Guinea Pig Testes Research Articles

The ultrastructural localization of the enzymes related to steroid hormone metabolism in the guinea-pig testis

A study of the ultrastructural localization of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD), glucose-6-phosphate dehydrogenase (G-6-PD), beta-hydroxybutyrate dehydrogenase (beta-HBD), NADH diaphorase (NADH-D) and NADPH diaphorase (NADPH-D) in the guinea-pig testis is reported. The procedures employed included short immersion or perfusion fixation with aldehydes followed by incubation of small blocks in a tetrazolium salt or a ferricyanide medium. The effects of incubation conditions were investigated, and a reaction medium for the ultracytochemical demonstration of 11 beta-HSD is described. Using suitable controls, evidence for the specificity of the cytochemical reactions is presented. It was found that all the enzymes studied were present in both the Leydig and Sertoli cells of the guinea-pig testis and that the intracellular distribution pattern for each enzyme was independent of the cell type. Using tetrazolium salt techniques, both 3 beta-HSD and 11 beta-HSD activities were localized on or in membranes of smooth endoplasmic reticulum and within the mitochondria. With the ferricyanide techniques, G-6-PD activity was found to be associated mainly with the smooth endoplasmic reticulum membranes, while beta-HBD activity was limited to mitochondria. With both the tetrazolium salt and ferricyanide techniques, the reaction products for NADH-D and NADPH-D activities showed localizations which were similar to those observed for the steroid dehydrogenases.

Allergic orchitis lesions are adoptively transferred from vasoligated guinea pigs to syngeneic recipients.

Histopathology typical of allergic orchitis developed in testes of inbred guinea pigs 16 months after vasoligation. A similar histopathology was found in unoperated testes after unilateral vasoligation. Peritoneal exudate cells from vasoligated guinea pigs transferred identical lesions to syngeneic recipients. The testicular lesions in long-term vasoligated guinea pigs have an immunological basis.

Compartmentalization of testosterone and androstenedione in guinea pig testis.

Decapsulated guinea pig testes were superfused with a mixture of [3H]testosterone (T) and [14C]androstenedione (A). Steady state isotopic data from these experiments were used to estimate, among other parameters, the uptake of steroids, the tissue concentrations of labeled T and A derived from each of the superfused tracers, and the ratio of labeled T and A released by the tissue to the medium. Concentrations of endogenous T and A were measured in testicular samples and, after supervision of decapsulated testis with buffer without steroids, in tissue and superfusate. The ratios of concentrations of T and A in tissue as well as of T and A released to the medium were not the same for labeled (exogenous) and unlabeled (endogenous) steroids. These findings indicate that superfused tracers do not adequately reflect the fate of the endogenously produced androgens. The results from this study can be interpreted on the basis of a model representing two or more spaces of distribution of T and A not equally accessi...

Binding of Thyroid Stimulators to Guinea Pig Testis and Thyroid*

We have demonstrated TSH binding sites in membranes prepared from guinea pig testis. The binding of TSH to testicular homogenate was localized to the interstitial tissue and showed similar characteristics to the binding of TSH to thyroid membranes with high affinity (Ka = 2.5 X 10(10) M-1) but limited capacity (approximately 0.9-1.7 pg/mg equivalent testis homogenate). In addition, thyroid-stimulating antibodies inhibited the binding of TSH to both testicular and thyroid homogenate but failed to inhibit the binding of hCG to the testis preparation. These data suggested that the TSH-binding site was distinct from the hCG/LH binding site. Testicular and thyroid TSH-binding sites also appeared to demonstrate a similar degree of cross-reactivity with a crude preparation of hCG. In conclusion, our findings suggest that thyroid-stimulators may influence gonadal function. Furthermore, the ability of hCG to inhibit the binding of TSH to thyroid membranes provides a possible mechanism for the known thyroid-stimulating properties of hCG.

Androgen and Estrogen Synthesis in the Fetal Guinea Pig Gonad12

Bisected fetal guinea pig ovaries and testes were examined in vitro for their ability to synthesize androgens from [1-’ CI sodium acetate and estrogens from either I i-� HI androstenedione or 11 ,2-� HI androstenedione. With [1-’ � Cl sodium acetate as the substrate, hexane-soluble steroids, androstenedione and testosterone were synthesized by fetal testes from Day 30 to 65 of development. To a lesser extent, ovaries also synthesized hexane-soluble steroids, but only when ovaries from various stages of gestation were pooled was the synthesis of ‘4C-androstenedione detected. Radioactive testosterone was not detected in ovarian incubations and at no time was 4C-dihydrotestosterone produced by fetal ovaries or testes. More radioactive testosterone and androstenedione (d/min X mg tissue) appeared in Day 30 testicular incubations than in incubations of organs from later stages of development. In addition, between Days 30 and 50 of gestation more testosterone than androstenedione was recovered from the medium, but by Day 60-65 androstenedione predominated. Total activity (d/min X gonad) generally paralleled organ weight and increased with advancing gestation. The rate of 3H20 formation was utilized as an index of 1i-� HI androstenedione aromatization. Ovaries between 30 and 65 days of gestation were considerably more active (d/min X mg tissue or d/min X gonad) in synthesizing 3H20 (2.3-0.3% of the radioactive substrate) than corresponding testes in which activity was not significantly different than that noted for abdominal muscle. When ovaries were incubated with l1,2-� I androstenedione for 60 mm, the rate of synthesis of H2 0, 3H-estrone and 3H-estradiol-17� was constant. In this case tritiated estrogens accounted for 16-23% of the total product radioactivity. The results indicate that fetal guinea pig testes and ovaries contain the necessary enzymes for de novo androgen and estrogen synthesis at a time during development when anatomical and behavioral phenotypes are differentiated.

Species differences in steroidogenesis following the in vitro incubation of decapsulated testes.

ABSTRACT Decapsulated testes of rats, guinea pigs and rabbits were incubated with or without labelled precursors and the steroids formed under various experimental conditions were analysed. Also a decapsulated human testis was incubated with labelled acetate. Cholesterol, testosterone and androstenedione were isolated in a radiochemically homogeneous form following the incubation of carbon-14-labelled sodium acetate with decapsulated testes of all 3 animal species. No other labelled steroid was detected following the incubation of rat testes. Guinea pig testes also converted labelled acetate to pregnenolone. Rabbit and human testes converted labelled acetate to cholesterol, pregnenolone, dehydroepiandrosterone, androstenediol, testosterone and androstenedione. When decapsulated testes of rats, guinea pigs and rabbits were incubated with carbon-14 labelled pregnenolone as a precursor, radiochemically homogeneous progesterone, 17-hydroxypregnenolone, dehydroepiandroster-one, androstenediol, androstenedione and testosterone were isolated in each experiment. Using radioimmunoassay techniques, preformed steroids together with steroids formed from endogenous precursors were analysed following the incubation of rat, guinea pig and rabbit testes in the absence and in the presence of human chorionic gonadotrophin (HCG). Marked (seasonal?) variations were observed between the results of experiments conducted at different times. Incubation of guinea pig testes in the absence as well as in the presence of HCG resulted in a major increase in pregnenolone levels. No such finding was encountered when rat and rabbit testes were incubated. The addition of HCG resulted in at least a 10-fold increase in testosterone formation by the testes of all three species. The addition of HCG to the incubation medium induced a marked rise in dihydrotestosterone levels in the rabbit testes but had no effect whatsoever on the levels of this steroid in guinea pig testes. It is concluded that considerable species differences exist in the steroid metabolism of decapsulated testes incubated in vitro.

Testosterone synthesis [formula omitted] by the fetal testis of the guinea pig

Synthesis and secretion of testosterone by testes of guinea pig fetuses were studied in organ culture. The amount of testosterone secreted into the culture medium was estimated by radio-immunoassay. It was demonstrated that testosterone was already secreted by testes explanted at 25 days. The amount of testosterone secreted during the first day in culture by testes from fetuses of different ages (25–30 days) increased with fetal age. The amount of testosterone extracted from fetal guinea pig testes at ages used for explantation (25–30 days) were much lower than the amount secreted during the first 24 hours in culture. During subsequent days in culture, an increase in the amount of testosterone secreted was observed only for testes explanted at 25 days. The amount secreted by testes from older fetuses (26–30 days) stayed constant or decreased. Aminogluthetimide phosphate (100 μM) decreased significantly testosterone secretion by testes explanted at 25 days. These findings indicate that the capacity for biosynthesis of testosterone is present in fetal guinea pig testes at 25 days and increases during the subsequent days.

Metabolic Effects of Unilateral Orchiectomy on the Guinea Pig Testis

Six months after unilateral orchiectomy, there was a significant increase in the RNA and DNA content of contralateral testes as compared with the testes of age-matched normal guinea pigs. The ratio of RNA to DNA was not affected by orchiectomy, suggesting that the compensatory growth was secondary to hyperplasia rather than hypertrophy. Tritiated thymidine uptake by the contralateral testis was elevated at 2 weeks and 2,4 and 6 months after orchiectomy, confirming that increased metabolic activity occurred in the remaining testis after hemicastration.

Direct visualization of contractile proteins in peritubular cells of the guinea-pig testis using antibodies against highly purified actin and myosin

Antibodies against actin and myosin from smooth muscle, which may react with contractile elements from both muscular and muscle-like cells, were applied to fresh frozen sections of adult guinea-pig testis. Sections stained with an antibody against pectoralis (striated) muscle myosin or with non-immune globulin were used for controls. Peritubular cells of the lamina propria surrounding seminiferous tubulus contained large amounts of actin and myosin as judged by the intensity of immunofluorescence. Sertoli cells did not stain with the antibodies. Our results support the concept of peritubular cells being the critical force for the contractility of seminiferous tubules.

Changes in the concentrations of testosterone and androstenedione in the plasma and testis of the guinea-pig from birth to death.

Testosterone and androstenedione concentrations in the plasma and testis of male guinea-pigs were estimated by gas chromatography at intervals (36 stages) from birth to death. Four main periods of androgenic activity were recognized. The neonatal period, from birth to Day 16, is characterized by a precocious but transient peak in plasma testosterone concentration at Days 2 and 3. The pubertal period from Days 16 to 90 can be subdivided into a prepubertal period starting on Day 16 and marked by a sudden linear increase in plasma and testicular testosterone concentration together with an increase in testicular and seminal vesicle weight, and a postpubertal period, from Day 50 (the time of hormonal puberty) to Day 90, characterized by high and stable androgen levels while testicular and genital tract development continues. Adulthood spans the period between Months 3-6 and Month 24; plasma and testicular concentrations of testosterone and androstenedione are stable but lower than those observed during puberty. The period of senescence occurs between Months 24 and 28 and is marked by a fall in testosterone secretion and involution of the seminiferous tubules and accessory sex glands.

Experimental allergic aspermatogenic orchitis IV. Chemical properties of sperm glycoproteins isolated from guinea pig testes

Of four glycoproteins isolated from guinea pig testes, two were aspermatogenic (types I and IV) and two (types II and III) were inactive. The glycoproteins were rich in carbohydrate, varying from 41.5% to 49.5% carbohydrate by weight. Each glycoprotein had a unique amino acid composition, but in general low levels of tyrosine, tryptophan, and basic amino acids were found along with relatively high contents of serine, threonine, glutamic acid, and proline. Types I and IV glycoproteins were remarkably stable; their aspermatogenic activity was not affected by urea, trypsin, or heating at 100 °C in water or in 1 M HCl for 15 min. Carbohydrate analysis revealed little difference in the monosaccharide compositions of types I and IV glycoproteins, except that only the type I contained sialic acid. In contrast, types II and III glycoproteins lacked sialic acid and fucose and contained much less mannose. Both N-acetylglucosamine and N-acetylgalactosamine were present in all four glycoproteins, and they dominated in the types II and III. Fucose and at least 20–25% of the galactose appeared to occupy terminal positions in type IV glycoprotein as shown by their release after 15 min hydrolysis in 1 M HCl. All of the glycoproteins contained a relatively high percentage of galactose by weight, from 12.6 to 19.3%. The molecular weights of the glycoproteins were estimated by sodium dodecyl sulfate gel electrophoresis to be 47 000, 105 000 and 18 000 respectively for the types I, II and IV; type III glycoprotein showed two major bands, with molecular weights of 41 500 and 22 800. All the above molecular weight values are probably overestimated because of high carbohydrate content. The molecular weight of type IV glycoprotein was found to be 13 000 by ultracentrifugation; a corrected value of 29 000 was calculated for type I glycoprotein.

Changes in testis of guinea pig after vasectomy

Thirty adult guinea pigs were bilaterally vasectomized and subsequently sacrificed at one, three, and six months after operation. Cell counts were performed on five seminiferous tubules from each animal to identify and quantitate changes in spermatogenesis. Forty-three per cent of the animals had alterations in spermatogenesis that were characterized by generalized hypospermatogenesis and presence of multinucleated spermatids. There was a high incidence (93 per cent) of spermagglutinating antibodies in the vasectomized group. Sperm antibodies were not detected in the normal and sham-operated animals. Mean testicular weights and seminiferous tubule diameters were significantly reduced in the hypospermatogenic animals. The interstitial tissue of the vasectomized and sham-operated animals was morphologically indistinguishable from that of unoperated animals.

Experimental Allergic Aspermatogenic Orchitis

Abstract Four glycoproteins (GP1, 2, 3, and 4) rich in carbohydrate were isolated from guinea pig testes. GP1, 2, and 4 (one or more) were localized in the sperm acrosome by the indirect immunofluorescence technique. Purification consisted of delipidation with chloroform-methanol (2:1), acid extraction at pH 3.0, precipitation with 85% saturated ammonium sulfate, extraction with 5% trichloroacetic acid, and either gel filtration on agarose or ultrafiltration. The final purification steps were isoelectrofocusing or gel filtration on Sephadex G-75 followed by preparative slab gel electrophoresis at pH 8.6. Each glycoprotein appeared homogeneous by gel electrophoresis at pH 2.7 and 8.6, and by immunoelectrophoresis. The crude glycoprotein fraction from the agarose column was resolved into the three major components, GP1, 2, and 3, distinguished by their isoelectric points (pI 3.9, 4.4, and 5.0, respectively), electrophoretic mobilities at pH 8.6, and reactivities with antiserum in immunoelectrophoresis. GP4, isolated by ultrafiltration and Sephadex G-75 chromatography, was differentiated by the same criteria. Approximately 5 mg each of purified GP1, 3, and 4 and 2 to 3 mg of GP2 were isolated from 1000 g of wet guinea pig testes. GP1, 2, and 4 induced precipitating antibody in rabbits and goats. GP1 and GP4 induced allergic aspermatogenic orchitis in guinea pigs, an autoimmune disease characterized by infiltration of mononuclear cells around and within the seminiferous tubules, followed by extensive destruction of the germinal epithelium. The course of the disease induced by 2 μg of either GP1 or GP4 was essentially identical in time course and pathology to that induced by whole testicular homogenates or 1 μg of purified acrosomal protein (AP1).

Experimental allergic aspermatogenic orchitis. 1. Isolation of a spermatozoal protein (AP1) which induces allergic aspermatogenic orchitis.

A unique highly soluble aspermatogenic protein (AP1) was isolated from guinea pig testes and was shown by immunofluorescence to occupy the outer surface of the sperm acrosome. This protein is a potent inducer of allergic orchitis and aspermatogenesis; as little as 0.2 mug induced orchitis in 60 percent of guinea pig tested. The AP1 protein, relatively small and neutral, is stable under acid conditions, but at pH 8.6 shows a variety of forms due either to aggregation or polymorphism. The purified AP1 protein appeared homogeneous by polyacrylamide gel electrophoresis at pH 2.7 and in sodium dodecyl sulfate and by immunoelectrophoresis using rabbit antisera to either the purified protein or the testes extract. It also showed a single band on immunodiffusion over a wide concentration range. The purification procedure consisted of delipidation with chloroform/methanol (2/1); acid extraction at pH 3.0; precipitation with 85 percent saturated ammonium sulfate; trichloroacetic acid extraction and gel filtration on Bio-Gel A-1.5; gel filtration on Bio-Gel P-10; chromatography on CM52 cellulose; and preparative gel electrophoresis at pH 2.7. Approximately 20 mg of purified AP1 protein were obtained from 5000 g of wet guinea pig testes. The AP1 protein induced an autoimmune disease characterized by infiltration of mononuclear cells around and within the seminiferous tubules (orchitis), followed by extensive damage and destruction of the germinal cells (aspermatogenesis). The course of the disease induced by this protein (0.5 to 1 mug) was essentially identical with that seen with whole testicular tissue or other purified fractions.

Effect of vasectomy on guinea pig testes

The effects of vasectomy on the guinea pig testes were histologically assessed at weekly intervals. Degenerative changes were seen at eight weeks postvasectomy which were noted to be completely reversed in the sixteen-week specimens.

Histochemical studies on the Leydig-cell-leucine aminopeptidase activity in the guinea-pig testis.

The activity pattern and functional significance of leucine aminopeptidase (LAP) have been studied in the testes of oestrogen\x=req- treated, cryptorchid and HCG-treated guinea-pigs. The enzyme was present only in the Leydig cells and showed marked fluctuations in its activity pattern, which correlated closely with alterations in the functional activities of the Leydig cells. Hyperfunctioning Leydig cells exhibited enhanced LAP activity, whereas hypofunctioning of these intertubular cells resulted in a diminution in the enzyme activity. The LAP activity in the intertubular Leydig cells may be regarded as a cytochemical marker enzyme in the assessment of Leydig cell function in the guinea-pig testes under varying experimental conditions.

Infertility in female guinea pigs injected with testis.

The degree of fertilization failure and embryo loss was measured in female guinea pigs injected with guinea pig testis homogenate and Freund’s adjuvant. Controls were injected with saline and adjuvant. The fertilization rate was 91.6% in 26 control animals and 88.0% in 36 experimental animals. Embryo survival was estimated by comparing the number of normal fetuses at 25 or 60 days of gestation with the number of corpora lutea adjusted for fertilization rate. The average percentage of embryos surviving in 46 antigen-treated females was 45.8 compared to 72.7 in 19 controls. Embryo loss usually involved whole litters and occurred early enough for the females to return to estrus by 15.8 ± 3.2 days. Prebreeding estrous cycles on the same animals averaged 14.8 ± 1.0 days. These results indicated that, in guinea pigs injected with testis and adjuvant, the major cause of infertility is early embryonic death. The mechanism is not known but is presumed to involve a reaction of antibodies in the uterus with antigens borne by the embryo.

Drechslera hawaiiensis: causative agent of a fatal fungal meningo-encephalitis.

Cultural studies of a fatal disease of unknown origin with severe neurological symptoms in a human led to the isolation of Drechslera hawaiiensis from the base of the brain. In histological sections abundant, dematiaceous, septate mycelial elements were present. Animals were successfully infected with the isolate, lesions developing in the liver and spleen of mice and the testes of guinea-pigs. Experimental infections were also produced in animals with 6 plant isolates of D. hawaiiensis. The genus Drechslera can be differentiated from the superficially similar genera Bipolaris and Helminthosporium by its conidiophore development.

Nature of the Spermagglutinin in the Normal Serum of Rabbits

THE agglutination of homologous spermatozoa1,2 by heat-inactivated rabbit serum (56° C for 30 min) seems to be an immunological reaction. Although complement fixing activity of normal rabbit serum by rabbit spermatozoa was reported3,4, antibody-like activity was not proved. Edwards3 obtained neither immobilization of spermatozoa nor a positive reaction with passive cutaneous anaphylaxis and precipitin tests. Immunofluorescence6 and mixed conglutination7 gave similar results for spermagglutinin activity in normal rabbit serum. Germinal cells of guinea-pig testis have been lysed by the animal's own serum8, but contrary findings have been reported9–11 in which reaction between serum and homologous spermatozoa by immunofluorescence was negative. I have now investigated whether the spermagglutinin activity of normal rabbit serum is associated with antibody protein.

Aspermatogenesis

The testes of guinea pigs varying in ages up to 10 weeks and the testes of guinea pigs rendered aspermatogenic by immunization with antigen were assayed for RNase, DNase, cathepsin, acid phosphatase, <i>β</i>-glucuronidase, hyaluronidase, sorbitol dehydrogenase, lactic dehydrogenase and leucine amino peptidase. The principal findings were: (1) RNase, DNase and cathepsin were significantly elevated in the aspermatogenic testes. This finding is related to the lysosomal membrane damage mediated by antibody and is discussed from the point of view of cell damage; (2) hyaluronidase is proposed as a marker for aspermatogenic antigen since it appears when ASA is found and is lost when the antigen is lost; (3) the levels of hyaluronidase and other enzymes in aspermatogenic testes indicate a conversion from the mature to the immature type of testes.