Characteristics of the various type A, intermediate (In) and B spermatogonia were determined in C57BL/6J mice using transmission electron microscopy. Spermatogonia were photographed at all stages of the cycle of the seminiferous epithelium. Over 450 images were taken. Spermatogonia could be classified into As, Apr, Aal, A1 cells, A2 cells, A3 cells, A4 cells, intermediate type and type B cells primarily on the basis of nuclear and nucleolar characteristics. The most primitive spermatogonia (As, Apr, Aal) had mottled chromatin; A1 cells contained homogeneously finely granular chromatin throughout the nucleus; A2, A3, A4 and intermediate type spermatogonia had progressively increasing amounts of chromatin encrusting the nuclear envelope; type B spermatogonia had less heterochromatin along the nuclear envelope, although the particles were more dense and rounded than in intermediate type spermatogonia. Mitochondrial size and position of Golgi complexes varied in different types of spermatogonia. These data show that types of spermatogonia can be differentiated such that these characteristics can be used in functional studies.
H Chiarini-Garcia and LD Russell
H Chiarini-Garcia, AM Raymer and LD Russell
The relationships and distribution of spermatogonia were studied as a function of the stage of the seminiferous epithelium cycle in rats. Primitive spermatogonia in the mouse are located along regions of the basal lamina that face the interstitium. Before studying the distribution of spermatogonia in rats, it was necessary to characterize the various types of spermatogonia, as recently performed for mice. The Strauss' linear index (Li) selectivity method was then used and spermatogonia of the A(single) (A(s)) to A(aligned) (A(al)) lineage were preferentially found to be located in regions opposing the interstitium at stages V, VII and IX of the spermatogenic cycle. Because relatively little tubule-to-tubule contact occurs in rats, the aim of this study was to determine whether tubule-to-tubule contact or tubule proximity (or alternatively, the amount of interstitium) was an important factor in spermatogonial position. In this regard, another method (tubule proximity) was devised to determine spermatogonial position that accounted for the presence of adjacent tubules. This method showed that the position of tubules, rather than tubule contact, was more accurate than the Li method in determining the location of spermatogonia in the rat. The results also showed a non-random distribution of spermatogonia resembling that of the mouse, and that tubule-to-tubule contact is not essential for the positioning of spermatogonia. In conclusion, the results of this study strongly indicate that the most primitive type A spermatogonia (A(s), A(paired) and A(al)) in rats are present in niches located in those areas of the seminiferous tubules that border the interstitial tissue.
C A Rezende-Melo, A L Caldeira-Brant, Al Drumond-Bock, G M Buchold, G Shetty, F R.c.l. Almeida, M M Matzuk, K. Hara, S Yoshida, M L Meistrich and H Chiarini-Garcia
The existence of cytoplasmic passages between germ cells and their potential function in the control of the spermatogenic process has long been an intriguing question. Evidence of the important role of such structures, known as intercellular bridges (ICB), in spermatogenesis has been implicated by the failure of spermatogenesis in Tex14 mutant mice, which lack the ICBs, to progress past the pachytene spermatocyte stage. Using these Tex14 mutants, the present study evaluated, for the first time, the behavior and synchrony of the spermatogonial lineage in the absence of ICBs. Our data suggest that the absence of these cytoplasmic connections between cells affects the expansion of the undifferentiated type A (Aundiff) spermatogonia compartment and their transition to A1, resulting in a significant numerical reduction of differentiating A1 spermatogonia, but did not interfere with cell amplification during subsequent mitotic steps of differentiating spermatogonia from A1 through intermediate (In). However, beginning at the type B spermatogonia, the synchrony of differentiation was impaired as some cells showed delayed differentiation compared to their behavior in a normal seminiferous epithelium cycle. Thus although spermatogonial development is able to proceed, in the absence of ICBs in Tex14-/- mutants, the yield of cells, specific steps of differentiation, the synchrony of the cell kinetics, and the subsequent progression in meiosis are quantitatively lower than normal.
A L Caldeira-Brant, L M Martinelli, M M Marques, A B Reis, R Martello, F R C L Almeida and H Chiarini-Garcia
Human spermatogonial stem cells (SSCs) are an essential source to maintain spermatogenesis as an efficient process for daily sperm production with high self-renewal capacity along adulthood. However, the phenotype and the subpopulation that represent the real reserve SSC for the human testis remain unknown. Moreover, although SSC markers have been described for undifferentiated spermatogonia (Adark and Apale), the existence of a specific subtype that could be identified as the actual/true SSC has not yet been fully determined. Herein we evaluated spermatogonial morphology, kinetics, positioning regarding blood vasculature in relation to protein expression (UTF1, GFRA1, and KIT) as well as proliferative activity (MCM7) and identified a small subpopulation of Adark with nuclear rarefaction zone (AdVac) that behaves as the human reserve SSC. We show that AdVac is the smallest human spermatogonial population (10%), staying quiescent (89%) and positioned close to blood vessels throughout most of the stages of the seminiferous epithelium cycle (SEC) and divides only at stages I and II. Within this AdVac population, we found a smaller pool (2% of A undifferentiated spermatogonia) of entirely quiescent cells exhibiting a high expression of UTF1 and lacking GFRA1. This finding suggests them as the real human reserve SSC (AdVac UTF1+/GFRA1-/MCM7-). Additionally, Adark without nuclear vacuole (AdNoVac) and Apale have similar kinetic and high proliferative capacity throughout the SEC (47%), indicating that they are actively dividing undifferentiated spermatogonia. Identification of human stem cells with evident reserve SSC functionality may help further studies intending to sort SSCs to treat male diseases and infertility.