Immunolocalization of clusterin in the ram testis, rete testis, and excurrent ducts

Clusterin, a glycoprotein that elicits cell aggregation, has previously been isolated from ram rete testis fluid, and has been partially characterized. In experiments reported, we have used monoclonal antibodies against clusterin in combination with indirect immunofluorescence microscopy to investigate the distribution of clusterin in the adult ram testis, rete testis, and excurrent ducts. Tissue blocks (5 mm3) were fixed in periodate/lysine/paraformaldehyde containing 0.1% glutaraldehyde and, after embedding, 5-microM sections were prepared for immunolocalization. In the testis, 2 basic patterns were observed: 1) strong to moderate staining for clusterin in the adluminal region with little staining in the basal region of the seminiferous epithelium and germinal cells; and 2) moderate staining throughout the seminiferous epithelium between germinal cells. In the rete testis, strong clusterin staining was localized intracellularly in the rete epithelial cells, most often associated with the luminal surface. In the epididymis, intracellular clusterin was localized in some principal cells of the caput epididymidis. The luminal surfaces and spermatozoa within the lumen were strongly positive. In the vas deferens, clusterin staining was associated with the luminal surface only. The presence of clusterin was clearly detected in unwashed isolated epididymal spermatozoa, but not in spermatozoa washed with phosphate-buffered saline containing 0.05% Tween 20.     

Rabbit testis proacrosin: Immunological similarities between testis,sperm proacrosins and the initially formed testis acrosin

Anti-rabbit proacrosin IgG was prepared from goat serum following immunization with a homogeneous preparation of rabbit testis proacrosin. The “auto-activation” products of purified testis proacrosin were separated into 68,000 and 34,000 molecular weight (mol wt) acrosins by Sephadex G-100 column chromatography. Immunodiffusion analysis of testis and epididymal sperm proacrosins and acrosins on agarose gel against goat anti-rabbit testis proacrosin showed immunological identity between rabbit testis and sperm proacrosins and the initial testis acrosin (mol wt 68,000). However, the 34,000 mol wt form of testis acrosin showed weaker reaction with the antibody and only partial identity with the proacrosin and the 68,000 mol wt form of acrosin. These results suggest that there is no major structural difference between testis and sperm proacrosins and between proacrosin and the 68,000 mol wt acrosin, but such a structual change occurs when the 34,000 mol wt acrosin is formed.     

Postnatal development of the testis in the Mongolian gerbil, Meriones unguiculatus

The testicular development in gerbils was studied over 16-week periods starting from birth. Testicular weight and seminiferous tubule diameter increased considerably between 1 and 11 weeks of age. At 16 weeks the testicular weight was about 540 mg. Spermatogenesis commenced at about 2 weeks when mitoses first appeared in spermatogonia. Spermatozoa appeared in a few of the seminiferous tubules by 7 weeks and consistently so in all of the tubules at 10 weeks. Epididymal spermatozoa appeared first in the cauda epididymis at 10 weeks and were consistently present at 12 weeks. Formation of mature Leydig cells with a grouped perivascular arrangement appeared by 3 weeks and continuously so thereafter. From these results, it is evident that the male Mongolian gerbil is almost sexually matured by 10 to 12 weeks of age.     

Aromatase in the human testis

Low levels of testicular estrogen synthesis have been reported in a number of species, but the cellular localization has not been unequivocally established. To study aromatase in the human testis, we have combined immunocytochemistry with direct measurement of enzyme activity in the testicular 6μm cryosections. Thus, the functionality of the immunoreaction and its sensitivity can be assessed in quantitative terms. Testes were obtained from immediate autopsy from men aged 18–53 years, from surgery from two patients with prostatic cancer (67 and 74 years) and from two normal children aged 8 months and 3 years at autopsy. Benign testicular sex cord tumors were also examined from two unrelated patients aged 5 and 8 years with gynecomastia and diagnosed with Peutz-Jeghers syndrome. Our results consistently showed low to moderate staining intensity of immunoreactive aromatase in comparison to that of normal human placental cryosections. Immunoreactive aromatase was only present in the interstitial Leydig cells and absent from the Sertoli cells of all normal adult testes showing spermatogenesis. Aromatase activity correlated well with the intensity of the immunostain. However, there was no obvious relationship between the level of aromatase activity and increasing age. Generally higher levels were present in testes of young men (18–22 years). No immunostain in any cell type was detected in one 33-year-old patient with testicular cancer. In the testes of the two normal prepubertal boys, no immunostaining was observed. However, intensely stained Sertoli cells as well as high aromatase activity were observed in the testicular tumors of the patients with Peutz-Jeghers syndrome. Our results suggest that Leydig cells are the source of aromatase in normal men but that Sertoli cells may express this enzyme under abnormal conditions. The combined methods for measuring enzyme activity and immunoreactive aromatase are suitable for application to tissues expressing low levels of aromatase.     

Cultivation in vitro of the testis cord of the newt, Triturus pyrrhogaster

Testis cords of Triturus pyrrhogaster were cultivated in vitro on (a) medium with chick embryo extract and calf serum, (b) medium with newt gonad extract, (c) Trowell 's medium T8 and (d) liquid synthetic medium 199. Of the four media utilized, medium 199 gave the best result for long-term maintenance of the normal histological structures of the testis cords. Addition of insulin (5 μ/ml) to medium 199 resulted in a remarkable improvement for the maintenance of the testis cord and the migration of columnar cells of the peritoneal epithelium into the primordial germinal tissue occurred as in the intact testis of this animal. Trowell 's medium T8 was proved inadequate. Medium with chick embryo extract and calf serum retained most of the germ cells healthy but caused gradual decrease in height of the columnar cells. Testis cords cultivated on the same medium in combination with Xenopus testis maintained normal histological structure for 18 days, whereas, those kept in contact with Xenopus ovary showed involution within the same period. Newt testis extract brought about transformation of somatic elements of the germinal tissue into fibroblastlike cells which was followed by the disintegration of germ cells. Ovary extract did not cause selective destruction on the somatic or germinal elements.     

Tissue, cellular, and subcellular distribution of 241Pu in the rat testis

The distribution and localization of 241Pu in rat testes were determined by quantitative autoradiography. Rats were given an intravenous injection of 241Pu citrate and tissues were collected 1 week later. The tissue distribution of 241Pu was determined by light microscope autoradiography. Significant concentrations of 241Pu were observed in the interstitial tissue but not in seminiferous tubules. The cellular distribution and subcellular localization of 241Pu were determined by electron microscope autoradiography. Within the interstitial tissue, 241Pu was concentrated in macrophages. There was no preferential localization of 241Pu in any other interstitial tissue components. Within interstitial tissue macrophages, 241Pu was concentrated in the lysosomal system of the cell. Other organellar compartments of the cell did not preferentially incorporate 241Pu. The association of 241Pu with the macrophage lysosomal system may explain the long retention times of Pu in testes as observed in experimental studies.     

Glucocorticoids, androgens, testis mass, and the energetics of vocalization in breeding male frogs

Male advertisement vocalization in frogs is known to be one of the energetically most expensive activities of ectothermic vertebrates. Glucocorticoids have marked effects on energy metabolism, and, generally, plasma concentrations of glucocorticoids increase during the course of prolonged exercise bouts. Androgen concentrations are also known to vary considerably among breeding male frogs. Intraspecific and interspecific comparisons were used to test for a relationship among androgen concentration, corticosterone concentration, testis mass, and the energetics of vocalization in natural populations of calling male frogs. The results of this study indicate that: (1) intraspecific variation in androgen and corticosteroid concentrations in breeding male frogs is positively correlated as a result of both interindividual variation in the amount of performed vocalization and the relationship between calling effort of an individual male and the level of calling in other males, (2) interspecific variation in corticosteroid concentration of calling male frogs is correlated with the relative energy expended in the species-specific vocalization, and (3) when differences in testis mass are controlled for, vocalization effort is correlated with androgen concentration among species of breeding male frogs. These findings are in contrast to some recent work reported from laboratory experiments on calling frogs.     

The rete testis of the goat, a morphological study

The rete testis of the goat can be divided into three parts, septal, mediastinal and extratesticular. The septal rete is short, relatively straight and connects the terminal part of the seminiferous tubules with the mediastinal rete. The mediastinal rete is a labyrinth of intercommunicating channels that occupies about two-thirds of the central axis of the testis. The extratesticular rete is located outside the testis at its extremitas capitata and forms sac-like dilations up to 2 mm in diameter. The rete testis, regardless of its location, is lined by simple cuboidal to simple squamous epithelium that invariably contains a few intra-epithelial lymphocytes and macrophages. The epithelial cells possess few microvilli and a centrally located flagellum at the luminal border. With the exception of a few small vesicles in the apical cytoplasm, morphological features associated with absorption such as coated vesicles, canaliculi, vacuoles and multivesicular bodies are absent. However, basolateral interdigitations between adjacent cells, another feature of absorptive epithelium, are frequently noticed. Most cytoplasmic organelles except mitochondria and free ribosomes are poorly developed, suggesting that caprine rete epithelial cells are not associated with protein/glycoprotein secretion. There is no evidence of sperm phagocytosis by the rete epithelium, but luminal macrophages containing sperm fragments are occasionally encountered. The structural-functional relationships of the rete epithelial cells are discussed.     

Ultrastructure of spermatogonia, spermatocytes, and sertoli cells in the testis of the crayfish, Procambarus paeninsulanus

The testes of the crayfish, Procambarus paeninsulanus, were prepared for light and transmission electron microscopy. During early stages of spermatogenesis, when the spermatogonia are dividing, processes from a single Sertoli cell extend between numerous spermatogonia. As the cells enter meiosis, many points of contact can be observed between the Sertoli cell processes and spermatocytes. These desmosome-gap junctions are maintained between the germ and Sertoli cells until the early spermatid stage.     

Hormonal regulation of beta-endorphin in the testis

It is well established that beta-endorphin has a regulatory influence on the reproductive function at the level of the hypothalamic-pituitary axis. However, recent immunohistochemical evidence demonstrated that beta-endorphin is also present in the Leydig cells of fetal, neonatal and adult mice and hamsters. In addition, beta-endorphin synthesis was localized in the Leydig cells of adult rats, leading to the hypothesis of a direct function of the peptide in the reproductive organs. Our interest was to investigate the role of beta-endorphin at testicular level. We have demonstrated the presence of high-affinity opioid binding sites (Kd in the nanomolar range) in tubular homogenates and Sertoli cells in culture of adult (50 days) and immature (18 days post-natal) rat testes. Also, chronic beta-endorphin treatment of the Sertoli cells significantly inhibited basal and FSH-stimulated androgen-binding protein production, this effect being prevented by the universal opiate antagonist naloxone. No opiate binding was observed on Leydig cell cultures. Furthermore, we have demonstrated that acute or chronic beta-endorphin treatment does not affect testosterone production by Leydig cells in vitro, consistent with the absence of receptors on these cells. On the other hand, fetal Leydig cells (21 days fetal life) in culture produced considerable amounts of beta-endorphin. Also, fetal Leydig cells represented a preferred in vitro system to study beta-endorphin release since in adult cell culture a marked degradation of the peptide was detected (greater than 50%). beta-endorphin accumulation for 3 and 5 days was markedly increased by inhibitors of steroid biosynthesis (1.5-fold); a significant reduction by GnRH at both days (by 50-30%) was observed, while by dexamethasone the reduction was only noted after 5 days of treatment (by 50%). Acute stimulation (3 h) of control cells with hCG enhanced by 10-12-fold the beta-endorphin secretion. The hormone stimulation of beta-endorphin production was not mediated by testosterone. On the contrary, inhibition of Leydig cells steroid biosynthesis markedly increased basal and hCG-stimulated beta-endorphin production (150-200%), suggesting autocrine negative modulation of Leydig cell beta-endorphin by androgen and/or its metabolites. In contrast, dexamethasone reduced basal and hCG-stimulated beta-endorphin production (by 50%). Altogether these findings indicate that beta-endorphin produced within the Leydig cells may behave as a paracrine inhibitor of the Sertoli cell function and demonstrate that the peptide production is under direct control by gonadotropins and is modulated by steroids.     

Local control mechanisms in the testis

The gonads are unique organs in that they harbor the cells of the germline and consequently provide the local environment necessary for the normal development and differentiation of gametes. Since the local requirements for germ differentiation differ considerably from those of somatic cells the structural and physiological organization of the gonad is complex and compartmentalized. An elaborate network of local paracrine interactions between the somatic and gametogenic elements appears to be essential for normal germ cell development in mammals. This is especially true for the testis where meiosis is continuous throughout adulthood and where the spermatogenic cycle of the seminiferous epithelium is strictly controlled in time and space. The present paper reviews briefly the rapidly expanding field of testicular paracrinology. Special emphasis is given to the role of intra- and intercompartmental paracrine communication in the development of the male gamete.     

Lipid metabolism in the testis of the ram

1. Analysis of rams testes revealed that phosphatidylcholine was the major phospholipid and accounted for about 40% of the total. Only small amounts of choline plasmalogen were present. 2. The ratio of phosphatidylcholine to choline plasmalogen in the testis was very different from that occurring in the spermatozoa. This result was in contrast with those for rat testis and rat spermatozoa (obtained from the head of the epididymis), where the ratio of the two lipids was very similar. 3. Infusions of [(32)P]orthophosphate into the testicular artery of rams resulted in incorporation of radioactivity into most phospholipids; phosphatidylinositol labelling accounted for 68% and 39% of the radioactivity after infusions lasting 3hr. and 5hr. respectively. 4. With the exception of phosphatidic acid the specific radioactivity of phosphatidylinositol was higher than that of any other lipid. 5. After the infusion of [U-(14)C]glucose, triglycerides accounted for about 60% of the radioactivity in testicular neutral lipids, whereas diglycerides had only about 15% of the radioactivity. 6. Palmitic acid (16:0) was the major component both in neutral lipids and phospholipids of ram testes. 7. The effects of gonadotrophic hormones (luteinizing hormone and follicle-stimulating hormone) on the incorporation of [(32)P]orthophosphate into total testicular phospholipids in vivo were also examined.