The cycle of the seminiferous epithelium in the Australian Swamp buffalo

The relative frequencies of stages and substages of the Swamp buffalo seminiferous epithelium were determined using a morphological classification. Duration of one cycle of the seminiferous epithelium was determined from radiolabelling studies using tritiated thymidine. Mean (+/-SD) duration of the cyle of the seminiferous epithelium of five Swamp buffalo was 8.74 +/- 0.18 d. Mean (+/-SEM) relative frequencies of stages and substages of the seminiferous epithelial cycle in ten bulls were Stage 1a, 7.27 +/- 0.72; Stage 1b, 8.11 +/- 0.85; Stage 1c, 8.54 +/- 1.13; Stage 2a, 5.9 +/- 0.79; Stage 2b, 7.49 +/- 0.78; Stage 3a, 9.05 +/- 0.66; Stage 3b, 9.69 +/- 1.11; Stage 4a, 5.04 +/- 0.44; Stage 4b, 4.8 +/- 0.69; Stage 5, 1.86 +/- 0.23; Stage 6, 8.81 +/- 0.84; Stage 7, 10.64 +/- 1.2; Stage 8a, 6.87 +/- 0.96; and Stage 8b, 5.93 +/- 0.72.     

Renewal and proliferation of spermatogonia during spermatogenesis in the Japanese quail,Coturnix coturnix japonica

Summary Four different types of spermatogonia were identified in the seminiferous tubules of the Japanese quail: a dark type A (A_d), 2 pale A type (A_p1 and A_p2), and a type B. A model is proposed describing the process of spermatogonial development in the quail. The A_d spermatogonia are considered to be the stem cells. Each divides to produce a new A_d spermatogonium and a A_p1 spermatogonium during Stage IX of the cycle of the seminiferous epithelium. An A_p1 spermatogonium produces two A_p2 spermatogonia during Stage II of the cycle, A_p2 spermatogonia produce four type B spermatogonia during Stage VI of the cycle, and type B spermatogonia produce eight primary spermatocytes during Stage III of the cycle. Consequently, 32 spermatids can result from each division of an A_d spermatogonium. Spermatogonial development in the quail differs from the process described in mammals in that there are fewer mitotic divisions and they are all synchronized with the cycle of the seminiferous epithelium. It is suggested that the fewer mitotic divisions explain why a smaller area of the seminiferous tubule is occupied by a cellular association in the quail than in mammals like the rat, ram and bull. The duration of spermatogenesis from the division of the A_d spermatogonia to sperm release from the seminiferous epithelium was estimated to be 12.77 days.     

Identification of stage-specific proteins synthesized by rat seminiferous tubules

Experiments were conducted to determine how the cycle of the seminiferous epithelium influenced synthesis and secretion of proteins by seminiferous tubules. Tubular segments were treated with collagenase and then cultured with [35S]methionine. These myoid cell-depleted tubules isolated from different stages of the epithelial cycle exhibited, at Stages VI and XII, two distinct peaks of secretion of total radiolabeled proteins. Two-dimensional gel electrophoresis indicated that the patterns of secreted proteins from these two stages were remarkably different, while those from other stages were intermediate between those at the peaks. At least 15 proteins were secreted cyclically, many of them previously unrecognized products of the seminiferous epithelium. One product, designated Cyclic Protein-2 (CP-2), exhibited a pronounced cycle of secretion, its peak at Stage VI being 30-fold greater than at its nadir at Stages XII-XIV. Further investigation indicated that CP-2 did not appear to originate from myoid cells or dispersed germ cells but could be recovered from Sertoli cell-enriched cultures prepared from Stage VI tubules. Protein secretion by tubular segments was also characterized by immunoprecipitation with two polyspecific antisera directed against Sertoli cell products. Five secretory proteins were identified which had cycles different from one another and from CP-2. In contrast to secreted products, the synthesis of most cellular proteins by tubular segments remained relatively constant throughout the cycle. It is concluded: 1) segments of the seminiferous epithelium secrete proteins into the culture medium which are distinct from cellular proteins; 2) the synthesis of many of these proteins varies with the epithelial cycle; and 3) several of the secreted proteins are of Sertoli cell origin, including a newly identified protein, CP-2. This indicates that the morphology and the protein synthetic capacity of the seminiferous epithelium are coordinated over space and time.     

The Cycle of the Seminiferous Epithelium of the Turkish Hamster (Mesocricetus brandti)

The Turkish hamster ( Mesocricetus brandti ) has become a desirable species for experimentation in testicular function, photoperiod, reproductive hormones and hibernation. Basic data on the kinetics of the seminiferous epithelium have not yet been published. In the present study, the cycle of the seminiferous epithelium was divided into eight stages based on the overall cellular associations of 1540 cross sections of tubules. The mean relative frequencies for stages 1 through 8 were 5.9, 3.3, 11.7, 6.7, 7.2, 28.5, 21.6 and 15.1%, respectively. The absolute duration of the cycle of the seminiferous epithelium was determined by administration of ³H-thymidine, removal of testes at intevals after injection and autoradiography. The mean duration of one cycle was estimated at 8.0 days and the duration of stages 1 through 8 was 0.5, 0.3, 0.9, 0.5, 0.6, 2.3, 1.7 and 1.2 days, respectively. The duration of meiotic prophase was 11.5 days and of spermiogenesis was 13.8 days. The life span of preleptotene cell was estimated at 1.21 days, leptotene, 0.73 days, zygotene, 0.94 days and pachytene, 7.37 days. The total cycle length of spermatogenesis as usually calculated was 32.0 days.     

Spermatogenesis in white-lipped peccaries (Tayassu pecari)

We describe here morphological and functional analyses of the spermatogenic process in sexually mature white-lipped peccaries. Ten sexually mature male animals, weighing approximately 39 kg were studied. Characteristics investigated included the gonadosomatic index (GSI), relative frequency of stages of the cycle of seminiferous epithelium (CSE), cell populations present in the seminiferous epithelium in stage 1 of CSE, intrinsic rate of spermatogenesis, Sertoli cell index, height of seminiferous epithelium and diameter of seminiferous tubules, volumetric proportion of components of the testicular parenchyma and length of seminiferous tubules per testis and per gram of testis. The GSI was 0.19%, relative frequencies of pre-meiotic, meiotic and post-meiotic phases were, respectively 43.6%, 13.8% and 42.6%, general rate of spermatogenesis was 25.8, each Sertoli cell supported an average 18.4 germinative cells, height of seminiferous epithelium and diameter of seminiferous tubules were, respectively, 78.4 microm and 225.6 microm, testicular parenchyma was composed by 75.8% seminiferous tubules and 24.2% intertubular tissue, and length of seminiferous tubules per gram of testis was 15.8m. These results show that, except for overall rate of spermatogenesis, the spermatogenic process in white-lipped peccaries is very similar to that of collared peccaries, and that Sertoli cells have a greater capacity to support germinative cells than most domestic mammals.     

Stages and duration of the cycle of the seminiferous epithelium in oncilla (Leopardus tigrinus, Schreber, 1775)

Six adult Leopardus tigrinus (oncilla) were studied to characterize stages of the seminiferous epithelium cycle and its relative frequency and duration, as well as morphometric parameters of the testes. Testicular fragments were obtained (incisional biopsy), embedded (glycol methacrylate), and histologic sections examined with light microscopy. The cycle of the seminiferous epithelium was categorized into eight stages (based on the tubular morphology method). The duration of one seminiferous epithelium cycle was 9.19 d, and approximately 41.37 d were required for development of sperm from spermatogonia. On average, diameter of the seminiferous tubules was 228.29 μm, epithelium height was 78.86 μm, and there were 16.99 m of testicular tubules per gram of testis. Body weight averaged 2.589 kg, of which 0.06 and 0.04% were attributed to the testis and seminiferous tubules, respectively. In conclusion, there were eight distinct stages in the seminiferous epithelium, the length of the seminiferous epithelium cycle was close to that in domestic cats and cougars, and testicular and somatic indexes were similar to those of other carnivores of similar size.     

Cycle of the seminiferous epithelium and its duration in the zebu, Bos indicus

The cycle of the seminiferous epithelium was studied in Nelore zebu bulls 4–6 years old. The stages of the cycle were determined according to the shape and position of spermatid nuclei and the presence of meiotic divisions in cross-sections of seminiferous tubules. The relative frequencies of stages 1 to 8 were, respectively: 31.3 ± 0.5, 12.2 ± 0.7, 21.2 ± 0.5, 8.8 ± 0.6, 4.2 ± 0.4, 5.7 ± 0.6, 6.3 ± 0.5 and 10.3 ± 0.5. The duration of the cycle was estimated by autoradiography using tritiated thymidine injected directly into the testes. The mean duration of one cycle was estimated to be 14.0 ± 0.4 days.     

Frequency of the stages in the cycle of the seminiferous epithelium in the rat

This study determined the optimum number of tubules to be counted per testis cross section, and the number of animals per treatment group, when changes in stage frequencies in the cycle of the seminiferous epithelium are criteria for assessing effects of treatment on spermatogenesis. A data base of 9,672 observed and staged tubules was collected from testicular cross sections of 15 Sprague-Dawley rats. A significant variation between animals was found for the frequencies of Stages I, II, IV, VI, VIII, and XIII. Computer simulation was used to randomly select different combinations of animal and tubule numbers from the observed data. Stage frequency means from each simulation experiment were compared statistically to observed mean frequencies. A model that used data from all 14 stages was analyzed. The following conclusions were made: a) a minimum of 200 tubule cross sections/testis is recommended for estimating stage frequencies; b) for a fixed number of tubules scored, the number of animals sampled is more important than the number of tubules per animal in reducing variance; c) to detect a difference of 2 standard deviations from the mean with a 2% error rate and examining 200 tubules/testis, at least 12 animals must be used per group when assessing all 14 stages; d) when individual stages are examined using 10 animals per group, only Stage VII has 80% or greater power of test (alpha = 0.05) to detect a frequency difference; e) pooling stages into 3-4 groups is recommended to improve the power of detecting a treatment difference.     

A method for quantifying synchrony in testes of rats treated with vitamin A deprivation and readministration

Using a variation of a previously published method for manipulating vitamin A levels, we obtained synchronized rat testes and determined the frequency of stages of the seminiferous epithelium in each rat. In this study, we have demonstrated a method for quantitative analysis of the synchrony. The degree of synchronization was expressed as a fraction of the cycle of the seminiferous epithelium, and thus in terms not influenced by the different durations of the stages of this cycle. The median stage about which the tubules were synchronized was calculated. This method may be used to compare the effects of different synchronizing treatments, which may be subtle, and to study various aspects of spermatogenesis in the synchronized testes. For example, the duration of the cycle of the seminiferous epithelium in synchronized testes is estimated to be 12.5 days.     

Testosterone micromilieu in staged rat seminiferous tubules

Endogenous testosterone concentrations in rat seminiferous tubules were measured in relation to different stages of the cycle of the seminiferous epithelium. For this purpose, the seminiferous tubules were mechanically separated from the interstitial tissue on a cooled (1 degree C) petri dish under a stereomicroscope without added medium. After recognition of the stages of the cycle by transillumination, the specimens were rapidly transferred by dry forceps into test tubes for testosterone radioimmunoassay. The results of the dry dissection method were compared with measurements on tubules that were kept after separation in phosphate buffered saline (PBS, pH 7.4), in order to reveal the possible leakage of testosterone from the tubules. The maximal concentration of testosterone per unit length of seminiferous tubule was found in stages VII and VIII of the cycle (288 +/- 60 fmol/cm, mean +/- SEM, n = 12), and the minimal in stages IX-XII (219 +/- 57 fmol/cm, P less than 0.01). If the levels were correlated with unit volumes of the seminiferous tubules, identical concentrations of testosterone (521-542 fmol/mm3, approx. 500 nmol/l) were found in the different stages of the cycle. Despite the similarity of testosterone concentrations in the different parts of the seminiferous tubules the local concentrations of biologically active (i.e. free) testosterone may be modulated by extracellular and intracellular androgen binding components.     

Stability of spermatogenic synchronization achieved by depletion and restoration of vitamin A in rats

Studies of synchronization of spermatogenesis following vitamin A deficiency have suggested that this may provide an in vivo model for the study of stage-dependent changes in hormonal action and protein secretion within the seminiferous epithelium. However, until now, no information on the stability or durability of this condition has been available. In this study, 200 seminiferous tubules from each of 40 rats (including controls) were classified according to their spermatogenic stage after withdrawal and replenishment of vitamin A. Following 15 wk withdrawal and subsequent replenishment of vitamin A, spermatogenesis was initiated in a synchronous fashion. This synchrony remained stable for more than 10 cycles of the seminiferous epithelium (2.5 spermatogenic cycles). In association with the extended period of vitamin A deficiency, a proportion of tubules (30%) showed morphological characteristics of either Sertoli cells only or Sertoli cells plus spermatogonia with occasional pachytene spermatocytes. During the 11-wk period of observation in this study, no significant change in proportions of damaged tubules were observed. Testicular testosterone concentrations, although elevated with respect to controls, showed no correlation with the stage of the cycle of the seminiferous epithelium observed, whereas pituitary and serum follicle-stimulating hormone levels were elevated, probably due to the number of damaged tubules observed. The persistence of synchrony in spermatogenesis following vitamin A treatment suggests that this model is applicable for studies of paracrine actions within the testis. However, the decreased ratio of synchrony observed with time may provide evidence that duration of the individual stages of the cycle of the seminiferous epithelium might be subject to temporal variation, leading to a progressive desynchronization of spermatogenesis in this model system.     

Stage specific effect during one seminiferous epithelial cycle following ethylene glycol monomethyl ether exposure in rats.

Stage specific effect of single oral dose (500 mg/kg body wt) of ethylene glycol monomethyl ether (EGME) was characterised during one cycle of seminiferous epithelium in rats. Maximum peritubular membrane damage and germinal epithelial distortion were observed at stages IX-XII. Cell death occurred during conversion of zygotene to pachytene spermatocytes (stage XIII) and between dividing spermatocytes and step I spermatids (stage late XIII-XIV). Profound effect was noted during first meiotic division than during second meiotic division. Presence of multinucleated secondary spermatocytes indicated cytokinesis arrest. The spermatogenesis was delayed and consequently frequency of tubules at stages I-VIII was reduced by day 10. Many of the tubules were devoid of round spermatids on day 12. Possibly, EGME (or it's metabolite) distorted the barrier system at stages IX-XIV and damaged the cells mostly at stages XII-early XIV.     

Kinetics of spermatogenesis in the white-lipped peccary (Tayassu pecari)

This study aimed to characterize the stages of the seminiferous epithelium cycle by the tubular morphology method, and to determine the number of differentiated spermatogonia generations in the adult white-lipped peccary. Twenty adult white-lipped peccaries, obtained from commercial slaughterhouse, were used. Fragments of the testicular parenchyma were fixed in 3% glutaraldehyde and embedded into a methacrylate resin. The number of germ and Sertoli cells was estimated by the analysis of cell populations in 50 transversal sections of seminiferous tubules in different stages of the cycle. The tubular morphology method allowed the identification of cellular associations characteristic of the eight stages of the seminiferous epithelium cycle in white-lipped peccaries. The results showed the presence of six generations of differentiated spermatogonia in white-lipped peccaries, and that the cell composition of the eight stages of the seminiferous epithelium cycle in this species is very similar to that described for collared peccaries.     

The length of the cycle of seminiferous epithelium in goats (Capra hircus).

This is the first report in literature showing the length of the seminiferous epithelium cycle in goats. In the present study, the duration of spermatogenesis was estimated using intratesticular injections of tritiated thymidine. Animals were castrated at 4 h, 7 days, and 11 days after injections. The duration of each spermatogenic cycle in goats is 10.6 +/- 0.5 days (SEM). Considering that the total duration of spermatogenesis takes about 4.5 cycles of seminiferous epithelium, spermatogenesis was estimated to last 47.7 days. The approximate primary spermatocytes life span is 14.1 days, while spermiogenesis in goats lasts 14.9 days. Staging in goats was based on the tubular morphology, where 8 stages of the cycle are yielded for all species. The relative stage frequencies in goats, based on 400 seminiferous tubule cross sections for each animal were as follows: stage 1: 15.8 +/- 1.0%; stage 2: 12.8 +/- 0.5%; stage 3: 20.5 +/- 0.9%; stage 4: 10.7 +/- 0.7%; stage 5: 11.6 +/- 0.6%; stage 6: 9.3 +/- 1.1%; stage 7: 7.6 +/- 0.4%; stage 8: 11.7 +/- 0.6%. The pre-meiotic, meiotic and post-meiotic phases' relative frequencies were 49.1%, 10.7% and 40.2%, respectively. The duration of spermatogenesis in goats is very similar to that found in rams.     

Renewal of spermatogonia in the monkey (Macaca fascicularis)

Populations of different types of spermatogonia and their mitotic activity were analyzed in the monkey Macaca fascicularis: 3 adults aged 5-6 yr and 3 young aged 2-3 mo. Two young and two adult monkeys received injections of 3H-thymidine for radioautographic study of the relationships between Type A spermatogonia: dark Type A (Ad), pale Type A (Ap) and transition Type A (At). In the adult the number of Ad and At spermatogonia did not change significantly throughout the seminiferous epithelium cycle. The number of Ap spermatogonia doubled at Stage VII, and half divided at Stage IX to give rise to B1 spermatogonia. The durations of the seminiferous epithelium cycle and spermatogenesis were estimated as 10.5 days and 42 days respectively. In the young and adult monkeys, some Ap spermatogonia and a lesser number of At spermatogonia were labeled one h after injection of precursor. At longer intervals after injection, the number of labeled At spermatogonia increased significantly, and some Ad as well as Ap spermatogonia were also labeled. These results indicate that Ap spermatogonia are renewal stem cells, and Ad spermatogonia are reserve stem cells. The differences in labeling after isotope exposure suggest that Ap cells may give rise successively to At and Ad cells.     

Relative volume of Sertoli cells in monkey seminiferous epithelium: a stereological analysis.

Techniques of quantitative stereology have been utilized to determine the relative volume occupied by the Sertoli cells and germ cells in two particular stages (I and VII) of the cycle of the seminiferous epithelium. Sertoli cell volume ranged from 24% in stage I of the cycle to 32% in stage VII. Early germ cells occupied 3.4% in stage I (spermatogonia) and 8.7% in stage VII (spermatogonia and preleptotene spermatocytes). Pachytene spermatocytes occupied 15% (Stage I) and 24% (stage VII) of the total volume of the seminiferous epithelium. In stage I the two generations of spermatids comprised 58% of the total epithelium by volume, whereas in stage VII, after spermiation, the acrosome phase spermatids occupied 35% of the total seminiferous epithelial volume.     

Synchronization of the seminiferous epithelium after vitamin A replacement in vitamin A-deficient mice

The effect of vitamin A deficiency and vitamin A replacement on spermatogenesis was studied in mice. Breeding pairs of Cpb-N mice were given a vitamin A-deficient diet for at least 4 wk. The born male mice received the same diet and developed signs of vitamin A deficiency at the age of 14-16 wk. At that time, only Sertoli cells and A spermatogonia were present in the seminiferous epithelium. These spermatogonia were topographically arranged as single and paired cells and as clones of 4, 8 and more cells. A few mitoses of single, paired, and clones of 4 A spermatogonia were found, which were randomly distributed over the seminiferous epithelium. When vitamin A-deficient mice were treated with retinol-acetate combined with a normal vitamin A-containing diet, spermatogenesis restarted again synchronously. Only a few successive stages of the cycle of the seminiferous epithelium were present up to at least 43 days after vitamin A replacement. After 20 days, 98.3% of the seminiferous tubules were synchronized, showing pachytene spermatocytes as the most advanced cell type, mostly being in epithelium stages IX-XII. After 35 and 43 days, spermatogenesis was complete in 99.6% of the tubular cross sections, and most tubular cross sections were in stages IV-VII of the cycle of the seminiferous epithelium. The degree of synchronization was comparable or even higher than found in rats. The rate of development of the spermatogenic cells between 8 and 43 days after vitamin A replacement seemed to be similar to that in normal mice. Assuming that the rate of development of the spermatogenic cells is also normal during the first 8 days after vitamin A replacement, it can be deduced that the preleptotene spermatocytes, present after 8 days, were A spermatogonia in the beginning of stage VIII at the moment of vitamin A replacement. These results indicate that the mouse can be used as a model to study epithelial stage-dependent processes in the testis.     

Isolation and staging of horse seminiferous tubules by transillumination

Stages of the spermatogenic cycle in the horse were determined by trans-illumination of enzymically isolated, seminiferous tubules and were verified by whole-mounted tubules observed by Nomarski optics and by conventional histology. Isolated tubules were obtained from young (less than 2 years) and adult (4-10 years) horses by enzymic digestion. Dispersed tubules were separated into three different groups based on the presence, size, and intensity of a dark region in the centre of the tubules: (1) pale--homogeneously light, (2) spotty--light on the periphery with a wide spotty region in the central two-thirds, or (3) dark--an intensely dark, narrow region through the central one-third. Seminiferous tubules from young stallions separated easily, but were only of the homogeneously light pattern as they lacked mature spermatids. After observation by Nomarski optics and bright-field microscopy, pale tubules under transillumination largely contained Stages I and II, spotty tubules contained Stages V and VI, and dark tubules contained Stages VII and VIII of the spermatogenic cycle. In-vitro incorporation of [3H]thymidine in spermatogonia and preleptotene/leptotene primary spermatocytes of these tubules confirmed the viability of germ cells in isolated tubules, and ultrastructural analysis confirmed excellent preservation of normal structure of seminiferous epithelium in isolated tubules. Hence, segments of seminiferous tubules in specific stages of the spermatogenic cycle can be obtained from enzymically digested horse testes when viewed by transillumination.     

Passive immunization with anti-laminin immunoglobulin G modifies the integrity of the seminiferous epithelium and induces arrest of spermatogenesis in the guinea pig

In the testis, the base of the Sertoli cells is in contact with the basement membrane matrix, in which the laminins constitute the major noncollagenous components. We have previously demonstrated that antibodies against a preparation enriched in basement membranes of seminiferous tubules (STBM) or a noncollagenous fraction of STBM passively transferred induced modifications to the basement membranes and focal sloughing of the seminiferous epithelium in the rat. In the present report, we tested the effect of passive immunization with anti-laminin IgG on the limiting membrane of the seminiferous tubules, spermatogenesis, and maintenance of the blood-testis barrier in the adult guinea pig. Rabbit antibodies to laminin 1 (IgG fraction) were injected in adult male guinea pigs (GP). Nonimmunized GP and GP immunized with normal rabbit serum IgG were used as controls. Measurements of variations in the diameter and lumen of the tubules and in the size of individual components of the tubular limiting membrane showed that the highest percentage of tubules with reduced lumen occurred 30 days after passive immunization with anti-laminin, when the limiting membrane was thickest and lesions to the seminiferous epithelium were most severe. The lesions included thickening of the limiting membrane, infolding in the basal lamina, deposits of immune complexes coincident with sloughing of pachytene spermatocytes and spermatids, and vacuolization of the Sertoli cells. Mononuclear cell infiltration of the tubules was rare. Permeability tracer studies revealed that Sertoli cell tight junctions remained impermeable. Fifty and 80 days after treatment, the basement membrane of the tubules and the progression of the spermatogenesis were normal. Passive immunization with anti-laminin IgG provided a valuable experimental model for the in vivo study of the influence of the basement membrane on the issue of spermatogenesis and the integrity of the seminiferous epithelium.