Stage-specific degeneration of germ cells in the seminiferous tubules of non-obese diabetic mice

The cause of fertility problems in insulin-dependent diabetes is largely unknown. To evaluate the role of autoimmunity-associated phenomena in the testis as a possible cause of the derangement in spermatogenesis, the stage-specific apoptosis of germ cells in the insulitis phase of pre-diabetes was quantified in the testes of non-obese diabetic (NOD) mice. The seminiferous epithelium of normal BALB/c and NOD mice contained cells positive for in-situ end-labelling (ISEL) of DNA. ISEL-positive germ cells formed clusters in the seminiferous epithelium of the NOD mice in marked contrast to the seminiferous epithelium of the BALB/c mice, which contained only individual cells positive for ISEL. ISEL-positive cells were present in the basal and luminal compartments of the epithelium. Ultrastructural analysis and demonstration of externalized phosphatidyl serine confirmed that the cells were undergoing apoptosis. The ultrastructurally apoptotic cells included spermatogonia, spermatocytes and spermatids. In cytological squash preparations of segments of seminiferous tubules from NOD mice aged 17–20 weeks, the number of ISEL-positive cells/mm tubule was significantly lower in segments at stages I–II of the seminiferous epithelial wave but higher at stages III–IV in comparison to BALB/c mice. The numbers of ISEL-positive cells/mm tubule in the other stages were similar in the two strains of mice. Analysis of ³²P-3' -end labelled DNA from the testes showed that the BALB/c mice had relatively more DNA fragmentation than did the NOD mice. These data suggest that autoimmune insulitis in the NOD mice is associated with increased amounts and abnormal stage distribution of apoptosis in the seminiferous epithelium, resulting in derangement of spermatogenesis.     

Proliferation and apoptosis in the seminiferous epithelium of photoinhibited Syrian hamsters (Mesocricetus auratus)

In the hamster, male reproductive quiescence is accomplished via testicular atrophy and the germinal epithelium is regressed to spermatogonia and spermatocytes after 8-14 weeks of short photoperiods. However, the cellular mechanisms involved in this process have not been elucidated. As it is suggested that the regulation of seasonal testicular activity is characterized by coordinated shifts in the relationships between mitosis, meiosis and apoptosis, the changes in the proliferative and apoptotic activity in the seminiferous epithelium of photoinhibited Syrian hamster were examined and compared with those maintained in natural photoperiod. The proliferative activity was studied using BrdU immunostaining, and germ cell apoptosis was assessed by in situ TUNEL labelling and transmission electron microscopy. A significant increase in the rate of apoptosis (percentage of TUNEL-positive spermatogonia + spermatocytes) was observed in photoinhibited animals (2.84 +/- 0.16) compared with those exposed to natural photoperiod (0.77 +/- 0.03, p < 0.05). The majority of apoptotic germ cells were spermatocytes and in some occasions spermatogonia. Germ cell apoptosis was confirmed by morphological characteristics: condensation of the chromatin and nuclear fragmentation. The rate of proliferation (percentage of BrdU-positive spermatogonia + preleptotene spermatocytes) was significantly higher in photoinhibited hamsters (42.7 +/- 2.6) compared with animals exposed to natural photoperiod (31.1 +/- 1.6, p < 0.05). After the exposure to a short photoperiod the apoptotic index positively correlated with the proliferative index (r = 0.8150, p < 0.05). In conclusion, the seminiferous epithelium of photoinhibited Syrian hamsters is characterized by an increased rate of apoptosis associated to an enhanced rate of proliferation.     

Ageing and testicular function in Octodon degus

Summary. The present study analyses cell loss and proliferation which account for the decrease in the number of germ cell populations in the senile male Octodon degus.
This is a good model to study ageing in wild animals, since it has recently been incorporated as a laboratory animal but still has a high degree of genetic heterogeneity, thus representing a situation found in natural systems. The cell loss from pachytene spermatocytes to round spermatids is estimated by cell counts in the cross section of seminiferous tubules. DNA testicular synthesis is measured by scintillation counting and the index of labelling of spermatogonia by radioautography of testes comparing sexually mature young animals and senile animals. Other determinations in both groups are testis weight, thickness of the albuginea and tubular wall, daily sperm production, percentage of depleted seminiferous tubules and nuclear cell diameters of germ cells. The results suggest a decrease in the number of cell population in the senile animals resulting from an increase in physiological cell loss coupled with a decreased proliferative spermatogonial activity. There is also a decreased yield of meiosis in terms of round spermatid production. Lowered testosterone levels both in plasma and testicular parenchymal fluid are found in senile animals. All these senescent changes reflect an altered remodelling activity of the seminiferous epithelium and presumably also of Leydig cells.     

Apoptosis and molecular pathways in the seminiferous epithelium of aged and photoinhibited Syrian hamsters (Mesocricetus auratus)

Aging and short photoperiod exposure induce germ cell apoptosis in the Syrian hamster; however, the specific germ cells affected and the molecular pathways triggered have not been elucidated. We analyzed germ cell apoptosis and the expression of the Fas/Fas-L system, Bcl-2 family, and p53 in aged and photoinhibited hamsters and compared with those young maintained in natural photoperiod. Aging increased apoptosis in spermatogonia and spermatocytes, but in photoinhibited hamster testes only an increase in apoptotic spermatocytes was observed. Apoptosis was higher in aged hamsters in stages I-IV, V-VI and VII-VIII. Aging increased apoptosis of spermatogonia in stages I-IV and V-VI. Apoptotic pachytene spermatocytes were significantly higher in stages I-IV, V-VI, and VII-VIII in aging. Apoptotic preleptotene and pachytene spermatocytes were higher in aging, but no differences were observed in leptotene-zygotene. Fas-L was expressed by Sertoli cells, of young, aged, and photoinhibited hamsters. Bcl-x(L) was strongly expressed in germ cells on young hamsters and slightly in aging and after short photoperiod exposure. Spermatocytes of photoinhibited hamsters were intensively stained with Fas, Bax, Bcl-xs/L, and p53. In conclusion, aging increases apoptosis in spermatogonia and spermatocytes, depending on the stage of the seminiferous epithelium cycle, whereas after a short photoperiod exposure only an increase in apoptotic spermatocytes is observed. The results suggest that Fas, Bcl-x(L), Bax, and p53 participate in germ cell apoptosis induction after short photoperiod exposure, whereas only Bcl-x(L) is involved in aging.     

Stage-dependent topographical relationship of spermatogonia and early spermatocytes to Sertoli-Sertoli interspaces in the rat testis

In the rat seminiferous epithelium the Sertoli-Sertoli interspaces can be regarded as ending at the surface of basal germ cells as well as at the basement membrane. The relationship between these basal endings (BEs) of the Sertoli-Sertoli interspaces and the basal germ cells was examined using the electron microscope. The number of BEs in a given area of the seminiferous epithelium was counted and found to be constant throughout the cycle of the seminiferous epithelium. Undifferentiated spermatogonia had contact with very few BEs while differentiating type A spermatogonia at stage VII and later during the cycle seemed to be associated with a maximum of BEs. As type A spermatogonia developed to intermediate spermatogonia, type B spermatogonia, and preleptotene spermatocytes, the average number of BEs per cell decreased exponentially. Thin, fingerlike cell projections emerged from the differentiating type A and intermediate spermatogonia along the basement membrane. These projections were associated with BEs. It is concluded that a specific stage-dependent topographical arrangement is established between Sertoli cells and germ cells long before the first synchronous division of type A spermatogonia takes place. The significance of this arrangement is discussed.     

Quantitative parameters of seminiferous epithelium in secretory and excretory oligoazoospermia

Testicular biopsy specimens from infertile men (sperm count, less than 10(6)/ml) were evaluated on 1-micron thick sections, and counts of stem cells and differentiated spermatogonia, primary spermatocytes, early and late spermatids, and Sertoli cells were compared to counts in six fertile men. Biopsy specimens were also compared for the appearance of seminiferous tubule wall, blood vessels, and interstitium. Infertile men were grouped according to the following diagnoses: hypospermatogenesis (n = 5), spermatocyte arrest of spermatogenesis (n = 5), and obstruction of the genital tract (n = 7). A low productivity of spermatogenesis in cases of hypospermatogenesis appeared to be due to an exaggerated degeneration of primary spermatocytes and to a yield of abnormal spermatids. A block of meiosis in spermatocyte arrest was associated with a degeneration of primary spermatocytes and with a reduced number of staminal spermatogonia. Abnormal spermiogenesis was observed in cases of obstruction of the genital tract and was associated with an increase in stem cell spermatogonia. A thickening of seminiferous tubule and blood vessel walls could be responsible for the limited functional capacity of Sertoli cells, causing altered spermiogenesis in cases of excretory azoospermia. A severe primitive failure of Sertoli cells in secretory oligoazoospermia could account for a deranged maturation and degeneration of premeiotic and postmeiotic germ cells.     

Adverse effects of chronic GnRH antagonist administration on seminiferous epithelium in adult rats.

Seminiferous epithelium in adult rats was studied by light and electron microscopy after 5 weeks of chronic administration of GnRH antagonist (Ac-D2 Nal 1, D4ClPhe 2, DTrp 3, DArg 6, DAla 10; GnRH code-103-289-10, National Institutes of Health, USA). In these rats, the epithelium showed significant accumulation of vacuoles in more than 80% of the tubules, along with germ cell degeneration and nuclear pyknosis. Disruption in the process of spermatogenesis was also very much evident. In most of the tubules studied (greater than 90%), germ cell development was arrested beyond the pachytene spermatocyte stages. The vacuoles in the seminiferous epithelium were different sizes and when magnified were seen to consist of a thickened outer margin of solid nonfibrous coat within the Sertoli cell cytoplasm. Associated changes in the interstitium showed increased intertubular space but no inflammatory type of response. In actual cell counts, the decrease in the average number of macrophages was 32% and in Leydig cells 23%, while the total number of all types of cells in the interstitium was 30% less than that of the controls. Following the treatment, weights of testis, epididymis, seminal vesicle, and ventral prostate were drastically reduced. Rats treated with testosterone supplementation (60 micrograms/rat day) to GnRH antagonist recovered testicular and epididymal weights to approximately 57% and seminal vesicle and ventral prostate weights by 82.9 and 84%, respectively. Normalcy returned to the tubular epithelium and the interstitial cell counts were restored to original levels.     

In vivo analysis of germ cell apoptosis reveals the existence of stage-specific `social' control of germ cell death in the seminiferous epithelium

It has become clear in recent years that programmed cell death is regulated during development by signals from other cells. Nevertheless, compared to the `social' control of cell proliferation, relatively little is known about the `social' control of cell death in other systems. Since in a previous study we showed that induced germ cell apoptosis occurs at specific stages of the spermatogenic cycle, in this study we aimed to ascertain the existence of supracellular control of germ cell death during spermatogenesis. Therefore, the TUNEL technique has been used to analyse whether all of the different germ cell types are induced to die at these specific stages in animals injected intratesticularly with one of several inducers of apoptosis. Our findings suggest that all of the investigated agents trigger apoptosis in all the diverse progenies of germ cells existing at stages I, XII or XIV of the spermatogenic cycle. In contrast, at most other stages the number of apoptotic cells was similar to that found in control animals. These data are consistent with the existence of an intercellular control of germ cell death during spermatogenesis. We conclude that the seminiferous epithelium provides a suitable in vivo model to study the mechanisms underlying the `social' control of apoptosis.     

The cycle of the seminiferous epithelium in humans: a need to revisit?

Understanding the dynamics of spermatogenesis is central to clinical andrology or to probing environmental effects on human testes. This review considers what is known about renewal and proliferation of spermatogonia, how germ cells are organized in cellular associations constituting the cycle of the seminiferous epithelium, relative frequencies of cellular associations, durations of the cycle of the seminiferous epithelium and spermatogenesis, and measurement of daily sperm production. Daily sperm production (DSP) per testis tends to decline with advancing age. Regardless of age, there is substantial loss of potential sperm from degeneration of spermatocytes, but not spermatids. DSP per gram testis parenchyma or DSP per testis cannot be predicted on the basis of testis size or age of a man. The review shows why our 1960s data base is neither robust nor precise and suggests how deficiencies might be rectified. New cellular associations should be defined, with none representing >15% of the cycle of the seminiferous epithelium. Then determine when A(pale)-spermatogonia become committed to proliferate or how many mitotic divisions occur thereafter. Restudy the duration of spermatogenesis because the accepted value might be in error by approximately 6 days. Restudying human spermatogenesis will benefit clinicians, toxicologists, and epidemiologists probing testis function by direct evaluations or indirectly via evaluations of quantity and quality of sperm ejaculated. It also will benefit scientists interested in renewal and proliferation of spermatogonia, or a spermatogonium as a prototype stem cell.     

Cellular regulation of follicle-stimulating hormone (FSH) binding in rat seminiferous tubules

Stage-specific binding of follicle-stimulating hormone (FSH) was measured in rat seminiferous tubules. The binding in single-point assays was over 3-fold higher (P less than 0.05) in stages XIII to I than in stages VI to VII of the epithelial cycle. No difference was found between the equilibrium association constants (Ka) of FSH binding in stages XIV to IV (10 +/- 1.9 X 10(9) 1/mol) and VII to VIII (9.2 +/- 0.6 X 10(9) 1/mol, mean +/- SEM, n = 5). In another experiment, the testes were dosed locally with 3 Gy of 4 MV x-irradiation to selectively lower the number of spermatogonia. After irradiation, FSH binding in staged seminiferous tubule segments was measured when the desired types of spermatogenic cells were reduced in number. Seven days after irradiation when differentiating spermatogonia and preleptotene spermatocytes were reduced in number, FSH binding was decreased in all stages of the cycle, but the cyclic variation remained. Seventeen days after irradiation when intermediate and type B spermatogonia and spermatocytes up to diplotene of stage XIII showed low numbers, FSH binding was decreased in all stages of the cycle and the stage-dependent variation disappeared. At 38 days when pachytene spermatocytes and early spermatids were reduced in number, similar results were found. But at 52 days postirradiation when all spermatids were low in number, FSH binding was slightly elevated compared with days 17 and 38. There were no significant differences in serum FSH or LH levels between irradiated and non-irradiated animals. These findings suggest that all spermatogenic cell types may stimulate FSH binding in the Sertoli cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pattern of Sertoli cell degeneration in cryptorchid prepubertal tests

Seventy-three testicular biopsies from 54 children (aged 2 months-14 years) with undescended testes were examined by light and electron microscopy. The biopsies included abdominal, inguinally fixed, inguinally moveable, and retractile testes. Alterations in Sertoli cell morphology were found in all biopsies. The alterations included dilated elements of rough endoplasmic reticulum, vacuolization of the cytoplasm, mitochondria with poorly preserved cristae, increase in electron density of the matrix, elongation of the nuclei, and irregularities of the nuclear membrane. According to the numerical appearance of these cells and to the extent of lesions in single Sertoli cells, seven phases in the continuous process of tubular alteration were distinguished. The most severe tubular damaged (phase VII) occurred when the seminiferous epithelium consisted exclusively of necrotic cells. All phases of tubular alterations were seen regularly in each of the biopsies investigated. Germ cells occurred only in phases I-IV and were never observed in tubules in phases V-VII. Significant differences became evident between inguinal and retractile testes by morphometric evaluation. It was demonstrated that the number of germ cells per cross-sectioned tubule (S/T value) correlated negatively with the percentage of tubules in phases V-VII. In contrast to inguinal testes, a complete absence of Sertoli cells and an S/T value less than 0.1 were never found in retractile testes and the percentage of tubules in phases V-VII was reduced significantly compared with inguinal testes. Our findings indicate that (i) maldescended testis in patients between 1 and 15 years-of-age is associated with a special pattern of Sertoli cell degeneration; (ii) Sertoli cell degeneration is a continuous process, which can lead eventually to complete dissolution of the seminiferous epithelium; (iii) total degeneration is not related to age but is dependent on testicular position; (iv) a defined phase of degeneration excludes germ cell development, and therefore enhanced Sertoli cell degeneration in cryptorchid testes must also account for the reduction in germ cell number.     

Spermatogenic cycle length and spermatogenic efficiency in the gerbil (Meriones unguiculatus)

The gerbil (Meriones unguiculatus) is a rodent native of the arid regions of Mongolia and China. Because the gerbil can be easily bred in laboratory conditions, this species has been largely used as an experimental model in biomedical research. However, there is still little information concerning the testis structure and function in the gerbil. In this regard, we performed a detailed morpho-functional analysis of the gerbil testis and estimated the spermatogenic cycle length utilizing 3H-thymidine as a marker for germ cell progression during their evolution through the spermatogenic process. The stage frequencies of the XII stages characterized according to the acrosome formation and development were (I-XII) 13.8, 10.1, 8.1, 7.8, 4.0, 11.2, 7.5, 7.1, 5.9, 7.6, 8.1, and 8.9. The mean duration of each seminiferous epithelium cycle was determined to be 10.6 +/- 1.0 days and the total duration of spermatogenesis, based on 4.5 cycles, was approximately 47.5 days. The volume density of tubular and interstitial compartments was approximately 92% and 8%, respectively. Based on the volume occupied by seminiferous tubules in the testis and the tubular diameter, about 9 and 18 m of seminiferous tubules were found per testis and per gram of testis, respectively. Twelve primary spermatocytes were formed from each type A1 spermatogonia. The meiotic index was 2.8, indicating that 30% of cell loss occurs during meiosis. The number of Leydig and Sertoli cells per gram of the testis was 28 million and each Sertoli cell was able to support approximately 13 spermatids. The daily sperm production per gram of testis (spermatogenic efficiency) was 33 million. Taken together, these data indicate that, mainly due to the high seminiferous tubule volume density and Sertoli cell support capacity for germ cells, the gerbil presents high spermatogenic efficiency compared with other mammalian species already investigated. The data obtained in the present study might provide the basis for future research involving the reproductive biology in this species.     

BCG-induced orchitis: Structural changes during the degeneration of seminiferous tubules of rats and rabbits

Summary The effect of BCG-induced orchitis on the structure of the seminiferous tubules in rats and rabbits was investigated by light and electron microscopy. The formation of cavities between Sertoli cells and the displacement of the cells of the spermatogenic cycle are the earliest changes to be observed. Individual Sertoli cells degenerate and separate from spermatocytes and spermatids. The latter form multinuclear complexes by a broadening of the intercellular bridges. The nuclei of spermatids undergo ring-like chromatin condensation in the rat and swelling in the rabbit. After the loss of spermatocytes and spermatids from the germinal epithelium, the remaining Sertoli cells have a very irregular shape and contain many residual bodies, which are probably derived from previously phagocytosed spermatids. They often contain crystalline inclusions. The nuclei of Sertoli cells show small chromatin condensations. In the rabbit, the tubular wall increases considerably in diameter. In the vicinity of a granuloma in the interstitium caused by BCG inflammatory cells accumulate around the wall of the seminiferous tubules. Although the basal lamina seems to be an obstacle, penetration of macrophages into the tubular lumen could be observed. However, this occurred only after the degeneration of the germinal epithelium.     

Local regulation of Leydig cells by the seminiferous tubules. Effect of short-term cryptorchidism

Unilateral cryptorchism was induced in adult rats for 24 h, and its effect on testicular morphology and intratesticular testosterone concentration after hCG-stimulation were studied. In seminiferous, tubules from abdominal testes an increased number of degenerating germ cells was noted in stages XIV-III of the spermatogenic cycle and Sertoli cells contained an increased amount of lipid droplets in stages XIV-VIII. However, germ cells and Sertoli cells from tubules at other stages of the cycle appeared unaffected. In scrotal testes the size of peritubular Leydig cells varied in phase with the spermatogenic cycle. The largest cells were found adjacent to stage VII-VIII and the smallest adjacent to stage XI-XII. In abdominal testes no stage-dependent variation in the size of peritubular Leydig cells was seen. Perivascular Leydig cells were of equal size in abdominal and scrotal testes. The testicular testosterone concentration following stimulation with a low dose of hCG was significantly lower in abdominal testes. It is suggested that the seminiferous tubules locally modulate Leydig cell function and that the stage specific stimulatory influence from stage VII-VIII is rapidly lost during experimental cryptorchidism.     

The spermatogenic cycle in the blue fox (Alopex lagopus): relative frequency and absolute duration of the different stages

The spermatogenic cycle of the blue fox was divided into eight distinct stages, based on an analysis of different cell associations of the seminiferous epithelium. The criteria used for classification of the stages were the type of spermatogonia, the occurrence of meiotic figures, and the shape and location of spermatids. The relative frequencies of the stages I to VIII were 25.7, 9.8, 8.7, 5.9, 13.8, 9.9, 10.6 and 15.5%, respectively. The duration of one cycle of the seminiferous epithelium was 12.0 +/- 0.2 days as determined from the progression of 5-bromo-2-deoxyuridine (BrdU)-labelled cells at various time intervals. The absolute duration of stages I to VIII was calculated to be 3.1, 1.2, 1.0, 0.6, 1.7, 1.2, 1.3 and 1.9 days, respectively. The estimated life span of primary spermatocytes was 19.2 days, of secondary spermatocytes less than 0.6 days, of spermatids with round nuclei 9.2 days and of spermatids with elongated nuclei 8.9 days.     

On the Postnatal Development of the Terminal Segment of the Seminiferous Tubules in Normal and Germ Cell Depleted Rat Testes

The development of the terminal segment of the seminiferous tubules was studied in 5 to 50 days old normal rats. At the age of 5, 10, and 15 days the terminal segment contained fewer gonocytes or spermatogonia than did the corresponding seminiferous tubule. The differentiation of the terminal segment was obvious at 20 days of age due to the high number of germ cells in the seminiferous tubules, where the epithelium became stratified at this stage. The blood-testis barrier in the terminal segment was chiefly established between 15 and 20 days of age as revealed by the lanthanum tracer technique.
To study the effect of the germ cells on the differentiation, the germ cell depleted testes of prenatally irradiated rats were also studied. The modified Sertoli cells of the terminal segment were more vacoulated and had fewer lipid droplets and inter-Sertoli cell junctions than did the Sertoli cells of the seminiferous tubules. The ultrastructure of the modified Sertoli cells of the terminal segment was similar in adult normal and adult SCO (Sertoli cell only) rats. The amount of lipid droplets in the Sertoli cells of SCO rats showed considerable variation among different tubular cross-sections within one testis.     

Potential daily sperm production, Sertoli cell number, and seminiferous tubule characteristics in beef bulls fed conventional or gossypol-enriched diets.

The objective of this study was to determine the amount of germ cell degeneration throughout spermatogenesis in the bull and to determine if gossypol-enriched diets enhance that degeneration. Twenty-six adult beef bulls in two different sets of treatment groups were chosen for the study. The first set of bulls were broken into three groups, receiving soybean meal (<0.03 g gossypol per bull, per day), cottonseed meal (1.8 g gossypol per bull, per day), or whole cottonseed (16 g gossypol per bull per day). The second set of bulls were fed 4 g of gossypol per day for 180 days and then were placed in their respective treatment groups of 0 g or 4 g of gossypol per day for 72 days to evaluate the effect of continued gossypol treatment versus stopping treatment. Hence, one group received gossypol for 180 days, followed by withdrawal for 72 days. The treated group received gossypol up to castration at 250 days. In both sets of bulls, significant germ cell degeneration occurred between B spermatogonia and primary spermatocytes (i.e., at the end of mitosis and into the beginning of meiosis), with no subsequent degeneration throughout the rest of spermatogenesis. For both sets of bulls, the gossypol-enriched diet fed had no effect on the nuclear volume or number per gram parenchyma of any germ cell or Sertoli cell. Therefore, gossypol did not augment the amount of germ cell degeneration that occurred between B spermatogonia and homogenate-resistant (mature) spermatids in either set of bulls. Gossypol-enriched diets also had no effect on volume density, diameter, luminal diameter, or basement membrane thickness of seminiferous tubules for the first set of bulls. The second set of bulls, with continued diets enriched with gossypol, had a larger number of B spermatogonia per gram than did bulls removed from the gossypol-enriched diet. In conclusion, the bull did experience significant degeneration of germ cells between B spermatogonia and pachytene primary spermatocytes, with no subsequent loss throughout the rest of spermatogenesis, and the gossypol-enriched diet did not further enhance germ cell degeneration.     

The effects of aging on the seminiferous epithelium and the blood-testis barrier of the Brown Norway rat.

Steroidogenesis and spermatogenesis decrease in aging Brown Norway rats. We therefore hypothesized that there must be accompanying morphological changes taking place in the seminiferous tubules of the aging testis. The testes of Brown Norway rats ranging in age from 3 to 24 months were prepared for light and electron microscopy. To assess the integrity of the blood-testis barrier with age, a lanthanum nitrate study was done. The normal seminiferous tubules present in rats at 3 and 12 months of age were largely replaced at 24 months by fully regressed tubules that were virtually devoid of germ cells and contained large intercellular spaces. An electron-microscopic study of these regressed tubules showed a complete loss of cyclical variations of the organelles of the Sertoli cells. The nucleus was more irregularly shaped and was present at various levels in the epithelium. The endoplasmic reticulum was a loose, vesiculated network that was unlike the elaborate, tubular, anastomotic network noted in young animals. The lysosomes were large, oddly-shaped, and contained lipidic inclusions, in contrast to the distinct membrane-bound lysosomes and dense core bodies found in the young animals. Adjacent Sertoli cell processes encompassed large, empty intercellular spaces, possibly occupied previously by germ cells. The typical Sertoli-Sertoli junctions of the blood-testis barrier in the young animal were rarely seen at 24 months and were replaced by focal contact points, usually between three Sertoli cell processes. In the aged animals, lanthanum nitrate permeated the basal and adluminal compartments, extending between Sertoli cell processes and entering the intercellular spaces and lumen. In summary, during aging, there is a breakdown of the blood-testis barrier, and there are striking changes in the appearance of Sertoli cells. These results suggest a possible intrinsic limitation that prevents stem cells from renewing themselves, whether because of a degeneration of immunological origin or because of a lack of Sertoli cell support.     

DNA-flow cytometry of defined stages of rat seminiferous epithelium: effects of 3 Gy of high-energy X-irradiation

Testes of adult Sprague-Dawley rats were irradiated locally by 3 Gy of 4 MeV X-rays produced by a linear accelerator. This type and dose of radiation gives an even distribution through the testis and selectively kills the proliferating spermatogonia. The seminiferous tubular cells were quantified by DNA flow cytometry at defined stages of the epithelial cycle at 7, 17, 22, 38, 52, and 80 days after irradiation. The flow cytometric technique was modified by using frozen instead of fresh samples. Freezing did not alter cell numbers when compared with fresh samples. At 7 days post-irradiation no significant changes were observed in any cell population by DNA flow cytometry, whereas histological analysis revealed a reduction in intermediate and type B spermatogonia. At 17 and 22 days post-irradiation, the number of cells at meiotic prophase (4C) was decreased, particularly in stages II-V of the cycle. In stages VII-VIII, cell numbers were 40 and 31%, and in stages IX-XIII, 24 and 43% of that in non-irradiated controls at 17 and 22 days, respectively. At 38 days after irradiation, both 4C and 1C (haploid) cells were decreased in number. The 4C cells were reduced to 24, 17, and 13% of that in non-irradiated controls in stages II-V, VII-VIII, and IX-XIII of the cycle, respectively. The corresponding numbers of 1C cells were 5, 17, and 4%. At 52 days after irradiation, 1C cells had declined to 38 and 19% of control values in stages II-V and IX-XIII, respectively. In stages II-V, 1C' cells (haploid cells with condensed nuclei) declined to 28% of controls at 52 days. The present data provide a quantitative basis for the use of X-ray-irradiated rat testes as a model system in experiments pursuing interactions between Sertoli cells and spermatogenic cells.     

Expression of the proliferation-associated Ki-67 antigen in bovine testicular tubular cells during the seminiferous epithelial cycle, demonstrated with the MIB-1 antibody

Summary. Ki-67 expression in the seminiferous tubule of the bovine testis was studied by immunohistochemistry during the seminiferous epithelial cycle using the monoclonal antibody MIB-1. Spermatogonial proliferation is most obvious in stages 5–7, and 8, when B-spermatogonia divide. A lower rate of spermatogonial propagation is observed preceding or during meiosis in stages 1–4. The MIB-1 antibody also gives positive results with some post-spermatogonial tubular cells. Preleptotenes passing through S-phase in stage 1 reveal positive nuclei. During prophase of meiosis I pachytenes react strongly, diplotenes react in an attenuated manner, while leptotenes and zygotenes stay negative. Secondary spermatocytes seen in stage 4 are positive as are the chromosomes during meta- and anaphase of the meiotic divisions. Post-meiotic spermatids are also decorated but stop Ki-67 expression abruptly at the end of stage 4. Sertoli cells are negative.