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Assuming that spermatogonial multiplication in primates will not be fundamentally different from that in non-primates it can be deduced that Ap and Ad spermatogonia in primates are analogous to the undifferentiated spermatogonia in non-primates. Ap and Ad spermatogonia consist of single, pairs and chains of cells, among which the single cells likely are the stem cells. The single Ap are the active stem cells while the single Ad only become active after cell loss. After cell loss, Ad spermatogonia without division transform into Ap spermatogonia. The human testis is a very sensitive indicator of radiation. However, the full effect on stem cells of radiation or other insults will only become visible in the ejaculate after 3 to 5 months. When no cell loss is inflicted, damaged to Ad stem cells may remain hidden for years. Damage to spermatocytes and spermatids will become visible much earlier. However, these cells heavily depend on a normal Sertoli cell function. Hence, effects found on spermatocytes and spermatids may be caused by a direct action on these cells but also via a deteriorated Sertoli cell or Leydig cell function. The number of Sertoli cells per testis can only be affected by treatment before puberty. Leydig cell numbers can also be affected in the adult, but this cell population is more vulnerable when treatment takes place before adulthood. However, the functional capacity of the Leydig cell population as a whole only decreases after severe cell loss.

Mature male rats, gamma-irradiated in utero, were hypophysectomized. In an effort to maintain the seminiferous epithelium, some animals were treated with exogenous androgen while in other animals the seminiferous epithelium was allowed to regress without hormonal treatment. These Sertoli cell-enriched (SCE) males were evaluated for 7 weeks following hypophysectomy. In SCE males the average initial weight of each testis was 300 mg which declined to 110 mg at 7 weeks post-hypophysectomy. Concomitantly, seminiferous tubule diameter decreased from 130 microns to approximately 89 microns. Numerous cells were detached from the lamina propria and were observed in the centre of the tubule. Two layers of Sertoli cell nuclei were frequently observed in the regressed seminiferous tubules. Many of these nuclei appeared to be less differentiated, i.e. the nuclei were smaller with smaller nucleoli and more heterochromatin. In contrast, hypophysectomized animals treated with testosterone propionate during the last 5 weeks of the 7 week observational period, retained a tissue weight of about 270 mg/testis (a 5-10% decline in weight compared with normal untreated controls). Also, these animals had seminiferous tubule diameters of 132 microns. Finally, the Sertoli cells which comprised primarily a single layer inside the seminiferous tubules had larger nuclei with finely granulated chromatin and large nucleoli. Protein changes in SCE testes, (+/-) androgen, following hypophysectomy were analysed using polyacrylamide gels containing SDS. Prominent changes in the protein profile as separated by molecular weight were observed and were attributable to androgen stimulation. These changes were probably occurring in Sertoli cells since the Sertoli cell represents about 70% of the total cell population in the gamma-irradiated model. It is concluded that testosterone is responsible for major changes in mature Sertoli cells, although potential contributions of other cell types such as myoid cells and Leydig cells are considered.

Sertoli and Leydig cell functions were evaluated in men with testicular damage due either to cytotoxic chemotherapy (CCT) or radiotherapy (XRT). Serum immunoactive inhibin, follicle-stimulating hormone (FSH), luteinizing hormone (LH) and testosterone concentrations were measured in 15 men (19-50 years) who had received 6-10 courses of combination CCT (mustine, vinblastine, procarbazine and prednisolone) for Hodgkin's disease 1-8 years earlier and 18 men (21-49 years) who had undergone unilateral orchidectomy for testicular seminoma followed by XRT (30 Gy) to the remaining testis, 1-4 years earlier. Normal men (n = 16, 19-36 years) acted as controls. Median inhibin (422 U/l) and testosterone (16.0 nmol/l) levels in the CCT-treated group were not significantly different from controls, whereas median FSH (14.5 IU/l) and LH (10.0 IU/l) levels were higher (p less than 0.0001 and p less than 0.001) than normal (2.9 and 5.5 IU/l). The median inhibin/FSH (I/FSH) ratio in the patients was lower (p less than 0.0001) than in the controls (33.8 vs. 187.0) as was the testosterone/LH (T/LH) ratio (1.7 vs. 3.8, p less than 0.001). In the XRT-treated group, both median inhibin (194.5 U/l) and testosterone (12.7 nmol/l) levels were lower (p less than 0.0001 and p less than 0.01) than normal (532.8 U/l and 20.0 nmol/l) in the presence of greatly elevated FSH (26.0 IU/l) and LH (14.5 IU/l) levels. In conclusion, CCT-induced testicular damage is associated with subtle Sertoli and Leydig cell dysfunction demonstrated by the reduced I/FSH and T/LH ratios; however, compensatory mechanisms maintain normal testosterone and inhibin levels.(ABSTRACT TRUNCATED AT 250 WORDS)

The Sertoli's and Leidig's cells (sustentocytes and glandulocytes) exposed to ionizing radiation were studied cytologically and histochemically. The experiments were made on white male mice which were irradiated in dosage from 50 to 400 r. The DNP of the nuclei of sustentocytes and glandulocytes were investigated at different periods of the spermatogenesis cycle and under hormonal loading. The irradiation was established to result in changes in the chromatine state of both types of cells, the Sertoli's cells being more resistant to radiation. Injection of testosterone at different periods after irradiation causes a decreased dissociation of DNP. Inspite of the apparent morphological stability to ray effects the sustentocytes and glandulocytes are highly reactive elements.

The ultrastructure of the Sertoli and Leydig cells of rats under conditions of a long-term inner radiation has been studied. The inner radiation (intravenous injection of 75Se-selenomethionine) induced considerable alterations in the ultrastructure of these cells that, in their turn, led to functional changes in the cells. The ultrastructure of the Sertoli and Leydig cells was seen restored in 18 months after an injection of 75Se-selenomethionine. The importance of these alterations in the course of spermatogenesis is discussed.

Male Sprague Dawley rats were continuously irradiated at a dose-rate of either 5 or 7 cGy/day, up to a total dose of 900 cGy. Changes in spermatogenesis with irradiation and the recovery of the testis during 33 weeks after irradiation were studied. No clear dose-rate effect with testicular weight occurred. During the irradiation time, increased dose and dose-rate induced a decrease in A spermatogonia and preleptotene spermatocyte number. In our experimental conditions germ cell production did not plateau, as shown by the increasing number of tubular cross sections devoid of germ cells beyond 500 cGy. The recovery of seminiferous epithelium occurred essentially within nine weeks. It was not dose-rate dependent and was still incomplete after 33 weeks. This lack of recovery might be due to limited compensatory division ability of the stem cells. Clusters of Sertoli cells were observed in the lumen of the seminiferous tubules; impaired function of these cells could also prevent the complete recovery of the seminiferous epithelium. By 16 weeks after the end of irradiation 67% of 5 cGy/day irradiated rats and 34% of 7 cGy/day irradiated rats recovered fertility.

Is the presence of germinal cells necessary for the Sertoli cells to acquire normal features? To respond to this question we have studied the development of the Sertoli cells in rats irradiated at the end of the foetal life. In the prenatal irradiated rats, the lumen of the seminiferous tubules appears later than in the control rats. The Sertoli cells show numerous flexuose apical processes, with central microtubule bundles. These processes regress progressively after the 40th day of life when the tubular lumen appears; numerous junctional complexes differentiate with the same structure as those of control animals. There are important dilatations of the intercellular spaces. The cytoplasmic organelles show a normal development up to the 40th day of life. After this period, the rough endoplasmic reticulum and the Golgi apparatus clearly regress while important dilatations appear in the smooth endoplasmic reticulum and persist in the adult animal. From the 35th day on, the basal lamina of the seminiferous tubules is irregular and multilayered. The differentiation of the Sertoli cells seems to be independent of the presence of germinal cells until the 40th day of life and presents several particularities; thereafter the Sertoli cells show signs of regression.

The ultrastructure of the seminiferous tubules was studied in rats that had been subjected to whole body irradiation on the 19th day of gestation. The seminiferous tubules from 3 months-old irradiated animals are devoid of germ cells and contain only Sertoli cells. Compared with controls of the same age, the seminiferous tubule basal membrane is thickened and multilayered and several alterations are observed in the Sertoli cells. The most characteristic of these alterations are: (a) an abnormal number of nuclear heterochromatin clumps, (b) the presence of numerous cytoplasmic vacuoles and various sized lipid droplets, (c) elaborate interdigitations and junctions between adjacent cells, and (d) the presence of anomalous ectoplasmic specializations disposed perpendicularly to the Sertoli cell membrane.

To study cell to cell communications within the testis of adult Sprague-Dawley rats, we used acute whole body neutron plus gamma-irradiation (0.99 Gray of neutron and 0.24 Gray of gamma-rays, 3 min; Exp A) over 7-121 days postirradiation and chronic whole body gamma-irradiation (7 cGy/day 60Co gamma-rays; Exp B) over 14-84 days of irradiation and 7-86 days postirradiation. Neither irradiation protocol had an effect on the body weight of the animals. Neutron plus gamma-rays induced dramatic damages to spermatogonia, preleptotene spermatocytes, spermatozoa, and, to a lesser extent, pachytene spermatocytes. In contrast, gamma-rays induced a selective destruction of spermatogonia. Subsequently, in both experiments a maturation-depletion process led to a marked decrease in all germ cell types. A complete or near complete recovery of the different germ cell types and spermatozoa took place during the two postirradiation periods. Under both irradiation protocols Sertoli cells number was unchanged. Androgen-binding protein and FSH levels were normal in spite of the disappearance of most germ cells from spermatogonia to early spermatids. However, the decline of androgen-binding protein as well as the rise of FSH and their subsequent recovery were highly correlated to the number of late spermatids and spermatozoa. Moreover, it appeared that spermatocytes may also interfere with the production of inhibin (Exp B). With neither irradiation was Leydig cell function altered, except in Exp B in which elevated LH levels were temporarily observed. Correlation analysis suggested a relationship between preleptotene spermatocytes and Leydig cell function. In conclusion, this study establishes that chronic gamma-irradiation is particularly useful in the study of intratesticular paracrine regulation in vivo and provides further support to the concept that late spermatids play a major role in controlling some aspects of Sertoli cell function in the adult rat.

The effects of retinoid addition of vitamin A-depleted (UV-irradiated) culture medium on uridine metabolism in cultured Sertoli cells have been studied. After vitamin A depletion, a consistent 2- to 4-fold enhancement of [3H]uridine incorporation into RNA was observed. Several lines of evidence indicate that this enhancement is the result of an increase in the specific activity of the uridine-labeled precursors of RNA. Although vitamin A depletion did not affect either uridine uptake or alter cellular RNA content, a 5-fold increase in the specific activity of UMP was found in vitamin A-depleted cells. This increase results because the cellular content of uracil nucleosides plus nucleotides is selectively lowered in vitamin A-depleted cells. The decreased content of uridine derivatives could be accounted for by a 45-57% decrease in the activity of glutamine-dependent carbamylphosphate synthetase in vitamin A-depleted cells. The effects of vitamin A deprivation on uridine incorporation, as well as carbamylphosphate synthetase activity, could be completely restored to or above control values by supplementing vitamin A-depleted cell culture medium with either retinol or retinoic acid. This effect of vitamin A depletion appears to be highly specific. Under the same conditions, no gross alteration in either the pattern or extent of synthesis of cellular or secreted proteins, glycoproteins, glycosaminoglycans, and lipids was observed. In addition, vitamin A depletion/repletion had no effect on the growth rate or morphology of the cells.

Peripheral concentrations of FSH in the male rat seem to be regulated in part by a protein hormone, inhibin, which originates from the testes. In an attempt to ascertain which type of testicular cell secretes inhibin, groups of male rats were irradiated prenatally or on days 4, 6 or 8 of postnatal life, and killed at 21, 51 or 81 days of age together with castrated and intact controls. The concentrations of FSH and LH in the pituitary gland, and FSH, LH and testosterone in the plasma were estimated for each animal, and the numbers of each class of intratubular cell in the testes were calculated. Rats irradiated neonatally had fewer Sertoli cells than controls at all ages studied, while the numbers of Sertoli cells in rats irradiated prenatally were higher than those in controls on day 21. The number of spermatogenic cells was usually decreased in rats irradiated postnatally. In the rats irradiated prenatally normal numbers of spermatogenic cells were found at day 51. Numbers of spermatogenic cells were significantly correlated with the number of Sertoli cells at the ages of 51 and 81 days. The concentrations of FSH in the plasma usually increased in the postnatally irradiated animals on days 21 and 51, but not on day 81; prenatal irradiation did not result in altered levels of FSH at any age. Peripheral levels of LH and testosterone were not affected by irradiation. The concentration of FSH in the plasma was negatively correlated with the number of Sertoli cells in all age groups, whereas significant correlations between the level of FSH and the number of spermatogenic cells were only found at days 51 and 81. It is concluded from these data that the Sertoli cell is the most likely source of inhibin.

Young adult Sprague-Dawley rats were continuously whole-body irradiated with gamma rays at a dose-rate of 7 cGy/day for 92 days. Plasma LH, FSH and testosterone concentrations and testicular histology were quantified at different times during exposure. Irradiation selectively decreased spermatogonial numbers until 17 days of irradiation, following which a maturation depletion was observed. By the end of the exposure all germ cell types were reduced in number to about 10% of the control values. No significant changes were found in testosterone concentration nor in the weights of testosterone dependent accessory sex organs, LH plasma concentration increased slightly but not significantly at the end of irradiation. A significant increase in plasma FSH concentration occurred after the numbers of a spermatogonia and preleptotene spermatocytes had been reduced, when number of stage VII pachytene spermatocytes decreased to 36% of control values, whereas numbers of round spermatids and Sertoli cells were respectively 86% and 100% of the control values. These results suggest a possible role of pachytene spermatocytes in the regulation of inhibin production by the testis.

The importance of the morphological study of cell death has recently been emphasized by the recognition that the ultrastructural features of dying cells allow categorization of the death as either apoptosis or necrosis. This classification enables inferences to be drawn about the mechanism and biological significance of the death occurring in a particular set of circumstances. In this study, Sertoli cell death induced in the immature testis of three and four day old rats by 5 Gy (500 rads) x-irradiation was described by light and transmission electron microscopy with the objective of categorizing the death as apoptosis or necrosis. The testes were examined 1, 2, 3, 4, 8, and 24 h after irradiation. Following irradiation, there was a wave of apoptosis of the Sertoli cells starting in three to four hours and reaching a peak between four and eight hours. At 24 hours, only 61% of the expected number of Sertoli cells remained. These findings are in accord with recent ultrastructural reports that ionizing radiation induces cell death by apoptosis in rapidly proliferating cell populations. New insights into the pathogenesis of radiation-induced cell death might thus be expected to stem from future elucidation of the general molecular events involved in triggering apoptosis.

Gonadal function in most animal species exhibits considerable annual fluctuations, with gametogenesis and fertility often being confined to a short and rigidly controlled breeding season. In males, production of androgenic steroids by the testis is usually maximal immediately before and during the breeding season. In the golden hamster, seasonal regression of the testes is associated with decrease in the total content of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and prolactin (PRL) receptors, and similar findings have been reported for other mammalian species. However, the concentration of LH and FSH receptors per unit of testis weight is typically elevated rather than suppressed during testicular regression. Reduction in the number of testicular LH and PRL receptors in adult golden hamsters exposed to short photoperiod is due primarily to suppression of pituitary PRL release under these circumstances. Regulation of seasonal changes in testicular FSH binding, as well as regulation of the levels of LH, PRL and FSH receptors in other seasonally breeding species remain to be elucidated. Reduction in the content of LH receptors in the testes is accompanied by reduced capacity to produce androgens in response to LH stimulation. Although these events are likely to be causally related, other mechanisms are also involved. In particular, seasonal regression is accompanied by reduced capacity of the testes to convert C21 steroid precursors into biologically active androgens. Seasonal loss of FSH receptors was reported to be accompanied by increased rather than reduced responsiveness of the Sertoli cells to FSH, thus resembling the situation in immature animals. It can be concluded that alterations in the ability of the testes to bind pituitary gonadotropins and to respond to gonadotropic stimulation are among the mechanisms responsible for seasonal shifts between gonadal activity and quiescence.

Continuous low-dose gamma-irradiation of mature rats induced a progressive degeneration of the germ cells. Blood FSH increased by 127, 176 and 214%, respectively, after 55, 70 and 85 days of treatment when compared to FSH levels in control rats (8.50 +/- 0.60 ng/ml); conversely, serum LH and testosterone levels were unchanged. The Sertoli cell function was affected by the treatment from 70 days on, as attested by androgen binding protein (ABP) and transferrin secretions which diminished 35-40%. Serum ABP levels were not altered, whatever the duration of irradiation, even though epididymal ABP contents (as well as concentrations) diminished 34-60% when compared to those of the controls. Moreover, in purified Leydig cells, LH-stimulated intracellular cAMP levels, which were decreased by seminiferous tubule medium (STM) from control rats, were enhanced in presence of STM from treated animals. Testosterone output was stimulated 9-fold in presence of oLH and further increased (46-76%) from stages XIV-V by STM prepared from control and irradiated rats, respectively. After 85 days the STM effects on both cAMP and testosterone syntheses were zero. These results demonstrate a probable alteration of Sertoli cell function after irradiation, but also a role of the germ cells in the regulation of the synthesis of ABP, transferrin and Sertoli cell paracrine factors.

The effects of continuous gamma-irradiation of adult rats at two low-dose rates (7 cGy and 12 cGy/day; up to a total dose of 9.1 Gy and 10.69 Gy 60Co gamma-ray, respectively) were investigated. Over a period of 3-131 days of irradiation, groups of experimental and control animals were killed. Body weight, testis, epididymis, prostate and seminal vesicle weights, the number of germ cells and Sertoli cells, tubular ultrastructure, epididymal and testicular levels of biologically active androgen-binding protein (ABP), and the plasma concentrations of follicle-stimulating hormone (FSH), luteinizing hormone (LH) and testosterone were monitored. Irradiation had no effect on body weight, whereas testicular and epididymal weight began to decrease following 35 and 50 days of irradiation at 7 and 12 cGy, respectively. At 7 cGy the target cells of the gamma-rays were essentially A spermatogonia, whereas at 12 cGy A spermatogonia and preleptotene spermatocytes were primarily affected. This resulted in a progressive and sequential dose-related reduction in the number of pachytene spermatocytes, round spermatids and late spermatids (LS). Under both irradiation procedures the Sertoli cell number remained unchanged whereas partial (7 cGy) or no change (12 cGy) was seen at the Leydig cell level. Whatever the irradiation protocol, from the time LS numbers decreased, vacuolisation of the Sertoli cell cytoplasm progressively occurred, followed by thickening and folding of the peritubular tissue. Moreover, in parallel to the drop in the number of these germ cell types, ABP production fell whereas FSH levels rose. A highly significant positive correlation was found between LS numbers and these Sertoli cell parameters. This study supports our previous concept of a control of certain important aspects of Sertoli cell function by late spermatids in the adult rat.