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.     

Biochemical and morphological characterization of the intra-acrosomal antigen SP-10 from human sperm

The human sperm protein SP-10 was previously defined as a "primary vaccine candidate" by a World Health Organization Taskforce on Contraceptive Vaccines. By one- and two-dimensional immunoblots, we show that SP-10, extracted from ejaculated human sperm, demonstrated a polymorphism of immunogenic peptides from 18 to 34 kDa, a pattern that was conserved from individual to individual and was not altered by reducing agents. The majority of the antigenic peptides possessed isoelectric points of approximately 4.9. Immunocytochemistry on testis sections indicated that SP-10 was localized to round spermatids and spermatozoa within the adluminal compartment of the seminiferous epithelium. Immunofluorescence showed that SP-10 was not associated with the surface of acrosome-intact, ejaculated sperm. Light and electron microscopic immunocytochemistry localized SP-10 throughout the acrosome, and electron microscopic evidence demonstrated a bilaminar array in association with the inner aspect of the outer acrosomal membrane and the outer aspect of the inner acrosomal membrane. After induction of the acrosome reaction with the ionophore A23187, SP-10 remained displayed on the sperm head in association with the inner acrosomal membrane and equatorial segment. The results indicate that the MHS-10 monoclonal antibody may be used as a marker of acrosome development in the human and as a probe to evaluate acrosome status. The results also support the hypothesis that inhibition of sperm-egg interaction by anti-SP-10 monoclonal antibody may occur as a result of antigen exposure following the acrosome reaction.     

Morphological characterization of the testicular cells and seminiferous epithelium cycle in six species of Neotropical bats

We know little about the process of spermatogenesis in bats, a great and diverse clade of mammals that presents different reproductive strategies. In the present study, spermatogenesis in six species of Neotropical bats was investigated by light microscopy. On the basis of chromatin condensation, nuclear morphology, relative position to the basal membrane and formation of the flagellum, three types of spermatogonia were recognized: dark type A (A_d), pale type A (A_p), and type B; the development of spermatids was divided into seven steps. With the exception of Myotis nigricans, the seminiferous epithelium cycle of the other five species studied was similar to those of other mammals, showing gradual stages by the tubular morphology method. Asynchrony was observed in the seminiferous epithelium cycle of M. nigricans, shown by overlapping stages and undefined cycles. The frequencies found in the three phases of the cycle were variable with the greatest frequency occurring in the postmeiotic phase (>50%) and the least in the meiotic phase (<10%). The similarities observed in the five species of Phyllostomidae appeared to be related to their phylogenetic relationship and shorter divergence times, whereas the differences in M. nigricans appeared to be related to its greater phylogenetic distance because the Vespertilionidae family diverged earlier. J. Morphol., 2009. © 2009 Wiley‐Liss, Inc.     

The cycle of the seminiferous epithelium in Landrace boars

The present study describes the morphological features of the eight stages of the seminiferous epithelium in Landrace boars according to the tubular morphology method, as well as their relative frequency, length, and duration. In Landrace boars the pre-meiotic stages occupied the 31.9 +/- 19.9% of the spermatogenic cycle and had a total length of 1788.8 +/- 1153.0 microm and a duration of 2.78 days; they were mainly characterised by the presence of leptotene and pachytene spermatocytes and round spermatids. Meiotic stages, with a relative frequency of 16.4 +/- 6.8%, a length of 787.1 +/- 603.1 microm and a duration of 1.41 days, contained spermatocytes in advanced meiosis I and/or in meiosis II and elongating spermatids grouped in bundles. Post-meiotic stages occupied the 50.6 +/- 20.4% of the spermatogenic cycle and had a length of 2096.8 +/- 1175.0 microm and a duration of 4.37 days; the most important event of these stages was the spermiation, which included the complete remodelling of sperm head and tail and the releasing of spermatozoa into the lumen, as well as the formation of residual bodies. From data obtained we concluded that both germ cell associations of the stages maintain constant among Landrace boars, and that the relative frequency, length and duration of the stages were directly dependent of the cytological transformations on the seminiferous tubules.     

Localization of cellular retinol-binding protein mRNA in rat testis and epididymis and its stage-dependent expression during the cycle of the seminiferous epithelium

Anatomical localization of cellular retinol-binding protein (CRBP) mRNA was examined in normal rat testis and epididymis and also in retinoid-deficient rat testis. In situ hybridization was performed with 35S-labeled rat CRBP cRNA probes on frozen tissue sections. In normal testis, CRBP mRNA was mainly localized in the Sertoli cells and to some extent in peritubular cells. A distinct cyclic variation of the relative levels of hybridizable CRBP mRNA was observed during the spermatogenic cycle. The peak of CRBP mRNA content was seen in the stages of the cycle that preceded those in which peak CRBP protein content had been observed previously in our laboratory by immunohistochemistry. No appreciable amount of CRBP mRNA was observed in the interstitial space or in the lumen of the tubules. CRBP mRNA displayed the same anatomical localization in the retinoid-deficient testis, but the level of hybridizable CRBP mRNA was substantially reduced. A strong hybridization signal for CRBP mRNA was seen in proximal epididymis and was strikingly localized in the ductular epithelium. CRBP mRNA was not detectable in the distal portion of the epididymis. These studies provide information about the cell-specific expression of CRBP synthesis within the testis and epididymis and about its cyclic variation and regulation.     

Duration of the cycle of the seminiferous epithelium and its stages in the rhesus monkey (Macaca mulatta)

Doses of 1 Gy or more of X-irradiation killed all B spermatogonia present in the testis, and during the first 3 weeks after irradiation, virtually no new B spermatogonia were formed. The number of Apale spermatogonia decreased during the first cycle of the seminiferous epithelium while the number of Adark spermatogonia only began to decrease during the second cycle after irradiation. In this study, the duration of the cycle of the seminiferous epithelium in the rhesus monkey was estimated to be 10.5 days (SE = 0.2 days). This was determined following the depletion of germinal cells in the seminiferous epithelium during the first 3 weeks after irradiation. The duration of each of the 12 stages of the cycle was also determined. Our observations of the progress of germinal cell depletion revealed that after a dose of X-irradiation sufficient to kill all B spermatogonia, all spermatocytes disappeared from the testis within about 17 days, and all spermatids within about 31 days.     

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.     

Changes in the volume of Sertoli cells during the cycle of the seminiferous epithelium in the rat

Volume density of Sertoli cells in mature rats was estimated in different stages of the cycle of the seminiferous epithelium. Manual point-counting on electron micrograph montages revealed a more than 40% increase in Sertoli cell volume from just before to just after spermiation. It is suggested that this variation should be considered in studies of stage-dependent cyclic variations in activity of Sertoli cells and/or germ cells.     

Spatial arrangement of the stages of the cycle of the seminiferous epithelium in the Japanese quail, Coturnix coturnix japonica

The spatial arrangement of the stages of the cycle of the seminiferous epithelium of the Japanese quail was investigated by preparing three-dimensional reconstructions of a seminiferous tubule from each of 3 quails. It was found that the stages were not distributed at random, but were arranged in a wave which spiralled helically along a seminiferous tubule. Adjacent stages in space were always adjacent numbers in the cycle of the seminiferous epithelium. Complete spermatogenetic waves were found in which all 10 stages of the cycle were in sequential order. However, in most waves the sequential order of stages was disturbed by the occurrence of modulations. The area of a cellular association varied from 4600 to 41,600 microns 2 with a mean +/- s.e.m. (3 animals) of 17,902 +/- 2614 microns 2. The number of Sertoli cells involved in an association ranged from 4 to 35, with a mean +/- s.e.m. (3 animals) of 13.5 +/- 2.8. The findings support our earlier suggestion that the kinetics of spermatogenesis in the quail are fundamentally similar to the pattern which has been described for mammals.     

Development of the stages of the cycle in mouse seminiferous epithelium after transplantation of green fluorescent protein-labeled spermatogonial stem cells

To study the mechanism of male germ cell differentiation, testicular germ cells carrying green fluorescent protein (GFP) as a transgene marker were transplanted into infertile mouse testis. Fluorescence-positive seminiferous tubule segments colonized with GFP-labeled donor germ cells were isolated and measured, and differentiated germ cells were analyzed in living squashed preparations. Cell associations in normal stages of the seminiferous epithelial cycle were also studied and used as a reference. Two months after transplantation, the average length of the colonies was 1.3 mm. The cell associations of transplanted colonies were consistent with those of normal stages of the cycle. However, stages of the cycle were not necessarily identical in different colonies. Three months after transplantation, the average length of transplanted colonies was 3.4 mm, and the cell association in every portion of a colony was similar to that of the corresponding stage of the cycle. Even in long fused colonies made by transplantation of a higher concentration of male germ cells, the cell association patterns in various regions of a single colony were similar and consistent with those of some of the normal stages of the cycle. Development of different stages inside the colony was observed by 6 mo after transplantation. These results indicate that the commencement of spermatogonial stem cell differentiation occurs randomly to develop different stages of the cycle in different colonies. Then, each colony shows one single stage of the cycle for a long time, even if it becomes a very large colony or fuses with other colonies. These observations indicate the existence of some kind of synchronization mechanism. By 6 mo, however, normal development of the stages of the cycle appeared in seminiferous tubules.     

Enrichment of the stages of the seminiferous epithelium in vitamin A-replaced-vitamin A-deficient rats

Morphometric study revealed that, at 40 days after the start of vitamin A replacement, A1 spermatogonia and preleptotene spermatocytes appeared in more than 70% of the whole mounts of seminiferous tubules of vitamin A-deficient rats. By 42 days, the appearance of these cell types was reduced by 50%, and A2 and A3 spermatogonia were predominant. By 46 days, A1-A3 spermatogonia appeared in less than 30% of the tubular length while A4, intermediate and B spermatogonia became the major cell types in the basement compartment of seminiferous tubules. The predominance of spermatogonia noted at given times was corroborated by higher frequencies of tubular cross-sections of stages in which that particular type of spermatogonium resides. These results indicate that seminiferous tubules of vitamin A-replaced-vitamin A-deficient rats are 'enriched' for particular stages. Tracing the development of [3H]thymidine-labelled preleptotene spermatocytes revealed normal kinetics of germ cell differentiation in these animals. Furthermore, the spermatogonial proliferations in the vitamin A-replaced-vitamin A-deficient rats were quantitatively normal. We suggest that vitamin A replacement may result in temporal suppression of the differentiation of A2-B spermatogonia, leading to a stimulation or synchronization of certain groups of undifferentiating spermatogonia which undergo active proliferation simultaneously. These synchronized populations of spermatogonia continue to proliferate and differentiate, thus resulting in the stage-enrichments noted at later times.     

Spermatogonia and the cycle of the seminiferous epithelium in the nine-banded armadillo

Summary The cycle of the seminiferous epithelium of the nine-banded armadillo can be divided into ten stages. As in most mammals, only one stage is observed per tubular cross-section. The process of spermiogenesis can be divided into thirteen steps according to the development of the acrosomal system and the flagellum. Four generations of spermatogonia are observed in the germinal epithelium: 1) stem cells, 2) type A, 3) intermediate, and 4) type B spermatogonia. The stem cell is characterized by a highly irregular nucleus and the presence of glycogen in its cytoplasm. The type A spermatogonium contains an oblong nucleus with one or two shallow infoldings of the nuclear membrane. The intermediate spermatogonium contains an ovoid nucleus characterized by one or two nuclei and heterochromatin scattered in the nucleoplasm. The nucleus of the type B spermatogonium is more spherically shaped with a centrally placed nucleolus and heterochromatin associated with the nuclear envelope.The author wishes to acknowledge the technical assistance of Teri Lane