Spermatogonial behavior in marmoset: a new generation, their kinetics and niche

Spermatogonial behavior in marmoset: a new generation, their kinetics and niche Abstract STUDY QUESTION Could a more detailed evaluation of marmoset spermatogonial morphology, kinetics and niches using high-resolution light microscopy (HRLM) lead to new findings? SUMMARY ANSWER Three subtypes of marmoset undifferentiated spermatogonia, which were not evenly distributed in terms of number and position along the basal membrane, and an extra premeiotic cell division not present in humans were identified using HRLM. WHAT IS KNOWN ALREADY The seminiferous epithelium cycle (SEC) of marmosets is divided into nine stages when based on the acrosome system, and several spermatogenic stages can usually be recognized within the same tubular cross-section. Three spermatogonial generations have been previously described in marmosets: types Adark, Apale and B spermatogonia. STUDY DESIGN, SIZE, DURATION Testes from five adult Callithrix penicillata were fixed by glutaraldehyde perfusion via the cardiac route and embedded in Araldite plastic resin for HRLM evaluation. Semi-thin sections (1 μm) were analyzed morphologically and morphometrically to evaluate spermatogonial morphology and kinetics (number, mitosis and apoptosis), spermatogenesis efficiency and the spermatogonial niche. PARTICIPANTS/MATERIALS, SETTING, METHODS Shape and nuclear diameter, the presence and distribution of heterochromatin, the granularity of the euchromatin, as well as the number, morphology and degree of nucleolar compaction were observed for morphological characterization. Kinetics analyses were performed for all spermatogonial subtypes and preleptotene spermatocytes, and their mitosis and apoptosis indexes determined across all SEC stages. Spermatogenesis parameters (mitotic, meiotic, Sertoli cell workload and general spermatogenesis efficiency) were determined through the counting of Adark and Apale spermatogonia, preleptotene and pachytene primary spermatocytes, round spermatids, and Sertoli cells at stage IV of the SEC. MAIN RESULTS AND THE ROLE OF CHANCE This is the first time that a study in marmosets demonstrates: the existence of a new spermatogonial generation (B2); the presence of two subtypes of Adark spermatogonia with (AdVac) and without (AdNoVac) nuclear rarefaction zones; the peculiar behavior of AdVac spermatogonia across the stages of the SEC, suggesting that they are quiescent stem spermatogonia; and that AdVac spermatogonia are located close to areas in which blood vessels, Leydig cells and macrophages are concentrated, suggesting a niche area for these cells. LARGE SCALE DATA Not applicable. LIMITATIONS, REASONS FOR CAUTION The C. penicillata spermatogonial kinetics evaluated here consider spermatogonial number across the SEC and their mitotic and apoptotic figures identified in HRLM sections. Therefore, caution is required when comparing absolute values between species. Although morphometric evaluation has suggested that AdVac spermatogonia are stem cells, a functional proof of this is still missing. It is known that parameters of the spermatogenic process in C. penicillata have similarities with those of the common marmoset C. jacchus, however, a detailed study of spermatogonial morphology, kinetics and niche has not yet been performed in C. jacchus, and a full comparison of the two species is not possible. WIDER IMPLICATIONS OF THE FINDINGS Our findings in C. penicillata contribute to a better understanding of the spermatogonial behavior and spermatogenesis efficiency in non-human primates. Given the phylogenetic closeness of the marmoset to the human species, similar processes might occur in humans. Therefore, marmosets may be an excellent model for studies regarding human testicular biology, fertility and related disorders. STUDY FUNDING/COMPETING INTEREST(S) Experiments were partially supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) and Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq). The authors declare that there are no conflicts of interest. spermatogenesis, spermatogenesis efficiency, spermatogonia, spermatogonial kinetics, spermatogonial niche, testis, marmoset, primate Introduction Spermatogenesis is a well co-ordinated cyclic process organized in different cellular associations. The process encompasses a proliferative phase in which spermatogonia produce spermatocytes after successive mitotic divisions, a meiotic phase, in which spermatocytes go through meiotic division giving rise to spermatids, and a differentiation phase in which spermatids mature into spermatozoa (Russell et al., 1990). The co-ordinated development of these three complex phases is essential for fertility. The first phase, the mitotic or proliferative step, is directly related to the quantitative success of spermatogenesis since it is strictly dependent on spermatogonial homeostasis, their correct number, and well-balanced mitotic and apoptotic behavior (Schlatt and Ehmcke, 2014). Spermatogonia reside on the basement membrane of the seminiferous epithelium and proliferate to maintain undifferentiated spermatogonial stem cells (SSCs) by self-renewal and to produce progeny differentiating spermatogonia that undergo considerable amplification through mitosis. In rodents, Chiarini-Garcia et al. described the spermatogonial biology in terms of morphology, kinetics and niche (Chiarini-Garcia and Russell, 2001; Chiarini-Garcia et al., 2003). They showed that undifferentiated spermatogonia are not randomly distributed in the basal compartment of the seminiferous tubules. In fact, these cells preferentially stay close to interstitial areas, where blood and lymphatic vessels, Leydig cells, macrophages and other stromal and transient cells are concentrated. Subsequently, it was revealed that a key factor that influences the behavior of undifferentiated spermatogonia in rodents is their position near blood vessels (Yoshida et al., 2007). Although these findings are well established in rodents, more research should be done in human and non-human primates, as information on these species is scarce. Research involving non-human primates would help to understand the role of spermatogonial behavior in human reproductive biology, owing to their close phylogenetic relation. Certainly, important lessons about human spermatogenesis have been drawn from biomedical research using the common marmoset—Callithrix jacchus—due to its relatively early sexual maturity, high fecundity ratio (Weinbauer et al., 2001; Sharpe et al., 2003), and similarity in spermatogenesis to human (Wistuba et al., 2003; 2007). Some authors subdivided the marmoset's seminiferous epithelium cycle (SEC) into nine stages involving three spermatogonial subtypes, identified as Adark, Apale and B spermatogonia (Holt and Moore, 1984; Millar et al., 2000). In addition, some aspects of spermatogonial biology and spermatogenesis, such as their mitotic, apoptotic and efficiency rates (Millar et al., 2000; Weinbauer et al., 2001; Leal and Franca, 2006) and molecular signatures of germ cells (Lin et al., 2012) have been determined. Despite these advances, little is known about spermatogonial biology related to kinetics and niche in any species of marmosets. As a result, research designed for eventual human application remains focused on rodents, even though the use of marmosets as an experimental model presents several advantages. With the intention to fill this gap, the present study evaluated the spermatogonial behavior of the black-tufted marmoset Callithrix penicillata, by determining the spermatogonial kinetics (number, mitosis and apoptosis), spermatogonial niche and aspects of spermatogenesis efficiency. To this end, we used a processing technique called high-resolution light microscopy (HRLM), which reveals morphological details of germ cells and has been successfully used in rodents. Materials and Methods Ethical approval Animal care and experimental procedures were approved by the Ethics Committee on Animal Experimentation (CETEA) at the Federal University of Minas Gerais, Brazil. The experiments were carried out in accordance with the approved guidelines (077/03). Animals and tissue preparation Five sexually mature black-tufted marmoset C. penicillata, weighing 293 ± 20 g, provided by the Brazilian Institute of Environment and Renewable Natural Resources (IBAMA) were used. They were sedated with sodium thiopental (50 mg/kg; Cristália, Brazil) and testes were fixed by heart perfusion. Initially, the vascular bed was rinsed with 0.9% saline solution and then perfused with 4% glutaraldehyde (biological grade; Electron Microscopy Sciences, Hatfield, PA, USA) in 0.05 M phosphate buffer pH 7.3. Next, the testes, weighing 571 ± 22 mg were cut into small 1–2 mm thick fragments, which were re-fixed by immersion in the same buffered 4% glutaraldehyde for 24 h at 4°C. The fragments were post-fixed in reduced osmium (osmium tetroxide 1% and potassium ferrocyanide in 1.5% phosphate buffer 0.05 M, pH 7.3) for 90 min. After ethanol and acetone dehydration, testicular fragments were embedded in Araldite resin, sectioned at 1 μm thickness, and stained with toluidine blue-borate for histomorphometric evaluation under HRLM (Chiarini-Garcia and Meistrich, 2008). Germ cell morphology and stages of the SEC After morphological characterization using HRLM, the germ cells were arranged in nine recurrent associations involving spermatogonia, spermatocytes and spermatids, constituting the nine stages of the SEC. The stages of the SEC were based on previous descriptions (Holt and Moore, 1984; Millar et al., 2000), which consider structural changes of the acrosomal system during spermatid differentiation (Supplementary Fig. S1). Given that the seminiferous epithelium in marmosets may present more than one stage per seminiferous tubule section, the frequencies (%) of the nine stages were determined by measuring the area occupied by each one. These areas were determined in at least 50 digital images of the seminiferous tubules per animal, obtained through the Image J software (National Institutes of Health, Bethesda, MD, USA). The spermatogonial morphology was described by considering nuclear features such as shape and diameter, the presence and distribution of heterochromatin, euchromatin granularity, and number, morphology, and degree of nucleolar compaction. To describe morphological aspects of the different spermatogonial subtypes, photomicrographs of all spermatogonia were taken in each of the nine stages of the SEC. At least 50 digital images from each spermatogonial subtype per animal and per stage of the SEC were taken using a Digital Q-Color 3 camera attached to an Olympus BX-51 microscope (Olympus, Tokyo, Japan), using the software Image-Pro Express (Media Cybernetics, Rockville, MD, USA). The images were adjusted/treated for resolution (600 dpi), sharpness (at 140%, radius set at 7 pixels, and threshold set at 0.0), and contrast/gray level (sigmoid curve) using Adobe Photoshop (Adobe Systems, Inc., Mountain View, CA, USA). The photomicrographs were set in plates using the Adobe Illustrator software (Adobe Systems, Inc., Mountain View, CA, USA). Spermatogonial kinetics To determine spermatogonial kinetics, the numbers of each type A undifferentiated (AdVac, A dark spermatogonia with nuclear rarefaction zone; AdNoVac, A dark spermatogonia without nuclear rarefaction zone and Apale, A pale spermatogonia) and type differentiating spermatogonia (B1 and B2) and preleptotene spermatocytes were counted in the nine stages of the SEC. Sertoli cell nucleoli (SCN) were counted in the same seminiferous tubule sections. The numbers of each spermatogonial subtype were corrected for section thickness and nuclear or nucleolar diameter, according to Abercrombie (1946) and Amann and Almquist (1962). The nuclear diameters (average of two perpendicular axes) of each spermatogonial subtype and preleptotene, as well as nucleoli diameter of Sertoli cells, were measured in all stages of the SEC. To do so, the diameter of the ten biggest profiles of nuclei/nucleoli from selected cells were measured for each animal using a graduated ruler fitted in a ×10 eyepiece calibrated with a micrometer ruler, in a final magnification of ×1000. The numbers of each spermatogonial subtype and preleptotene were equalized in each stage in relation to 100 SCN. Mitotic and apoptotic spermatogonia The number of mitotic and apoptotic spermatogonia positioned close to the basal membrane was obtained from at least 30 sectioned seminiferous tubules per animal. In addition, the SCN numbers were determined and the obtained value used to equalize the number of mitosis and apoptosis in 100 SCN. Given that the nuclear morphology changes during mitotic and apoptotic processes, it was not possible to identify the spermatogonial subtype. Therefore, the number of mitotic and apoptotic figures obtained corresponded to the sum of all dividing or dying spermatogonia (Adark, Apale, B1 and B2) observed at each stage of the SEC. Indexes of spermatogenesis To determine spermatogenesis efficiency in marmosets, the total number of Adark (AdVac plus AdNoVac) and Apale spermatogonia, preleptotene and pachytene primary spermatocytes, round spermatids, and SCN were counted in stage IV of the SEC in at least 50 sections per animal. The stage IV was chosen due to the presence, in the same region, of all generations of germ cells necessary to calculate the indexes evaluated. The obtained numbers were corrected according to Abercrombie (1946) and Amann and Almquist (1962) and the following indexes were determined according to Chiarini-Garcia et al. (2017): - Mitotic index (to determine the coefficient of spermatogonial mitosis efficiency): preleptotene spermatocytes/Adark spermatogonia. - Meiotic index (to obtain the rate of germ cell apoptosis/loss during meiosis): round spermatid/pachytene spermatocyte. - Sertoli cell workload (to estimate the number of spermatids supported by each Sertoli cell): round spermatid/SCN. - Spermatogenesis efficiency (to determine the number of spermatids produced after a completed cycle from a single Adark spermatogonium, estimating the overall spermatogenesis rate): round spermatid/Adark spermatogonia. In addition, we calculated the mitotic index and spermatogenesis efficiency based on the number of Adark subtypes (AdVac and AdNoVac) separately. This analysis aimed to predict each Adark subtype being considered as SSC. These indexes were determined as follows: - mitotic index: preleptotene spermatocytes/AdVac or AdNoVac spermatogonia and - spermatogenesis efficiency: round spermatid/AdVac or AdNoVac spermatogonia. Spermatogonial niche To investigate spermatogonial positioning related to blood vessels (venules and capillaries; Fig. 1), 30 digital photomicrographs of the testicular parenchyma were taken per animal, and seminiferous tubules and adjacent interstitial tissue were observed. The Image-Pro Express software was used to determine distance of germ cells to the closest blood vessel. The distance of ~400 germ cells per animal, including spermatogonia (AdVac, AdNoVac, Apale, B1 and B2) and preleptotene primary spermatocytes was determined in each of the nine stages of the SEC. In addition, the position of all spermatogonial subtypes was determined in relation to the interstitial or tubule contact areas, based on the previous description in rodent by Chiarini-Garcia et al. (2003). To do so, 30 photomicrographs of the testicular parenchyma from each animal were obtained. Subsequently, spermatogonial subtypes were identified and their percentage determined as adjacent to the interstitium or tubule contact area (Fig. 1). Figure 1 View largeDownload slide Scheme used to determine the position of Callithrix penicillata marmoset spermatogonia based on their proximity to blood vessels and tubule-interstitium and tubule-tubule contact. Figure 1 View largeDownload slide Scheme used to determine the position of Callithrix penicillata marmoset spermatogonia based on their proximity to blood vessels and tubule-interstitium and tubule-tubule contact. Statistical analysis All variables were tested for normality using the univariate procedure of the Statistical Analysis System (SAS Institute, Cary, NC, USA). Next, they were analyzed using the general linear model (GLM) procedure of SAS. In the event where significant treatment effects were established, multiple comparisons were performed using the probability of difference (pdiff) between means, adjusted by Tukey–Kramer (P < 0.05). Biometrical values (body and testes weight) were presented as mean ± SD and all other morphometrical data reported as mean ± SEM. All data in the present study were obtained from five animals. Results Morphological characterization of spermatogonia Undifferentiated and differentiating spermatogonia morphologically described through HRLM are shown in Figs 2 and 3, respectively. The spermatogonial subtypes were distinguished according to nuclear size and shape, euchromatin and heterochromatin density and distribution, nucleolar number, morphology and position, and spermatogonial location along the stages of the SEC. Two different Adark spermatogonia were identified with regards to the presence or absence of nuclear chromatin rarefaction zone (vacuole). These were named Adark with vacuole (AdVac; Fig. 2a–d) and Adark without vacuole (AdNoVac; Fig. 2e–h). Type Apale spermatogonia (Fig. 2i–l) were characterized by their finely stained euchromatin and bigger size in comparison with predecessor cells. Although previous studies (Holt and Moore, 1984; Millar et al., 2000; Weinbauer et al., 2001; Leal and França, 2006) have described one generation of type B spermatogonia in marmosets, in the present study we described, for the first time, two morphological subtypes of B spermatogonia, which we named as B1 (Fig. 3a–d) and B2 (Fig. 3e–h). Type B2 spermatogonia were bigger than B1 and presented darkly stained euchromatin with irregular nuclear rarefaction zones. Type B2 spermatogonia mitosis generates the preleptotene primary spermatocytes, which were smaller and presented more heterochromatin flakes (Fig. 3i–l). Figure 2 View largeDownload slide Morphological characterization of marmoset (C. penicillata) undifferentiated spermatogonia under high-resolution light microscopy. Note the nuclear morphological description of Adark spermatogonia containing nuclear rarefaction zones (AdVac; a–d), Adark spermatogonia without nuclear rarefaction zone (AdNoVac; e–h), and Apale spermatogonia (i–l), considering nuclear diameter (mean ± SEM), nuclear shape, nucleolus, heterochromatin and euchromatin. 2° = secondary spermatocyte; Es = elongated spermatid; L = leptotene primary spermatocyte; P = pachytene primary spermatocyte; S = Sertoli cell. Scale Bar: 6 μm. Figure 2 View largeDownload slide Morphological characterization of marmoset (C. penicillata) undifferentiated spermatogonia under high-resolution light microscopy. Note the nuclear morphological description of Adark spermatogonia containing nuclear rarefaction zones (AdVac; a–d), Adark spermatogonia without nuclear rarefaction zone (AdNoVac; e–h), and Apale spermatogonia (i–l), considering nuclear diameter (mean ± SEM), nuclear shape, nucleolus, heterochromatin and euchromatin. 2° = secondary spermatocyte; Es = elongated spermatid; L = leptotene primary spermatocyte; P = pachytene primary spermatocyte; S = Sertoli cell. Scale Bar: 6 μm. Figure 3 View largeDownload slide Morphological characterization of differentiating spermatogonia (B1 and B2) and preleptotene primary spermatocytes in the marmoset (C. penicillata) by HRLM. Observe detailed nuclear morphological description of B1 (a–d), B2 (e–h), and preleptotene primary spermatocytes (Pl; i–l) considering nuclear diameter (mean ± SEM), nuclear shape, nucleolus, heterochromatin and euchromatin. Scale Bar: 6 μm. Figure 3 View largeDownload slide Morphological characterization of differentiating spermatogonia (B1 and B2) and preleptotene primary spermatocytes in the marmoset (C. penicillata) by HRLM. Observe detailed nuclear morphological description of B1 (a–d), B2 (e–h), and preleptotene primary spermatocytes (Pl; i–l) considering nuclear diameter (mean ± SEM), nuclear shape, nucleolus, heterochromatin and euchromatin. Scale Bar: 6 μm. Spermatogonial numbers and kinetics Even though AdVac and AdNoVac spermatogonia numbers were relatively constant across the nine stages of the SEC (Fig. 4a), the number of AdVac (which corresponds to 21% of the total Adark population) was smaller and showed lower numerical variation. This behavior suggests that AdVac and AdNoVac may represent two distinct populations. On the other hand, the number of Apale spermatogonia clearly varied along the SEC, decreasing from stage IV to VIII and progressively increasing thereafter. From Apale spermatogonia to preleptotene spermatocytes, consecutive spermatogonia numerical waves (increase/decrease) were observed. The number of successor spermatogonia was approximately double that of the predecessor cells, clearly showing kinetic processes and providing evidence in support of two type B spermatogonia generations (Fig. 4b). While the number of Apale spermatogonia decreased after stage IV, the number of B1 spermatogonia progressively increased, peaking at stage VIII and then decreasing. In turn, the number of B2 spermatogonia increased and reached a peak at stage II, sharply decreasing subsequently, while preleptotene spermatocytes increased sharply at stage III (Fig. 4b). To confirm the number of differentiating B spermatogonia generations, the number of preleptotene primary spermatocytes (204; peak at stage IV) was divided by the number of type B1 spermatogonia (63; peak at stage VIII), thus leading to a ratio of 3.2, which is slightly lower than the theoretical value of 4 (not considering expected spermatogonial apoptosis). This finding indicated two spermatogonial divisions, leading to the assumption of two subtypes of B spermatogonia in marmosets. Figure 4 View largeDownload slide Kinetics of marmoset (C. penicillata) spermatogonia (AdVac, AdNoVac, Apale, B1, B2) and Pl across the nine stages of the SEC. In (a), observe that while the numbers of Adark undifferentiated spermatogonia (AdNoVac and AdVac) are constant across the seminiferous epithelium cycle (SEC), the number of Apale spermatogonia decreases from stages V to VIII. Every dot represents a mean ± SEM from five animals. (b) Shows that the numbers of differentiating spermatogonia (B1 and B2) and Pl are progressively twice as high in subsequent generations, supporting the existence of two generations of type B spermatogonia. Every dot represents a mean ± SEM from five animals. (c) The specific stages in which mitosis and apoptosis occur. Different letters (a, b) indicate statistical differences for spermatogonial mitosis (P-value < 0.05 by Tukey–Kramer test.) and upper spermatogonial types indicate where mitosis normally takes place. Every bar represents a mean ± SEM from five animals. (d) Figures of spermatogonial mitosis in anaphase (An) and metaphase (Me). (e) Apoptotic figures (arrowhead). Sg = spermatogonia; TP = tunica propria; SCNu = Sertoli cell nucleoli; S, Sertoli cell. Scale Bar: 3 μm. Figure 4 View largeDownload slide Kinetics of marmoset (C. penicillata) spermatogonia (AdVac, AdNoVac, Apale, B1, B2) and Pl across the nine stages of the SEC. In (a), observe that while the numbers of Adark undifferentiated spermatogonia (AdNoVac and AdVac) are constant across the seminiferous epithelium cycle (SEC), the number of Apale spermatogonia decreases from stages V to VIII. Every dot represents a mean ± SEM from five animals. (b) Shows that the numbers of differentiating spermatogonia (B1 and B2) and Pl are progressively twice as high in subsequent generations, supporting the existence of two generations of type B spermatogonia. Every dot represents a mean ± SEM from five animals. (c) The specific stages in which mitosis and apoptosis occur. Different letters (a, b) indicate statistical differences for spermatogonial mitosis (P-value < 0.05 by Tukey–Kramer test.) and upper spermatogonial types indicate where mitosis normally takes place. Every bar represents a mean ± SEM from five animals. (d) Figures of spermatogonial mitosis in anaphase (An) and metaphase (Me). (e) Apoptotic figures (arrowhead). Sg = spermatogonia; TP = tunica propria; SCNu = Sertoli cell nucleoli; S, Sertoli cell. Scale Bar: 3 μm. Spermatogonial mitosis and apoptosis Spermatogonial mitoses were seen in all stages of the SEC, appearing more frequently in the stages just before the peaks of each spermatogonial generation, from Apale up to preleptotene spermatocytes (compare Fig. 4b and c). Three peaks of mitoses can be observed: at stages VI and VII, in which type Apale cells are splitting into B1 spermatogonia; at stages IX and I, in which type B1 spermatogonia give rise to B2; and at stages III and IV, in which B2 cells divide into preleptotene spermatocytes. The mitotic figures were determined by the appearance of condensed chromosomes in three phases: prophase, metaphase and anaphase (Fig. 4d). Apoptosis at the base of seminiferous tubules was observed across all stages of the SEC, albeit with variable numbers; they were more frequent in stages in which a higher number of spermatogonial mitosis was observed (IX–IV, Fig. 4c). Apoptotic bodies were morphologically characterized as showing bordering and blabbing of chromatin, nucleus fragmentation, cytoplasm condensation, and the presence of vacuoles (Fig. 4e). The mitotic peaks confirmed the presence of two generations of type B spermatogonia and demonstrated that the numbers of these cells in the peaks did not correspond to twice the number of predecessor cells because of apoptosis, as theoretically expected. Spermatogenesis efficiency The spermatogenesis indexes of marmoset were determined by germ cell counts at stage IV of the SEC. Initially, the indexes were determined considering total Adark spermatogonia as a single population (Advac plus AdNoVac). The mitotic index, showing how many preleptotene spermatocytes were produced by mitosis from a single Adark spermatogonium, was 8.4 (Table I). The number of round spermatids produced from each pachytene spermatocyte that entered the meiotic phase (meiotic index) was 3.0. The Sertoli cell workload for marmosets, which estimates the number of round spermatid supported by each Sertoli cell, was 6.3. In terms of general spermatogenesis efficiency, 45.4 elongated spermatids were produced from each Adark spermatogonium. Additionally, the Adark subtypes—AdVac and AdNoVac—were considered separately as SSCs to evaluate the mitotic index and spermatogenesis efficiency. Both indexes were ~2-fold higher when AdVac spermatogonia were considered as SSC (Table I). This finding reinforces the hypothesis that AdVac and AdNoVac cells represent different populations of A undifferentiated spermatogonia in marmosets, as previously observed in the kinetic analysis. Table I The spermatogenesis indexes for the marmoset Callithrix penicillata, as determined by germ cell counts at stage IV of the seminiferous epithelium cycle. SSC Mitotic Meiotic Sertoli cell workload Spermatogenesis efficiency Total Adark 8.4 ± 0.9 3.0 ± 0.1 6.3 ± 2.1 45.4 ± 12.0 AdVac 28.2 ± 4.4 NA NA 147.5 ± 21.3 AdNoVac 12.2 ± 1.6 NA NA 66.5 ± 21.4 SSC Mitotic Meiotic Sertoli cell workload Spermatogenesis efficiency Total Adark 8.4 ± 0.9 3.0 ± 0.1 6.3 ± 2.1 45.4 ± 12.0 AdVac 28.2 ± 4.4 NA NA 147.5 ± 21.3 AdNoVac 12.2 ± 1.6 NA NA 66.5 ± 21.4 All data were obtained from five marmosets and expressed as mean ± SEM. SSC, stem spermatogonial cell; Adark, type A dark spermatogonia; AdVac, type A dark spermatogonia with nuclear rarefaction zone; AdNoVac type A dark spermatogonia without nuclear rarefaction zone. NA, not available. Table I The spermatogenesis indexes for the marmoset Callithrix penicillata, as determined by germ cell counts at stage IV of the seminiferous epithelium cycle. SSC Mitotic Meiotic Sertoli cell workload Spermatogenesis efficiency Total Adark 8.4 ± 0.9 3.0 ± 0.1 6.3 ± 2.1 45.4 ± 12.0 AdVac 28.2 ± 4.4 NA NA 147.5 ± 21.3 AdNoVac 12.2 ± 1.6 NA NA 66.5 ± 21.4 SSC Mitotic Meiotic Sertoli cell workload Spermatogenesis efficiency Total Adark 8.4 ± 0.9 3.0 ± 0.1 6.3 ± 2.1 45.4 ± 12.0 AdVac 28.2 ± 4.4 NA NA 147.5 ± 21.3 AdNoVac 12.2 ± 1.6 NA NA 66.5 ± 21.4 All data were obtained from five marmosets and expressed as mean ± SEM. SSC, stem spermatogonial cell; Adark, type A dark spermatogonia; AdVac, type A dark spermatogonia with nuclear rarefaction zone; AdNoVac type A dark spermatogonia without nuclear rarefaction zone. NA, not available. Spermatogonial niche At stages II, III and IV, AdVac spermatogonia were observed closer to blood vessels in comparison with AdNoVac and Apale (Fig. 5a), albeit with evident variability (16–41 μm). Regarding AdNoVac and Apale, the distance from blood vessels was similar and showed lower variation (30–40 μm) across all the stages of the cycle (Fig. 5a). On the other hand, B1 and B2 spermatogonia and preleptotene spermatocytes presented considerable variation in their distance from the nearest blood vessels (20–60 μm; Fig. 5b). We observed that, while only 10% of AdVac spermatogonia were positioned adjacent to tubule contact areas, the other 90% were preferentially located in the regions of the seminiferous tubules in contact with the interstitium (Fig. 5c), where Leydig cells, macrophages and blood vessels were concentrated. On the other hand, while 35% of AdNovac, Apale and B1 spermatogonia were positioned close to tubule contact, the remaining 65% were adjacent to the interstitium. Finally, type B2 spermatogonia and preleptotene spermatocytes were seen randomly positioned (50–60%) (Fig. 5c). The different positioning of Advac and AdNovac in regards to the proximity to blood vessels and interstitial areas, observed for the first time here, suggests that these different populations of Adark spermatogonia present distinct behaviors in the testicular parenchyma. Figure 5 View largeDownload slide Spermatogonial niche in the marmoset C. penicillata. The distance of each spermatogonial generation to the nearest blood vessel across all nine stages of the SEC was determined. In (a), observe that while the distance (μm) of AdNoVac and Apale undifferentited spermatogonia are similar, undifferentiated spermatogonia with nuclear rarefaction (AdVac) are closer to blood vessels in most of the stages. Different letters (a,b) showed P-value < 0.05 by Tukey–Kramer test. In (b), note that differentiating spermatogonia (B1 and B2) and preleptotene spermatocytes are frequently further away from the blood vessels in comparison with undifferentiated spermatogonia. (c) percentage of undifferentiated (AdVac, AdNoVac, Apale) and differentiating (B1, B2) spermatogonia and preleptotene primary spermatocytes (Pl) in regions of the seminiferous epithelium adjacent to the interstitium, in each stage of the SEC. Note that while Advac undifferentiated spermatogonia are preferentially adjacent to the interstitial area, undifferentiated (AdNoVac, Apale) and differentiating (B1, B2) spermatogonia are randomly distributed. Letters a and b indicate a statistical difference in comparison with other spermatogonia at the same stage of the SEC. For all three figures, every dot represents a mean ± SEM from five animals. Figure 5 View largeDownload slide Spermatogonial niche in the marmoset C. penicillata. The distance of each spermatogonial generation to the nearest blood vessel across all nine stages of the SEC was determined. In (a), observe that while the distance (μm) of AdNoVac and Apale undifferentited spermatogonia are similar, undifferentiated spermatogonia with nuclear rarefaction (AdVac) are closer to blood vessels in most of the stages. Different letters (a,b) showed P-value < 0.05 by Tukey–Kramer test. In (b), note that differentiating spermatogonia (B1 and B2) and preleptotene spermatocytes are frequently further away from the blood vessels in comparison with undifferentiated spermatogonia. (c) percentage of undifferentiated (AdVac, AdNoVac, Apale) and differentiating (B1, B2) spermatogonia and preleptotene primary spermatocytes (Pl) in regions of the seminiferous epithelium adjacent to the interstitium, in each stage of the SEC. Note that while Advac undifferentiated spermatogonia are preferentially adjacent to the interstitial area, undifferentiated (AdNoVac, Apale) and differentiating (B1, B2) spermatogonia are randomly distributed. Letters a and b indicate a statistical difference in comparison with other spermatogonia at the same stage of the SEC. For all three figures, every dot represents a mean ± SEM from five animals. Discussion Spermatogonial biology has been extensively studied for decades, and most of the current knowledge derives from studies using rodents as experimental models. Little is known about the spermatogonial behavior in primates, and particularly in the genus Callithrix sp, which is phylogenetically close to human and, therefore, more likely to mimic human spermatogonial biology. In this sense, the current study focused on describing the spermatogonial behavior and spermatogenesis efficiency of the marmoset and some of our findings were similar to what has been reported in humans up to now. The germ cell morphology has been described in marmosets, and it is known that they present similar features when compared with human germ cells (Nihi et al., 2017) after similar fixative and embedding procedures for HRLM. The two marmoset subtypes of Adark spermatogonia, with and without nuclear rarefaction zones, described herein have been previously observed in humans, by Nihi et al. (2017). However, while various small areas of nuclear rarefaction can be seen dispersed in the marmoset nucleoplasm, in human the nuclear rarefaction was seen mainly as a single rounded zone. On the other hand, Apale spermatogonia, characterized by their slightly stained and homogeneously distributed euchromatin, were similar in both species. After morphometric evaluations in marmosets, previous studies (Holt and Moore, 1984; Millar et al., 2000; Weinbauer et al., 2001) speculated, but did not confirm, the existence of two morphological subtypes of B spermatogonia. Thus, the description of only one generation of B spermatogonia for this species remained in the literature until now. Using the HRLM method, we were able to clearly distinguish two subtypes of B spermatogonia: type B1 (smaller, with lightly stained chromatin) and type B2 (larger, with darkly stained chromatin and the presence of small nuclear vacuoles). Moreover, after a kinetic spermatogonial evaluation across all stages of the SEC (discussed below), we confirmed, for the first time, the existence of two generations of type B spermatogonia in marmosets. The accurate identification of the morphological subcellular details of germ cells seen at the light microscopy level was possible due to the HRLM method here applied (Chiarini-Garcia and Meistrich, 2008). By using paraffin embedding, and even plastic embedding as with glycol methacrylate resin, the precise identification of morphological details is not possible, as demonstrated in the human testis by Chiarini-Garcia et al. (2017). Having morphologically identified undifferentiated (AdVac, AdNoVac and Apale) and differentiating (B1 and B2) spermatogonia in marmosets, we evaluated their behavior along the nine stages of the SEC. The number of total Adark spermatogonia, which are considered the reserve stem cells that remain quiescent through normal spermatogenesis (Clermont, 1966a,b), was constant across the marmoset SEC. Unexpectedly, when the Adark subtypes (AdVac and AdNoVac) were evaluated separately, we observed that AdVac cells were less frequent and had lower numerical variation than AdNoVac at all stages of the SEC. These findings suggested that Adark spermatogonia may constitute a stable population throughout the healthy spermatogenic process. Unlike Adark spermatogonia, Apale cells showed evident population variation along the SEC, which demonstrates their constant activity to maintain spermatogenesis (Simorangkir et al., 2005; Ehmcke et al., 2005; Ehmcke and Schlatt, 2006). Their number increased progressively after stage VIII and until stage IV of the following SEC when their number started to decrease until reaching stage VIII again. Interestingly, the decrease in the number of AdNoVac spermatogonia after stage IX coincided with the increase in the number of Apale spermatogonia. This suggested that AdNoVac may be the active Adark spermatogonia, which are proliferating and giving rise to Apale. However, whether AdNoVac split into Apale, or Apale remains self-renewing over these stages was not clear. Despite the new findings, the role of each of these cells during the normal spermatogonial cycle is still unclear. Although our kinetic data suggest that AdVac may be a more quiescent subtype of Adark spermatogonia, further experimental studies and/or molecular fingerprint evaluations are necessary to confirm this behavior. In this sense, previous studies in humans have demonstrated that stem spermatogonia are a heterogeneous cell population (Neuhaus et al., 2017) and that Adark spermatogonia with nuclear vacuoles express stem cell markers and present low proliferative activity, which associates them with an undifferentiated state (von Kopylow et al., 2012a,b). Regarding the kinetics of differentiating type B spermatogonia, we observed that the reduction of the Apale population after stage V also corresponded to the increase in the number of B1 spermatogonia, thereby suggesting that Apale were splitting into B1 spermatogonia. The alternation in the spermatogonial population dynamics, whereby a decrease in one cell population coincides with the increase of the following generation, strongly supports the existence of different generations. This behavior is observed among Apale, B1 and B2 spermatogonia and Pl spermatocyte. The variation in the number of spermatogonial mitosis and apoptosis events at specific stages coincides with each spermatogonial peak, thus also supporting the presence of these spermatogonial generations. Therefore, our kinetic analysis demonstrated that marmosets present four spermatogonial generations (Adark, Apale, B1 and B2) with two subtypes of Adark spermatogonia (AdVac and AdNoVac). We therefore conclude that the number of spermatogonial generations in marmosets is not the same as in humans, as the latter present three spermatogonial generations, and two undifferentiated (Adark and Apale) and one differentiating (B) spermatogonia populations (Clermont, 1963; Nihi et al., 2017). In this sense, care should be taken for a comparison between the two species regarding spermatogonial expansion. For the first time in primates, the present study investigated the position of the spermatogonial generations in specific regions of the seminiferous epithelium, which may characterize a particular region of stimulation/inhibition corresponding to a niche, which could be responsible for controlling differentiation or self-renewal. For this purpose, we evaluated marmoset spermatogonial proximity to the interstitial regions and blood vessels, which are considered niches in other species (Chiarini-Garcia et al., 2001; Yoshida et al., 2007; review in Potter and DeFalco, 2017). Indeed, different positioning was observed for AdVac and AdNoVac undifferentiated spermatogonia. Spermatogonia with nuclear vacuoles (AdVac) were preferentially observed closer to the interstitial regions and, in specific stages of the SEC, closer to blood vessels in opposition with the AdNoVac spermatogonia. Such positioning of undifferentiated spermatogonia usually indicates the presence of controlling paracrine and juxtacrine factors (Wang et al., 2015; Potter and DeFalco, 2017). In our view, this positioning suggests that AdVac spermatogonia may be subjected to controlling factors that co-ordinate their position and function; and we identified, for the first time in primates, the presence of spermatogonial niches. In contrast, types AdNoVac, Apale, B1 and B2 spermatogonia and preleptotene spermatocytes were found randomly distributed in terms of proximity to interstitial areas and blood vessels. The random positioning observed for differentiating spermatogonia (B1 and B2) is similar to that observed in the same differentiating cell types in rodents (A1, A2, A3, A4, In and B) (Chiarini-Garcia and Russell, 2001; Chiarini-Garcia et al., 2003). However, while in rodents all undifferentiated spermatogonia are distributed in niche areas, in marmosets the undifferentiated AdNoVac and Apale spermatogonia were distributed regardless of the presence of niche regions. The AdVac spermatogonia behavior in marmosets, e.g. proximity to interstitium and blood vessels around the spermiation event (stage IV) and away from those structures at subsequent stages (V and VI), was similar to the previous findings concerning type A undifferentiated spermatogonia (Asingle, Apared and Aaligned) in mouse (Chiarini-Garcia et al., 2001) and rat (Chiarini-Garcia et al., 2003). This repeatable pattern among mammalian species could mean a special functional change in the seminiferous epithelium during the spermiation process, resulting in active migration of stem spermatogonia. However, further studies are required to determine the meaning of this behavior and address the regulatory factors that could control the spermatogonial niches. To evaluate the efficiency of specific steps in spermatogenesis, distinct indexes can be used to compare different species and determine specific characteristics in the spermatogenic process. To evaluate the proliferative step, the mitotic index for marmoset was determined and reached a value of 8.4. The meiotic index showed that three round spermatids were produced by each diplotene spermatocyte that entered meiosis. Sertoli cells could support six round spermatids; and the overall efficiency of spermatogenesis, represented by the number of round spermatids produced by each Adark spermatogonia, was 45. This finding is consistent with previous studies in marmosets (Wistuba et al., 2003; Luetjens et al., 2005), despite the different methodologies employed. In humans, these indexes were all lower: mitotic and meiotic indexes <2; Sertoli workload = 4; and overall efficiency of the spermatogenesis <5 (Chiarini-Garcia et al., 2017). Thus, our findings show that marmosets present a higher spermatogenesis efficiency for all indexes evaluated when compared to humans. These indexes were determined considering the whole pool of Adark spermatogonia as stem cells, regardless of the presence or absence of nuclear vacuoles. However, when analyzing them as separated SSC, the values for mitotic index and spermatogenesis efficiency were both more than threefold higher in AdVac. Parameters of the spermatogenic process of the black-tufted marmoset C. penicillata (Leal and França, 2006) present similarities with those of common marmosets C. jacchus (Holt and More, 1984; Millar et al., 2000; Weinbauer et al., 2001) in terms of tubular diameter, number of stages per tubular cross-sections, number of germ cells, duration of premeiotic and postmeiotic phases and apoptotic rates. However, as detailed studies of spermatogonial morphology, kinetics and niche have never been performed so far in C. jacchus, it is impossible to say if their spermatogenic processes are fully comparable. In conclusion, the present study showed that black-tufted marmosets have four spermatogonial generations (Adark, Apale, B1 and B2). Additionally, we showed that Adark spermatogonia can be separated into two subtypes (AdVac and AdNoVac) that show different kinetic distribution along the stages of the SEC and distinct positioning in the seminiferous epithelium. Functional studies further exploring AdVac spermatogonia and the chromatin rarefaction zones may help to improve the current understanding of the primates SSCs. Supplementary data Supplementary data are available at Molecular Human Reproduction Online. Acknowledgements We thank the Centro de Microscopia (UFMG, Brazil) and Centro de Aquisição e Processamento de Imagens—CAPI (UFMG, Brazil) for the use of their facilities. Authors’ roles Tissue collection and data acquisition and analysis: A.L.C.-B., D.A.-F. and L.E.-G.; data interpretation: A.L.C.-B., D.A.-F., L.E.-G. and H.C.-G.; article drafting and critical revision: A.L.C.-B., F.R.C.L.A. and H.C.G.; research conception, design and coordination: H.C.-G. Approval of the final version: all authors. Funding This study was partially supported by the following Brazilian Foundations: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), and Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq). Conflicts of Interest None of the authors has received any benefits that are relevant to this study from sources other than the research foundations listed above. References Abercrombie M . Estimation of nuclear population from microtome sections . Anat Rec 1946 ; 94 : 239 – 247 . Google Scholar CrossRef Search ADS PubMed Amann R , Almquist J . Reproductive capacity of dairy bulls. VIII. Direct and indirect measurement of testicular sperm production . J Dairy Sci 1962 ; 45 : 774 – 781 . Google Scholar CrossRef Search ADS Chiarini-Garcia H , Hornick JR , Griswold MD , Russell LD . Distribution of type A spermatogonia in the mouse is not random . Biol Reprod 2001 ; 65 : 1179 – 1185 . 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For Permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Molecular Human Reproduction Oxford University Press

Spermatogonial behavior in marmoset: a new generation, their kinetics and niche

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com
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1360-9947
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1460-2407
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10.1093/molehr/gay017
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Abstract

Abstract STUDY QUESTION Could a more detailed evaluation of marmoset spermatogonial morphology, kinetics and niches using high-resolution light microscopy (HRLM) lead to new findings? SUMMARY ANSWER Three subtypes of marmoset undifferentiated spermatogonia, which were not evenly distributed in terms of number and position along the basal membrane, and an extra premeiotic cell division not present in humans were identified using HRLM. WHAT IS KNOWN ALREADY The seminiferous epithelium cycle (SEC) of marmosets is divided into nine stages when based on the acrosome system, and several spermatogenic stages can usually be recognized within the same tubular cross-section. Three spermatogonial generations have been previously described in marmosets: types Adark, Apale and B spermatogonia. STUDY DESIGN, SIZE, DURATION Testes from five adult Callithrix penicillata were fixed by glutaraldehyde perfusion via the cardiac route and embedded in Araldite plastic resin for HRLM evaluation. Semi-thin sections (1 μm) were analyzed morphologically and morphometrically to evaluate spermatogonial morphology and kinetics (number, mitosis and apoptosis), spermatogenesis efficiency and the spermatogonial niche. PARTICIPANTS/MATERIALS, SETTING, METHODS Shape and nuclear diameter, the presence and distribution of heterochromatin, the granularity of the euchromatin, as well as the number, morphology and degree of nucleolar compaction were observed for morphological characterization. Kinetics analyses were performed for all spermatogonial subtypes and preleptotene spermatocytes, and their mitosis and apoptosis indexes determined across all SEC stages. Spermatogenesis parameters (mitotic, meiotic, Sertoli cell workload and general spermatogenesis efficiency) were determined through the counting of Adark and Apale spermatogonia, preleptotene and pachytene primary spermatocytes, round spermatids, and Sertoli cells at stage IV of the SEC. MAIN RESULTS AND THE ROLE OF CHANCE This is the first time that a study in marmosets demonstrates: the existence of a new spermatogonial generation (B2); the presence of two subtypes of Adark spermatogonia with (AdVac) and without (AdNoVac) nuclear rarefaction zones; the peculiar behavior of AdVac spermatogonia across the stages of the SEC, suggesting that they are quiescent stem spermatogonia; and that AdVac spermatogonia are located close to areas in which blood vessels, Leydig cells and macrophages are concentrated, suggesting a niche area for these cells. LARGE SCALE DATA Not applicable. LIMITATIONS, REASONS FOR CAUTION The C. penicillata spermatogonial kinetics evaluated here consider spermatogonial number across the SEC and their mitotic and apoptotic figures identified in HRLM sections. Therefore, caution is required when comparing absolute values between species. Although morphometric evaluation has suggested that AdVac spermatogonia are stem cells, a functional proof of this is still missing. It is known that parameters of the spermatogenic process in C. penicillata have similarities with those of the common marmoset C. jacchus, however, a detailed study of spermatogonial morphology, kinetics and niche has not yet been performed in C. jacchus, and a full comparison of the two species is not possible. WIDER IMPLICATIONS OF THE FINDINGS Our findings in C. penicillata contribute to a better understanding of the spermatogonial behavior and spermatogenesis efficiency in non-human primates. Given the phylogenetic closeness of the marmoset to the human species, similar processes might occur in humans. Therefore, marmosets may be an excellent model for studies regarding human testicular biology, fertility and related disorders. STUDY FUNDING/COMPETING INTEREST(S) Experiments were partially supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) and Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq). The authors declare that there are no conflicts of interest. spermatogenesis, spermatogenesis efficiency, spermatogonia, spermatogonial kinetics, spermatogonial niche, testis, marmoset, primate Introduction Spermatogenesis is a well co-ordinated cyclic process organized in different cellular associations. The process encompasses a proliferative phase in which spermatogonia produce spermatocytes after successive mitotic divisions, a meiotic phase, in which spermatocytes go through meiotic division giving rise to spermatids, and a differentiation phase in which spermatids mature into spermatozoa (Russell et al., 1990). The co-ordinated development of these three complex phases is essential for fertility. The first phase, the mitotic or proliferative step, is directly related to the quantitative success of spermatogenesis since it is strictly dependent on spermatogonial homeostasis, their correct number, and well-balanced mitotic and apoptotic behavior (Schlatt and Ehmcke, 2014). Spermatogonia reside on the basement membrane of the seminiferous epithelium and proliferate to maintain undifferentiated spermatogonial stem cells (SSCs) by self-renewal and to produce progeny differentiating spermatogonia that undergo considerable amplification through mitosis. In rodents, Chiarini-Garcia et al. described the spermatogonial biology in terms of morphology, kinetics and niche (Chiarini-Garcia and Russell, 2001; Chiarini-Garcia et al., 2003). They showed that undifferentiated spermatogonia are not randomly distributed in the basal compartment of the seminiferous tubules. In fact, these cells preferentially stay close to interstitial areas, where blood and lymphatic vessels, Leydig cells, macrophages and other stromal and transient cells are concentrated. Subsequently, it was revealed that a key factor that influences the behavior of undifferentiated spermatogonia in rodents is their position near blood vessels (Yoshida et al., 2007). Although these findings are well established in rodents, more research should be done in human and non-human primates, as information on these species is scarce. Research involving non-human primates would help to understand the role of spermatogonial behavior in human reproductive biology, owing to their close phylogenetic relation. Certainly, important lessons about human spermatogenesis have been drawn from biomedical research using the common marmoset—Callithrix jacchus—due to its relatively early sexual maturity, high fecundity ratio (Weinbauer et al., 2001; Sharpe et al., 2003), and similarity in spermatogenesis to human (Wistuba et al., 2003; 2007). Some authors subdivided the marmoset's seminiferous epithelium cycle (SEC) into nine stages involving three spermatogonial subtypes, identified as Adark, Apale and B spermatogonia (Holt and Moore, 1984; Millar et al., 2000). In addition, some aspects of spermatogonial biology and spermatogenesis, such as their mitotic, apoptotic and efficiency rates (Millar et al., 2000; Weinbauer et al., 2001; Leal and Franca, 2006) and molecular signatures of germ cells (Lin et al., 2012) have been determined. Despite these advances, little is known about spermatogonial biology related to kinetics and niche in any species of marmosets. As a result, research designed for eventual human application remains focused on rodents, even though the use of marmosets as an experimental model presents several advantages. With the intention to fill this gap, the present study evaluated the spermatogonial behavior of the black-tufted marmoset Callithrix penicillata, by determining the spermatogonial kinetics (number, mitosis and apoptosis), spermatogonial niche and aspects of spermatogenesis efficiency. To this end, we used a processing technique called high-resolution light microscopy (HRLM), which reveals morphological details of germ cells and has been successfully used in rodents. Materials and Methods Ethical approval Animal care and experimental procedures were approved by the Ethics Committee on Animal Experimentation (CETEA) at the Federal University of Minas Gerais, Brazil. The experiments were carried out in accordance with the approved guidelines (077/03). Animals and tissue preparation Five sexually mature black-tufted marmoset C. penicillata, weighing 293 ± 20 g, provided by the Brazilian Institute of Environment and Renewable Natural Resources (IBAMA) were used. They were sedated with sodium thiopental (50 mg/kg; Cristália, Brazil) and testes were fixed by heart perfusion. Initially, the vascular bed was rinsed with 0.9% saline solution and then perfused with 4% glutaraldehyde (biological grade; Electron Microscopy Sciences, Hatfield, PA, USA) in 0.05 M phosphate buffer pH 7.3. Next, the testes, weighing 571 ± 22 mg were cut into small 1–2 mm thick fragments, which were re-fixed by immersion in the same buffered 4% glutaraldehyde for 24 h at 4°C. The fragments were post-fixed in reduced osmium (osmium tetroxide 1% and potassium ferrocyanide in 1.5% phosphate buffer 0.05 M, pH 7.3) for 90 min. After ethanol and acetone dehydration, testicular fragments were embedded in Araldite resin, sectioned at 1 μm thickness, and stained with toluidine blue-borate for histomorphometric evaluation under HRLM (Chiarini-Garcia and Meistrich, 2008). Germ cell morphology and stages of the SEC After morphological characterization using HRLM, the germ cells were arranged in nine recurrent associations involving spermatogonia, spermatocytes and spermatids, constituting the nine stages of the SEC. The stages of the SEC were based on previous descriptions (Holt and Moore, 1984; Millar et al., 2000), which consider structural changes of the acrosomal system during spermatid differentiation (Supplementary Fig. S1). Given that the seminiferous epithelium in marmosets may present more than one stage per seminiferous tubule section, the frequencies (%) of the nine stages were determined by measuring the area occupied by each one. These areas were determined in at least 50 digital images of the seminiferous tubules per animal, obtained through the Image J software (National Institutes of Health, Bethesda, MD, USA). The spermatogonial morphology was described by considering nuclear features such as shape and diameter, the presence and distribution of heterochromatin, euchromatin granularity, and number, morphology, and degree of nucleolar compaction. To describe morphological aspects of the different spermatogonial subtypes, photomicrographs of all spermatogonia were taken in each of the nine stages of the SEC. At least 50 digital images from each spermatogonial subtype per animal and per stage of the SEC were taken using a Digital Q-Color 3 camera attached to an Olympus BX-51 microscope (Olympus, Tokyo, Japan), using the software Image-Pro Express (Media Cybernetics, Rockville, MD, USA). The images were adjusted/treated for resolution (600 dpi), sharpness (at 140%, radius set at 7 pixels, and threshold set at 0.0), and contrast/gray level (sigmoid curve) using Adobe Photoshop (Adobe Systems, Inc., Mountain View, CA, USA). The photomicrographs were set in plates using the Adobe Illustrator software (Adobe Systems, Inc., Mountain View, CA, USA). Spermatogonial kinetics To determine spermatogonial kinetics, the numbers of each type A undifferentiated (AdVac, A dark spermatogonia with nuclear rarefaction zone; AdNoVac, A dark spermatogonia without nuclear rarefaction zone and Apale, A pale spermatogonia) and type differentiating spermatogonia (B1 and B2) and preleptotene spermatocytes were counted in the nine stages of the SEC. Sertoli cell nucleoli (SCN) were counted in the same seminiferous tubule sections. The numbers of each spermatogonial subtype were corrected for section thickness and nuclear or nucleolar diameter, according to Abercrombie (1946) and Amann and Almquist (1962). The nuclear diameters (average of two perpendicular axes) of each spermatogonial subtype and preleptotene, as well as nucleoli diameter of Sertoli cells, were measured in all stages of the SEC. To do so, the diameter of the ten biggest profiles of nuclei/nucleoli from selected cells were measured for each animal using a graduated ruler fitted in a ×10 eyepiece calibrated with a micrometer ruler, in a final magnification of ×1000. The numbers of each spermatogonial subtype and preleptotene were equalized in each stage in relation to 100 SCN. Mitotic and apoptotic spermatogonia The number of mitotic and apoptotic spermatogonia positioned close to the basal membrane was obtained from at least 30 sectioned seminiferous tubules per animal. In addition, the SCN numbers were determined and the obtained value used to equalize the number of mitosis and apoptosis in 100 SCN. Given that the nuclear morphology changes during mitotic and apoptotic processes, it was not possible to identify the spermatogonial subtype. Therefore, the number of mitotic and apoptotic figures obtained corresponded to the sum of all dividing or dying spermatogonia (Adark, Apale, B1 and B2) observed at each stage of the SEC. Indexes of spermatogenesis To determine spermatogenesis efficiency in marmosets, the total number of Adark (AdVac plus AdNoVac) and Apale spermatogonia, preleptotene and pachytene primary spermatocytes, round spermatids, and SCN were counted in stage IV of the SEC in at least 50 sections per animal. The stage IV was chosen due to the presence, in the same region, of all generations of germ cells necessary to calculate the indexes evaluated. The obtained numbers were corrected according to Abercrombie (1946) and Amann and Almquist (1962) and the following indexes were determined according to Chiarini-Garcia et al. (2017): - Mitotic index (to determine the coefficient of spermatogonial mitosis efficiency): preleptotene spermatocytes/Adark spermatogonia. - Meiotic index (to obtain the rate of germ cell apoptosis/loss during meiosis): round spermatid/pachytene spermatocyte. - Sertoli cell workload (to estimate the number of spermatids supported by each Sertoli cell): round spermatid/SCN. - Spermatogenesis efficiency (to determine the number of spermatids produced after a completed cycle from a single Adark spermatogonium, estimating the overall spermatogenesis rate): round spermatid/Adark spermatogonia. In addition, we calculated the mitotic index and spermatogenesis efficiency based on the number of Adark subtypes (AdVac and AdNoVac) separately. This analysis aimed to predict each Adark subtype being considered as SSC. These indexes were determined as follows: - mitotic index: preleptotene spermatocytes/AdVac or AdNoVac spermatogonia and - spermatogenesis efficiency: round spermatid/AdVac or AdNoVac spermatogonia. Spermatogonial niche To investigate spermatogonial positioning related to blood vessels (venules and capillaries; Fig. 1), 30 digital photomicrographs of the testicular parenchyma were taken per animal, and seminiferous tubules and adjacent interstitial tissue were observed. The Image-Pro Express software was used to determine distance of germ cells to the closest blood vessel. The distance of ~400 germ cells per animal, including spermatogonia (AdVac, AdNoVac, Apale, B1 and B2) and preleptotene primary spermatocytes was determined in each of the nine stages of the SEC. In addition, the position of all spermatogonial subtypes was determined in relation to the interstitial or tubule contact areas, based on the previous description in rodent by Chiarini-Garcia et al. (2003). To do so, 30 photomicrographs of the testicular parenchyma from each animal were obtained. Subsequently, spermatogonial subtypes were identified and their percentage determined as adjacent to the interstitium or tubule contact area (Fig. 1). Figure 1 View largeDownload slide Scheme used to determine the position of Callithrix penicillata marmoset spermatogonia based on their proximity to blood vessels and tubule-interstitium and tubule-tubule contact. Figure 1 View largeDownload slide Scheme used to determine the position of Callithrix penicillata marmoset spermatogonia based on their proximity to blood vessels and tubule-interstitium and tubule-tubule contact. Statistical analysis All variables were tested for normality using the univariate procedure of the Statistical Analysis System (SAS Institute, Cary, NC, USA). Next, they were analyzed using the general linear model (GLM) procedure of SAS. In the event where significant treatment effects were established, multiple comparisons were performed using the probability of difference (pdiff) between means, adjusted by Tukey–Kramer (P < 0.05). Biometrical values (body and testes weight) were presented as mean ± SD and all other morphometrical data reported as mean ± SEM. All data in the present study were obtained from five animals. Results Morphological characterization of spermatogonia Undifferentiated and differentiating spermatogonia morphologically described through HRLM are shown in Figs 2 and 3, respectively. The spermatogonial subtypes were distinguished according to nuclear size and shape, euchromatin and heterochromatin density and distribution, nucleolar number, morphology and position, and spermatogonial location along the stages of the SEC. Two different Adark spermatogonia were identified with regards to the presence or absence of nuclear chromatin rarefaction zone (vacuole). These were named Adark with vacuole (AdVac; Fig. 2a–d) and Adark without vacuole (AdNoVac; Fig. 2e–h). Type Apale spermatogonia (Fig. 2i–l) were characterized by their finely stained euchromatin and bigger size in comparison with predecessor cells. Although previous studies (Holt and Moore, 1984; Millar et al., 2000; Weinbauer et al., 2001; Leal and França, 2006) have described one generation of type B spermatogonia in marmosets, in the present study we described, for the first time, two morphological subtypes of B spermatogonia, which we named as B1 (Fig. 3a–d) and B2 (Fig. 3e–h). Type B2 spermatogonia were bigger than B1 and presented darkly stained euchromatin with irregular nuclear rarefaction zones. Type B2 spermatogonia mitosis generates the preleptotene primary spermatocytes, which were smaller and presented more heterochromatin flakes (Fig. 3i–l). Figure 2 View largeDownload slide Morphological characterization of marmoset (C. penicillata) undifferentiated spermatogonia under high-resolution light microscopy. Note the nuclear morphological description of Adark spermatogonia containing nuclear rarefaction zones (AdVac; a–d), Adark spermatogonia without nuclear rarefaction zone (AdNoVac; e–h), and Apale spermatogonia (i–l), considering nuclear diameter (mean ± SEM), nuclear shape, nucleolus, heterochromatin and euchromatin. 2° = secondary spermatocyte; Es = elongated spermatid; L = leptotene primary spermatocyte; P = pachytene primary spermatocyte; S = Sertoli cell. Scale Bar: 6 μm. Figure 2 View largeDownload slide Morphological characterization of marmoset (C. penicillata) undifferentiated spermatogonia under high-resolution light microscopy. Note the nuclear morphological description of Adark spermatogonia containing nuclear rarefaction zones (AdVac; a–d), Adark spermatogonia without nuclear rarefaction zone (AdNoVac; e–h), and Apale spermatogonia (i–l), considering nuclear diameter (mean ± SEM), nuclear shape, nucleolus, heterochromatin and euchromatin. 2° = secondary spermatocyte; Es = elongated spermatid; L = leptotene primary spermatocyte; P = pachytene primary spermatocyte; S = Sertoli cell. Scale Bar: 6 μm. Figure 3 View largeDownload slide Morphological characterization of differentiating spermatogonia (B1 and B2) and preleptotene primary spermatocytes in the marmoset (C. penicillata) by HRLM. Observe detailed nuclear morphological description of B1 (a–d), B2 (e–h), and preleptotene primary spermatocytes (Pl; i–l) considering nuclear diameter (mean ± SEM), nuclear shape, nucleolus, heterochromatin and euchromatin. Scale Bar: 6 μm. Figure 3 View largeDownload slide Morphological characterization of differentiating spermatogonia (B1 and B2) and preleptotene primary spermatocytes in the marmoset (C. penicillata) by HRLM. Observe detailed nuclear morphological description of B1 (a–d), B2 (e–h), and preleptotene primary spermatocytes (Pl; i–l) considering nuclear diameter (mean ± SEM), nuclear shape, nucleolus, heterochromatin and euchromatin. Scale Bar: 6 μm. Spermatogonial numbers and kinetics Even though AdVac and AdNoVac spermatogonia numbers were relatively constant across the nine stages of the SEC (Fig. 4a), the number of AdVac (which corresponds to 21% of the total Adark population) was smaller and showed lower numerical variation. This behavior suggests that AdVac and AdNoVac may represent two distinct populations. On the other hand, the number of Apale spermatogonia clearly varied along the SEC, decreasing from stage IV to VIII and progressively increasing thereafter. From Apale spermatogonia to preleptotene spermatocytes, consecutive spermatogonia numerical waves (increase/decrease) were observed. The number of successor spermatogonia was approximately double that of the predecessor cells, clearly showing kinetic processes and providing evidence in support of two type B spermatogonia generations (Fig. 4b). While the number of Apale spermatogonia decreased after stage IV, the number of B1 spermatogonia progressively increased, peaking at stage VIII and then decreasing. In turn, the number of B2 spermatogonia increased and reached a peak at stage II, sharply decreasing subsequently, while preleptotene spermatocytes increased sharply at stage III (Fig. 4b). To confirm the number of differentiating B spermatogonia generations, the number of preleptotene primary spermatocytes (204; peak at stage IV) was divided by the number of type B1 spermatogonia (63; peak at stage VIII), thus leading to a ratio of 3.2, which is slightly lower than the theoretical value of 4 (not considering expected spermatogonial apoptosis). This finding indicated two spermatogonial divisions, leading to the assumption of two subtypes of B spermatogonia in marmosets. Figure 4 View largeDownload slide Kinetics of marmoset (C. penicillata) spermatogonia (AdVac, AdNoVac, Apale, B1, B2) and Pl across the nine stages of the SEC. In (a), observe that while the numbers of Adark undifferentiated spermatogonia (AdNoVac and AdVac) are constant across the seminiferous epithelium cycle (SEC), the number of Apale spermatogonia decreases from stages V to VIII. Every dot represents a mean ± SEM from five animals. (b) Shows that the numbers of differentiating spermatogonia (B1 and B2) and Pl are progressively twice as high in subsequent generations, supporting the existence of two generations of type B spermatogonia. Every dot represents a mean ± SEM from five animals. (c) The specific stages in which mitosis and apoptosis occur. Different letters (a, b) indicate statistical differences for spermatogonial mitosis (P-value < 0.05 by Tukey–Kramer test.) and upper spermatogonial types indicate where mitosis normally takes place. Every bar represents a mean ± SEM from five animals. (d) Figures of spermatogonial mitosis in anaphase (An) and metaphase (Me). (e) Apoptotic figures (arrowhead). Sg = spermatogonia; TP = tunica propria; SCNu = Sertoli cell nucleoli; S, Sertoli cell. Scale Bar: 3 μm. Figure 4 View largeDownload slide Kinetics of marmoset (C. penicillata) spermatogonia (AdVac, AdNoVac, Apale, B1, B2) and Pl across the nine stages of the SEC. In (a), observe that while the numbers of Adark undifferentiated spermatogonia (AdNoVac and AdVac) are constant across the seminiferous epithelium cycle (SEC), the number of Apale spermatogonia decreases from stages V to VIII. Every dot represents a mean ± SEM from five animals. (b) Shows that the numbers of differentiating spermatogonia (B1 and B2) and Pl are progressively twice as high in subsequent generations, supporting the existence of two generations of type B spermatogonia. Every dot represents a mean ± SEM from five animals. (c) The specific stages in which mitosis and apoptosis occur. Different letters (a, b) indicate statistical differences for spermatogonial mitosis (P-value < 0.05 by Tukey–Kramer test.) and upper spermatogonial types indicate where mitosis normally takes place. Every bar represents a mean ± SEM from five animals. (d) Figures of spermatogonial mitosis in anaphase (An) and metaphase (Me). (e) Apoptotic figures (arrowhead). Sg = spermatogonia; TP = tunica propria; SCNu = Sertoli cell nucleoli; S, Sertoli cell. Scale Bar: 3 μm. Spermatogonial mitosis and apoptosis Spermatogonial mitoses were seen in all stages of the SEC, appearing more frequently in the stages just before the peaks of each spermatogonial generation, from Apale up to preleptotene spermatocytes (compare Fig. 4b and c). Three peaks of mitoses can be observed: at stages VI and VII, in which type Apale cells are splitting into B1 spermatogonia; at stages IX and I, in which type B1 spermatogonia give rise to B2; and at stages III and IV, in which B2 cells divide into preleptotene spermatocytes. The mitotic figures were determined by the appearance of condensed chromosomes in three phases: prophase, metaphase and anaphase (Fig. 4d). Apoptosis at the base of seminiferous tubules was observed across all stages of the SEC, albeit with variable numbers; they were more frequent in stages in which a higher number of spermatogonial mitosis was observed (IX–IV, Fig. 4c). Apoptotic bodies were morphologically characterized as showing bordering and blabbing of chromatin, nucleus fragmentation, cytoplasm condensation, and the presence of vacuoles (Fig. 4e). The mitotic peaks confirmed the presence of two generations of type B spermatogonia and demonstrated that the numbers of these cells in the peaks did not correspond to twice the number of predecessor cells because of apoptosis, as theoretically expected. Spermatogenesis efficiency The spermatogenesis indexes of marmoset were determined by germ cell counts at stage IV of the SEC. Initially, the indexes were determined considering total Adark spermatogonia as a single population (Advac plus AdNoVac). The mitotic index, showing how many preleptotene spermatocytes were produced by mitosis from a single Adark spermatogonium, was 8.4 (Table I). The number of round spermatids produced from each pachytene spermatocyte that entered the meiotic phase (meiotic index) was 3.0. The Sertoli cell workload for marmosets, which estimates the number of round spermatid supported by each Sertoli cell, was 6.3. In terms of general spermatogenesis efficiency, 45.4 elongated spermatids were produced from each Adark spermatogonium. Additionally, the Adark subtypes—AdVac and AdNoVac—were considered separately as SSCs to evaluate the mitotic index and spermatogenesis efficiency. Both indexes were ~2-fold higher when AdVac spermatogonia were considered as SSC (Table I). This finding reinforces the hypothesis that AdVac and AdNoVac cells represent different populations of A undifferentiated spermatogonia in marmosets, as previously observed in the kinetic analysis. Table I The spermatogenesis indexes for the marmoset Callithrix penicillata, as determined by germ cell counts at stage IV of the seminiferous epithelium cycle. SSC Mitotic Meiotic Sertoli cell workload Spermatogenesis efficiency Total Adark 8.4 ± 0.9 3.0 ± 0.1 6.3 ± 2.1 45.4 ± 12.0 AdVac 28.2 ± 4.4 NA NA 147.5 ± 21.3 AdNoVac 12.2 ± 1.6 NA NA 66.5 ± 21.4 SSC Mitotic Meiotic Sertoli cell workload Spermatogenesis efficiency Total Adark 8.4 ± 0.9 3.0 ± 0.1 6.3 ± 2.1 45.4 ± 12.0 AdVac 28.2 ± 4.4 NA NA 147.5 ± 21.3 AdNoVac 12.2 ± 1.6 NA NA 66.5 ± 21.4 All data were obtained from five marmosets and expressed as mean ± SEM. SSC, stem spermatogonial cell; Adark, type A dark spermatogonia; AdVac, type A dark spermatogonia with nuclear rarefaction zone; AdNoVac type A dark spermatogonia without nuclear rarefaction zone. NA, not available. Table I The spermatogenesis indexes for the marmoset Callithrix penicillata, as determined by germ cell counts at stage IV of the seminiferous epithelium cycle. SSC Mitotic Meiotic Sertoli cell workload Spermatogenesis efficiency Total Adark 8.4 ± 0.9 3.0 ± 0.1 6.3 ± 2.1 45.4 ± 12.0 AdVac 28.2 ± 4.4 NA NA 147.5 ± 21.3 AdNoVac 12.2 ± 1.6 NA NA 66.5 ± 21.4 SSC Mitotic Meiotic Sertoli cell workload Spermatogenesis efficiency Total Adark 8.4 ± 0.9 3.0 ± 0.1 6.3 ± 2.1 45.4 ± 12.0 AdVac 28.2 ± 4.4 NA NA 147.5 ± 21.3 AdNoVac 12.2 ± 1.6 NA NA 66.5 ± 21.4 All data were obtained from five marmosets and expressed as mean ± SEM. SSC, stem spermatogonial cell; Adark, type A dark spermatogonia; AdVac, type A dark spermatogonia with nuclear rarefaction zone; AdNoVac type A dark spermatogonia without nuclear rarefaction zone. NA, not available. Spermatogonial niche At stages II, III and IV, AdVac spermatogonia were observed closer to blood vessels in comparison with AdNoVac and Apale (Fig. 5a), albeit with evident variability (16–41 μm). Regarding AdNoVac and Apale, the distance from blood vessels was similar and showed lower variation (30–40 μm) across all the stages of the cycle (Fig. 5a). On the other hand, B1 and B2 spermatogonia and preleptotene spermatocytes presented considerable variation in their distance from the nearest blood vessels (20–60 μm; Fig. 5b). We observed that, while only 10% of AdVac spermatogonia were positioned adjacent to tubule contact areas, the other 90% were preferentially located in the regions of the seminiferous tubules in contact with the interstitium (Fig. 5c), where Leydig cells, macrophages and blood vessels were concentrated. On the other hand, while 35% of AdNovac, Apale and B1 spermatogonia were positioned close to tubule contact, the remaining 65% were adjacent to the interstitium. Finally, type B2 spermatogonia and preleptotene spermatocytes were seen randomly positioned (50–60%) (Fig. 5c). The different positioning of Advac and AdNovac in regards to the proximity to blood vessels and interstitial areas, observed for the first time here, suggests that these different populations of Adark spermatogonia present distinct behaviors in the testicular parenchyma. Figure 5 View largeDownload slide Spermatogonial niche in the marmoset C. penicillata. The distance of each spermatogonial generation to the nearest blood vessel across all nine stages of the SEC was determined. In (a), observe that while the distance (μm) of AdNoVac and Apale undifferentited spermatogonia are similar, undifferentiated spermatogonia with nuclear rarefaction (AdVac) are closer to blood vessels in most of the stages. Different letters (a,b) showed P-value < 0.05 by Tukey–Kramer test. In (b), note that differentiating spermatogonia (B1 and B2) and preleptotene spermatocytes are frequently further away from the blood vessels in comparison with undifferentiated spermatogonia. (c) percentage of undifferentiated (AdVac, AdNoVac, Apale) and differentiating (B1, B2) spermatogonia and preleptotene primary spermatocytes (Pl) in regions of the seminiferous epithelium adjacent to the interstitium, in each stage of the SEC. Note that while Advac undifferentiated spermatogonia are preferentially adjacent to the interstitial area, undifferentiated (AdNoVac, Apale) and differentiating (B1, B2) spermatogonia are randomly distributed. Letters a and b indicate a statistical difference in comparison with other spermatogonia at the same stage of the SEC. For all three figures, every dot represents a mean ± SEM from five animals. Figure 5 View largeDownload slide Spermatogonial niche in the marmoset C. penicillata. The distance of each spermatogonial generation to the nearest blood vessel across all nine stages of the SEC was determined. In (a), observe that while the distance (μm) of AdNoVac and Apale undifferentited spermatogonia are similar, undifferentiated spermatogonia with nuclear rarefaction (AdVac) are closer to blood vessels in most of the stages. Different letters (a,b) showed P-value < 0.05 by Tukey–Kramer test. In (b), note that differentiating spermatogonia (B1 and B2) and preleptotene spermatocytes are frequently further away from the blood vessels in comparison with undifferentiated spermatogonia. (c) percentage of undifferentiated (AdVac, AdNoVac, Apale) and differentiating (B1, B2) spermatogonia and preleptotene primary spermatocytes (Pl) in regions of the seminiferous epithelium adjacent to the interstitium, in each stage of the SEC. Note that while Advac undifferentiated spermatogonia are preferentially adjacent to the interstitial area, undifferentiated (AdNoVac, Apale) and differentiating (B1, B2) spermatogonia are randomly distributed. Letters a and b indicate a statistical difference in comparison with other spermatogonia at the same stage of the SEC. For all three figures, every dot represents a mean ± SEM from five animals. Discussion Spermatogonial biology has been extensively studied for decades, and most of the current knowledge derives from studies using rodents as experimental models. Little is known about the spermatogonial behavior in primates, and particularly in the genus Callithrix sp, which is phylogenetically close to human and, therefore, more likely to mimic human spermatogonial biology. In this sense, the current study focused on describing the spermatogonial behavior and spermatogenesis efficiency of the marmoset and some of our findings were similar to what has been reported in humans up to now. The germ cell morphology has been described in marmosets, and it is known that they present similar features when compared with human germ cells (Nihi et al., 2017) after similar fixative and embedding procedures for HRLM. The two marmoset subtypes of Adark spermatogonia, with and without nuclear rarefaction zones, described herein have been previously observed in humans, by Nihi et al. (2017). However, while various small areas of nuclear rarefaction can be seen dispersed in the marmoset nucleoplasm, in human the nuclear rarefaction was seen mainly as a single rounded zone. On the other hand, Apale spermatogonia, characterized by their slightly stained and homogeneously distributed euchromatin, were similar in both species. After morphometric evaluations in marmosets, previous studies (Holt and Moore, 1984; Millar et al., 2000; Weinbauer et al., 2001) speculated, but did not confirm, the existence of two morphological subtypes of B spermatogonia. Thus, the description of only one generation of B spermatogonia for this species remained in the literature until now. Using the HRLM method, we were able to clearly distinguish two subtypes of B spermatogonia: type B1 (smaller, with lightly stained chromatin) and type B2 (larger, with darkly stained chromatin and the presence of small nuclear vacuoles). Moreover, after a kinetic spermatogonial evaluation across all stages of the SEC (discussed below), we confirmed, for the first time, the existence of two generations of type B spermatogonia in marmosets. The accurate identification of the morphological subcellular details of germ cells seen at the light microscopy level was possible due to the HRLM method here applied (Chiarini-Garcia and Meistrich, 2008). By using paraffin embedding, and even plastic embedding as with glycol methacrylate resin, the precise identification of morphological details is not possible, as demonstrated in the human testis by Chiarini-Garcia et al. (2017). Having morphologically identified undifferentiated (AdVac, AdNoVac and Apale) and differentiating (B1 and B2) spermatogonia in marmosets, we evaluated their behavior along the nine stages of the SEC. The number of total Adark spermatogonia, which are considered the reserve stem cells that remain quiescent through normal spermatogenesis (Clermont, 1966a,b), was constant across the marmoset SEC. Unexpectedly, when the Adark subtypes (AdVac and AdNoVac) were evaluated separately, we observed that AdVac cells were less frequent and had lower numerical variation than AdNoVac at all stages of the SEC. These findings suggested that Adark spermatogonia may constitute a stable population throughout the healthy spermatogenic process. Unlike Adark spermatogonia, Apale cells showed evident population variation along the SEC, which demonstrates their constant activity to maintain spermatogenesis (Simorangkir et al., 2005; Ehmcke et al., 2005; Ehmcke and Schlatt, 2006). Their number increased progressively after stage VIII and until stage IV of the following SEC when their number started to decrease until reaching stage VIII again. Interestingly, the decrease in the number of AdNoVac spermatogonia after stage IX coincided with the increase in the number of Apale spermatogonia. This suggested that AdNoVac may be the active Adark spermatogonia, which are proliferating and giving rise to Apale. However, whether AdNoVac split into Apale, or Apale remains self-renewing over these stages was not clear. Despite the new findings, the role of each of these cells during the normal spermatogonial cycle is still unclear. Although our kinetic data suggest that AdVac may be a more quiescent subtype of Adark spermatogonia, further experimental studies and/or molecular fingerprint evaluations are necessary to confirm this behavior. In this sense, previous studies in humans have demonstrated that stem spermatogonia are a heterogeneous cell population (Neuhaus et al., 2017) and that Adark spermatogonia with nuclear vacuoles express stem cell markers and present low proliferative activity, which associates them with an undifferentiated state (von Kopylow et al., 2012a,b). Regarding the kinetics of differentiating type B spermatogonia, we observed that the reduction of the Apale population after stage V also corresponded to the increase in the number of B1 spermatogonia, thereby suggesting that Apale were splitting into B1 spermatogonia. The alternation in the spermatogonial population dynamics, whereby a decrease in one cell population coincides with the increase of the following generation, strongly supports the existence of different generations. This behavior is observed among Apale, B1 and B2 spermatogonia and Pl spermatocyte. The variation in the number of spermatogonial mitosis and apoptosis events at specific stages coincides with each spermatogonial peak, thus also supporting the presence of these spermatogonial generations. Therefore, our kinetic analysis demonstrated that marmosets present four spermatogonial generations (Adark, Apale, B1 and B2) with two subtypes of Adark spermatogonia (AdVac and AdNoVac). We therefore conclude that the number of spermatogonial generations in marmosets is not the same as in humans, as the latter present three spermatogonial generations, and two undifferentiated (Adark and Apale) and one differentiating (B) spermatogonia populations (Clermont, 1963; Nihi et al., 2017). In this sense, care should be taken for a comparison between the two species regarding spermatogonial expansion. For the first time in primates, the present study investigated the position of the spermatogonial generations in specific regions of the seminiferous epithelium, which may characterize a particular region of stimulation/inhibition corresponding to a niche, which could be responsible for controlling differentiation or self-renewal. For this purpose, we evaluated marmoset spermatogonial proximity to the interstitial regions and blood vessels, which are considered niches in other species (Chiarini-Garcia et al., 2001; Yoshida et al., 2007; review in Potter and DeFalco, 2017). Indeed, different positioning was observed for AdVac and AdNoVac undifferentiated spermatogonia. Spermatogonia with nuclear vacuoles (AdVac) were preferentially observed closer to the interstitial regions and, in specific stages of the SEC, closer to blood vessels in opposition with the AdNoVac spermatogonia. Such positioning of undifferentiated spermatogonia usually indicates the presence of controlling paracrine and juxtacrine factors (Wang et al., 2015; Potter and DeFalco, 2017). In our view, this positioning suggests that AdVac spermatogonia may be subjected to controlling factors that co-ordinate their position and function; and we identified, for the first time in primates, the presence of spermatogonial niches. In contrast, types AdNoVac, Apale, B1 and B2 spermatogonia and preleptotene spermatocytes were found randomly distributed in terms of proximity to interstitial areas and blood vessels. The random positioning observed for differentiating spermatogonia (B1 and B2) is similar to that observed in the same differentiating cell types in rodents (A1, A2, A3, A4, In and B) (Chiarini-Garcia and Russell, 2001; Chiarini-Garcia et al., 2003). However, while in rodents all undifferentiated spermatogonia are distributed in niche areas, in marmosets the undifferentiated AdNoVac and Apale spermatogonia were distributed regardless of the presence of niche regions. The AdVac spermatogonia behavior in marmosets, e.g. proximity to interstitium and blood vessels around the spermiation event (stage IV) and away from those structures at subsequent stages (V and VI), was similar to the previous findings concerning type A undifferentiated spermatogonia (Asingle, Apared and Aaligned) in mouse (Chiarini-Garcia et al., 2001) and rat (Chiarini-Garcia et al., 2003). This repeatable pattern among mammalian species could mean a special functional change in the seminiferous epithelium during the spermiation process, resulting in active migration of stem spermatogonia. However, further studies are required to determine the meaning of this behavior and address the regulatory factors that could control the spermatogonial niches. To evaluate the efficiency of specific steps in spermatogenesis, distinct indexes can be used to compare different species and determine specific characteristics in the spermatogenic process. To evaluate the proliferative step, the mitotic index for marmoset was determined and reached a value of 8.4. The meiotic index showed that three round spermatids were produced by each diplotene spermatocyte that entered meiosis. Sertoli cells could support six round spermatids; and the overall efficiency of spermatogenesis, represented by the number of round spermatids produced by each Adark spermatogonia, was 45. This finding is consistent with previous studies in marmosets (Wistuba et al., 2003; Luetjens et al., 2005), despite the different methodologies employed. In humans, these indexes were all lower: mitotic and meiotic indexes <2; Sertoli workload = 4; and overall efficiency of the spermatogenesis <5 (Chiarini-Garcia et al., 2017). Thus, our findings show that marmosets present a higher spermatogenesis efficiency for all indexes evaluated when compared to humans. These indexes were determined considering the whole pool of Adark spermatogonia as stem cells, regardless of the presence or absence of nuclear vacuoles. However, when analyzing them as separated SSC, the values for mitotic index and spermatogenesis efficiency were both more than threefold higher in AdVac. Parameters of the spermatogenic process of the black-tufted marmoset C. penicillata (Leal and França, 2006) present similarities with those of common marmosets C. jacchus (Holt and More, 1984; Millar et al., 2000; Weinbauer et al., 2001) in terms of tubular diameter, number of stages per tubular cross-sections, number of germ cells, duration of premeiotic and postmeiotic phases and apoptotic rates. However, as detailed studies of spermatogonial morphology, kinetics and niche have never been performed so far in C. jacchus, it is impossible to say if their spermatogenic processes are fully comparable. In conclusion, the present study showed that black-tufted marmosets have four spermatogonial generations (Adark, Apale, B1 and B2). Additionally, we showed that Adark spermatogonia can be separated into two subtypes (AdVac and AdNoVac) that show different kinetic distribution along the stages of the SEC and distinct positioning in the seminiferous epithelium. Functional studies further exploring AdVac spermatogonia and the chromatin rarefaction zones may help to improve the current understanding of the primates SSCs. Supplementary data Supplementary data are available at Molecular Human Reproduction Online. Acknowledgements We thank the Centro de Microscopia (UFMG, Brazil) and Centro de Aquisição e Processamento de Imagens—CAPI (UFMG, Brazil) for the use of their facilities. Authors’ roles Tissue collection and data acquisition and analysis: A.L.C.-B., D.A.-F. and L.E.-G.; data interpretation: A.L.C.-B., D.A.-F., L.E.-G. and H.C.-G.; article drafting and critical revision: A.L.C.-B., F.R.C.L.A. and H.C.G.; research conception, design and coordination: H.C.-G. Approval of the final version: all authors. Funding This study was partially supported by the following Brazilian Foundations: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), and Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq). Conflicts of Interest None of the authors has received any benefits that are relevant to this study from sources other than the research foundations listed above. References Abercrombie M . Estimation of nuclear population from microtome sections . Anat Rec 1946 ; 94 : 239 – 247 . Google Scholar CrossRef Search ADS PubMed Amann R , Almquist J . Reproductive capacity of dairy bulls. VIII. Direct and indirect measurement of testicular sperm production . J Dairy Sci 1962 ; 45 : 774 – 781 . Google Scholar CrossRef Search ADS Chiarini-Garcia H , Hornick JR , Griswold MD , Russell LD . Distribution of type A spermatogonia in the mouse is not random . Biol Reprod 2001 ; 65 : 1179 – 1185 . 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Organization of seminiferous epithelium in primates: relationship to spermatogenic efficiency, phylogeny, and mating system . Biol Reprod 2003 ; 69 : 582 – 591 . Google Scholar CrossRef Search ADS PubMed Wistuba J , Stukenborg J-B , Luetjens CM . Mammalian spermatogenesis . Func Dev Embryol 2007 ; 1 : 99 – 117 . Yoshida S , Sukeno M , Nabeshima Y . A. vasculature-associated niche for undifferentiated spermatogonia in the mouse testis . Science 2007 ; 317 : 1722 – 1726 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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Molecular Human ReproductionOxford University Press

Published: Apr 11, 2018

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