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ABSTRACT
This study was designed to investigate the differences in testosterone concentrations measured in testicular extracellular interstitial fluid obtained with a push–pull cannula or by post-mortem drip-collection. In the first experiment, testosterone-filled silicone elastomer capsules (2–16 cm lengths) or empty 2 cm capsules were implanted s.c. in adult male rats for 1 week. Animals were then anaesthetized and interstitial fluid was collected with a push–pull cannula for 1 h from one testis in each animal. Testicular and peripheral venous blood were then sampled and supernatant fluid was collected from the dispersed cells of the same testis. The contralateral testis in each animal was removed, and post-mortem interstitial fluid obtained by drip-collection for 20 h at 4 °C. In animals given empty capsules, testosterone concentrations in drip-collected interstitial fluid were significantly (P < 0·01) greater than testicular and peripheral venous blood levels, testicular fluid levels, and levels in interstitial fluid calculated from push–pull cannula samples. The concentrations of testosterone calculated in interstitial fluid collected with a push-pull cannula were never significantly greater than testicular venous blood levels. In animals with testosterone-filled capsules, testosterone concentrations measured in drip-collected interstitial fluid were similar to those calculated from push–pull cannulae samples, and to testicular venous blood levels. In a second experiment, a group of adult male rats was pretreated with aminoglutethimide to block steroidogenesis. Two hours later, interstitial fluid was drip-collected from the testes of these animals and from a group of vehicle-treated controls. For each animal, one testis was placed on ice and interstitial fluid collected for only 10 min, while the contralateral testis was kept at 4 °C and interstitial fluid collected for 20 h. In control animals, testosterone concentrations in interstitial fluid collected for 20 h were significantly (P < 0·01) greater than those in interstitial fluid collected for 10 min. In animals pretreated with aminoglutethimide, testosterone concentrations at 10 min and 20 h were not significantly different, and did not differ from those in control samples collected for 10 min.
We believe these results support earlier suggestions that the post-mortem drip-collection technique may give misleading results, and indicate that testosterone synthesis and secretion continues after isolation of the testis. This may also be the case with other Leydig cell products.
Journal of Endocrinology (1989) 121, 303–309
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ABSTRACT
We have used a push–pull cannula to collect interstitial fluid from the testes of anaesthetized rats at various times after a single injection of human chorionic gonadotrophin (hCG; 50 IU), and compared the levels of testosterone in this fluid with the levels in testicular and peripheral venous blood collected at the same times. Following hCG injection, significant increases in testosterone concentrations were observed in all fluids with notable peaks occurring in interstitial fluid at 2, 8 and 24 h, in testicular venous blood at 2, 8 and 30 h, and in peripheral venous blood at 2, 8, 24 and 72 h. The results demonstrate for the first time that changes in testosterone concentrations in interstitial fluid can be different from those in testicular venous blood. In addition, when testosterone levels in interstitial fluid were compared with levels in testicular venous blood at each time-point, the results suggested that the partitioning of testosterone between these two compartments can be regulated. Furthermore, the changes in both interstitial fluid and testicular venous blood levels of testosterone do not always parallel those in peripheral venous blood, suggesting that changes in testicular blood flow and peripheral clearance rates of testosterone may also be important in the control of circulating testosterone concentrations.
Journal of Endocrinology (1989) 121, 311–316
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ABSTRACT
Testosterone concentrations have been measured in testicular interstitial fluid (IF), and in blood plasma sampled from various parts of the rat testis and spermatic cord, to assess (1) the most accurate method for determination of the intratesticular levels of testosterone, and (2) the route of secretion of testosterone from the testis. In untreated adult rats, testosterone concentrations were highest in blood collected from veins on the surface of the testis (269·50 ± 30·63 (s.e.m.) nmol/l), but were reduced by 56% on average in blood collected from veins at the proximal end of the spermatic cord (123·06±24·75 nmol/l), and were reduced considerably in peripheral venous blood (4·55 ± 0·55 nmol/l). Similar changes occurred in adult rats in which steroidogenesis was either stimulated (by treatment with human chorionic gonadotrophin; hCG) or inhibited (by treatment with aminoglutethimide; AMG), and in rats of various ages during sexual maturation. The reduction in testosterone levels during passage of blood from the testis up the spermatic cord is probably due mainly to dilution by incoming arterial blood which transfers to venous blood via anastomoses in the spermatic cord. Venous-arterial transfer of testosterone in the cord contributed to this in only a minor way. Concentrations of testosterone in testicular IF were always greater than testicular venous concentrations in control, developing and hCG-stimulated rats, but were comparable in rats treated with AMG to suppress Leydig cell steroidogenesis. These and other results demonstrate that the method of drip-collection of IF results in over-estimation of the actual intratesticular levels of testosterone. However, the present study also suggests that testicular venous blood probably provides the most accurate indication of the concentrations of testosterone in IF (and therefore in the testis) at any given time.
Journal of Endocrinology (1989) 122, 323–329
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ABSTRACT
Levels of immunoactive and bioactive inhibin were measured in venous blood collected at a point just before (testicular venous) and after (spermatic venous) its passage through the mediastinal venous plexus over the anterior pole of the rete testis, and compared with levels in peripheral venous blood and testicular interstitial fluid (IF). In 15 control rats, levels of inhibin were highest in IF (8900 ± 432 ng/l; mean ± SEM) and lowest in peripheral (290 ± 32 ng/l) and testicular (288 ± 34 ng/l) venous blood, whilst levels in spermatic venous blood (633 ± 99 ng/l) were always higher (P<0.002) than the levels in testicular venous blood. The latter difference was either reduced or abolished after disruption of spermatogenesis by local heating of the testes 8, 14, or 21 days previously, and by ligation of the efferent ducts for 6 h or more, but was not affected by acute removal of the epididymis. It is concluded that inhibin secreted into seminiferous tubule fluid may be reabsorbed from the rete testis and this may be the major route by which it reaches the peripheral bloodstream in rats with normal spermatogenesis.
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ABSTRACT
Regulation of testicular interstitial fluid (IF) volume has been investigated in adult male rats in which the Leydig cells were selectively destroyed with a single i.p. injection of ethane dimethane sulphonate (EDS). Following this treatment, some animals also received testosterone supplementation by s.c. injection every 3 days, beginning either from the time of EDS injection, or 3–12 days afterwards. The volume of IF obtained by drip collection was determined, and testosterone and gonadotrophin concentrations measured in blood and in IF. Testosterone levels in IF and serum became undetectable by 3 days after EDS treatment. IF volume was reduced by 50% (P < 0·01) to reach a minimum level between 6 and 9 days after treatment. However, this decline was prevented in the absence of Leydig cells by supplementation with testosterone from the time of EDS injection, a treatment which also kept gonadotrophins at minimum or undetectable levels. Furthermore, the reduced IF volume seen up to 9 days after treatment with EDS alone could be restored to control levels within 3 days by a single injection of testosterone. The results obtained demonstrate that androgens, but not Leydig cells or gonadotrophins, are required for the maintenance of interstitial fluid volume in the adult rat testis. It is suggested that the seminiferous tubules may mediate this response, through an androgen-dependent mechanism.
Journal of Endocrinology (1989) 120, 215–222
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ABSTRACT
During normal sexual maturation of the male rat there is a progressive change in the route of secretion of inhibin by the Sertoli cell, from a predominantly basal route of secretion in prepuberty to a predominantly apical route of secretion in adulthood. This change may be monitored by comparing the levels of inhibin in testicular (TV), spermatic and peripheral (PV) venous blood and the levels in testicular interstitial fluid (IF). This study has assessed the role of germ cells in effecting this change by assessing (a) the effect of total germ cell depletion by X-irradiation of the males in utero, and (b) the effect of selective germ cell depletion in adulthood using the testicular toxicant, methoxyacetic acid (MAA).
Female rats were X-irradiated on day 20 of gestation to produce male offspring whose testes were germ-cell deficient. Blood and IF samples were collected from groups of these offspring and age-matched controls at 35 and 100 days of age. In blood and IF samples, inhibin concentrations were significantly higher at 35 days of age than at 100 days. The absence of germ cells in X-irradiated animals did not affect the age-related fall in inhibin levels, nor the change in the predominant route of secretion of inhibin from the testis into blood. Testosterone was almost undetectable in 35-day-old controls, but was raised significantly by 100 days of age. In X-irradiated animals, testosterone levels were increased significantly at 35 days of age, and the levels in most samples were increased even more substantially by 100 days of age. However, PV levels of testosterone in 100-day-old X-irradiated animals were significantly lower than in controls. LH and FSH levels were raised in X-irradiated animals compared with their age-matched controls, but FSH levels in X-irradiated animals still fell with age, as in the controls.
The role of specific germ cell types in regulating the route of secretion of inhibin from the normal adult testis was studied after depletion (80–100%) of pachytene and later spermatocytes by a single oral administration of MAA (650 mg/kg) to adult rats. At 3 days after MAA treatment, coincident with the loss of pachytene spermatocytes, plasma inhibin levels were increased significantly in blood and IF samples, and this was associated with a dramatic change in the route of secretion of inhibin from the testis, with increased secretion of this peptide via the base of the Sertoli cell into IF and TV blood. However, previous studies suggest that this may be a consequence of direct stimulation by MAA, rather than the absence of pachytene spermatocytes. By 21 days after MAA treatment, when late-stage spermatids are absent, plasma inhibin levels were reduced significantly compared with controls, although the route of secretion of inhibin from the testis was comparable with that of controls. By 42 days, when a normal germ cell complement has been restored, plasma concentrations and the route of secretion of inhibin from the testis were similar to controls.
It is concluded that: (1) the presence of germ cells is not necessary for the maturational changes in the rate and route of secretion of inhibin by the Sertoli cell; these changes are most likely a consequence of formation of the blood–testis barrier, (2) in the normal adult testis, MAA-induced depletion of the most mature germ cell types affects the rate, but not the route, of inhibin secretion, whilst depletion of pachytene spermatocytes affects both parameters; the latter may indicate an early effect of MAA on the functional competence of the blood–testis barrier.
Journal of Endocrinology (1992) 132, 439–448
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Abstract
The aims of this study were to determine the plasma concentrations of follistatin in rams and to assess if the testis contributes to circulating follistatin and if there is uptake or production of follistatin by the head in rams. Catheters were inserted in the carotid artery, jugular vein and spermatic vein of intact rams during the non-breeding season (experiment 1; n=5) and breeding season (experiment 2; n=4). In experiment 1, blood samples were collected from 5 rams every 10 min for 4 h, commencing 20–60 min after surgery. After 2 h of sampling 1 μg gonadotrophin-releasing hormone (GnRH) was injected intravenously. In experiment 2, blood samples were collected from 4 of the rams used in experiment 1 by venipuncture 30 and 15 min before surgery and every 15 min throughout surgery. Commencing 1 h after surgery, matched samples were taken from each of the vessels every 10 min for 4 h (1–4 h after surgery), then every hour for 20 h (4–24 h after surgery) and then every 10 min for 4 h (24–28 h after surgery). In both experiments, follistatin secretion was non-pulsatile and there were no significant differences between the concentrations of follistatin in any of the vessels. There was a significant (P<0·05) increase in the concentrations of follistatin in each of the vessels throughout the 4 h of 10-min sampling in both experiments. In experiment 2 plasma concentrations of follistatin in the jugular vein were significantly (P<0·05) lower before surgery than at other stages of the experiment. During the non-breeding season (experiment 1) the concentrations of follistatin in all vessels were about 2-fold higher (P<0·001) than during the breeding season (experiment 2). Concentrations of follistatin were measured in the testicular tissue of the ram, bull, monkey and rat and were found to be 13·6, 2·1, 2·5, 0·8 ng/g testis respectively. In experiment 3, blood samples were collected every 15 min for 4 h from castrated rams (n=6) in the absence of treatment with testosterone propionate (TP) and after 7 days of treatment with a physiological dose of TP during the breeding and non-breeding seasons. There was no effect of stage of breeding season or TP on the plasma concentrations of follistatin and these concentrations in the castrated rams were similar to the concentrations in the intact rams in experiment 2. In experiment 4, the function of Leydig cells was stimulated by administration of human chorionic gonadotrophin but this had no effect on plasma concentrations of follistatin.
These experiments show that the concentrations of follistatin in the plasma of rams are measurable, that the testis is not the major contributor to circulating follistatin and that there is no significant uptake or production of follistatin by the head in rams. It appears that the contribution of the testis to circulating follistatin may vary with the stage of the breeding season, being greater during the non-breeding season than the breeding season. The gonadotrophins and testosterone do not appear to have a direct effect on the secretion of follistatin in rams. The increase in concentrations of circulating follistatin during surgery and more frequent blood sampling suggest a stress-related effect on the production of follistatin.
Journal of Endocrinology (1996) 149, 55–63
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Abstract
Interleukin-1 (IL-1) is a multifunctional cytokine with profound effects on ovarian function. The effects of IL-1 on ovarian steroidogenesis have been demonstrated in several species. IL-1 mRNA levels are increased in the thecal layer of the ovulating follicle and IL-1β has been shown to induce ovulations in vitro. In this study we have investigated the presence and distribution of the mRNAs for type I IL-1 receptor (IL-1RtI) and for the naturally occurring IL-1 receptor antagonist (IL-1ra) in ovaries of adult cycling rats, to elucidate the target cells for IL-1 action. We have demonstrated the presence of mRNA for both substances by in situ hybridisation and reverse transcription PCR. mRNA for IL-1RtI was not found in primordial follicles but was abundant in the granulosa and thecal layer in developing follicles with stronger signals in the granulosa layer. In the preovulatory and ovulatory follicles, there was a further increase in the signal for IL-1RtI mRNA in the thecal layer compared with the granulosa layer. Corpora lutea were weakly positive at all stages and atretic follicles were largely negative. No mRNA was detected in oocytes of any stage. mRNA for IL-1ra showed a similar distribution to that of IL-1RtI. The changes in distribution suggest an action of IL-1 on rat granulosa cells during follicular development and on thecal cells during ovulation.
Journal of Endocrinology (1997) 152, 11–17