Evaluation of the seminiferous epithelial cycle, spermatogonial kinetics and niche in donkeys (Equus asinus)

Kinetics of spermatogonia as well as localization in niches have been described in rodents, but rarely in large animals or in species of economical interest. In this regard, and envisioning the possibility of spermatogonial transplantation from donkeys (Equus asinus) to mules (Equus mulus mulus), many variables that may contribute for an enhanced understanding of the spermatogonial biology in donkeys were investigated. Testes from five adult donkeys were routinely processed for high-resolution light microscopy. Donkey seminiferous epithelium can be divided in XII stages based on the development of the acrosomal system. In addition, spermatogonial morphology and morphometric analysis were performed allowing the characterization of two groups of spermatogonia: undifferentiated (A_und) and differentiating (A₁, A₂, A₃, B₁ and B₂). A_und spermatogonia were present along all XII stages of the seminiferous epithelium cycle of this species, whereas differentiating spermatogonia were only at specific stages. Number of differentiating spermatogonia gradually increased as the cycle progressed, despite the apparent rigid regulation of the balance between mitosis and apoptosis throughout the spermatogenic process. Understanding of spermatogonial biology and kinetics in donkeys, revealed that type A_und spermatogonia are located in specific microenvironments, the spermatogonial niches. The present results enhance understanding of spermatogonial biology in donkeys providing information about subtypes, morphology, number and mitosis/apoptosis along the seminiferous epithelium cycle.     

Feedback Regulation of the Proliferation of the Undifferentiated Spermatogonia In the Chinese Hamster By the Differentiating Spermatogonia

In the seminiferous epithelium the differentiating spermatogonia proliferate following a very strict synchronous pattern, and undergo the S phase during parts of particular epithelial stages. the undifferentiated spermatogonia do not divide synchronously and display maximum proliferative activity in stages XI-III. Hence the S-phase-specific cytotoxic agent Ara-C kills different proportions of these two cell types dependent on the epithelial stage. We have studied the effect of several combinations of degrees of cell loss to both compartments on proliferation of the undifferentiated spermatogonia. It was found that when the differentiating spermatogonia are removed, the proliferation of the undifferentiated spermatogonia is not inhibited at epithelial stage III, as seen in controls. However, when the undifferentiated spermatogonia were already arrested in G₁, removal of the differentiating spermatogonia did not evoke proliferation again. When the population of undifferentiated spermatogonia was reduced in an area where the differentiating spermatogonia were left intact, the inhibition of the proliferation of undifferentiated spermatogonia took place around stage III as usual. It is concluded that in the normal adult seminiferous epithelium, the length of the period of active proliferation of the undifferentiated spermatogonia is regulated by negative feedback from the differentiating spermatogonia.     

The pluripotency factor LIN28 marks undifferentiated spermatogonia in mouse

Background  

Life-long production of spermatozoa depends on spermatogonial stem cells. Spermatogonial stem cells exist among the most primitive population of germ cells – undifferentiated spermatogonia. Transplantation experiments have demonstrated the functional heterogeneity of undifferentiated spermatogonia. Although the undifferentiated spermatogonia can be topographically divided into A_s (single), A_pr (paired), and A_al (aligned) spermatogonia, subdivision of this primitive cell population using cytological markers would greatly facilitate characterization of their functions.     

A quantitative study of spermatogonial multiplication and stem cell renewal in the C3H/101 F1 hybrid mouse

In whole mounts of seminiferous tubules of C3H/101 F₁ hybrid mice, spermatogonia were counted in various stages of the epithelial cycle. Furthermore, the total number of Sertoli cells per testis was estimated using the disector method. Subsequently, estimates were made of the total numbers of the different spermatogonial cell populations per testis.

The results of the cell counts indicate that the undifferentiated spermatogonia are actively proliferating from stage XI until stage IV. Three divisions of the undifferentiated spermatogonia are needed to obtain the number of A₁ plus undifferentiated spermatogonia produced each epithelial cycle. Around stage VIII almost two-thirds of the A_pr and all of the A_al spermatogonia differentiate into A₁ spermatogonia. It was estimated that there are 2.5 × 10⁶ differentiating spermatogonia and 3.3 × 10⁵ undifferentiated spermatogonia per testis. There are about 35,000 stem cells per testis, constituting about 0.03% of all germ cells in the testis. It is concluded that the undifferentiated spermatogonia, including the stem cells, actively proliferate during about 50% of the epithelial cycle.     

Feedback regulation of the proliferation of the undifferentiated spermatogonia in the Chinese hamster by the differentiating spermatogonia

In the seminiferous epithelium the differentiating spermatogonia proliferate following a very strict synchronous pattern, and undergo the S phase during parts of particular epithelial stages. The undifferentiated spermatogonia do not divide synchronously and display maximum proliferative activity in stages XI-III. Hence the S-phase-specific cytotoxic agent Ara-C kills different proportions of these two cell types dependent on the epithelial stage. We have studied the effect of several combinations of degrees of cell loss to both compartments on proliferation of the undifferentiated spermatogonia. It was found that when the differentiating spermatogonia are removed, the proliferation of the undifferentiated spermatogonia is not inhibited at epithelial stage III, as seen in controls. However, when the undifferentiated spermatogonia were already arrested in G1, removal of the differentiating spermatogonia did not evoke proliferation again. When the population of undifferentiated spermatogonia was reduced in an area where the differentiating spermatogonia were left intact, the inhibition of the proliferation of undifferentiated spermatogonia took place around stage III as usual. It is concluded that in the normal adult seminiferous epithelium, the length of the period of active proliferation of the undifferentiated spermatogonia is regulated by negative feedback from the differentiating spermatogonia.     

EFFECT OF A SPERMATOGONIAL CHALONE ON THE GROWING RAT TESTIS

Immature rats were used in an experiment to test the possible influence of a spermatogonial chalone on the expanding spermatogonial population in their developing testes. An extract from adult rat testes was injected intraperitoneally into 33-day-old rats and control animals were injected with an equal amount of saline. Two groups of normal adult rats similarly injected with the testicular extract and saline solutions served as additional controls. Following these injections, all animals were administered a dose of ³H-thymidine 10 hr before sacrifice. An analysis of the labeling indices of the various types of spermatogonia revealed that in young rats injected with testicular extract the percentage of labeled type A spermatogonia was significantly lower than in control animals. In contrast, the labeling indices of Intermediate and type B spermatogonia were similar in the two groups of young rats. In the two groups of adult animals, there was no difference in the labeling indices of type A or of other types of spermatogonia. These data indicated that the saline extract of adult testes contained a substance, a spermatogonial chalone, inhibiting specifically the proliferation of some type A spermatogonia. The results also support the concept that a spermatogonial chalone may intervene, through its action on the spermatogonial stem cell population, to arrest the growth of the seminiferous tubules as the animal reaches maturity.     

SPERMATOGENETIC CLONES DEVELOPING FROM REPOPULATING STEM CELLS SURVIVING A HIGH DOSE OF AN ALKYLATING AGENT

We investigated stem cell renewal and differentiation in 10- and 15-days-old spermatogonial clones developing in mouse seminiferous epithelium after an extremely large cell loss, inflicted by high doses of the alkylating agent Myleran. The spermatogonial clones arise from cells that resemble the A_is spermatogonia but have a larger nuclear diameter. In spite of their mitotic activity these ‘re-populating stem cells’ lie mainly isolated or in pairs. This is explained by migration and differentiation. Migration appeared to occur at random in all directions along the basement membrane of the seminiferous tubule. After one or more divisions of the stem cells, a second type of cell appears, which is called the ‘differentiating spermatogonium’. The time elapsing before this type of cell appears, depends on the dose of Myleran: the larger the dose the later differentiation starts. A relation could be demonstrated between the stage of the cycle of the seminiferous epithelium and the start of differentiation. Differentiating cells were found isolated or in groups of two, four, eight or sixteen cells. Hence we concluded that at least up to their fourth division differentiating cells divide synchronously without degenerations. Three types of division of repopulating stem cells were distinguished, producing (1) two repopulating stem cells, (2) one repopulating stem cell and one cell starting spermatogonial differentiation, or (3) two differentiating cells. Type 1 divisions were found most frequently.     

Regulation of proliferation and differentiation in spermatogonial stem cells: the role of c-kit and its ligand SCF

To study self-renewal and differentiation of spermatogonial stem cells, we have transplanted undifferentiated testicular germ cells of the GFP transgenic mice into seminiferous tubules of mutant mice with male sterility, such as those dysfunctioned at Steel (Sl) locus encoding the c-kit ligand or Dominant white spotting (W) locus encoding the receptor c-kit. In the seminiferous tubules of Sl/Sl(d) or Sl(17H)/Sl(17H) mice, transplanted donor germ cells proliferated and formed colonies of undifferentiated c-kit (-) spermatogonia, but were unable to differentiate further. However, these undifferentiated but proliferating spermatogonia, retransplanted into Sl (+) seminiferous tubules of W mutant, resumed differentiation, indicating that the transplanted donor germ cells contained spermatogonial stem cells and that stimulation of c-kit receptor by its ligand was necessary for maintenance of differentiated type A spermatogonia but not for proliferation of undifferentiated type A spermatogonia. Furthermore, we have demonstrated that their transplantation efficiency in the seminiferous tubules of Sl(17H)/Sl(17H) mice depended upon the stem cell niche on the basement membrane of the recipient seminiferous tubules and was increased by elimination of the endogenous spermatogonia of mutant mice from the niche by treating them with busulfan.     

The spermatogonial stem cell niche in the collared peccary (Tayassu tajacu)

In the seminiferous epithelium, spermatogonial stem cells (SSCs) are located in a particular environment called the "niche" that is controlled by the basement membrane, key testis somatic cells, and factors originating from the vascular network. However, the role of Leydig cells (LCs) as a niche component is not yet clearly elucidated. Recent studies showed that peccaries (Tayassu tajacu) present a peculiar LC cytoarchitecture in which these cells are located around the seminiferous tubule lobes, making the peccary a unique model for investigating the SSC niche. This peculiarity allowed us to subdivide the seminiferous tubule cross-sections in three different testis parenchyma regions (tubule-tubule, tubule-interstitium, and tubule-LC contact). Our aims were to characterize the different spermatogonial cell types and to determine the location and/or distribution of the SSCs along the seminiferous tubules. Compared to differentiating spermatogonia, undifferentiated spermatogonia (A(und)) presented a noticeably higher nuclear volume (P < 0.05), allowing an accurate evaluation of their distribution. Immunostaining analysis demonstrated that approximately 93% of A(und) were GDNF receptor alpha 1 positive (GFRA1(+)), and these cells were preferentially located adjacent to the interstitial compartment without LCs (P < 0.05). The expression of colony-stimulating factor 1 was observed in LCs and peritubular myoid cells (PMCs), whereas its receptor was present in LCs and in GFRA1(+) A(und). Taken together, our findings strongly suggest that LCs, different from PMCs, might play a minor role in the SSC niche and physiology and that these steroidogenic cells are probably involved in the differentiation of A(und) toward type A(1) spermatogonia.     

The sensitivity of quiescent and proliferating mouse spermatogonial stem cells to X irradiation.

The radiosensitivity of spermatogonial stem cells to X rays was determined in the various stages of the cycle of the seminiferous epithelium of the CBA mouse. The numbers of undifferentiated spermatogonia present 10 days after graded doses of X rays (0.5-8.0 Gy) were taken as a measure of stem cell survival. Dose-response relationships were generated for each stage of the epithelial cycle by counting spermatogonial numbers and also by using the repopulation index method. Spermatogonial stem cells were found to be most sensitive to X rays during quiescence (stages IV-VII) and most resistant during active proliferation (stages IX-II). The D0 for X rays varied from 1.0 Gy for quiescent spermatogonial stem cells to 2.4 Gy for actively proliferating stem cells. In most epithelial stages the dose-response curves showed no shoulder in the low-dose region.     

CELL CYCLE PROPERTIES OF DIFFERENTIATING SPERMATOGONIA IN ADULT SPRAGUE-DAWLEY RATS

The 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.     

Sex-inversion of the hermaphroditic, protogynous teleost Coris julis L. (Labridae)

Sex-inversion of Coris julis , a protogynous hermaphrodite, was studied using histological and cytological criteria. Four stages were recognized: oocyte and oogonial atresia; occurrence of spermatogonia with primordial germ cells (PGC) in the ovarian wall; onset of spermatogonial proliferation; considerable proliferation of spermatogonia and PGC with build-up of seminiferous tubules. Spermatogonia arise from PGC, undifferentiated and bipotential cells, in which mitotic activity was detected.     

Essential role of mouse Dead end1 in the maintenance of spermatogonia

Dead end is a vertebrate-specific RNA-binding protein implicated in germ cell development. We have previously shown that mouse Dead end1 (DND1) is expressed in male embryonic germ cells and directly interacts with NANOS2 to cooperatively promote sexual differentiation of fetal germ cells. In addition, we have also reported that NANOS2 is expressed in self-renewing spermatogonial stem cells and is required for the maintenance of the stem cell state. However, it remains to be determined whether DND1 works with NANOS2 in the spermatogonia. Here, we show that DND1 is expressed in a subpopulation of differentiating spermatogonia and undifferentiated spermatogonia, including NANOS2-positive spermatogonia. Conditional disruption of DND1 depleted both differentiating and undifferentiated spermatogonia; however, the numbers of A_single and A_paired spermatogonia were preferentially decreased as compared with those of A_aligned spermatogonia. Finally, we found that postnatal DND1 associates with NANOS2 in vivo, independently of RNA, and interacts with some of NANOS2-target mRNAs. These data not only suggest that DND1 is a partner of NANOS2 in undifferentiated spermatogonia as well as in male embryonic germ cells, but also show that DND1 plays an essential role in the survival of differentiating spermatogonia.     

E-cadherin can be expressed by a small population of rat undifferentiated spermatogonia in vivo and in vitro

Spermatogonial stem cells (SSCs) maintain gamete production in the testes throughout adult life by balancing self-renewal and differentiation. In vitro culture of SSCs is a crucial technique for gene manipulation of SSCs to generate transgenic animals, for transplantation of SSCs to restore male fertility for infertile man, and for generation of pluripotent stem cells from SSCs to differentiate into various cell lineages. Isolation of highly purified SSCs is an all-important component for development of these techniques. However, definitive markers for SSCs, which purify SSCs (100% enrichment), are unknown. SSCs of many species can colonize the mouse testis; thus, we reasoned that same molecules of SSCs are conserved between species. In mouse, undifferentiated spermatogonia express the surface marker E-cadherin. The hypothesis tested in this work was that E-cadherin (also known as CDH1) can be expressed by undifferentiated spermatogonia of rat testes. In this paper, cross-section immunohistochemistry and whole-mount immunohistochemistry of rat seminiferous tubules were conducted to show that E-cadherin-positive cells were small in number and there are single, paired, and aligned spermatogonia attached along the basement membrane. During in vitro culture period, the undifferentiated rat spermatogonial colonies co-expressed E-cadherin and glial-derived neurotrophic factor family receptor alpha-1 or E-cadherin and promyelocytic leukemia zinc finger. Data collected during the study demonstrate that E-cadherin is expressed by a small population of rat undifferentiated spermatogonia both in vivo and during in vitro culture period.     

Spermatogenetic clones developing from repopulating stem cells surviving a high dose of an alkylating agent.

We investigated stem cell renewal and differentiation in 10- and 15-days-old spermatogonial clones developing in mouse seminiferous epithelium after an extremely large cell loss, inflicted by high doses of the alkylating agent Myleran. The spermatogonial clones arise from cells that resemble the Ais spermatogonia but have a larger nuclear diameter. In spite of their mitotic activity these 'repopulating stem cells' lie mainly isolated or in pairs. This explained by migration and differentiation. Migration appeared to occur at random in all directions along the basement membrane of the seminiferous tubule. After one or more divisions of the stem cells, a second type of cell appears, which is called the 'differentiating spermatogomium'. The time elapsing before this type of cell appears, depends on the dose of Myleran: the larger the dose the later differentiation starts. A relation could be demonstrated between the stage of the cycle of the seminiferous epithelium and the start of differentiation. Differentiating cells were found isolated or in groups of two, four, eight or sixteen cells. Hence we concluded that at least up to their fourth division differentiating cells divide synchronously without degenerations. Three types of division of repopulating stem cells were distinguished, producing (1) two repopulating stem cells, (2) one repopulating stem cell and one cell starting spermatogonial differentiation, or (3) two differentiating cells. Type 1 divisions were found most frequently.     

SPERMATOGONIAL CHALONE(S): EFFECT ON THE PHASES OF THE CELL CYCLE OF TYPE A SPERMATOGONIA IN THE RAT

Adult rats with X-irradiated testes were used to analyze the effect of the spermatogonial chalone(s) on the phases of the cell cycle of type A spermatogonia. Twelve days after irradiation, the animals were used in two experiments designed to test the existence of hypothetical G₂ and S phase chalones. For the G₂ assay, rats injected twice with testicular extract (Group I), liver extract (Group II) or physiological saline (Group III) were killed 10 hr after the initial injection. Mitoses of type A, Intermediate and type B spermatogonia were counted in whole mounts of dissected seminiferous tubules. To test for an S phase inhibitor, two groups of rats were given multiple injections of either testicular extract (Group IV) or saline solution (Group V). Twenty-two hr after the first injection they were injected with [³H]thymidine and killed 2 hr later. Silver grains over labelled type A nuclei were counted in radioautographed sections of testes from these animals. The average grain counts were identical in Groups IV and V, indicating that the testicular extract did not affect type A spermatogonia during the S phase. Counts of type A mitoses in Groups I, II and III revealed that in the animals injected with the testicular extract (Group I) the number of divisions was 50% lower than in the control groups (Groups II and III). In contrast, mitotic activity of differentiating spermatogonia (In + B) was similar in all three groups of animals. This result is attributed to a testicular chalone which specifically inhibits type A spermatogonia during the G₂ phase of the cell cycle. Indirect evidence for a G₁ spermatogonial chalone is also presented, as a result of an analysis of published data (Clermont & Mauger, 1974).     

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.