Efficacy of perfluorodecalin as an oxygen carrier for mouse and rat testes perfused in vitro

Perfluorodecalin is a superior artificial oxygen carrier because of its high oxygen dissolving capacity, low toxicity, and short retention times within tissues. However, the instability of perfluorodecalin emulsions has hindered its application in blood substitutes. The present study addressed two questions. First, is perfluorodecalin deleterious to the endocrine function of testes? This question was examined by comparing testosterone secretion by testes perfused in vitro with medium incorporating either perfluorodecalin or erythrocytes as oxygen carriers. Second, can stable emulsions of perfluorodecalin be attained with the new surfactant Butronic U-1? This question was approached by determining the stability of perfluorodecalin emulsions containing either Butronic U-1 or Pluronic F-68, a proven ineffective surfactant. The experimental results support the efficacy of perfluorodecalin emulsions as oxygen carriers for mouse and rat testes perfused in vitro. Perfluorodecalin emulsions formed with Butronic U-1 were stable during the 4-hr perfusions but not during long-term storage.     

In vitro perfusion of isolated mouse testes: a model system for investigating testicular steroidogenesis

1. A procedure was developed to perfuse isolated mouse testes in vitro and investigate testosterone secretion. 2. The perfused testis preparation was validated by determining organ edema, glucose uptake, enzymatic and morphological indices of cellular integrity, and testosterone secretion in the absence and presence of LH and other pituitary peptides. 3. The results establish that this procedure can be exploited as a model system to investigate biochemical, physiological, and genetic aspects of steroidogenesis and Leydig cell function in the mouse, and presumably other small rodents as well.     

Testosterone response of cryptorchid and hypophysectomized rats to human chorionic gonadotrophin (hCG) stimulation

The testosterone responses to a single injection of hCG (100 i.u.) in hypophysectomized (hypox.), cryptorchid or sham-operated rats were followed over a 5-day period. In sham-operated rats, hCG induced a biphasic rise in serum testosterone, peaks being observed at 2 and 72 h. Reduced testis weights, elevated FSH and LH levels and reduced serum testosterone levels were found after 4 weeks of cryptorchidism, but hCG stimulation resulted in a normal 2 h peak in serum testosterone. However, the secondary rise at 72 h in cryptorchid rats was significantly lower than sham-operated rats. Reduced testis weight and undetectable serum FSH and LH levels together with decreased testosterone levels were found 4 weeks after hypophysectomy. Serum testosterone levels rose 2 h after hCG in comparison to hypox. controls but this peak was significantly reduced compared with sham-operated rats. The second rise in serum testosterone began on day 2, peaking on day 4 at levels comparable to that seen in sham-operated rats after hCG. The in vitro basal and hCG stimulated secretion of testosterone by cryptorchid testes was greater than that secreted by normal rat testes (518.0 +/- 45.9 and 3337.6 +/- 304.1 pmol per testis per 4 h compared with 223.6 +/- 24.9 and 1312.9 +/- 141.4 pmol per testis per 4 h for normal rat testes). In cryptorchid animals a single injection of 100 i.u. hCG resulted in a pattern of in vitro refractoriness similar to normal rats, lasting from 12 h to 2 days, during which testosterone secretion was reduced to near basal levels. The in vitro basal and hCG-stimulated secretion of testosterone by hypox. rat testes was severely diminished compared with normal rat testes. The temporal pattern of in vitro secretion of testosterone from hypox. rat testes mimicked the in vivo serum testosterone pattern seen in these animals. This study demonstrates important differences in the in vivo and in vitro testosterone response to hCG after testicular damage.     

Effect of Physalis peruviana L. on Cadmium-Induced Testicular Toxicity in Rats

Cadmium (Cd) stimulates the production of reactive oxygen species and causes tissue damage. We investigated here the protective effect of Physalis peruviana L. (family Solanaceae) against cadmium-induced testes toxicity in rats. Twenty-eight Wistar albino rats were used. They were divided into four groups (n?=?7). Group 1 was used as control. Group 2 was intraperitoneally injected with 6.5 mg/kg body weight (bwt) of cadmium chloride for 5 days. Group 3 was orally treated with 200 mg/kg bwt of methanolic extract of physalis (MEPh). Group 4 was pretreated with MEPh before cadmium for 5 days. Changes in body and testes weights were determined. Oxidative stress markers, antioxidant enzymes, and testosterone level were measured. Histopathological changes of testes were examined, and the immunohistochemical staining for the proapoptotic (caspase-3) protein was performed. The injection of cadmium caused a significant decrease in body weight, while a significant increase in testes weight and testes weight index was observed. Pretreatment with MEPh was associated with significant reduction in the toxic effects of Cd as shown by reduced testicular levels of malondialdehyde, nitric oxide, and caspase-3 expression and increased glutathione content, and the activities of superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase, and testosterone were also increased. Testicular histopathology showed that Cd produced an extensive germ cell apoptosis, and the pretreatment of MEPh in Cd-treated rats significantly reduced Cd-induced testicular damage. On the basis of the above results, it can be hypothesized that P. peruviana L. has a protective effect against cadmium-induced testicular oxidative stress and apoptosis in the rat.     

Effects of cadmium chloride on testicular steroidogenesis and fertility of male rats

Effects of a single subcutaneous injection of 1 or 5 mg/rat of cadmium chloride (CdCl2) on circulating steroids and fertility were studied over a period of 120 days in fertile male rats. Androgens and fertility returned to normal 120 days after 1 mg CdCl2 but males treated with 5 mg showed none to poor restoration of some of these parameters. The in vitro release of testosterone (T), 5 alpha-dihydrotestosterone (5 alpha-DHT) and androstenedione (delta 4A) by the decapsulated tests from CdCl2 treated males was significantly reduced whereas progesterone (delta 4P) was accumulated in significantly higher amounts into the incubation medium. When testes from CdCl2 treated males were incubated in vitro with hCG, a dose and time dependent stimulation of steroidogenesis was evident. Since the testes regained the steroidogenic capacity but the males remained sterile 120 days after 5 mg CdCl2 treatment, it appeared that CdCl2 induced a permanent damage to the germinal components of the testes.     

Oxygen supply to immobilized cells

Summary Oxygen supply is a critical point in technical processes when aerobic cells are used in immobilized preparations. This report concerns the use of hemoglobin or emulsions of perfluorochemicals (completely fluorinated organic compounds) to carry oxygen to immobilized cells. Both methods work well and do not seem to harm the cells. The perfluorochemical method of improving oxygen supply showed a higher operational stability than the hemoglobin method.Part of this work was presented at the Biochemical Engineering meeting in St. Barbara, CA. USA, 18–24 Sept. 1982     

Regulation of thyroid hormones on the production of testosterone in rats

The effects of a thyroidectomy and thyroxine (T4) replacement on the spontaneous and human chorionic gonadotropin (hCG)-stimulated secretion of testosterone and the production of adenosine 3',5'-cyclic monophosphate (cAMP) in rat testes were studied. Thyroidectomy decreased the basal levels of plasma luteinizing hormone (LH) and testosterone, which delayed the maximal response of testosterone to gonadotropin-releasing hormone (GnRH) and hCG in male rats. T4 replacement in thyroparathyroidectomized (Tx) rats restored the concentrations of plasma LH and testosterone to euthyroid levels. Thyroidectomy decreased the basal release of hypothalamic GnRH, pituitary LH, and testicular testosterone as well as the LH response to GnRH and testosterone response to hCG in vitro. T4 replacement in Tx rats restored the in vitro release of GnRH, GnRH-stimulated LH release as well as hCG-stimulated testosterone release. Administration of T4 in vitro restored the release of testosterone by rat testicular interstitial cells (TICs). The increase of testosterone release in response to forskolin and androstenedione was less in TICs from Tx rats than in that from sham Tx rats. Administration of nifedipine in vitro resulted in a decrease of testosterone release by TICs from sham Tx but not from Tx rats. The basal level of cAMP in TICs was decreased by thyroidectomy. The increased accumulation of cAMP in TICs following administration of forskolin was eliminated in Tx rats. T4 replacement in Tx restored the testosterone response to forskolin. But the testosterone response to androstenedione and the cAMP response to forskolin in TICs was not restored by T4 in Tx rats. These results suggest that the inhibitory effect of a thyroidectomy on the production of testosterone in rat TICs is in part due to: 1) the decreased basal secretion of pituitary LH and its response to GnRH; 2) the decreased response of TICs to gonadotropin; and 3) the diminished production of cAMP, influx of calcium, and activity of 17beta-HSD. T4 may enhance testosterone production by acting directly at the testicular interstitial cells of Tx rats.     

Regulation of infant and developing rat testicular gonadotropin and prolactin receptors and steroidogenesis by treatments with human chorionic gonadotropin, gonadotropin-releasing hormone analogs, bromocriptine, prolactin, and estrogen

Infant (5-day-old) male rats were treated with hormonal regimens to alter their exposure to gonadotropins, prolactin (Prl), and estrogen, and the response of testicular endocrine functions was measured. Human chorionic gonadotropin (hCG) or a potent gonadotropin-releasing hormone agonist analog (GnRH-A) resulted in a short-lived decrease of testicular receptors (R) for luteinizing hormone (LH), but no deleterious effects were found on testicular capacity to produce testosterone (T), which is a typical response of the adult testis. Only GnRH-A, through probable direct testicular action, induced a relative blockade of C21 steroid side-chain cleavage that was observed in vitro upon hCG stimulation. Human chorionic gonadotropin treatment, but not GnRH-A treatment, increased testicular Prl-R. GnRH antagonist analog (GnRH-Ant) treatment did not affect testicular LH-R, but decreased Prl-R and testicular T production. Decrease of serum Prl by bromocriptine had no effect on testicular LH-R or Prl-R, but slightly decreased T production in vitro. Ovine Prl increased binding sites for LH/hCG. The postnatal rats were insensitive to negative effects of diethylstilbestrol when monitored by testis weight, T, and LH-R. In conclusion, the responses to changes in the hormonal environment differed greatly between infant and adult testes. Mainly positive effects of elevated gonadotropin and Prl levels were seen on infant rat Leydig cell functions. Likewise, decreased tropic hormone levels, and exposure to estrogen, were ineffective in bringing about the inhibitory actions seen in the adult.     

Testicular testosterone concentration and in vitro response to HCG in normal and in testosterone immunized rabbits.

The testosterone concentration, the in vitro response to HCG and the percentage Leydig cells in testes of normal and of testosterone-3-BSA immunized rabbits were determined. Following immunization all three parameters increased in the same order of magnitude (1.8-2.6fold). The results indicate that active immunization with testosterone has no deleterious effects on the endocrine capacity of the Leydig cells. The observed functional and morpholigical alterations of the testes are due solely to increased trophic hormone secretion from the pituitary caused by antibody binding of circulating androgens. The basic testosterone concentration in the testes of the control rabbits were in the range of values reported for other species.     

Testosterone metabolism by incubated rat testes after chronic LHRH treatment

Adult male rats were injected 4 or 8 days with LHRH agonist. After sacrifice the testes were incubated in vitro with or without [4-14C]testosterone. After LHRH-administration the endogenously produced amounts of testosterone and of 7 alpha-hydroxytestosterone, the main testosterone metabolite normally found on incubation of adult rat testes, were drastically reduced when compared with controls. hCG, injected to rats 2 h before sacrifice, increased steroid production. In the LHRH-treated rats, however, the amounts of testosterone and of 7 alpha-hydroxytestosterone produced were much less while an important formation of 5 alpha-androstanediol was observed. The testes of LHRH treated rats metabolized [4-14C]testosterone to a large extent to 5 alpha-reduced and unextractable metabolites while the formation of 7 alpha-hydroxylated metabolites was much reduced. It is concluded that prolonged LHRH treatment provokes not only a depression of the testosterone production but has also an influence on the testicular metabolism pattern of testosterone resulting in a proportionally increased production of 5 alpha-reduced steroids and unextractable metabolites while the formation of 7 alpha-hydroxylated steroids is inhibited.     

Regulation of testicular and Sertoli cell gamma-glutamyl transpeptidase by follicle-stimulating hormone

Hormonal deprivation achieved by hypophysectomy or gonadotropin-releasing hormone (GnRH)-antagonist treatment of immature rats resulted in markedly lower testicular gamma-glutamyl transpeptidase (GGT) activity than in the testes of age-matched controls. When begun 15 days after hypophysectomy, follicle-stimulating hormone (FSH) treatment significantly increased testicular GGT above that in testes from hypophysectomized controls in a time- and dose-dependent manner. In contrast, testosterone propionate had only a small effect. Testicular GGT was higher in adult hypophysectomized rats treated with FSH from the time of surgery than in untreated hypophysectomized rats; testosterone propionate treatment had no effect. GGT activity in Sertoli cells isolated from GnRH antagonist-treated or hypophysectomized immature rats was also lower than in cells from control rats. FSH treatment from the day of hypophysectomy resulted in Sertoli cell GGT values equivalent to those from intact controls. These data indicate that FSH regulates GGT activity in rat testis and Sertoli cells.     

Effect of female sexual displays on the endocrine physiology and behaviour of male white-crowned sparrows, Zonotrichia leucophrys

In photoperiodic birds, endocrine responses to behavioural interactions between males and females may be involved in temporally "fine-tuning" the onset of reproduction to yearly variations in the environment. This study examined the endocrine and behavioural responses of male White-crowned sparrows ( Zonotrichia leucophrys ) to changes in the endocrine state of the female, as signalled by changes in her behaviour. Males on different photoperiodic regimes were paired with oestrogen-treated, sexually receptive females. Males exposed to gonadostimulatory long days mounted and copulated with oestrogen-treated females even before gonadal development was complete. These males had higher plasma levels of testosterone and luteinizing hormone and maintained enlarged testes longer than control males paired with untreated, nonreceptive females. Males maintained on nonstimulatory short days also mounted oestrogen-treated females; however, testes of these males remained nonfunctional and their plasma levels of testosterone and luteinizing hormone were basal. Thus, reproductive function of photostimulated males is profoundly affected by changes in the endocrine state and behaviour of the female. However, male sexual behaviours are expressed in response to visual and auditory stimuli from the female regardless of male hormonal condition or photoperiodic treatment.     

Taenia taeniaeformis: inhibition of rat testosterone production by excretory-secretory product of the cultured metacestode

In 3- to 5-month-old male Sprague-Dawley rats infected with the hepatic metacestode, Taenia taeniaeformis, the serum testosterone level was significantly lower than in comparable uninfected controls. By transmission electron microscopy, testicular Leydig cells of infected rats had less smooth endoplasmic reticulum than control Leydig cells. Cultured metacestodes isolated from the hepatic cysts secreted or excreted substances into the incubation medium. The effect of the excretory-secretory product on testosterone concentration in the sera and testes of 15-day-old rats was examined. Subcutaneous injection of 50-200 micrograms of excretory-secretory product/0.1 ml saline/rat for 2 days significantly reduced human chorionic gonadotropin-stimulated serum and testicular testosterone concentrations. Furthermore, the effect of the excretory-secretory product on isolated rat Leydig cell testosterone production was examined. Rat Leydig cells produced testosterone in vitro and, in the presence of 50 IU human chorionic gonadotropin/ml incubation medium, they responded with approximately 100% increase in testosterone production. Addition of 2-10 micrograms excretory-secretory product protein/ml of culture medium significantly reduced the testosterone production by rat Leydig cells in vitro. These results indicate that excretory-secretory product of cultured T. taeniaeformis metacestodes has a direct inhibitory effect on Leydig cell testosterone production under stimulation with human chorionic gonadotropin.     

Involvement of calcium-dependent mechanisms in T3-induced phosphorylation of vimentin of immature rat testis

Thyroid hormones have been shown to act at extra nuclear sites, inducing target cell responses by several mechanisms, frequently involving intracellular calcium concentration. It has also been reported that cytoskeletal proteins are a target for thyroid and steroid hormones and cytoskeletal rearrangements are observed during hormone-induced differentiation and development of rat testes. However, little is known about the effect of 3,5,3'-triiodo-L-thyronine (T3) on the intermediate filament (IF) vimentin in rat testes. In this study we investigated the immunocontent and in vitro phosphorylation of vimentin in the cytoskeletal fraction of immature rat testes after a short-term in vitro treatment with T3. Gonads were incubated with or without T3 and 32P orthophosphate for 30 min and the intermediate filament-enriched cytoskeletal fraction was extracted in a high salt Triton-containing buffer. Vimentin immunoreactivity was analyzed by immunoblotting and the in vitro 32P incorporation into this protein was measured. Results showed that 1 microM T3 was able to increase the vimentin immunoreactivity and in vitro phosphorylation in the cytoskeletal fraction without altering total vimentin immunocontent in immature rat testes. Besides, these effects were independent of active protein synthesis. The involvement of Ca2+-mediated mechanisms in vimentin phosphorylation was evident when specific channel blockers (verapamil and nifedipine) or chelating agents (EGTA and BAPTA) were added during pre-incubation and incubation of the testes with T3. The effect of T3 was prevented when Ca2+ influx was blocked or intracellular Ca2+ was chelated. These results demonstrate a rapid nongenomic Ca2+-dependent action of T3 in phosphorylating vimentin in immature rat testes.     

Differential metabolism of pregnenolone by testicular homogenates of humans and two species of macaques. Lack of synthesis of the human sex pheromone precursor 5,16-androstadien-3 beta-ol in nonhuman primates.

In previous reports we described the early time sequence in in vitro [4-14C] pregnenolone metabolism in human and rat testicular homogenates and, apart from a difference in the preferred route of the conversion of pregnenolone to testosterone, we demonstrated the presence of delta 16-synthetase activity in human but not in rat testes. In the study of testicular function higher monkeys are increasingly used as a model for human reproduction. The availability of testes from 2 different species of macaques (rhesus and crab eating monkeys) enabled us to compare the in vitro metabolism of pregnenolone in these testes with human testes. The pattern obtained in both monkey species were very similar, but completely different from those found in man. The delta 4 pathway was the preferred route for the conversion of pregnenolone to testosterone in the monkeys tested, the delta 5 pathway in the humans. delta 16-Synthetase activity, a prerequisite for the synthesis of the sex pheromone precursors 5,16-androstadien-3 beta-ol and 4,16-androstadien-3-one, was clearly measurable in the human but not in the monkey testicular homogenates. So far, man and boar are the only species harbouring delta 16-synthetase activity in their testes. These in vitro data indicate that the nonhuman primates studied are not suitable models for the study of human testicular function.     

Evidence for an androgen binding protein in the testis of a teleost fish (Salmo gairdneri R.): a potential marker of Sertoli cell function

A factor binding tritiated testosterone was detected using "steady-state" polyacrylamide-gel electrophoresis, in rainbow trout genital tract. It migrated with a Rf identical to that of rat ABP. This binding was thermolabile, and was competitively inhibited by unlabelled testosterone. The steroid binding protein was found in cytosols from trout testes which had been previously perfused to avoid blood contamination, trout seminal plasma and in testicular explants incubation media. Using a quantitative assay and a Scatchard analysis, 25-50 pmol binding sites per gram gonad were found in testis cytosol. Binding affinity constant for testosterone in the various samples was close to 4 x 10(8) M(-1). The dissociation of steroid-protein complex was rapid (t 1/2 approximately 1.5 min). Hormonal specificity was studied by the competition of 3H-T binding with several concentrations of unlabelled competitors and the following order for affinities was obtained: dihydrotestosterone approximately androstenedione greater than testosterone greater than oestradiol greater than 17 alpha, 20 beta DHP greater than 11KT greater than cyproterone acetate greater than cortisol. High testicular cytosol and seminal plasma concentrations and apparent in vitro production indicate that the testis may synthesize an ABP-like protein in the trout. Such a factor would provide a unique marker of Sertoli cell activity and regulation in various physiological or experimental situations.     

Abnormal secretion of reproductive hormones and antioxidant status involved in quinestrol-induced reproductive toxicity in adult male rat

This study aimed to evaluate the effects of quinestrol, a synthetic oestrogen homologue with reproductive toxicity, on the secretion of reproductive hormones and antioxidant status in adult male rat. Our results showed that quinestrol exposure significantly decreased the weight of the testis, epididymides, seminal vesicle, and prostate, as well as the sperm counts in the cauda epididymis of rats. Quinestrol significantly reduced the size of seminiferous tubules and the total number of spermatogenic cells. Serum testosterone, follitropin, and lutropin were also significantly reduced in a dose-related manner after quinestrol exposure. Meanwhile, the activity of superoxide dismutase, glutathione peroxidase, and total antioxide capacity significantly decreased, whereas the malondialdehyde and nitric oxide concentrations significantly increased in the testes. These findings revealed that endocrine disorders of reproductive hormones and oxidative stress may be involved in reproductive toxicity induced by quinestrol in adult male rats.