The seminiferous epithelium in the guinea fowl (Numida meleagris)

Summary Spermiogenesis and cellular associations in the seminiferous epithelium of the guinea fowl were studied and described in sexually active adult birds. PAS stain was found to be useful in the recognition of steps of spermatid differentiation only in the first early stages. Nuclear morphological changes were subsequently found to be more reliable in tracing steps of spermiogenesis. It was observed that haematoxylin-eosin stained tissue can be used in the study of spermiogenesis in the bird. Various stages of the seminiferous epithelium were observed in any cross-section of the seminiferous tubules. Distinct cellular associations were observed, but intermix of adjacent germ cells or heterogenous cellular associations were frequently encountered.     

Protective role of epithelium in the guinea pig airway

We developed an in vitro system to assess the role of the epithelium in regulating airway tone using the intact guinea pig trachea (J. Appl. Physiol. 64: 466-471, 1988). This method allows us to study the response of the airway when its inner epithelial surface or its outer serosal surface is stimulated independently. Using this system we evaluated how the presence of intact epithelium can affect pharmacological responsiveness. We first examined responses of tracheae with intact epithelium to histamine, acetylcholine, and hypertonic KCl when stimulated from the epithelial or serosal side. We then examined the effect of epithelial denudation on the responses to these agonists. With an intact epithelium, stimulation of the inner epithelial side always caused significantly smaller changes in diameter than stimulation of the outer serosal side. After mechanical denudation of the epithelium, these differences were almost completely abolished. In the absence of intact epithelium, the trachea was 35-fold more sensitive to histamine and 115-fold more sensitive to acetylcholine when these agents were applied to the inner epithelial side. In addition, the presence of an intact epithelium almost completely inhibited any response to epithelial side challenge with hypertonic KCl. These results indicate that the airway epithelial layer has a potent protective role in airway responses to luminal side stimuli, leading us to speculate that changes in airway reactivity measured in various conditions including asthma may result in part from changes in epithelial function.     

Antigen inhalation induces mast cells and eosinophils infiltration in the guinea pig esophageal epithelium involving histamine-mediated pathway

Antigen challenge in sensitized guinea pig esophagus in vitro induces mast cell degranulation and histamine release. This study tests the hypothesis that antigen inhalation in vivo induces infiltration of the esophageal epithelium by mast cells and eosinophils via a histamine pathway. Actively sensitized guinea pigs were exposed to inhaled 0.1% ovalbumin. One or 24 h after inhalation exposure, the esophagus was processed for immunofluorescent staining of mast cell tryptase and eosinophil major basic protein (MBP). Additional animals were pretreated with thioperamide, a histamine H4/H3 receptor antagonist. Total tryptase- and MBP-labeled cells and percent of positive cells in the epithelial layer were counted. The total number of mast cells was unchanged after inhalation challenge, but the percentage in the epithelium increased 1 h after challenge. The total number of eosinophils increased 1 h after challenge, and the percentage migrating to the epithelium increased by 24 h after challenge. Mast cell migration into the mucosal epithelium preceded that of eosinophils. Thioperamide inhibited mast cell and eosinophil migration. In conclusion, antigen inhalation in sensitized animals induces mast cells and eosinophils to infiltrate in the esophageal epithelium via histamine-mediated mechanism.     

New method of quantitative analysis of seminiferous epithelium in male guinea pigs

The aim of the study was the comparison of a newly developed method of quantitative analysis of the seminiferous epithelium (SE) cells with the traditional method based on stage classification according to Clermont. In 4000 sections of seminiferous tubules the following parameters have been evaluated: in procedure I--the frequency of cells at the particular stages of the SE cycle, in procedure II--the frequency of particular cell types forming SE. In the two procedures, 17,726 and 14,670 cells were assessed, respectively. In both procedures, the mean values of the studied cells per animal (I--886.3 +/- 15.4; II--733.5 +/- 96.9) and the distribution of the particular types of cells were similar. The new method of quantitative analysis is less laborious and cheaper. It may be applied instead of the method used so far.     

An intracellular trafficking pathway in the seminiferous epithelium regulating spermatogenesis: a biochemical and molecular perspective

During spermatogenesis in adult rat testes, fully developed spermatids (i.e. spermatozoa) at the luminal edge of the seminiferous epithelium undergo “spermiation” at stage VIII of the seminiferous epithelial cycle. This is manifested by the disruption of the apical ectoplasmic specialization (apical ES) so that spermatozoa can enter the tubule lumen and to complete their maturation in the epididymis. At the same time, the blood–testis barrier (BTB) located near the basement membrane undergoes extensive restructuring to allow transit of preleptotene spermatocytes so that post-meiotic germ cells complete their development behind the BTB. While spermiation and BTB restructuring take place concurrently at opposite ends of the Sertoli cell epithelium, the biochemical mechanism(s) by which they are coordinated were not known until recently. Studies have shown that fragments of laminin chains are generated from the laminin/integrin protein complex at the apical ES via the action of MMP-2 (matrix metalloprotease-2) at spermiation. These peptides serve as the local autocrine factors to destabilize the BTB. These laminin peptides also exert their effects on hemidesmosome which, in turn, further potentiates BTB restructuring. Thus, a novel apical ES-BTB-hemidesmosome regulatory loop is operating in the seminiferous epithelium to coordinate these two crucial cellular events of spermatogenesis. This functional loop is further assisted by the Par3/Par6-based polarity protein complex in coordination with cytokines and testosterone at the BTB. Herein, we provide a critical review based on the latest findings in the field regarding the regulation of these cellular events. These recent findings also open up a new window for investigators studying blood–tissue barriers.     

The epithelium of the middle ear in the guinea pig

Summary An electron microscopic study of aldehyde and osmium fixed normal guinea pig middle ear epithelium was made. Numerous branching microvilli occur between the cilia of the ciliated cells. The granules of the secretory cells are always surrounded by a membrane, and they vary in their content of electron dense substance. Half desmosomes are frequent in basal cells. The squamous epithelial cells of the bulla contain few microvilli and pinocytoric invaginations. In the basal part of the squamous epithelium dilations of the intercellular clefts often occur. The luminal part of the intercellular clefts are closed by multiple tight junctions.     

Antigenicity of penicillin G in the guinea pig

Antigenicity of penicillin G (PCG) was studied in guinea pigs. PCG 5 mg, 10 mg or 25 mg with Freund's complete adjuvant each on days 0, 7 and 21 was injected to a guinea pig: intramuscularly into both thighs and intracutaneously into four locations on the back. A remarkable antigenicity was induced in animals immunized with 25 mg although only low antigenicity in 5 mg and 10 mg. A maximum serum level of the antibody was observed about 2 weeks after last immunization and all of animals immunized with 25 mg died in active systemic anaphylaxis test. As mentioned above, it has been firstly demonstrated that a remarkable antigenicity of PCG can be produced by immunizing with a high dose of 25 mg in the guinea pig model in which PCG itself is used as immunogen.     

DHT deficiency perturbs the integrity of the rat seminiferous epithelium by disrupting tight and adherens junctions

In rats with a DHT deficiency induced by finasteride, morphological changes in the seminiferous epithelium were observed. The structural alterations were manifested by the premature germ cells sloughing into the lumen of seminiferous tubules. The etiology of this disorder could be connected with intercellular junctions disintegration. We showed in the immunohistochemical study the changes in expression of some proteins building tight and adherens junctions. The depression of N-cadherin, β-catenin and occludin immunoexpressions could be the reason for the release of immature germ cells from the seminiferous epithelium. However, the observed increase of the immunohistochemical reaction intensity of vinculin, one of the cadherin/catenin complex regulators, could be insufficient to maintain the proper function of adherens junctions. The hormonal imbalance appears to influence the pattern of expression of junctional proteins in the seminiferous epithelium. It could lead to untimely germ cells sloughing, and ultimately could impair fertility.     

Endocytosis and subsequent processing of 125I-labelled immunoglobulin G by guinea pig yolk sac in vitro.

We have developed conditions for studying the binding, uptake, degradation and transport of 125I-labelled IgG by yolk sac in vitro. Specific binding to tissue at 4 degrees C and to paraformaldehyde-treated tissue at 37 degrees C was time- and temperature-dependent and showed saturation kinetics (Kd,4 degrees C = 2.9 X 10(-6) M, Kd,37 degrees C = 5.3 X 10(-6) M). Uptake was studied at 37 degrees C using untreated tissue (K uptake = 13.3 X 10(-6) M) and was inhibited by preincubation with metabolic poisons but not with cycloheximide. Tissue that had been incubated with 125I-labelled IgG at 37 degrees C released radiolabelled degradation products and intact 125I-labelled IgG into the medium. Experiments with paraformaldehyde-treated and untreated tissue showed that release of intact 125I-labelled IgG was mostly the result of ligand dissociation from surface binding sites. However, more 125I-labelled IgG was released from untreated tissue than could be accounted for solely by loss of surface-bound ligand and the difference was presumed to reflect uptake, transport and exocytosis of 125I-labelled IgG. Degradation of 125I-labelled IgG was inhibited by leupeptin and lysosomotropic amines. These drugs had no detectable effect on 125I-labelled IgG release. The results suggest that degradation and transport of IgG are not intimately related and are consistent with a previously proposed model for IgG transport via coated vesicles which do not fuse with lysosomes and for non-selective uptake into another class of vesicle which does fuse with lysosomes.     

Extracellular matrix: recent advances on its role in junction dynamics in the seminiferous epithelium during spermatogenesis

Spermatogenesis takes place in the seminiferous epithelium of the mammalian testis in which one type A1 spermatogonium (diploid, 2n) gives rise to 256 spermatids (haploid, 1n). To accomplish this, developing germ cells, such as preleptotene and leptotene spermatocytes, residing in the basal compartment of the seminiferous epithelium must traverse the blood-testis barrier (BTB) entering into the adluminal compartment for further development into round, elongating, and elongate spermatids. Recent studies have shown that the basement membrane in the testis (a modified form of extracellular matrix, ECM) is important to the event of germ cell movement across the BTB because proteins in the ECM were shown to regulate BTB dynamics via the interactions between collagens, proteases, and protease inhibitors, possibly under the regulation of cytokines. While these findings are intriguing, they are not entirely unexpected. For one, the basement membrane in the testis is intimately associated with the BTB, which represents the basolateral region of Sertoli cells. Also, Sertoli cell tight junctions (TJs) that constitute the BTB are present side-by-side with cell-cell actin-based adherens junctions (AJ, such as basal ectoplasmic specialization [ES]) and intermediate filament-based desmosome-like junctions. As such, the relative morphological layout between TJs, AJs, and desmosome-like junctions in the seminiferous epithelium is in sharp contrast to other epithelia where TJs are located at the apical portion of an epithelium or endothelium, furthest away from ECM, to be followed by AJs and desmosomes, which in turn constitute the junctional complex. For another, anchoring junctions between a cell epithelium and ECM found in multiple tissues, also known as focal contacts (or focal adhesion complex, FAC, an actin-based cell-matrix anchoring junction type), are the most efficient junction type that permits rapid junction restructuring to accommodate cell movement. It is therefore physiologically plausible, and perhaps essential, that the testis is using some components of the focal contacts to regulate rapid restructuring of AJs between Sertoli and germ cells when germ cells traverse the seminiferous epithelium. Indeed, recent findings have shown that the apical ES, a testis-specific AJ type in the seminiferous epithelium, is equipped with proteins of FAC to regulate its restructuring. In this review, we provide a timely update on this exciting yet rapidly developing field regarding how the homeostasis of basement membrane in the tunica propria regulates BTB dynamics and spermatogenesis in the testis, as well as a critical review on the molecular architecture and the regulation of ES in the seminiferous epithelium.     

Loss of centrosome integrity induces p38-p53-p21-dependent G1-S arrest

Centrosomes organize the microtubule cytoskeleton for both interphase and mitotic functions. They are implicated in cell-cycle progression but the mechanism is unknown. Here, we show that depletion of 14 out of 15 centrosome proteins arrests human diploid cells in G1 with reduced Cdk2-cyclin A activity and that expression of a centrosome-disrupting dominant-negative construct gives similar results. Cell-cycle arrest is always accompanied by defects in centrosome structure and function (for example, duplication and primary cilia assembly). The arrest occurs from within G1, excluding contributions from mitosis and cytokinesis. The arrest requires p38, p53 and p21, and is preceded by p38-dependent activation and centrosomal recruitment of p53. p53-deficient cells fail to arrest, leading to centrosome and spindle dysfunction and aneuploidy. We propose that loss of centrosome integrity activates a checkpoint that inhibits G1-S progression. This model satisfies the definition of a checkpoint in having three elements: a perturbation that is sensed, a transducer (p53) and a receiver (p21).     

NIRF induces G1 arrest and associates with Cdk2

NIRF is a RING finger protein with a ubiquitin-like domain, a PHD finger, a YDG/SRA domain, and a RING finger domain. Previous study showed that NIRF is a nuclear protein expressed in association with cell proliferation. In this study, we further characterized NIRF functions in cell cycle regulation. Flow cytometric analysis showed that overexpression of NIRF induced an increase in G1 phase cells. Immunoprecipitation and immunoblotting experiments showed that NIRF bound to the inactive Cdk2-cyclin E complex. There existed phosphorylated NIRF in cells, and dephosphorylated NIRF interacted with Cdk2. NIRF was phosphorylated by Cdk2 in vitro. These results suggest that NIRF may participate in the G1/S transition regulation.     

Missing generations of spermatocytes and spermatids in seminiferous epithelium contribute to low efficiency of spermatogenesis in humans.

Daily sperm production per gram parenchyma (DSP/g) in humans is only 25 or 35% of that for most species including rats and nonhuman primates. To explain the low efficiency of spermatogenesis in humans, the number of generations of germ cells (spermatocytes and spermatids) and the number of these germ cells in each generation were determined for each spermatogenic stage in men with varied efficiencies. Testes were obtained at autopsy, fixed by vascular perfusion with glutaraldehyde, further fixed in osmium, and embedded in Epon 812 before 0.5-micron sections were stained with toluidine blue. Tubular cross sections were photographed, and spermatogenic stages were determined by two observers. Testes were divided into three groups on the basis of DSP/g. The number of generations of spermatocytes and spermatids was greater (p < 0.05) in the high (2.01 +/- 0.05) and intermediate (1.77 +/- 0.04) than in the low (1.45 +/- 0.15) DSP/g group. All groups had a lower number of generations of spermatocytes and spermatids compared to the optimum value of three. The number of these generations per cross section was related (r = 0.85; p < 0.01) to DSP/g in these men. The number per cross section of spermatocytes, spermatids, and the combined number of germ cells was higher (p < 0.01) in the high than in the low DSP/g group. The combined number of germ cells per cross section was related (r = 0.85; p < 0.01) to DSP/g. The combined number of germ cells was higher in the high versus the low DSP/g group in stages I through V, but this difference was significant only in stages IV and V.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenomatous polyposis coli (APC) is essential for maintaining the integrity of the seminiferous epithelium

Sertoli cells provide the microenvironment necessary for germ cell development and spermatogenesis; disruption of Sertoli cell morphology or function can lead to germ cell aplasia, which is observed in testicular dysgenesis syndrome. Mutation of the adenomatous polyposis coli (APC) gene has been associated with various human cancers, including testicular cancer, but its involvement in nonmalignant testicular pathologies has not been reported. We have developed a mouse model (APC(cko)) that expresses a truncated form of APC in Sertoli cells. Despite normal embryonic and early postnatal testicular development in APC(cko) mice, premature germ cell loss and Sertoli cell-only seminiferous tubules were observed in mutant testes without affecting Sertoli cell quiescence, apoptosis, or differentiation, which were confirmed by the absence of both proliferating cell nuclear antigen, DNA strand breaks, and anti-Müllerian hormone, respectively. We show that mutant Sertoli cells lose their apical extensions, which would normally enclose germ cells during various stages of spermatogenesis, and were unable to maintain the blood-testis barrier because of disrupted expression of junctional proteins. We also observed an up-regulation of Snail and Slug, markers suggestive of epithelial-mesenchymal transition in the Sertoli cells, but tumorigenesis was not observed. No comparable phenotype was observed with Sertoli cell-specific loss-of-function mutations in β-catenin, leading us to speculate that truncation of APC in Sertoli cells results in progressive degeneration of the seminiferous tubules by a mechanism that disrupts the integrity of Sertoli cell junctions independently of APC-regulated β-catenin activities and leads to development of a Sertoli cell-only phenotype.     

Functional role of nitric oxide in guinea pig tracheal epithelium

Nitric oxide (NO) may play an important regulatory role in airway function. We have, thus, investigated in vitro whether epithelium derived NO may modulate cholinergic neurotrasmission, via release of NO in guinea pig trachea, by using L-arginine (L-ARG), a precursor of NO synthesis, and L-N^G-nitro-arginine-methyl-ester (L-NAME), an inhibitor of NO synthase. Results show that L-ARG and L-NAME modify acetylcholine sensitivity in epithelium-intact smooth muscle preparations, suggesting a probable NO synthesis by tracheal guinea pig epithelium.