animals Article Follicular Atresia, Cell Proliferation, and Anti-Mullerian Hormone in Two Neotropical Primates (Aotus nancymae and Sapajus macrocephalus) Deise de Lima Cardoso 1,2 , Diva Anélie de Araújo Guimarães 1 , Pedro Mayor 2,3 , Maria Auxiliadora Pantoja Ferreira 1 , Leandro Nassar Coutinho 2 and Frederico Ozanan Barros Monteiro 2, * 1 Programa de pós-graduação em Ciência Animal, Universidade Federal do Pará, Rua Augusto Corrêa 01, Belém 66075-110, Brazil 2 Programa de Pós-Graduação em Saúde e Produção Animal na Amazônia, Universidade Federal Rural da Amazônia, Av. Perimetral 2501, Belém 66077-830, Brazil 3 Departament de Sanitat i Anatomia Animals, Facultat de Veterinària, Campus de la UAB, Universidade Autónoma de Barcelona, Plaça Cívica, 08193 Barcelona, Spain * Correspondence: frederico.monteiro@ufra.edu.br; Tel.: +55-(91)-99181-9493 Simple Summary: Reproductive processes in Neotropical primates are still poorly understood. Complications arising in these processes may result in a compromised reproductive condition for a significant portion of these species. We evaluated the follicular atresia, cell proliferation, and anti- Mullerian hormone presence in two Neotropical primates, and in three sexual phases (follicular, luteal, and gestational). Our results contribute to a greater understanding of the ovary morphofunctional processes that act in follicular selection, recruitment, and degeneration in these species. Abstract: This study evaluated the follicular atresia, cell proliferation, and anti-Mullerian hormone action in Aotus nancymae and Sapajus macrocephalus during three sexual phases (follicular, luteal, and gestational). Follicular quantification and immunolocalization of Caspase-3 protein, B-cell lymphoma Citation: Cardoso, D.d.L.; 2 (BCL-2), proliferating cell nuclear antigen (PCNA), and anti-Mullerian hormone (AMH) were Guimarães, D.A.d.A.; Mayor, P.; performed. A significant difference in the quantification between preantral and antral follicles, with a Ferreira, M.A.P.; Coutinho, L.N.; progressive decrease in the antrals, was identified. Protein and hormonal markers varied significantly Monteiro, F.O.B. Follicular Atresia, between follicle cell types (A. nancymae p = 0.001; S. macrocephalus, p = 0.002). Immunostaining in Cell Proliferation, and Anti-Mullerian the preantral and antral follicles was present in all sexual phases; for Caspase-3, in granulosa cells, Hormone in Two Neotropical oocytes, and stroma; for BCL-2, in granulosa cells, oocytes, and theca; and for PCNA and AMH, in Primates (Aotus nancymae and Sapajus oocytes and granulosa cells. The immunostaining for Caspase-3 was more expressive in the preantral macrocephalus). Animals 2023, 13, 1051. follicles (follicular phase, p < 0.05), while that for BCL-2 and PCNA was more expressive in the antral https://doi.org/10.3390/ follicles of the follicular phase. The AMH was more expressive in the primary and antral follicles of ani13061051 nonpregnant females, in both the follicular and luteal phases. Our results contribute to understanding Academic Editor: Giuseppina Basini the ovarian follicular selection, recruitment, and degeneration of these species. Received: 7 December 2022 Revised: 29 December 2022 Keywords: owl monkey; night monkey; capuchin monkey; ovary; folliculogenesis; apoptosis Accepted: 7 February 2023 Published: 14 March 2023 1. Introduction In recent years, there has been an increase in reproductive studies focusing on owl or Copyright: © 2023 by the authors. night monkeys (Aotus spp.) [1–6] and capuchin monkeys (Sapajus spp. and Cebus spp.) [4,7,8]. Licensee MDPI, Basel, Switzerland. There are behavioral differences between the primates, such as their mating systems. Owl This article is an open access article monkeys are monogamous [9], while capuchin monkeys are polygamous [10]. Their shared distributed under the terms and reproductive characteristics include the presence of a menstrual cycle [4] and the ability to conditions of the Creative Commons Attribution (CC BY) license (https:// easily reproduce in captivity [11]. In both species, it has been observed that during follicular creativecommons.org/licenses/by/ maturation, granulosa cells, theca cells, and oocytes develop a process of cellular death, 4.0/). Animals 2023, 13, 1051. https://doi.org/10.3390/ani13061051 https://www.mdpi.com/journal/animals Animals 2023, 13, 1051 2 of 13 resulting in follicular atresia and selection, which regulates the ovulation rate and restricts their reproductive potential [12,13]. Apoptosis can be initiated by activation signals such as cleaved Caspase-3, which is an effector protease responsible for cleaving structural and functional proteins in cells [14–16]. These mechanisms can be blocked by regulators such as BCL-2 proteins, which prevent the activation of caspases [17,18]. Proliferating cell nuclear antigen (PCNA) is impor- tant for DNA synthesis and repair during the cell cycle, therefore maintaining tissue integrity [19,20]. Thus, PCNA can be used to identify cell proliferation in the ovaries of nonhuman primates (NHP) [21]. Anti-Mullerian hormone (AMH) is a growth factor produced by granulosa cells that regulate folliculogenesis [22,23] and plays an inhibitory role in maintaining the primordial ovarian follicle pool [22]. AMH may serve as an indicator of the antral follicle reserve in humans and NHP [23,24]. The processes of follicular activation and selection in Aotus spp. and Sapajus spp. are still poorly understood [11]. Complications arising in these processes may result in a compromised reproductive condition for a significant portion of these species. There- fore, this study aims to describe the physiology related to follicular atresia through the immunostaining of Caspase-3, BCL-2, PCNA, and AMH proteins in the Ma’s night monkey and the large-headed capuchin. 2. Materials and Methods 2.1. Study Sites and Animals Genital organs from postpubertal Ma’s night monkeys (Aotus nancymae, n = 6) and large-headed capuchins (Sapajus macrocephalus, n = 6) were collected from two sites in the Peruvian Amazon. Ovaries from six Ma’s night monkeys kept in captivity at the Primate Center of the Instituto Veterinario de Investigaciones Tropicales y de Altura (IVITA, Iquitos, Peru) were collected. An additional six ovaries from large-headed capuchins were collected as waste organs from subsistence hunters in the Yavarí-Mirín River basin (YMR, S 04◦ 19.53; W 71◦ 57.33). All biological samples were free of charge and donated voluntarily. 2.2. Histological Procedure and Follicular Quantification Genital organs were examined for evidence of embryos or fetuses. Females with an embryo or fetus were considered pregnant. Nonpregnant females with ovaries containing active cyclic corpus luteum (CL) were classified as being in the luteal phase. Females with ovaries with large antral follicles and without cyclic CL were in the follicular phase of the menstrual cycle. The ovaries were fixed in 4% buffered formalin (v/v), dehydrated, embedded in paraffin wax, serially sectioned at 5 µm along the longitudinal axis, stained in hematoxylin and eosin, and analyzed under a photomicroscope. One section was selected for each series of ten sections cut. Ovarian follicles were classified following the criteria shown in Table 1. The total number of follicles was calculated using the following formula [25]: TN = number of follicles × total number of cuts per ovary × slice thickness (1) number of sections analyzed × nuclear diameter of each follicle Animals 2023, 13, 1051 3 of 13 Table 1. Criteria for ovarian follicle classification in Ma’s night monkeys (Aotus nancymae, n = 6) and large-head capuchins (Sapajus macrocephalus, n = 6) by Mayor et al. (2016, 2015b, 2019). Ovarian Follicles Follicle Preantral Antral Species Structures (Absence of Antrum) (Presence of Antrum) Primordial Primary Small Large Aotus Diameter (µm) 35.26 ± 7.13 109.35 ± 25.03 275.36 ± 67.57 728.51 ± 126.75 nancymae Layers 1 2–6 7–10 >10 Sapajus Diameter (µm) 33.76 ± 2.27 112.30 ± 31.43 238.84 ± 67.48 858.54 ± 149.05 macrocephalus Layers 1 2–6 7–10 >10 2.3. Immunohistochemistry for Caspase-3, BCL-2, PCNA, and AMH The sections of the ovaries were deparaffinized in xylene and rehydrated in decreasing concentrations of ethanol. The sections were washed in tween saline buffer solution (PBSw) and immersed in 3% hydrogen peroxide in methanol for 30 min. The slides were then incubated in sodium citrate buffer (0.1M pH6.0), heated to 70 ◦ C for 25 min, and incubated in 10% blocking buffer (10% goat serum + 3% bovine serum-BSA albumin + PBSw) for 1 h. Each slide was incubated with primary antibodies in the following dilutions: anti-cleaved Caspase-3 (1:200), anti-BCL-2 (1:200), anti-PCNA (1:100), and anti-AMH (1:100), for 12 h. Subsequently, the samples were post-incubated in peroxidase-conjugated anti-rabbit IgG secondary antibody (1:500) and developed in DAB solution (3,30 diaminobenzidine) for 5 min for photomicroscopic analysis. For the negative control, histological sections of the ovaries in the same analyzed phases were used, with the primary antibody replacement by PBSw. 2.4. Hormonal, Proliferative and Cell Apoptotic Quantification Immunostained oocytes, granulosa cells, and ovarian follicles (preantral and antral) were counted. A total of one thousand elements were counted per ovary. In each ovary, we calculated the percentage of the elements (ovarian follicles, granulosa cells, and oocytes) immunostained for cleaved Caspase-3, BCL-2, PCNA, and AMH [26,27]. 1. number of immunostained follicles/total follicles counted × 100; 2. number of immunostained granulosa cells/total of 1000 granulosa cells analyzed; 3. number of immunostained oocytes/number of oocytes counted × 100. All analyses were performed using NIS-Elements Microscope Imaging software (Nikon Corporation, Tokyo, Japan) and the Nikon Eclipse Ci-S H550S photomicroscope coupled to a digital camera (Nikon S-Ri1 DS-U3, Tokyo, Japan). 2.5. Statistical Analysis We compared the percentage of immunostained elements (ovarian follicles, granulosa cells, and oocytes) for Caspase-3, BCL-2, PCNA, and AMH counted in each ovary according to the sexual phase of females (follicular, luteal, and pregnant). Differences observed among these phases were assessed by a Kruskal–Wallis test, considering a significance of p < 0.05. Data were presented as mean and standard deviation. The number of granulosa cells, follicles, and oocytes for Caspase-3, BCL-2, PCNA, and AMH were evaluated by the Kruskal–Wallis test, indicating a significant difference between the values quantified by phases. To find the number of viable follicles by follicular types (preantral and antral), a Dunn test was applied, which demonstrated which repro- ductive periods were different from each other, following the assumptions of normality and homoscedasticity. A principal coordinate analysis (PCoA) was used to evaluate the relationship between the indicators of granulosa cells, follicles, and oocytes with positive staining for Caspase-3, BCL-2, PCNA, and AMH, while considering the sexual phases to elucidate the action Animals 2023, 13, 1051 4 of 13 of these proteins in the ovaries [28]. A permutational multivariate analysis of variance (PERMANOVA) was applied to assess the significance of the variation observed in this quantification [29]. The homogeneity of the dispersion and the difference between groups was determined by permutational analysis of multivariate dispersion (PERMDISP) [30]. All statistical analyses were performed using BioEstat 5.0 software (IDSM, MCT, CNPq, Belém, Pará, Brazil) and R Software (R Development Core Team, 2016). 3. Results 3.1. Follicular Quantification The number of follicles per follicular type did not vary between sexual phases (p > 0.05). Additionally, we observed a significant reduction in the number of follicles as follicular development progressed from primordial follicles to large antral follicles (Table 2). Table 2. Quantification of the ovaries of Ma’s night monkeys (Aotus nancymae, n = 6) and large-head capuchins (Sapajus macrocephalus, n = 6) according to the sexual phase. Ovarian Follicles Species Sexual Pre-Antral Antral Phases Primordial Primary Small Large Follicular 147.4 ± 6.6 a 83.9 ± 6.8 ab 17.6 ± 1.0 bc 5.0 ± 0.1 c Aotus Luteal 107.5 ±15.7 a 51.1 ± 1.6 ab 14.0 ± 0.5 bc 3.8 ± 0.1 c nancymae Gestational 123.2 ± 6.6 a 49.7 ± 6.2 ab 13.7 ± 0.1 bc 3.7 ± 0.1 c Follicular 141.0 ± 11.2 a 70.8 ± 1.6 ab 16.6 ± 0.7 bc 5.4 ± 0.1 c Sapajus Luteal 104.5 ±6.5 a 45.8 ± 2.3 ab 13.0 ± 0.5 bc 3.6 ± 0.07 c macrocephalus Gestational 135.1 ± 8.7 a 53.4 ± 1.8 ab 13.4 ± 0.4 bc 3.4± 0.06 c abc Different superscript letters on the same line indicate statistical difference. 3.2. Immunohistochemistry for Cleaved Caspase-3, BCL-2, PCNA, and AMH The immunoreactive pattern observed for cleaved Caspase-3, BCL-2, PCNA, and AMH was similar in the two species studied. Immunostaining for cleaved Caspase-3 was observed in oocytes and some granulosa cells of the preantral follicles (Figure 1A); it was also observed in the oocytes, granulosa cells, and in the internal and external theca of the antral follicles (Figure 1E). Strong immunostaining for BCL-2 and PCNA was observed in the oocytes and granulosa cells of the preantral and antral follicles (BCL-2: Figure 1B,F; PCNA: Figure 1C,G). Immunostaining for AMH was observed in granulosa cells of the Animals 2022, 12, x preantral follicles (Figure 1D), and in oocytes and granulosa cells of the antral follicles 5 of 13 (Figure 1H). Figure 1. Cont. Animals 2023, 13, 1051 5 of 13 Figure Photomicrographsofofovarian Figure1.1. Photomicrographs ovarian immunostaining immunostaining for for cleaved cleaved Caspase-3, Caspase-3, BCL-2, BCL-2, PCNA, PCNA, and AMH and AMH in granulosa in granulosacellscells and and oocytes of the oocytes of primordial and primary the primordial follicles and primary (A–D) follicles and small (A–D) and and small large and antral large follicles antral (E–H) follicles of Ma’s (E–H) nightnight of Ma’s monkeys (Aotus monkeys nancymae) (Aotus and large-head nancymae) capuchins and large-head (Sap- capuchins ajus macrocephalus). A: Negative control; Abbreviations: Po: Primordial follicle; Pm: Primary (Sapajus macrocephalus). A: Negative control; Abbreviations: Po: Primordial follicle; Pm: Primary follicle; T: Theca layers; Gc: Granulosa cells; An: Antrum; *: oocytes. Arrow: immunostained granulosa cells. follicle; T: Theca layers; Gc: Granulosa cells; An: Antrum; *: oocytes. Arrow: immunostained Hematoxylin–Eosin. Bar Scale: 25 µ m. granulosa cells. Hematoxylin–Eosin. Bar Scale: 25 µm. 3.3.Quantification 3.3. QuantificationofofImmunolabeled Immunolabeled Cells Cells for for Caspase-3-Cleaved, Caspase-3-Cleaved, BCL-2, BCL-2, PCNA, PCNA, and and AMH AMH Weobserved We observedsignificant significantdifferences differences inin thethe quantification quantification of of immunostained immunostained cells cells in in the the ovarian ovarian follicles follicles of both of both species species according according to the to the sexual sexual phase phase (p <(p0.05). < 0.05). Immunostain- Immunostaining ingCaspase-3 for for Caspase-3 and AMHand AMH in oocytes, in oocytes, granulosa granulosa cells, preantral cells, preantral and antral and antral follicles follicles was was higher higher in females in the follicular phase. Immunostaining for BCL-2 in females in the follicular phase. Immunostaining for BCL-2 and PCNA was mostly higher and PCNA was mostly higher in females in the follicular phase (p < 0.05); Table 3 shows these in females in the follicular phase (p < 0.05); Table 3 shows these differences according to differences according cell type and to the cellNPH. type and the NPH. In both species, the quantification analysis for PCNA showed a significant difference between granulosa cells in the follicular and gestational phases. Higher immunostaining (p < 0.05) was observed for the follicular phase and between oocytes and follicles in the follicular and luteal phases, with greater immunostaining for the gestational phase. In contrast, immunostaining for AMH revealed a statistically significant difference in granulosa cells, follicles, and oocytes between the follicular and luteal phases, with higher immunostaining for the follicular phase than the luteal phase (Table 3). In both the species studied, the percentage of immunostaining for Caspase-3 was higher in the preantral follicles than in the antral follicles (Figure 2). In contrast, the percentage of immunostaining for BCL-2, PCNA, and AMH was higher in the antral follicles than in the preantral follicles (p < 0.01) (Figure 2). Animals 2023, 13, 1051 6 of 13 Table 3. Number of ovarian follicles (Ov. follicles), granulosa cells, and oocytes immunostained for Caspase-3, BCL-2, PCNA, and AMH by sexual phases in Ma’s night monkeys (Aotus nancymae, n = 6) and large-head capuchins (Sapajus macrocephalus, n = 6). Sexual Phases Species Immunoreaction Quantification Follicular Luteal Gestational a b Ov. follicles 0.64 ± 0.07 0.27 ± 0.07 0.30 ± 0.04 ab Caspase-3 Granulosa cells 0.61 ± 0.02 a 0.44 ± 0.04 b 0.46 ± 0.02 ab Oocytes 0.79 ± 0.09 a 0.33 ± 0.08 b 0.37 ± 0.03 ab Ov. follicles 0.23 ± 0.04 0.19 ± 0.03 0.22 ± 0.04 BCL-2 Granulosa cells 0.51 ± 0.04 a 0.36 ± 0.03 ab 0.35 ± 0.03 b Aotus Oocytes 0.28 ± 0.04 0.23 ± 0.02 0.30 ± 0.07 nancymae Ov. follicles 0.24 ± 0.02 a 0.18 ± 0.01 b 0.19 ± 0.01 ab PCNA Granulosa cells 0.65 ± 0.04 a 0.42 ± 0.01 ab 0.36 ± 0.03 b Oocytes 0.32 ± 0.03 a 0.24 ± 0.03 b 0.26 ± 0.01 ab Ov. follicles 0.28 ± 0.03 a 0.16 ± 0.02 b 0.19 ± 0.03 ab AMH Granulosa cells 0.60 ± 0.07 a 0.32 ± 0.01 b 0.33 ± 0.02 ab Oocytes 0.32 ± 0.04 a 0.22 ± 0.03 b 0.25 ± 0.05 ab Ov. follicles 0.52 ± 0.13 a 0.24 ± 0.04 b 0.26 ± 0.05 ab Caspase-3 Granulosa cells 0.55 ± 0.03 a 0.39 ± 0.02 b 0.37 ± 0.04 ab Oocytes 0.77 ± 0.09 a 0.30 ± 0.03 b 0.30 ± 0.02 ab Ov. follicles 0.30 ± 0.02 a 0.17 ± 0.01 b 0.2 ±0.02 ab BCL-2 Granulosa cells 0.55 ± 0.04 a 0.34 ± 0.02 b 0.36 ± 0.03 ab Sapajus Oocytes 0.97 ± 0.24 a 0.26 ± 0.04 b 0.36 ± 0.01 ab macrocephalus Ov. follicles 0.24 ± 0.01 0.20 ± 0.01 0.23 ± 0.01 PCNA Granulosa cells 0.69 ± 0.03 a 0.48 ± 0.02 ab 0.38 ± 0.02 b Oocytes 0.28 ± 0.01 ab 0.24 ± 0.01 a 0.30 ± 0.03 b Ov. follicles 0.27 ± 0.02 a 0.16 ± 0.01 b 0.18 ± 0.03 ab AMH Granulosa cells 0.59 ± 0.04 a 0.31 ± 0.01 b 0.34 ± 0.03 ab Animals 2022, 12, x Oocytes 0.32 ± 0.03 a 0.19 ± 0.02 b 0.21 ± 0.03 ab 7 of 13 abc Different superscript letters on the same line indicate statistical difference. Figure Figure2. 2. Percentage of follicles (preantral (preantral and andantral) antral)immunostained immunostainedfor forCaspase-3, Caspase-3,BCL-2, BCL-2,PCNA, PCNA, and and AMH, according accordingtotothethesexual sexual phases phases (FOL—follicular, (FOL—follicular, LUT—luteal LUT—luteal and GES—gestational). and GES—gestational). (A) Ma’s (A) Ma’snight monkeys night monkeys (Aotus nancymae, (Aotus n = n6)=and nancymae, (B) (B) 6) and head-large capuchins head-large (Sapajus capuchins macrocephalus, (Sapajus n macrocephalus, n= =6).6). (*) (*) indicate thatthat indicate there is a is there statistically significant a statistically difference significant between difference preantral between and antral preantral and folli- antral cles, according follicles, to the according stage to the (Binomial stage Test.Test. (Binomial p < 0.01). p < 0.01). Significant differences Significant differences were were observed observed in in the the immunostaining immunostainingpatterns patternsofofgranulosa granulosa cells,follicles, cells, follicles, and and oocytes oocytes (Ma’s night monkeys: Pseudo Pseudo FF ==14.486 14.486pp==0.001; 0.001;large-headed large-headed capuchins: Pseudo capuchins: Pseudo F F== 8.32166; 8.32166; pp == 0.002). 0.002). In Inboth bothspecies, species,granulosa granulosacells cellswere werethe themost most immunoreactive for cleaved immunoreactive cleaved Caspase-3, Caspase-3,BCL-2, BCL-2,PCNA, PCNA,and andAMHAMH(Table (Table3 3and andFigure Figure3).3). There were There were greater greater differences differences observed between between the the quantification quantificationof ofgranulosa granulosacells cellsinin relation to follicles and oocytes (Pseudo F = 14.486; p = 0.001; Figure relation to follicles and oocytes (Pseudo F = 14.486; p = 0.001; Figure 3A,B). 3A,B). Significant differences were observed in the immunostaining patterns of granulosa cells, follicles, and oocytes (Ma’s night monkeys: Pseudo F = 14.486 p = 0.001; large-headed capuchins: Pseudo F = 8.32166; p = 0.002). In both species, granulosa cells were the most immunoreactive for cleaved Caspase-3, BCL-2, PCNA, and AMH (Table 3 and Figure 3). Animals 2023, 13, 1051 7 of 13 There were greater differences observed between the quantification of granulosa cells in relation to follicles and oocytes (Pseudo F = 14.486; p = 0.001; Figure 3A,B). Figure 3. Principal Figure 3. coordinate analysis Principal coordinate analysis (PCoA) (PCoA) of of the the variation variation in in the the follicular follicular cells, cells, follicle, follicle,and and oocytes in cleaved Caspase-3, BCL-2, PCNA, and AMH. (A) Ma’s night monkeys oocytes in cleaved Caspase-3, BCL-2, PCNA, and AMH. (A) Ma’s night monkeys (Aotus nancymae,(Aotus nancymae, nn = macrocephalus, nn ==6). (Sapajus macrocephalus, = 6) and (B) head-large capuchins (Sapajus 6). The The quantification quantification of immunohistochemical markers of immunohistochemical markers explained explained 94.52% 94.52% ofofthe thepositive positive immunostaining of follicles, granulosa cells, and oocytes for cleaved Caspase-3, immunostaining of follicles, granulosa cells, and oocytes for cleaved Caspase-3, BCL-2, BCL-2, PCNA, and AMH in Ma’s night monkeys (Figure 4A) and 94.85% PCNA, and AMH in Ma’s night monkeys (Figure 4A) and 94.85% for the same in large-for the same in large- headed capuchins (Figure 4B). In Ma’s night monkeys, the variables that contributed headed capuchins (Figure 4B). In Ma’s night monkeys, the variables that contributed most most to the formation of the first axis (75.83%) were BCL-2, PCNA, and AMH, which to the formation of the first axis (75.83%) were BCL-2, PCNA, and AMH, which were pos- were positively related to the axis. itively related to theFor theFor axis. second axis, cleaved the second Caspase-3 axis, cleaved contributed Caspase-3 positively contributed with positively 18.69%. In large-headed with 18.69%. In large-headedcapuchins, the variables capuchins, that contributed the variables most most that contributed to thetoformation the for- of the first axis (71.03%) were Caspase-3 and BCL-2. For the second axis (23.82%), mation of the first axis (71.03%) were Caspase-3 and BCL-2. For the second axis (23.82%), PCNA and AMH contributed positively. The results presented in Figure 3 confirmed PCNA and AMH contributed positively. The results presented in Figure 3 confirmed this this ordering Animals 2022, 12, x(Ma’s night monkeys: Pseudo F = 14.486; p = 0.001) and (large-headed capuchins: Pseudo 8 ordering (Ma’s night monkeys: Pseudo F = 14.486; p = 0.001) and (large-headed capuchins: FPseudo = 8.32166; p = 0.002). F = 8.32166; p = 0.002). Figure 4. Immunohistochemical variables (Caspase-3, Figure 4. Immunohistochemical BCL-2, variables PCNA, and (Caspase-3, AMH), BCL-2, sorted PCNA, byAMH), and the princi- sorted by the pal coordinate analysis (PCoA) according to follicular types. (A) Ma’s night monkeys (Aotus cipal coordinate analysis (PCoA) according to follicular types. (A) Ma’s night nancymae, monkeys (Aotu cymae, n =capuchins n = 6) and (B) head-large 6) and (B)(Sapajus head-large capuchins n(Sapajus macrocephalus, = 6). O:macrocephalus, n = 6). O: Oocytes; F: Follicles: G:Oocytes; Granu- F: Follic losa cells. Granulosa cells. 4. Discussion 4. Discussion A greater number of preantral A greater numberfollicles (primordial of preantral folliclesand primary) and (primordial was primary) observedwas in observ relation to antral follicles in the two species studied. These data are consistent with relation to antral follicles in the two species studied. These data are consistent with that previouslypreviously observedobserved in women in [25,31] womenand in capuchin [25,31] monkeys and in capuchin [32]. Preantral monkeys [32]. Preantral fol follicles, in the in early development stages, form the reserve pool. Such structures the early development stages, form the reserve pool. Such structures undergo undergo sele selection and dominance processes, in waves of follicular degeneration, which determine and dominance processes, in waves of follicular degeneration, which determine th productive potential of females [31,33]. Therefore, we believe that the quanti preantral follicles present is directly related to ovarian reserve maintenance in both cies in this study. This has been reported previously in women [31,33–35], NHP (rh [36], pigtailed monkeys [37], and capuchin monkeys [32], and in mice [38]. In these cies, the reserve of preantral follicles indicated the females reproductive life span. Animals 2023, 13, 1051 8 of 13 the reproductive potential of females [31,33]. Therefore, we believe that the quantity of preantral follicles present is directly related to ovarian reserve maintenance in both species in this study. This has been reported previously in women [31,33–35], NHP (rhesus) [36], pigtailed monkeys [37], and capuchin monkeys [32], and in mice [38]. In these species, the reserve of preantral follicles indicated the females reproductive life span. Immunostaining for Caspase-3 was observed in oocytes, granulosa cells, and theca cells from the primordial to preovulatory follicles in both species studied. Similar results have been described in the ovaries of women, with the same ovarian cellular compo- nents [39–41]. Thus, after the initial formation of the antrum, the activation of Caspase-3 represents the physiological effect of the follicle during atresia [42]. In the present study, the highest frequency of immunostaining for Caspase-3 was in the granulosa cells of the preantral and antral follicles. This indicates the occurrence of early follicle apoptosis in the oocytes and granulosa cells of the antral follicles [43,44]. In contrast, there was no immunostaining indicative of apoptosis in the preantral and non-atretic antral follicles; a similar result has been observed in the ovaries of rats [39,45]. This suggests that these follicles tend to develop. These findings indicate that apoptosis is evidence of follicular depletion and atresia during follicular dynamics in the ovaries of this study. These findings have also been observed in the ovaries of women and baboons [46]. According to Tilly et al. [47], apoptosis and the altered expression of the related genes are associated with ovarian cell degeneration in all vertebrate species. The same pattern was observed in the present study. The presence of BCL-2 in granulosa cells and oocytes in the three sexual phases tested in this study was relevant, as a similar result has been observed in the ovaries of women and rats [48]. However, when comparing BCL-2 immunostaining in Ma’s night monkey and large-headed capuchin ovaries, we observed a more significant value in the follicular phase, specifically in the antral follicles. Previous studies have found a greater expression of BCL-2 in the follicular phase of women [49,50] and suggest that this expression, in the menstrual cycle, is related to hormone regulation. Our results suggest that BCL-2 has a regulatory role in the cellular dynamics of the ovarian cycle in both species in this study. The results of the quantified granulosa cells, ovarian follicles, and oocytes immunos- tained for BCL-2 demonstrated its protective role against the advancement of the apoptotic process. This aligns with previous studies that have shown that BCL-2 acts as an ovarian anti-apoptotic factor [48,51–53]. Therefore, in this study, we suggest that the apoptotic process was suppressed by the action of BCL-2 to allow follicular repair or development, especially in the follicular phase. In contrast, we observed intense immunostaining for PCNA from the beginning of the follicular growth period. Similar results were observed in rats [54] and pigs [55]. According to Hirshfield [51], an intense proliferative activity of granulosa cells occurs shortly before the formation of the antrum. Thus, due to the increase in antral follicles, immunostaining for PCNA is reflected in the high rates of granulosa cell proliferation [55]. In the present study, intense immunostaining for PCNA was observed in oocytes from large antral follicles. A similar result was reported by Downey et al. [56], who linked this to DNA damage repair. The highest number of PCNA-positive granulosa cells, oocytes, and antral follicles occurred mostly in the follicular phase. These data corroborate the data previously observed in rat [54] and baboon ovaries [21]. Therefore, the results of the present study confirm that PCNA is an early marker of the onset of follicular growth, which contributes to a better understanding of the proliferative mechanisms involved in folliculogenesis of the species studied. In this study, immunostaining for AMH was observed in the granulosa cells of the preantral follicles, specifically the primary and antral follicles, particularly in the follicular phase. Such findings were also observed in women [57,58], mice [59–61], and NHP (bonnet macaque [23]; white-tufted-ear Marmoset [62]; squirrel monkey [63,64]; olive baboon [65]). AMH is produced by granulosa cells from follicles recruited to dominance and is likely involved in the regulation of follicular steroidogenesis [66]. Furthermore, AMH can act Animals 2023, 13, 1051 9 of 13 as an indirect serum marker of ovarian reserve and is considered a potential biomarker of ovarian follicular status [67–70]. Thus, we suggest that AMH acts as a regulator in the maturation of preantral and antral follicles during the ovarian cycle of the species in this study. The absence of immunostaining for AMH was observed in the primordial follicles in the species studied. However, in the primary follicles and small antrals, the highest expression of AMH occurred close to the antrum and in the large antrals around the oocyte. This aligns with previous observations in other species such as mice [60,71] and rats [72–74]. It has been reported that follicular growth exerts inhibitory feedback on immature follicles, such as primordial follicles [22,75–77]. Thus, our results confirm that the influence of AMH on the ovary occurs from the primary follicle, favoring the recruitment of follicles capable of development. 5. Conclusions The immunoreaction for cleaved Caspase-3, BCL-2, PCNA, and AMH indicates that the quantification of ovarian follicles, granulosa cells, and oocytes can assist in the evaluation of folliculogenesis in the ovaries of the species studied. These results aid our understanding of the action of proteins in folliculogenesis, which plays an important role in the gonadal remodeling process. This facilitates cell selection, inhibition, proliferation, repair, and elimination in follicular maturation. Therefore, our results improve the understanding of the events involved in ovarian dynamics and gamete production in these species. Author Contributions: D.d.L.C.: writing—original draft, investigation. D.A.d.A.G.: supervision, conceptualization, methodology, investigation. P.M.: supervision, visualization, investigation. M.A.P.F.: supervision, software. L.N.C.: supervision, funding acquisition. F.O.B.M.: supervision, visualization, investigation, writing—review and editing. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported by the Foundation for Research Support of Para (FAPESPA, protocol 005/2015), by the Coordination for the Improvement of Higher Education Personnel (CAPES, Scholarships and National Academic Cooperation Program in the Amazon–PROCAD/Amazônia, grant number 21/2018), and the National Council of Technological and Scientific Development (CNPq, grant number 305821/2017-2 and 176827/2022-6); and the Dean of Research and Graduate Studies (PROPESP) from the Federal University of Pará (Grant support 02/2023). Institutional Review Board Statement: The research protocol was evaluated and approved by the Ethics Committee for Experimentation from the Servicio Nacional Forestal y de Fauna Silvestre of Peru (protocol number 0350-2012-DGFFSDGEFFS), the Committee on Ethics in Research with Animals of the Federal Rural University of the Amazon, Belém, Pará, Brazil (CEUA/UFRA protocol 008/2016), and the Primate Center from Instituto Veterinario de Investigaciones Tropicales y de Altura (IVITA; Protocol No. 006/2014). The samples were sent to UFRA through the export license CITES/IBAMA (No. 14BR015991/DF). Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: We sincerely thank the community of Nueva Esperanza in the Yavarí-Mirín River who actively participated in data collection, showing the importance of communal participation in the development of wildlife management. 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