Mounts Of Seminiferous Tubules Research Articles

Computer Simulation of the Rodent Spermatogonial Stem Cell Niche1

A computer program has been developed that simulates the behavior of spermatogonial stem cells (SSCs) and their offspring inside and outside of the stem cell niche. Various parameters derived from previous morphological and cell kinetic studies have been used to set up an Excel-based computer program that simulates the proliferative activity of SSCs during the seminiferous epithelial cycle. SSCs and their offspring are depicted in a virtual piece of seminiferous tubule in which the daughter cells of self-renewing divisions of SSCs migrate away from each other, while after SSC differentiation a pair of cells is formed. Those SSC daughter cells that migrate out of the niche will very likely differentiate at their next division. Putting in physiologically acceptable parameters, the program renders numbers of spermatogonial cell types similar to those previously counted in whole mounts of seminiferous tubules. In this model, SSC numbers and numbers of differentiating cells remain constant for more than 50 virtual epithelial cycles, i.e., more than 1 yr of a mouse life and 2 yr of that of a Chinese hamster. The program can simulate various recent cell kinetic experiments and confirms, or offers alternative explanations for, the results obtained, showing its usefulness in spermatogenesis research.

Clonal organization of proliferating spermatogonial stem cells in adult males of two species of non-human primates, Macaca mulatta and Callithrix jacchus.

The present study examines the existence of clonogenic patterns in the proliferation and differentiation of spermatogonial stem cells in two species of non-human primates, the marmoset and the rhesus monkey. We developed a novel approach to detect proliferating spermatogonial clones in whole mounts of seminiferous tubules. Dual fluorescence labeling of bromodeoxyuridine and acrosin in conjunction with confocal microscopy allows the description of the clonogenic and spatial arrangement of proliferating spermatogonia at specific stages of the seminiferous epithelial cycle. Cross-sections of paraffin-embedded tissue were labeled by the same approach. For both monkey species we demonstrate the presence of proliferating spermatogonial clones of variable size at specific stages of the cycle of the seminiferous epithelium. Detailed analysis of the rhesus monkey reveals proliferating Apale spermatogonia at stages VII and IX of the cycle of the seminiferous epithelium, and of proliferating B spermatogonia at stages II, IV, VI, and XII. Proliferating Apale spermatogonia at stages VII and IX of the cycle are organized in pairs or quadruplets. B1 spermatogonia appear as quadruplets or eight-cell clones, and B2 spermatogonia as 8- or 16-cell clones. We conclude that spermatogenesis in the rhesus monkey is initiated by two divisions of duplets or quadruplets of Apale spermatogonia: a first division at stage VII, after which the clones of Apale spermatogonia separate, and a second division at stage IX, which leads to clones of B1 spermatogonia as well as pairs and quadruplets of Apale spermatogonia replenishing the seminiferous epithelium to maintain the original size of the A spermatogonial population.

Intercellular bridges and apoptosis in clones of male germ cells.

When an As spermatogonium divides to form a pair of Apr spermatogonia the two daughter cells stay interconnected by an intercellular bridge. These cytoplasmic bridges form after every subsequent division leading to large clones of interconnected germ cells. Cohorts of spermatogonia maintain synchronous development throughout spermatogenesis, which has been attributed to the presence of these intercellular bridges. To examine whether apoptotic signals are transduced through the intercellular bridges we studied germ cell apoptosis in whole mounts of seminiferous tubules from non-irradiated and irradiated mouse testes, using whole mount seminiferous tubules and confocal microscopy. This allowed us to use TUNEL staining of apoptotic germ cells and at the same time to study these apoptotic germ cells in their topographical context. Our results show that in response to ionizing radiation single spermatogonia within a clone can undergo apoptosis without affecting their neighboring cells. Additionally, also early spermatocytes were shown to undergo apoptosis individually. Both radiation-induced spermatogonial apoptosis and spontaneous apoptosis of spermatocytes are caused by DNA damage of individual cells. Degeneration of healthy spermatogonia because of regulatory signals, however, follows other death inducing mechanisms, which lead to apoptosis of chains of interconnected spermatogonia.

Arrest of Spermatogonial Differentiation in jsd/jsd, Sl17H/Sl17H, and Cryptorchid Mice

The nature of the spermatogenic arrest in cryptorchid C57Bl mice and in jsd/jsd and Sl17H/Sl17H mutant mice was identified by studying whole mounts of seminiferous tubules. In all three types of mice, virtually only A spermatogonia were found, topographically arranged in clones of 1 to 16 (rarely more) cells. These clonal sizes are typical for undifferentiated spermatogonia. The proportion of these cells lying in chains of more than 2 cells (50-70%) was comparable to that seen in epithelial stages VII-VIII in the normal epithelium. It is concluded that in all three types of mice, spermatogenesis is arrested at the point where the undifferentiated A spermatogonia, specifically A(al) spermatogonia, differentiate into the first generation of the differentiating-type spermatogonia, the A1 spermatogonia. The remaining A spermatogonia were proliferating, but no accumulation of spermatogonia was present, as spermatogonial apoptosis also took place. Spermatogonial clones of all sizes were seen to undergo apoptosis, but there were relatively many large apoptotic clones, indicating that the clones became more vulnerable when they became larger. In contrast to what is seen in the normal epithelium, odd-numbered clones, not composed of 2(n) cells, were present, as well as clumps of 2 or more spermatogonial nuclei in the same cytoplasm, in all three types of mice. This indicates a lack of integrity of spermatogonial clones, also observed in other situations with a relative paucity of cells on the basal membrane. It is concluded that the differentiation of the undifferentiated spermatogonia, affected in all three types of mice as well as in vitamin A-deficient animals, is a rather vulnerable point in the spermatogenic developmental pathway.

Alteration of mRNA transcript levels of rat testicular cells following procarbazine administration.

The present study examined the effects of a single 400-mg/kg dose of procarbazine upon various testicular mRNA transcript levels in adult Sprague-Dawley rats. Northern blot hybridization was employed to measure the steady-state mRNA levels of proteins specific for Sertoli cells (adrogen-binding protein [ABP] and transferrin) and spermatids (protamine 1 and transitional protein 2). In addition, mRNA transcript levels were determined for germ cell-specific hemiferrin and insulin-like growth factor-1 (IGF-1), which in adult rats is present predominantly in primary spermatocytes. Furthermore, the chronology of the effects of procarbazine upon spermatogonial populations was examined in whole mounts of seminiferous tubules. The effect of procarbazine upon spermatogenesis was first noted among mature A3, young A4, and B spermatogonia 48-72 hours after drug administration. These results indicate that A2 through intermediate spermatogonia were most susceptible to procarbazine. In addition, degeneration of spermatocytes and young spermatids was evident by 5 days. Northern blot analysis of testicular poly (A)+ RNA revealed that the steady-state levels of mRNA transcripts of the spermatid nuclear proteins (protamine 1, 700 bp; and transitional protein 2, 580 bp) remained relatively unchanged for 5 days after procarbazine administration but were significantly decreased by more than 70% by 7 days. On the other hand, the steady-state mRNA levels for the Sertoli cell proteins ABP (1.7 kb) and transferrin (2.7 kb) were decreased by 45% and 50% (P < 0.05), respectively, 2 days after procarbazine administration. Significant suppression of ABP mRNA levels persisted through day 5, with partial recovery noted on day 7.(ABSTRACT TRUNCATED AT 250 WORDS)

Response to Fission Neutron Irradiation of Spermatogonial Stem Cells in Different Stages of the Cycle of the Seminiferous Epithelium

Mice were irradiated with 1 Gy of fission neutrons. At intervals up to 15 days after irradiation undifferentiated spermatogonia were counted in whole mounts of seminiferous tubules in up to eight stages of the epithelial cycle. From Day 6 onward lower numbers of spermatogonia were found in the areas which were in stages IV-VII during irradiation than in those which were in stages IX-II. Minimal numbers in the former area were two to six times lower than those in the latter one. Areas which were in stages III or VIII gave intermediate values. It is concluded that the epithelial cycle can be divided into two parts with a different response to irradiation, that begin or end in stages III and VIII. Part III-VIII and part VIII-III comprise 45 and 55% of the epithelial cycle, respectively. In part VIII-III control levels were found again at Day 15, while in part III-VIII spermatogonial numbers were still very low. In controls it was found that part VIII-III corresponds to a period of high proliferative activity of the stem cells, while in part III-VIII the proliferative activity is very low. This may affect their radiosensitivity and/or their proliferative behavior after irradiation, resulting in different spermatogonial numbers in the two parts of the epithelial cycle. Unlike in normal epithelium, after irradiation giant cells, odd-numbered clones (not containing 2" cells), and clones of Apr and Aal , in which the composing cells clump together, were observed.

Cell cycle properties of differentiating spermatogonia in adult Sprague-Dawley rats.

ABSTRACTThe duration of the mitotic cycle and of its components was analysed for each of the six successive generations of differentiating spermatogonia (A1, A2, A3, A4, intermediate and B), using radioautographed whole mounts of seminiferous tubules from testes of adult Sprague‐Dawley rats. Cell cycles were determined from two successive waves of per cent labeled metaphases obtained during the period of 81 hr after a single dose of 3H‐thymidine. Except for the A1 spermatogonia, all spermatogonial types (A2 to B) had similar cell cycle durations of 41‐42.5 hr and comparable pre‐DNA synthesis phases (G1) of 11‐13 hr. Although the combined duration of DNA synthesis (S) and the post‐synthesis phase (G2) remained identical for all the cell types including A1, there was a progressive lengthening of the S period at the expense of G2 during the process of spermatogonial maturation. This change was most marked during the transition from A1 to A3 spermatogonia when the S period increased from 14 hr to 21 hr, and the G2 phase shortened from 13 hr to 7.5 hr. This feature seems to be unique to germ cells and may be associated with an increasing amount of heterochromatin in the nucleus. Excluding the development of type A1 cells, the entire process of spermatogonial maturation lasted for 208 hr. Combined data on cell cycle times indicated that every 313 hr or 13 days, a new sequence of spermatogonial differentiation was initiated by the A1 cells. This was equivalent to the duration of one ‘cycle’ of the seminiferous epithelium as measured by other techniques.

Role of spermatogonia in the repair of the seminiferous epithelium following x-irradiation of the rat testis.

AbstractIn the normal adult rat testis, type A0 spermatogonia do not appear to participate to a significant extent in the production of spermatocytes, while type A1 spermatogonia periodically initiate a series of divisions resulting in the production of spermatocytes and new type A1 spermatogonia.The behavior of type A0 and A1 spermatogonia was investigated following administration of a single dose of x‐rays (300 r) to the testis. Using whole mounts of seminiferous tubules, the type A0 and A1 cells were counted at various intervals after irradiation. At 8 and 13 days after irradiation, type A1 spermatogonia reached lowest values, i.e., 6% and 3% of non‐irradiated control, while type A0 reached the lowest value, i.e., 62% of control at eight days. Thereafter the numbers of type A0 and A1 progressively increased to return to normal at 39 days. It was thus concluded that the type A0 were comparatively more resistant to x‐irradiation than type A1 spermatogonia.To verify if the surviving type A0 proliferated in the irradiated testis, animals were injected with 3H‐thymidine three hours before they were sacrificed at various times after x‐irradiation. In irradiated testes the labeling indices of the surviving type A (A0, A1–A4) were the same as in the non‐irradiated testes except in stages V‐VI of the cycle of the seminiferous epithelium. While in the controls only 2% of type A cells were labeled at these two stages of the cycle, after irradiation the labeling index of type A reached a maximum of 31% at 13 days to return to control values by 39 days. Since at 13 days after irradiation type A0 spermatogonia were the predominant component of the spermatogonial population, it was concluded that these cells must have incorporated 3H‐thymidine and thereby contributed to the reconstruction of the spermatogonial population partially destroyed by irradiation.