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Search for other papers by D. W. LORDING in
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Search for other papers by D. M. DE KRETSER in
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Summary.
The interstitial cells of the albino rat testis were studied in animals ranging from the 15th day of fetal life until adulthood. The interstitial cells developed in two phases, a fetal phase from the 17th fetal day to the 2nd postnatal week and an adult phase from the 3rd week onwards. Lipid histochemical and ultrastructural techniques demonstrated the abundance of lipid droplets in the fetal interstitial cells and the paucity of lipid in the adult interstitial cells. The enzyme Δ5-3β-hydroxysteroid dehydrogenase (HSD) was studied in the interstitial cells throughout development using dehydroepiandrosterone (DHEA), pregnenolone and 17α-hydroxy pregnenolone as substrates. The enzyme activity was found with all substrates in both types of interstitial cells, DHEA producing the greatest reaction. The activity of HSD using pregnenolone on substrates was greater in the fetal interstitial cells.
The ultrastructural studies demonstrated the presence of abundant agranular endoplasmic reticulum in both the fetal and adult interstitial cells. The prominent cup-shaped mitochondria and lipid droplets present in the fetal cells were not present in the adult interstitial cells. The differences in the histochemical and ultrastructural features of the cells of each phase suggests that they represent separate generations of interstitial cells.
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Summary. The characteristics of the fetal and adult populations of Leydig cells from postnatal rat testes were compared by Percoll gradient centrifugation. A single peak of hCG binding, due to the presence of fetal Leydig cells, was obtained after purification of intertubular cells from 8-day-old animals. Two peaks of specific hCG binding were obtained after purification of intertubular cells from 15-day-old rats: it was confirmed by autoradiographic techniques that the hCG was bound by adult-like Leydig cells in one peak and fetal Leydig cells in the other. Similarly, intertubular cell preparations from 21- and 25-day-old rats resolved into two peaks of hCG binding; adult-like Leydig cells were observed in the first peak, but fetal Leydig cells were rarely observed in the second of these peaks. These results demonstrate the separation of two Leydig cell populations from intertubular cells obtained from animals aged up to 15 days. Thereafter the pattern of the hCG binding profile is similar but is not due to the presence of the same cell types. Therefore these results emphasize the necessity for morphological identification of cell types to permit the correct interpretation of the corresponding biochemical data.
Search for other papers by J. B. KERR in
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Search for other papers by D. M. DE KRETSER in
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Summary.
The formation, distribution and fate of lipid inclusions within the seminiferous tubules of the rat has been studied throughout the spermatogenic cycle. The occurrence of lipid inclusions within the Sertoli cell exhibited cyclic variation with the stages of the rat seminiferous cycle. At stage 9 of the cycle, residual bodies of maturing spermatids were phagocytosed by the Sertoli cell and released numerous lipid droplets which appeared to coalesce into large inclusions at the base of the Sertoli cell at stage 10. The Sertoli cell lipid inclusions persisted throughout the completion of meiosis (stages 11 to 14) and the formation of young spermatids (stages 1 to 2) and their numbers appeared to reach a peak at stages 12 to 13 of the cycle. The inclusions decreased markedly within the Sertoli cell cytoplasm during stage 2 and remained low until stage 9 when lipid from the residual bodies again became available to the cell. This cyclic variation of lipid inclusions within the Sertoli cell does not support previously held views that there is a gradual decline in Sertoli cell lipid during stages 10 to 14 of the spermatogenic cycle. A hitherto unnoticed finding was the presence of large lipid inclusions in the cytoplasm of late pachytene to diakinetic spermatocytes, and some observations suggest a transfer of these lipid inclusions from the Sertoli cells to primary spermatocytes.
Search for other papers by C. L. Au in
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Search for other papers by D. M. Robertson in
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Summary. The concentrations of inhibin in samples of rat testicular venous and arterial blood and interstitial fluid were measured by an in-vitro bioassay using pituitary cells in culture in which the standard was an ovine testicular lymph preparation (assigned potency 1 unit/mg). Inhibin levels were undetectable ( < 2 U/ml) in both blood samples but reached a mean concentration of 120 ± 7 U/ml in testicular interstitial fluid. After unilateral efferent duct ligation the rate of inhibin accumulation in seminiferous tubules was determined by the difference in the inhibin content of the ligated and unligated testes. Additionally, the rate of seminiferous tubule fluid production was obtained from the difference in weight between the ligated and non-ligated testes. In the 24 h after efferent duct ligation there were linear increases in inhibin (18·5 ± 1·0 U/h) and in seminiferous tubule fluid production (26 ± 1 μl/h), but there were no changes in serum FSH and LH levels.
Experimental induction of bilateral cryptorchidism led to a decrease in the inhibin content of the testis after 10 days. The rate of inhibin accumulation after efferent duct ligation declined more rapidly than the inhibin content, being significantly depressed in cryptorchid testes after 3 days, suggesting that this measurement is a more sensitive index of inhibin production than the determination of testicular inhibin content.
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Search for other papers by D. M. Robertson in
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Search for other papers by D. M. de Kretser in
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Summary. The testicular inhibin content showed an initial increase in the first 2–3 days after bilateral ligation of the efferent ducts of rats, followed by a subsequent decline to levels significantly below normal by 14 days, and reached 25% of control values at 42 days. Serum concentrations of FSH and LH were significantly increased at Day 6–7 after treatment and were still elevated after 42 days. The decline in testicular inhibin content at times associated with elevated FSH concentrations is consistent with the hypothesis of inhibin being involved in the feedback control of FSH secretion.
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The combined effects of transient neonatal hypothyroidism and neonatal hemicastration were investigated to see whether they were additive. Hypothyroidism was induced in litters of ten male rats for 25 days from the day of birth by administration of 0.1% (w/v) 6-propyl-2-thiouracil in the mother's drinking water; hemicastration was performed on the day of birth. Controls included both normal and sham-operated animals. Numbers of Sertoli cells and round spermatids were quantified at age 135 days using stereological methods. Sham-operation had no effect on testis mass, or numbers of Sertoli or germ cells. Transient neonatal hypothyroidism resulted in an increase in testicular mass of 27% (P <0.05), whereas neonatal hemicastration resulted in a 33% (P <0.05) increase over control; the combination of the two procedures resulted in a 62% (P < 0.05) increase. There were corresponding significant increases in the number of Sertoli cells: 82% with hypothyroidism, 18% with hemicastration and 123% with the combination of the two procedures. Numbers of round spermatids showed similar increases: 59% with hypothyroidism, 45% with hemicastration and 95% with the combination of the two procedures. It is concluded that the effects of the combination of transient neonatal hypothyroidism and hemicastration are additive with respect to testicular mass, and numbers of Sertoli and germ cells.
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Search for other papers by D. M. de Kretser in
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Summary. A single s.c. injection of hCG (100 i.u.) produced a biphasic serum testosterone response in adult male rats, peaks being noted at 2 h (24 ng/ml) and 3 days (16 ng/ml). The levels fell to control during the intervening interval (8 ng/ml), although there were elevated levels of serum hCG. Maintenance of high oestradiol levels by a s.c. injection of 50 μg oestradiol benzoate given on Day 2 after the initial hCG injection failed to prolong the refractory period and the secondary peak of testosterone (16 ng/ml) occurred on Day 3. Administration of the antioestrogen, tamoxifen (2 mg or 3 μg), 24 h before or simultaneously with hCG did not prevent testicular refractoriness in vivo because serum testosterone levels still declined after 2 h to reach a nadir at 2 days.
The basal in-vitro testosterone production by decapsulated testes from animals injected with hCG was enhanced at 2 h. Stimulation by hCG increased the amount of testosterone produced ( × 1·5 that in controls). By 12 h basal production decreased and there was no further increment in testosterone in the presence of hCG. This refractoriness to further hCG stimulation prevailed until Day 3, but the total production of testosterone fell so that at 24 h and 2 days testes were producing basal amounts of testosterone. Testes recovered from refractoriness at 4 and 5 days, when basal and stimulated testosterone production were greater than in controls. Injection of 50 μg oestradiol benzoate at 2 days did not prolong the in-vitro refractory period and 2 mg or 3 μg tamoxifen had no effect on the in-vitro steroidogenic activity, since testes were still refractory to further hCG stimulation from 12 h to 3 days. The results of the present study do not support the hypothesis that oestradiol is involved in the hCG-induced refractoriness of the Leydig cell. The nadir between the peaks of serum testosterone in vivo corresponds to the period during which the testis is refractory to in-vitro stimulation by hCG.
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Resident macrophages are maintained at a comparatively high, yet stable, tissue concentration in the adult rat testis. After destruction of Leydig cells by ethane dimethane sulphonate treatment, the number of resident macrophages increases briefly and then decreases to below normal values, but returns to normal after the reappearance of Leydig cells. The mechanisms by which the adult testicular macrophage population is maintained, either by monocyte recruitment or by mitosis of the resident macrophages, have not been examined. An immunohistochemical dual labelling approach using a specific monoclonal antibody for resident macrophages, ED2, and markers of mitotic activity (bromodeoxyuridine incorporation and expression of the proliferating cell nuclear antigen) was used to investigate resident macrophage proliferation in Bouin's-fixed paraffin wax-embedded adult rat testes. Detection of the normally fixation sensitive antigen recognized by ED2 was achieved by using a decreased fixation time and antigen retrieval. Peaks of resident macrophage mitotic activity were observed during the phases of macrophage proliferation immediately after ethane dimethane sulphonate treatment and during the recovery phase associated with Leydig cell restoration. These data demonstrate that resident macrophages have the capacity to proliferate within the adult rat testis and, thus, this population of resident macrophages is maintained, at least in part, by mitotic division in situ.
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Search for other papers by D. M. de Kretser in
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Summary. Rat testicular interstitial fluid and hydroxycholesterol both stimulated testosterone production by isolated Leydig cells in vitro in a dose-dependent manner, but the dose—response lines were not parallel. The addition of cycloheximide blocked the stimulation by interstitial fluid but not that of hydroxycholesterol. Use of the compounds SU 10603 and cyanoketone (which inhibit 3β-hydroxysteroid dehydrogenase and 17α-hydroxylase respectively) or aminoglutethimide (which acts on the cholesterol side-chain cleavage enzyme) showed that the stimulatory factor(s) in interstitial fluid stimulated steroidogenesis at the cholesterol side-chain cleavage enzyme, before the conversion of pregnenolone. This enzyme is rate-limiting in the synthesis of testosterone by Leydig cells and a site of action of LH; therefore, these results support the view that an interstitial fluid factor may be involved in the paracrine regulation of testicular steroidogenesis.
Search for other papers by B. Jégou in
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Search for other papers by D. M. de Kretser in
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Summary. Cryptorchidism for 28 or 10 days resulted in a severe disruption of spermatogenesis (assessed histologically or by fertility tests), Sertoli cell function (assessed by seminiferous tubule fluid production after efferent duct ligation, ABP levels, binding of 125I-labelled FSH to testis homogenates and serum FSH levels) and Leydig cell function (assessed by serum LH and testosterone levels, in-vitro testosterone production, binding of 125I-labelled hCG). Orchidopexy after 28 days of cryptorchidism resulted in a poor recovery of spermatogenesis since the majority of tubules were lined by Sertoli cells and a few spermatogonia. No recovery occurred in the indicators of Sertoli and Leydig cell function.
Orchidopexy after 10 days of cryptorchidism also resulted in a poor recovery of spermatogenesis, with a few animals showing partial recovery after 6 months. No recovery occurred in seminiferous tubule fluid production but partial recovery occurred in ABP content and production rate. Serum FSH, LH levels and in-vitro testosterone production by the testis remained elevated and did not change from the values found during cryptorchidism.
Fertility testing at 6 months revealed a small number of rats in which fertility was restored although the number of embryos was lower than in controls. In this group of animals there was a significant improvement in a number of indicators of Sertoli cell and Leydig cell function. These data provide further evidence to link the changes in Sertoli cell and Leydig cell function to the germ cell complement present in the testis.
