Histochemistry and ultrastructure of nerve fibres and contractile cells in the tunica albuginea of the rat testis

Whole-mounted preparations of the tunica albuginea of the rat testis were studied using light microscopy techniques for demonstration of cholinergic nerve fibres (Karnovski-Root method), catecholaminergic nerve fibres (De la Torre's method) and actin filaments (avidin-biotin-peroxidase method). An ultrastructural study of different regions of the albuginea was also performed. Cholinergic fibres were seen to penetrate into the albuginea with the testicular artery to form a broad network in the mediastinum testis, many fibres ending beneath the rete testis epithelium. Catecholaminergic fibres penetrated through the middle part of the cauda epididymis and formed a plexus in the albuginea covering the inferior testicular pole. This plexus gave rise to straight fibres which formed varicosities, some of them appeared related with mast cells. Actin-containing cells were only found beneath the rete testis epithelium. These cells were similar to myofibroblasts. The location of both cholinergic fibres and contractile cells among the rete testis channels suggests that these cells may be involved in the pumping of semen towards the ductuli efferentes and that their contractility may be regulated by cholinergic fibres.     

Electric activity of the testicular tunica albuginea during ejaculation: a canine study

We have shown in previous studies that electric waves at rest could be recorded from the testicle and originate from the tunica albuginea (TA) and not from the testicular tissue. In the current study, we investigated the hypothesis that the electric activity of the TA increases during ejaculation. Three electrodes were sutured to the TAs of 11 anesthetized male dogs. The slow waves were recorded at rest and on inducing ejaculation by an ejaculator applied to the glans penis. Basal electric waves were recorded from the testicle. Each wave consisted of a negative followed by a positive deflection with a mean frequency of 6.2 +/- 1.3 cycles/min, an amplitude of 0.59 +/- 0.06 mV, and a conduction velocity of 5.2 +/- 0.8 cm/sec. These wave variables showed a significant intermittent increase (P < 0.05) at intervals of 0.6-1.0 secs and occurred simultaneously with the bouts of ejaculation. The increase remained for 0.8-1.2 secs at each ejaculation bout. The number of bouts of increased electric waves varied from 3 to 5. Apparently, the TA is not an inert covering to the testicle, but it seems to have a functional activity. Recording resting electric waves of the TA presumably denotes that the TA possesses a resting tone that appears to support the testicular tissue. During ejaculation, the increased electric activity of the TA, which coincides with semen spurt episodes, presumably denotes TA contraction. The intermittent TA contractions seem to assist in massaging the testicular secretions to the epididymis and the vas deferens and augment testicular circulation. The effect of pathologic conditions of the TA on ejaculation needs to be studied.     

Fibrous pseudotumors of tunica albuginea,tunica vaginalis and epididymis: Report of two cases

We describe two rare cases of fibrous pseudotumor of the paratesticular region. In the first case, five nodules arising from the tunica albuginea of right testicle causing scrotal, enlargement raising after urinary tract infection were seen. In the second case, multiple nodules arising tunica albuginea, tunica vaginalis and epididymis raising after left varicocelectomy operation were observed. The histology showed a paucicellular fibroblastic proliferation of cells within a hyalinized collagenous fibrous stroma containing numerous thin-walled blood vessels accompanied by lymphocytes and plasma cells in tumor tissues in both cases. Tumors in both cases were successfully resected. After operation, both patients had an uneventful recovery without any complications.     

Immunocytochemical localization of protamine in the boar testis

Protamine was specifically demonstrated in spermatids and spermatozoa of the boar by immunoelectron microscopy, using anti-boar or anti-ram protamine antisera, and three different direct or indirect labelling techniques. The two isomers of the protamine could not be labelled separately. The protamine is present in the cytoplasm of elongating spermatids and it enters the nuclei throughout the elongation process after possible storage in the cytoplasm or in the nuclear envelope of spermatids, or both. These findings differ from previous observations in other species.     

Immunocytochemical localization of aromatase in rat testis

Summary The immunocytochemical localization of armatase in the testes of young and adult rats was investigated by an indirect-immunofluorescent method using antihuman placental aromatase-II cytochrome P-450 antibody. In both young (1 and 2 weeks old) and adult rats, only the Leydig cells in the interstitial tissue showed a positive immunoreaction for aromatase, while the germ cells and Sertoli cells in the seminiferous tubule were entirely negative. In addition, electron microscopy revealed that the Leydig cells in the testes of young as well as adult rats have a well-developed smooth endoplasmic reticulum, mitochondria with tubulovesicular cristae, and a few lipid droplets; these structures being characteristic of steroid secretory cells. On the basis of these results, we suggest that estrogens are mainly synthesized in Leydig cells of the testes.Supported by grants from the Ministry of Education, Science, and Culture, Japan, and USPHS HD 04945     

Immunocytochemical localization of aromatase in rat testis

The immunocytochemical localization of aromatase in the testes of young and adult rats was investigated by an indirect-immunofluorescent method using antihuman placental aromatase-II cytochrome P-450 antibody. In both young (1 and 2 weeks old) and adult rats, only the Leydig cells in the interstitial tissue showed a positive immunoreaction for aromatase, while the germ cells and Sertoli cells in the seminiferous tubule were entirely negative. In addition, electron microscopy revealed that the Leydig cells in the testes of young as well as adult rats have a well-developed smooth endoplasmic reticulum, mitochondria with tubulovesicular cristae, and a few lipid droplets, these structures being characteristic of steroid secretory cells. On the basis of these results, we suggest that estrogens are mainly synthesized in Leydig cells of the testes.     

A quantitative study of spermatogenesis in the developing rat testis

Quantitative (stereological) studies were performed to determine the number of germ cells in the developing rat testis. Sprague-Dawley rats aged 1-70 days were fixed by immersion or perfusion and embedded in Epon Araldite. Blocks of tissue were sectioned at 1.5 microns and stained with toluidine blue dye. Sections were systematically scanned and the areal density of nuclear profiles counted using an unbiased counting frame. Numerical density and absolute number of germ cells in the processed block were then estimated. Corrections for processing shrinkage were determined by comparing the volume of processed and unprocessed samples. The results demonstrate the necessity of determining absolute number rather than volume density (or areal density) in comparing germ cell numbers. In these experiments, spermatogonial numbers stabilized in the range 18.4-23.6 million per testis on Day 30. The number of primary spermatocytes that were first apparent on Day 15 increased rapidly to 54.6 million per testis on Day 30 and then slowly to 73.6 million on Day 70. Round spermatids were first apparent on Day 25 and increased rapidly to 85.7 million per testis on Day 40, then continued to increase to 151.9 million on Day 70. The study provides both methods and baseline data for future experiments involving manipulation of the spermatogenic potential of the testis.     

Immunocytochemical evidence for the presence of oxytocin in rat testis

Summary The presence of oxytocin, vasopressin and neurophysin in the testis of adult Wistar and Brattleboro rats has been examined immunocytochemically. After fixation in modified Bouin's solution, or Bouin's sublimate fixative, immunostaining was accomplished with the peroxidase-antiperoxidase method. The presence of immunoreactive oxytocin was demonstrated in 80% of the interstitial cell population of both rat strains while no staining was observed for vasopressin or neurophysin.     

Distribution of actin-filament bundles in myoid cells,Sertoli cells,and tunica albuginea of rat and mouse testes

Summary Frozen sections of the rat and mouse testes were stained with either FITC-phalloidin or NBD-phallacidin and viewed with conventional fluorescence and confocal laser microscopes in order to demonstrate the arrangment of actin-filament bundles in myoid cells, Sertoli cells and tunica albuginea. Myoid cells are rich in actin-filament bundles crossing at right angles. These bundles running in different directions can also be visualized by means of electron microscopy. Nerve fibers occur in the vicinity of myoid cells, suggesting a neural control of the cell. At Sertoli cell junctions actin filaments occur at the circumference of the cell, where they show a honeycomb pattern. The ratio of the number of Sertoli cells per myoid cell can be calculated by means of confocal microscopy; this technique may provide a new parameter for determining spermatogenic activity. In the tunica albuginea of the juvenile mouse testis, actin filaments are arranged in an alternate fashion.     

Some observations on the formation of the primary tunica albuginea in the ovary of the chick embryo (author's transl)

The ovarian cortex of chicken embryos of 10 to 14 days of incubation was observed in order to study the formation of the primary tunica albuginea and to discern in its cells an ultrastructural confirmation of an eventual steroidogenic capacity which has been proposed by some authors. From observations at the electron microscope, it is probable that the mesenchyme-like cells which constitute the primary tunica albuginea may originate from the somatic cells of the cortex, either from those of the germinal epithelium, or from those which, because of their position, may be termed prefollicular. There is not apparent ultrastructural confirmation of an active steroidogenic capacity in the albugineal cells. A possible physiological difference between clear cells and dark cells due to a different electron density, is furthermore discussed.     

Immunocytochemical localization of actin and tubulin in rat testis and spermatozoa

Using commercial monoclonal antibodies against actin and tubulin (alpha and beta), the respective antigens were localized on semithin and ultrathin sections of the rat testis. Tubulin immunofluorescence was found in the socalled manchette surrounding the heads of the maturating spermatids as well as the sperm tail. The distribution pattern varied with sperm development. Modified Sertoli cells found at the transition between the seminiferous tubules and the rete testis displayed much filamentous tubulin-reactive material. The immunofluorescence findings could be confirmed at the ultrastructural level using the indirect immunogold method. Actin immunofluorescence was demonstrated in vascular smooth muscle cells, interstitial macrophages and - most intensely - in peritubular cells. Inside the seminiferous tubules the Sertoli cell junctions and the ectoplasmic specializations of the Sertoli cells that follow the outer contour of spermatid heads displayed distinct actin immunofluorescence. In addition to the locations mentioned, actin-like immunoreactivity was visualized at the ultrastructural level in the chromatoid body and the subacrosomal space of spermatids as well as on the outer dense fibers of the sperm tail. Immunoblotting experiments with actin antibodies showed that in extracts from testicular spermatozoa, intact or fragmented into heads and tails, from isolated Sertoli cells grown in vitro, and from testis tissue in addition to authentic actin a protein was present in sperm tail extracts that strongly bound the actin antibody. This protein may be an actin-related protein and may be responsible for the actin-like immunoreactivity of the outer dense fibers of the sperm tail.     

Immunocytochemical localization of 17 beta-hydroxysteroid dehydrogenase in porcine testis

The immunocytochemical localization of 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) in porcine testes was examined by applying an indirect-immunofluorescence method using an antiporcine testicular 17 beta-HSD antibody. Only the Leydig cells located in the interstitial tissue exhibited a positive immunoreaction for 17 beta-HSD: the germ cells and Sertoli cells located in the seminiferous tubules were entirely negative. These results suggest that, in porcine testis, the biosynthesis of testicular testosterone, the final step of which is the conversion of androstenedione to testosterone, takes place in the Leydig cells.     

Immunocytochemical localization of the epidermal growth factor receptor in mouse testis

The distribution of the epidermal growth factor receptor (EGFR) in mouse testis was ascertained by immunocytochemical methodology using a polyclonal antibody (RK2) shown previously to recognize the cytoplasmic domain of the human (A431 cells), murine (Swiss 3T3 cells), and chicken (CK 109 cells) EGFR. Initial studies performed to determine the usefulness of this antibody as a probe of the murine EGFR in testis employed two murine cell lines, TM4 and MA10, of Sertoli cell and Leydig cell origin, respectively, in which a physiological response of EGF and specific binding of iodinated EGF has been demonstrated. Western blotting in membrane preparations of TM4 and MA10 revealed only one prominent band at 170 kDa. Immunocytochemical localization in TM4 and MA10 cells illustrated a plasma membrane distribution of the receptor. Western blotting of membrane fractions prepared from testis also revealed a specific band at 170 kDa. In the intact testis, the EGFR was immunolocalized specifically in Leydig cells and Sertoli cells only. These results suggest that the involvement of EGF action in spermatogenesis may occur at the level of the somatic components of the testes, principally in the Leydig and Sertoli cells.     

Immunocytochemical localization of actin and tubulin in rat testis and spermatozoa

Summary Using commercial monoclonal antibodies against actin and tubulin ( and ), the respective antigens were localized on semithin and ultrathin sections of the rat testis. Tubulin immunofluorescence was found in the socalled manchette surrounding the heads of the maturating spermatids as well as the sperm tail. The distribution pattern varied with sperm development. Modified Sertoli cells found at the transition between the seminiferous tubules and the rete testis displayed much filamentous tubulin-reactive material. The immunofluorescence findings could be confirmed at the ultrastructural level using the indirect immunogold method. Actin immunofluorescence was demonstrated in vascular smooth muscle cells, interstitial macrophages and — most intensely — in peritubular cells. Inside the seminiferous tubules the Sertoli cell junctions and the ectoplasmic specializations of the Sertoli cells that follow the outer contour of spermatid heads displayed distinct actin immunofluorescence. In addition to the locations mentioned, actin-like immunoreactivity was visualized at the ultrastructural level in the chromatoid body and the subacrosomal space of spermatids as well as on the outer dense fibers of the sperm tail.Immunoblotting experiments with actin antibodies showed that in extracts from testicular spermatozoa, intact or fragmented into heads and tails, from isolated Sertoli cells grown in vitro, and from testis tissue in addition to authentic actin a protein was present in sperm tail extracts that strongly bound the actin antibody. This protein may be an actin-related protein and may be responsible for the actin-like immunoreactivity of the outer dense fibers of the sperm tail.     

Immunocytochemical localization of a folicle stimulating hormone-like molecule in the testis.

A follicle-stimulating hormone (FSH)-like molecule was localized in normal adult rat testes as well as testosterone-treated hypophysectomized rat tests with an unlabeled antibody (anti-FSH), peroxidase-antiperoxidase complex technique. Anti-FSH bound specifically to ultrathin sections of acrosomes of spermatids and intranuclear bodies of early spermatids. Quantitation of staining intensity demonstrated that FSH, used as an absorbing antigen, would significantly reduce this binding. There was less anti-FSH binding to the acrosomes of spermatozoa in the body and tail of the epididymis as compared to the less mature germ cells located in the testis and head of the epididymis. The acrosomal and nuclear staining of spermatids taken from hypophysectomized animals was similar to staining observed in sham injected animals. Taken together, these results suggest that there is a molecule within the acrosome that is immunologically similar to FSH. Most importantly, these results emphasize the importance of conducting physiologic experiments in conjunction with immunocytochemical studies.