation using probes for three different regions of the Y chromosome. Patterns of expansion or contraction of signal are taken to reflect degrees of condensation of the related Y chromatin and hence its potential for genetic activity. For probe pHY2.1, which labels the distal non-fluorescent and fluorescent heterochromatin of the Y chromosome (Yq12), an expanded signal seen in gonocytes of the prepubertal testis is superseded by a condensed signal seen in adult germ cells at all but the zygotene stage of meiotic prophase when meiotic pairing takes place. In contrast, Sertoli cells show a condensed signal pre-pubertally but a greatly expanded signal in the adult testis. A totally condensed pHY2.1 signal is found in a chromosomally normal man with Sertoli-cell-only syndrome. It is hypothesised that control over at least some facets of spermatogenesis may not, in the adult, be autonomous to the germ cells, but rather may emanate from the Sertoli cells. Chromatin expansion at zygotene could, however, be important for pairing and crossing over in the XY bivalent, successful synapsis ensuring survival of spermatocytes into the post-meiotic stages.     

Sertoli cell proliferation in the adult testis--evidence from two fish species belonging to different orders

Germ cell survival and development critically depend on the cells' contact with Sertoli cells in the vertebrate testis. Fish and amphibians are different from mammals in that they show a cystic type of spermatogenesis in which a single germ cell clone is enclosed by and accompanied through the different stages of spermatogenesis by an accompanying group of Sertoli cells. We show that in maturing and adult testes from African catfish and Nile tilapia, Sertoli cell proliferation occurs primarily during spermatogonial proliferation, allowing the cyst-forming Sertoli cells to provide the increasing space required by the growing germ cell clone. In this regard, coincident with a dramatic increase in cyst volume and number of germ cells per cyst, in Nile tilapia, the number of Sertoli cells per cyst was strikingly increased from primary spermatogonia to spermatocyte cysts. In both African catfish and Nile tilapia, Sertoli cell proliferation is strongly reduced when germ cells have proceeded into meiosis, and stops in postmeiotic cysts. We conclude that Sertoli cell proliferation is the primary factor responsible for the increase in testis size and sperm production observed in teleost fish. In mammals, Sertoli cell proliferation in the adult testis is not observed under natural conditions. However, on the level of the individual spermatogenic cyst--similar to mammals--Sertoli cell proliferation ceases when germ cells have entered meiosis and when tight junctions are established between Sertoli cells. This suggests that fish are valid vertebrate models for studying Sertoli cell physiology.     

Sertoli-germ cells interactions in the human testis.

In previous histoimmunochemical studies we reported that transferrin (TF) and insulin-like growth factor I (IGF-I) are present in the cytoplasm of the Sertoli cells of the adult human testis. Receptors for TF were found mainly in adluminal germ cells and type I receptors for IGF-I both in Sertoli and germ cells. Using electron microscopy, evidence of transfer of both TF and IGF-I from the Sertoli to the germ cells through a receptor-mediated endocytosis mechanism was also found. In this paper we report the results of the histoimmunochemical localization of alpha inhibin in the human fetal, prepubertal and adult testis. In 8- to 14-week-old fetal testes a positive immunostaining was found mainly in the interstitial cells, whereas no staining was found in the germ cords. In the prepubertal testis the immunostaining was present in the Sertoli cells but not in the interstitial cells. In the adult human testis the immunostaining was present not only in the Sertoli cells but also in the spermatocytes and in several Leydig cells. Using electron microscopy and immunogold labeling the presence of alpha inhibin immunoreactivity was found in the rough endoplasmic reticulum and in the Golgi cisternae of both Sertoli and Leydig cells. Moreover we found evidence of transfer of alpha inhibin from the Sertoli to the germ cells through receptor-mediated endocytosis.     

Immunolocalization of albumin and transferrin in germ cells and Sertoli cells during rat gonadal morphogenesis and postnatal development of the testis

The localization of albumin and transferrin was examined immunohistochemically in germ cells and Sertoli cells during rat gonadal morphogenesis and postnatal development of the testis. These proteins appeared as early as the 13th day of gestation in migrating primordial germ cells before Sertoli cell differentiation. In the fetal testis, strong immunoreactivity was only detected in the gonocytes. In the prepubertal testis, spermatogonia, primary spermatocytes, and some Sertoli cells accumulate albumin and transferrin. At puberty, different patterns of immunostaining of the germ cells were observed at the various stages of the cycle of the seminiferous epithelium. Diplotene spermatocytes at stage XIII, spermatocytes in division at stage XIV, and round spermatids at stages IV–VIII showed maximal staining. Labeling was evident in the cytoplasm of adult Sertoli cells. Albumin and transferrin staining patterns paralleled each other during ontogenesis.     

Localisation du récepteur des androgènes dans le testicule

Whether or not germ cells contain the androgen receptor remains a matter of controversy. In the present study we performed biotinstreptavidin immunoperoxidase using an affinity purified rabbit polyclonal antibody made to a 21 amino acid peptide of the amino terminus of the rat AR to determine androgen receptor (AR) distribution in the rat and mouse testes. Specificity of the antibody was confirmed as follows: 1) Western immunoblots rendered a specific band at approximately 110 kD; 2) preadsorption of the antibody with the 21 amino acid peptide eliminated specifice immunostaining; 3) the intensity of staining in all AR positive cells diminished as a function of antisera dilution; 4) tissues known to express abundant AR (e.g., epididymis, prostate, seminal vesicles) all rendered a robust, nuclear AR immunostaining pattern in the epithelial cells; 5) prostate cell lines known to express AR immunostained positive with the antibody; 6) AR negative COS-7 cells became AR immunopositive when transfected with a vector expressing the rat AR and intracellular AR distribution was a function of androgens. AR immunostaining results revealed the following: Within the interstitial compartment of adult rats, AR was detected in some Leydig cells and all smooth muscle cells forming the walls of blood vessels, but endothelial cells were negative. In the seminiferous tubules AR was observed in all peritubular myoid cell nuelei, but not in the distal layer of Iymphatic endothelial cells. In Sertoli cells, nuclear AR immunostaining was stage specific; moderate AR immunostaining became evident at late stage IV of the cycle, reached a robust peak at stages VII-VIII, and then disappeared completely. Specific AR immunostaining was also discerned in the nuclei of stage XI elongated spermatids, in which nuclear elongation is apparent but chromatin condensation has not yet begun. With onset of chromatin condensation, nuclear AR immunostaining in elongated spermatids was not discerned concomitant with its detection in the cytoplasm. In general, similar observations have now been confirmed in the adult mouse testis, except that an Leydig cells were strongly AR positive. Nucleic acid in situ hybridization studies for AR were performed in adult rat testis using a 236 bp antisense cRNA probe (rat AR cDNA was provided by Dr. C. Chang, U. Wisconsin, Madison, WI) to confirm the AR immunostaining. A prominent hybridization signal at the base of the seminiferous epithelium was observed, in the area occupied by Sertofi and spermatogonia. This led us to re-examine the immunostaining results to determine if spermatogonia were also AR positive. Preliminary results are consistent with the interpretation that AR is present in certain spermatogonial populations. Taken together, these results concur with prior observations suggesting that AR is present in the somatic cells of the testis; thus, it is these cell types that likely respond to circulating androgens to control spermatogenesis. However, they raise anew the controversy of whether germ cells respond directly to androgens.     

Ultrastructure of germ cell development in the human fetal testis

Summary Electron-microscopic examination of the human fetal testis between 10 and 20 weeks gestation reveals the presence of two distinct cell types within the tubules: Sertoli cells and germ cells. The latter are distinguished by their spherical shape, smooth nuclear membranes, globular mitochondria and paucity of cytoplasmic organelles. The gonocytes, or primitive germ cells, occur as single cells in the central portions of the tubules. Their chromatin is finely granular and evenly dispersed. Nucleoli are centrally placed and of uniform electron density. Various stages in the migration of gonocytes to the tubular periphery are indicated by the extension of cytoplasmic processes toward the basal lamina. Bands of microtubules are present within the processes. Spermatogonia are arranged in pairs and groups at the tubular periphery. They lack the nucleolar and mitochondrial characteristics of adult spermatogonia. Except for slight changes in chromatin density and nucleolar structure, the fetal spermatogonia retain the ultrastructural characteristics of gonocytes. Intercellular bridges connect adjacent spermatogonia. Degeneration affecting large numbers of germ cells, but primarily gonocytes, begins with nuclear infolding and chromatin condensation and eventually involves both nuclear and cytoplasmic structures. The degenerated cells are removed by phagocytosis by adjacent Sertoli cells. Large phagosomes are present in the cytoplasm of many of the Sertoli cells.Supported by a grant from the Ford Foundation and by General Research Support Grant RR055511 from the National Institutes of Health. Technical assistance was provided by Mrs. Lucy A. Conner.     

RNA binding protein Musashi1 is expressed in sertoli cells in the rat testis from fetal life to adulthood.

The Musashi1 (Msi1) gene identified in mouse is a member of a subfamily of RNA binding proteins that are highly conserved across species. Msi1 expression is highly enriched in proliferative cells within the developing central nervous system. Within the testis, proliferation and differentiation of germ cells takes place within the seminiferous epithelium, where these cells are supported physically and functionally by Sertoli cells that do not themselves proliferate following the onset of puberty. RNA binding proteins expressed in testicular germ cells are essential for normal fertility. Preliminary data suggested the mRNA for Msi1 was present in ovary; therefore, we used an Msi1-specific cRNA and monoclonal antibody to investigate whether Msi1 was expressed in the testis. Msi1 mRNA was expressed in rat testis from birth until adulthood; in situ hybridization revealed silver grains within the seminiferous epithelium. Immunohistochemical studies demonstrated that at all ages examined (from Fetal Day 14.5 until adulthood) Msi1 protein was expressed in Sertoli cells. In fetal and adult rat ovaries, Msi1 was detected in granulosa cells and their precursors. In Sertoli cells, protein was detected in both cytoplasmic and nuclear compartments; in adult testes, the immunointensity of the nuclear staining was stage dependent, with highest levels of expression in Sertoli cells at stages I-VI. In rat gonads, the RNA binding protein Msi1 is expressed in both proliferating and nonproliferating Sertoli and granulosa cells.     

Intracellular distribution of poly(ADP-ribose) synthetase in rat spermatogenic cells

The highest activity of poly(ADP-ribose) synthetase was found in the testis among several rat tissues tested. Subcellular fractionation of the testis demonstrated that the synthetase was localized primarily in the nucleus and partially in the microsomal-ribosomal fraction. This result was confirmed by immunocytochemical staining with the enzyme-specific antibody. The synthetase was localized in the nuclei of interstitial cells, Sertoli cells, spermatogonia, and spermatocytes. In addition, round spermatids showed a granular staining in the cytoplasm, which was comparable in intensity with that in the nucleus. The cytoplasmic synthetase had a molecular weight of 115,000 and synthesized oligomers of ADP-ribose on itself (automodification). The synthetase activity in the isolated cytoplasmic fraction was stimulated about threefold by the addition of DNA and depressed by treatment with DNase I, suggesting the presence of endogenous activator DNA. A candidate DNA for such an activator was isolated from the microsomal-ribosomal fraction, and identified tentatively as mitochondrial DNA on the basis of its size and restriction fragment patterns.     

The interphase distribution of satellite DNA-containing heterochromatin in mouse nuclei

The distribution of sites capable of binding mouse satellite-complementary RNA in the cytological hybridization reaction has been examined in mouse liver and testis interphase nuclei. The approach taken has been to combine hybridization with semi-thin sectioning and autoradiography in order to obtain a clear picture of the relationship of satellite DNA-containing structures to the rest of the interphase nucleus. In liver nuclei, hybridization occurs primarily with blocks of heterochromatin associated with the nuclear envelope. The most prominent of these, in terms both of size and intensity of hybridization, is the nucleolar stalk and the rest of the nucleolus-associated heterochromatin. The nucleolar body itself is not labeled, nor is much of the peripheral condensed chromatin ; in fact, a polarized distribution of satellite DNA is evident. In Sertoli and spematid nuclei, satellite DNA is found in a small number of large heterochromatin blocks with which the nucleolus is associated; some of this material bears a relationship to the nuclear envelope in these cells also.     

Distribution of the microtubule-dependent motors cytoplasmic dynein and kinesin in rat testis.

To examine the possible role of microtubule-based transport in testicular function, we used immunofluorescent techniques to study the presence and localization of the microtubule mechanoenzymes cytoplasmic dynein (a slow-growing end-directed motor) and kinesin (a fast-growing end-directed motor) within rat testis. Cytoplasmic dynein immunofluorescence was observed in Sertoli cells during all stages of spermatogenesis, with a peak in apical cytoplasm during stages IX-XIV. Cytoplasmic dynein immunofluorescence was also localized within Sertoli cells to steps 9-14 (stages IX-XIV) germ cell-associated ectoplasmic specializations. In germ cells, cytoplasmic dynein immunofluorescence was observed in manchettes of steps 15-17 (stages I-IV) spermatids, and small, hollow circular structures were seen in the cytoplasm of step 17 and step 18 spermatids during stages V and VI. Kinesin immunofluorescence was observed in manchettes of steps 10-18 spermatids (stages X-VI). The stage-dependent apical Sertoli cell cytoplasmic dynein immunofluorescence, in conjunction with the previously reported orientation of Sertoli cell microtubules (slow-growing ends toward the lumen) and peak secretion of androgen-binding protein and transferrin, is consistent with the hypothesis that cytoplasmic dynein is involved in Sertoli cell protein transport and secretion. Further, the localization of cytoplasmic dynein and kinesin to manchettes is consistent with current hypotheses concerning manchette function.     

A reassessment of Y chromosomal behaviour in germ cells and Sertoli cells of the mouse as revealed by in situ hybridisation

In situ hybridisation experiments were carried out to reappraise the state of condensation of the Y chromosome in germ cells and Sertoli cells of the mouse. Previous work had suggested that all testicular cells showed a condensed Y chromosome prior to the adult stage. We now demonstrate that, although the Y chromosome is condensed in pre-pubertal Sertoli cells, it is greatly expanded in primordial germ cells (gonocytes). An expanded Y-signal is first seen in Sertoli cell nuclei at or around day 21 of postnatal development, coinciding with the first appearance of spermatids in the germinal epithelium.