Figure 1.
Stages of the seminiferous epithelium cycle (A) and their frequencies (B) in stallions.
A) The following symbols were used to designate specific germ cell types: A1, type A1 spermatogonia; A2, type A2 spermatogonia; B1, type B1 spermatogonia; B2, type B2 spermatogonia; P, pachytene spermatocyte; D, diplotene spermatocyte; M, meiotic figure; R, round spermatids; E, elongating/elongated spermatids; SC, Sertoli cell. Arabic numerals (1–12) indicate each step of the spermatid acrosome development. B) Note that stages I, VII and XII presented the highest frequencies, whereas the opposite was observed for stages II, III, IV and XI. White and black bars = 5 µm.
Figure 2.
Spermatogonial types and their kinetics in the stallion seminiferous epithelium cycle.
A) High-resolution light photomicrographies of spermatogonial cells: Aund and differentiated spermatogonia (type A1, A2, A3, B1 and B2), showing their nuclear size and details that allowed their morphological identification. B) Nuclear volume of the different spermatogonial types characterized showing that A2 presented the highest value, particularly in comparison to type B2 spermatogonia. C) Number (kinetics) of Aund and differentiated spermatogonial cells and preleptotene spermatocytes (Pl) per 1000 Sertoli cell nuclei. Note that, except for stages I and II, the values obtained for differentiated spermatogonia increased gradually, whereas the numbers of Aund were relatively stable, reaching their lowest level at stage VII. D) Illustration of the immunolocalization of active caspase-3 in differentiated type A (A1, A2, and A3) spermatogonial cells. Figures A and D, bar = 5 µm.
Figure 3.
Immunostaining evaluation of the presence of GFRA1 in equids.
A) As it can be noted, the expression of this marker was limited to the cytoplasm of Aund (arrowheads) and this pattern was similar for horse (A2–4), donkey (A6–8) and mule (A10–12). A1, A5 and A9 are the negative controls. B) Immunoblotting confirmed the expression of GFRA1 in the testis of horse [during the breeding (BS) and non-breeding (NBS) season], donkey and mule. C) Percentage of GFRA1(+) Aund cells showing that approximately 90% of these cells express this membrane receptor (*p<0.05). Figure A, bar = 10 µm.
Figure 4.
Immunostaining evaluation of the presence of PLZF in equids.
A) As it can be observed, the expression of this marker was present in the nucleus of Aund (arrowheads) and this pattern was similar for the three equid species investigated (horse, A2–4; donkey, A6–8; and mule, A10–12). Negative controls are shown in A1, A5 and A9. B) Expression of PLZF was confirmed by immunoblotting in the horse testis [during the breeding (BS) and non-breeding (NBS) season], donkey and mule. C) Approximately 80% (*p<0.05) of Aund express this transcription factor. Figure A, bar = 10 µm.
Figure 5.
Immunostaining evaluation of the presence of CSF1R in equids.
A) As it can be noted, the expression of this marker was limited to the cytoplasm of Aund (arrowheads) and this pattern was similar for horse (A2–4), donkey (A6–8) and mule (A10–12). A1, A5 and A9 are the negative controls. B) Immunoblotting confirmed the expression of CSF1R in the testis of horse [during the breeding (BS) and non-breeding (NBS) season], donkey and mule. C) Percentage of CSF1R(+) Aund cells showing that approximately 35% of these cells express this membrane receptor (*p<0.05). Figure A, bar = 10 µm.
Figure 6.
Qualitative evaluation of the co-localization of the three different spermatogonial markers used for horses.
Considering the co-expression of GFRA1 and PLZF the following pattern was observed: A) GFRA1(+) cells (A1; red arrowhead) presenting co-localization with PLZF (A2; yellow arrowhead), as evidenced in the merged figure (A3; white arrowhead); B) this panel illustrates GFRA1(+) cells (B1; red arrowhead) that do not present PLZF expression (B2), as shown in the merged figure (B3; white arrowhead). In relation to the co-expression of GFRA1 and CSF1R the following labeling pattern was observed: C) GFRA1(+) cells (C1; red arrowhead) also expressing CSF1R (C2; green arrowhead), shown in the merged figure (C3; white arrowhead); D) differently, some GFRA1(+) cells (D1; red arrowhead) do not present CSF1R (D2; white arrowhead in D3 merged figure). E) Summarization of the quantitative data obtained for Aund GFRA1, PLZF and CSF1R positive cells in horses, suggesting that these three proteins are differently expressed in this cell population. Yellow bar = 20 µm; White bar = 30 µm.
Figure 7.
Testis morphometry in horses, during the breeding and non-breeding season, and seminiferous tubules cross-sections subdivisions.
A) Whereas the seminiferous tubules (ST) volume density was not changed during the two periods evaluated, Leydig cells (LC) and connective tissue (CT) were the most prevalent components of the intertubular compartment during the breeding and non-breeding season. Seminiferous tubules cross-sections were subdivided into 4 different regions according to the prevalence of these aforementioned components (B). BV = blood vessels. Figure B, bar = 100 µm.
Figure 8.
Aund distribution in horses according to morphological (A–H) and immunostaining (I–P) criteria.
As indicated by red arrowheads, using both criteria, Aund cells were present in all four regions considered. However, independently of the breeding season, these cells were more frequently observed in the areas facing the interstitium, particularly nearby the blood vessels. TT = Tubule-Tubule contact; TI−BV = Tubule-Interstitium without blood vessels; TI+BV = Tubule-Interstitium with blood vessels; TIC = Tubule-Interstitium containing connective tissue. Figure A–D and H–K, bar = 10 µm.