Lonnie D. Russell

Sertoli cell junctions: morphological and functional correlates

Publisher Summary Modern cell biology has shown that cellular recognition and adhesion are not always manifested by junctions of a classical type or by morphological entities recognizable by standard transmission electron microscopy techniques. This chapter describes several junctions in which experimental data are the primary evidence for suggesting a junctional relationship between cells. The cyclic nature of spermatogenesis and the continually changing morphology of the Sertoli cell provide a dynamic framework in which junctions are periodically formed and eliminated. Desmosomes are important in maintaining the integrity of the seminiferous epithelium, a function, which should not be underemphasized, because many germ cell types appear only loosely arranged in the seminiferous epithelium. Based on circumstantial evidence, it is reasonable to suspect that, as yet, unrecognized junctional forms are present in the relationship of the Sertoli cell and germ cells and will be revealed in the future through methodological advances.

Morphological pattern elicited by agents affecting spermatogenesis by disruption of its hormonal stimulation

Abstract A variety of agents (clomiphene citrate, cyproterone acetate, estradiol 17-β, medrogestone, medroxyprogesterone, and methoxychlor) thought to disrupt the normal pathway by which hormones stimulate the testis were tested in rats to determine the early morphologic changes in the testis. After initial trial experiments at which dose and sacrifice intervals were determined, the earliest morphological abnormalities were assessed quantitatively and related to the particular spermatogenic stage in which these defects were initiated. All agents tested produced an identical morphological pattern of response displaying a significant increase in Stage VII degenerating cells (pachytene spermatocytes, step 7 and step 19 spermatids) as compared with vehicle-administered rats. The number of degenerating cells in other stages did not significantly change. The rise in degenerating cells in Stage VII, as seen in this study, was similar to that reported by Russell and Clermont (1972) utilizing hypophysectomized rats, and that reported by other investigators who have used a variety of agents to disrupt the hormonal stimulation of the seminiferous tubules. The data indicate that no matter how, or at what level the hormonal stimulation of the testis is interrupted, the morphological pattern of response is the same, and that such a pattern is useful in predicting the mechanism of action of agents suspected of interfering with hormone action.

Radiation-induced cell death in the mouse testis : Relationship to apoptosis

The killing of male germ cells by radiation and other toxicants has recently been attributed to apoptosis, but a critical evaluation of the presence of the different features of apoptosis has not been performed. In this study, mouse testes exposed to radiation were examined by light microscopy, electron microscopy and terminal transferase-mediated end labeling (TUNEL) to determine whether the cells were apoptotic according to several criteria. Testes were irradiated with single doses of gamma rays of up to 5 Gy. Although the maximum response was produced by 5 Gy, even 0.5 Gy induced marked changes. The numbers of abnormal spermatogonia reached a peak 12 h after irradiation and then declined, and the total number of spermatogonia began to decline at 12 h. These changes were most prominent among the B spermatogonia and early preleptotene spermatocytes. When examined by both light and electron microscopy, the majority of the abnormal spermatogonia showed condensation of nuclear chromatin and some showed features similar to necrosis, but the typical morphological characteristics of apoptosis, margination of chromatin and nuclear fragmentation, were rare. Many of the abnormal spermatogonia were TUNEL-positive, with the maximum number occurring at 12 h after irradiation. Although the morphological features of radiation-induced spermatogonial degeneration were not typical of apoptosis, the TUNEL staining, the rapid onset of degeneration and the sensitivity to low doses suggest that the mechanism of radiation-induced spermatogonial degeneration is closely related to apoptosis.

Enhancement of A Spermatogonial Proliferation and Differentiation in Irradiated Rats by Gonadotropin-Releasing Hormone Antagonist Administration

The initial changes in the numbers, proliferation, and differentiation of A spermatogonia in irradiated rats after the administration of a GnRH antagonist, which is known to induce differentiation in this system, were investigated. LBNF1 rats were given 6 Gy ofγ -irradiation; some were treated with the GnRH antagonist Cetrorelix beginning 15 weeks after irradiation. Although the spermatogonia in the irradiated rats without hormone treatment continue to proliferate (labeling and mitotic indexes of 24% and 18%, respectively), they underwent apoptosis (apoptotic indexes of 21% by the terminal transferase-mediated end labeling assay and 9% by nuclear morphology), resulting in a constant number of A spermatogonia. Whole mount analysis of clones of A spermatogonia revealed that larger clones were more likely to undergo apoptosis than mitosis. Hormone administration decreased the intratesticular testosterone concentration to 6% of the level in irradiated rats within 1 week. Concomitantly, there was a decrease in...

Gap junctions between sertoli and germ cells of rat seminiferous tubules

Ultrastructural observations of rat seminiferous tubules show clearly the presence of plasma membrane junctions between Sertoli and germ cells in the basal and adluminal compartments. Results obtained from the freeze fracture and thin section techniques were correlated in order to elucidate the nature of these intercellular junctions. We suggest that these intercellular membrane specializations are gap junctions which occur within regions of plasma membrane that also exhibit adherens-like modifications.

Isolation and Characterization of Membrane Vesicles from Human and Boar Spermatozoa: Methods Using Nitrogen Cavitation and Ionophore Induced Vesiculation

A method for isolation of plasma membrane vesicles from human and boar spermatozoa using nitrogen cavitation is described. The purity of the preparations were assessed by electron microscopy, marker enzyme assay and the sedimentation characteristics of fused plasma membrane-acrosomal membrane vesicles in sucrose gradients. PAGE-SDS profiles of plasma membrane polypeptides from boar spermatozoa were significantly different from those of human spermatozoa. Differences in electrophoretic profiles of polypeptides from different regions of the spermatozooon were also observed.

Cytoskeletal involvement in spermiation and sperm transport

The process of spermiation and sperm transport was studied using specific inhibitors of cytoskeletal elements. Within 12-24 hr after the intratesticular injection of taxol, a compound that acts to stabilize microtubules and inhibit microtubule-related processes, an unusually large number of microtubules was seen within the body of the Sertoli cell. At the same time, transport of elements within the seminiferous epithelium was affected. At the end of stage VI of the cycle, step 19 spermatids were maintained in the deep recesses of the Sertoli cell and not transported to the rim of the seminiferous tubule lumen. At stage VIII, residual bodies remained at, or near, the rim of the tubule and were not transported to the base of the tubule. They underwent only partial degradation at this site, indicating that there may have been two phases involved in their dissolution--one autophagic and one phagocytic, but the latter did not occur since the residual bodies were not transported to Sertoli lysosomes at the base of the tubule. The observations suggest that microtubules are involved in transport processes within the seminiferous epithelium. Within 1-12 hr after the intratesticular injection of 500 microM cytochalasin D, a compound which interferes with actin-related processes, normal appearing tubulobulbar complexes were not present. The tubular portion (distal tube) of the complex did not initiate development. It was assumed that filaments (which were identified as such using NBD-phallacidin and the S-1 fragment of myosin) played an important role in the development of this portion of the complex. Cells did not eliminate cytoplasm normally, as evidenced by an enlarged cytoplasmic droplet, further emphasizing the published role for tubulobulbar complexes in cytoplasmic elimination. Although sperm were released normally from stage VIII tubules, many remained within the tubular lumen and did not traverse the duct system. Cytochalasin did not inhibit fluid secretion by the Sertoli cell, as demonstrated by efferent duct ligation, but did alter myoid cell actin cytoskeletal organization, suggesting that myoid cell contractility is primarily responsible for transport of sperm. Overall, the observations suggest that cytoskeletal activity of the Sertoli cell is important for several aspects of the spermiation process as well as sperm transport.

Biological Activity and Enrichment of Spermatogonial Stem Cells in Vitamin A-Deficient and Hyperthermia-Exposed Testes from Mice Based on Colonization Following Germ Cell Transplantation

Abstract Spermatogenesis is a complex process in which spermatogonial stem cells divide and subsequently differentiate into spermatozoa. This process requires spermatogonial stem cells to self-renew and provide a continual population of cells for differentiation. Studies on spermatogonial stem cells have been limited due to a lack of unique markers and an inability to detect the presence of these cells. The technique of germ cell transplantation provides a functional assay to identify spermatogonial stem cells in a cell population. We hypothesized that vitamin A-deficient (VAD) and hyperthermically treated testes would provide an enriched in vivo source of spermatogonial stem cells. The first model, hyperthermic treatment, depends on the sensitivity of maturing germ cells to high temperatures. Testes of adult mice were exposed to 43°C for 15 min to eliminate the majority of differentiating germ cells. Treated donor testes were 50% of normal adult testis size and, when transplanted into recipients, resulted in a 5.3- and 19-fold (colonies and area, respectively) increase in colonization efficiency compared to controls. The second model, VAD animals, also lacked differentiating germ cells, and testes weights were 25% of control values. Colonization efficiency of germ cells from VAD testes resulted in a 2.5- and 6.2-fold (colonies and area, respectively) increase in colonization compared to controls. Hyperthermically treated mice represent an enriched source of spermatogonial stem cells. In contrast, the low extent of colonization with germ cells from VAD animals raises important questions regarding the competency of stem cells from this model.

Morphologic Characteristics of the Chemically Induced Acrosome Reaction in Human Spermatozoa

The morphologic changes accompanying the acrosome reaction in human spermatozoa, as it is induced by the antibiotic A23187 and calcium ions, are described. The reaction is shown to be similar to that observed in other species when the reaction occurs spontaneously or is induced by physiologic fluids. The reaction in human spermatozoa differs from the chemically induced reaction in other species in that plasma membrane microfilaments, prominent in the boar, and tubular-like elements prominent in boar, rabbit, and monkey sperm, are not observed. Motility remains high when human spermatozoa are treated with A23187 and calcium and it is possible that these agents may be useful in the study of certain causes of infertility.

The seasonal breeding hamster as a model to study structure-function relationships in the testis

The present study was undertaken to document morphological changes in the testis of the seasonally breeding golden hamster, an animal model which has been studied extensively from an endocrine standpoint but for which morphological data is inadequate. Germ cells, Sertoli cells and Leydig cells were studied during active and regressed state of gonadal activity by exposing the animals to long (16L:8D) and short photoperiods (6L:18D), respectively. Testis of the hamster exposed to short photoperiods displayed more than a ten-fold reduction in weight and decreased seminiferous tubule diameter. The seminiferous tubules contained primarily Sertoli cell and spermatogonia but also occasional spermatocytes and round spermatids. Leydig cells were decreased in size, a change which appeared to be primarily due to a decrease in cytoplasmic volume. The Leydig cell endoplasmic reticulum which was atypically saccular displayed both rough and smooth components and was decreased during short photoperiods. Mitochondria generally appeared larger and showed considerable structural heterogeneity. Short photoperiod-induced changes in the Sertoli cells included a marked reduction in cell height and an apparent reduction in cell volume, absence of lateral processes, presence of small, almost spheroidal nuclei with inconspicuous nucleoli, an increase in the amount of lipid and decreases in the amount of smooth endoplasmic reticulum and glycogen. The striking differences in the testicular structure between the active and regressed state of gonadal activity follows photoperiod-induced changes in endocrine parameters and suggests that the hamster would be an ideal model to study structure-function relationships in the testis, and especially those related to the Sertoli cell.