Introduction
The order Rodentia is the largest from the Mammalia class, representing about 40% of the mammalian species alive at present, with approximately 29 families, 443 genera and more than 2000 species that occur in almost all habitats (Nowak, Reference Nowak1999; Lange & Schmidt, Reference Lange, Schmidt, Cubas, Silva and Catão-Dias2007). The genus Proechimys (sub-order Hystricomorpha, family Echimyidae) represents one of the most diverse groups of neotropical rodents, with 25 species (Wilson & Reeder, Reference Wilson and Reeder2005). These rodents are terrestrial, nocturnal and called ‘spiny rat’ because of their rigid and prickly hair. Species from this genus are frugivorous but they also eat seeds and fungi (Eisenberg & Redford, Reference Eisenberg and Redford1999; Catzeflis & Patton, Reference Catzeflis and Patton2008), and reproduces throughout the year (Weir, Reference Weir1973; Nowak, Reference Nowak1999; Patton et al, Reference Patton, Da Silva and Malcolm2000).
The Proechimys guyannensis was described by Geoffroy (Reference Geoffroy Saint-Hilaire1803) and was successfully raised in the laboratory, being used for experimental research since 1960 (Hawking et al., Reference Hawking, Walker and Worms1964). A remarkable characteristic of this species is the high degree of development of the newborns (Arida et al., Reference Arida, Scorza, Carvalho and Cavalheiro2005; Scorza et al., Reference Scorza, Araujo, Leite, Torres, Otalora, Oliveira, Garrido-Sanabria and Cavalheiro2011). Due to its resistance to chronic epilepsy, this species has been used as a model for this disease (Cavalheiro, Reference Cavalheiro1995; Fabene et al., Reference Fabene, Bertini, Correia, Cavalheiro and Bentivoglio2001; Arida et al., Reference Arida, Scorza, Carvalho and Cavalheiro2005; Rocha et al., Reference Rocha, Arida, Carvalho, Scorza, Neri-Bazan and Cavalheiro2006; Silva et al., Reference Silva, Pimenta, Andersen, Schoorlemmer, Tufik and Cavalheiro2014), being also, as a natural host, used in studies involving infectious parasites (Everard & Tikasingh, Reference Everard and Tikasingh1973). Investigations related to ecology and evolution of P. guyannensis are also available in the literature (Steiner et al., Reference Steiner, Sourrouille and Catzeflis2000; Eler et al., Reference Eler, da Silva, Silva and Feldberg2012; Silva et al., Reference Silva, Eler, da Silva and Feldberg2012).
Although testis structure and organization can be very similar between mammals, each species may exhibit particular morphofunctional characteristics, such as those related to phylogenetic aspects and reproductive strategy/behaviour (Kerr et al, Reference Kerr, Loveland, O'Bryan, de Kretser and Neill2006; Setchell & Breed, Reference Setchell, Breed and Neill2006). Therefore, knowledge of male reproductive biology and physiology is fundamental for comparative and evolutionary studies (Wildt, Reference Wildt2005). This knowledge is also important to prevent species from extinction, as well as to improve species management and enhance male reproductive capacity in natural and artificial breeding programmes (Comizzoli et al, Reference Comizzoli, Mermillod and Mauget2000). The P. guyannensis reproduces throughout the year and its estimated age of puberty is around 83 days old (Tesh, Reference Tesh1970; Weir, Reference Weir1973; Nowak, Reference Nowak1999; Patton et al, Reference Patton, Da Silva and Malcolm2000; Madureira et al., Reference Madureira, Passos, Resende, Souza, Almeida and Zanini2014); however, its reproductive biology is poorly known. Therefore, the aims of the present study were to perform a detailed histological and stereological/morphometric analysis of testis structure and function, which allowed the estimation of key reproductive parameters such as duration of spermatogenesis and the SC and spermatogenic efficiencies for this species.
Materials and methods
Animals
Ten sexually mature (180 days old) males Proechimys guyannensis were used in the present study. The animals came from the vivarium of the Federal University of São João del-Rei (UFSJ), in Minas Gerais, Brazil. All procedures and protocols followed approved guidelines for the ethical treatment of animals (CETEA/UFMG).
BrdU injections and tissue preparation
To estimate the duration of spermatogenesis, two animals received intraperitoneal injections of 5-bromo-2′-deoxyuridine (BrdU; 150 mg/kg), a specific marker for cells that are synthesizing DNA. Two time intervals (1 h and 20 days) following BrdU injections were considered for each animal. At sacrifice, all 10 animals received an i.p. injection of sodium thiopental (50 mg/kg), and testes were removed, weighed and fixed by immersion in buffered glutaraldehyde or Bouin's fixative. Then, testes were cut transversally, routinely processed and embedded in plastic (glycol methacrylate) for histomorphometric analyses or in paraplast for BrdU analyses.
Immunostaining for BrdU
Immunohistochemical staining of cells in S-phase using anti-BrdU antibody was performed as briefly follows. Sections 5 µm thick were mounted on coated slides, dewaxed and rehydrated. Then, antigen retrieval was performed in citrate buffer (pH 6.0) for 5 min after boiling in a microwave oven. Slides were incubated in 30% hydrogen peroxide (Sigma Aldrich) for 30 min at room temperature, to block endogenous peroxidase activity. Nonspecific binding sites were blocked with 10% normal horse serum (VectorStain ABC kit, Vector Laboratories) in PBS before the addition of primary antibody anti-BrdU (BD Biosciences, R2232, 1:200) and incubated overnight at 4°C. After this procedure, the slides were exposed to horse anti-mouse secondary antibody (1:200, VectorStain ABC kit, Vector Laboratories) for 60 min at room temperature. Detection of the signal was performed by incubating the sections in streptavidin (Thermo Scientific, TS-125-HR) for 30 min at room temperature, followed by the reaction with peroxidase substrate 3,3′-diaminobenzidine (DAB, Sigma Aldrich) and counterstaining with hematoxylin (Merck). Following dehydration, sections were mounted and analyzed by light microscopy to detect the most advanced germ cell type labelled at the two different time intervals evaluated after BrdU injection.
Testis morphometry
Sections of 4-µm thickness obtained from plastic embedded testis were stained with toluidine blue. The volume densities of the testicular components were determined on images captured by light microscopy, using a 540-intersection grid from ImageJ software (National Institutes of Health, http://rsb.info.nih.gov/ij/). Fifteen randomly chosen fields/images (8100 points) were scored for each animal (n = 6) at ×400 magnification. Points were classified as one of the following: seminiferous epithelium, tunica propria, lumen, Leydig cell, connective tissue, blood or lymphatic vessels. The volume of each testis component was determined as the product of its volume density and testis parenchyma volume. For subsequent morphometric calculations, the specific density of testis tissue was considered to be 1.0 (França & Godinho, Reference França and Godinho2003; Leal & França, Reference Leal and França2006). To obtain a more precise measure of testis volume, the mean value of testis capsule (3.2%) was excluded from the testis weight. Seminiferous tubular diameter and height of the seminiferous tubule epithelium were measured at ×400 magnification using an ocular calibrated with a stage micrometer. Thirty round or nearly round tubular profiles were randomly chosen and measured for each animal. The total length of the seminiferous tubule (in metres) was obtained by dividing the seminiferous tubule volume by the square radius of the tubule multiplied by π (Johnson & Neaves, Reference Johnson and Neaves1981; França & Godinho, Reference França and Godinho2003; Auharek et al., Reference Auharek, Avelar, Lara, Sharpe and França2011).
Stages and length of the seminiferous epithelium cycle
Stages of the seminiferous epithelium cycle were characterized (n = 8) based on the development of the acrosomic system (Russell et al., Reference Russell, Ettlin, Sinha-Hikim and Clegg1990) and morphology of the spermatid nucleus. The relative stage frequencies were determined from the analysis of at least 200 seminiferous tubule cross-sections per animal, at ×1000 magnification, as described by Leal & França (Reference Leal and França2006). The histological sections used were those that presented high quality and more tubular cross-sections.
The duration of the spermatogenic cycle was estimated based on the stage frequencies and the most advanced germ cell type labelled at the two time periods used post-BrdU injections. The total duration of spermatogenesis took into account that approximately 4.5 cycles are necessary for this process to be completed, from type A spermatogonia to spermiation (Amann & Schanbacher, Reference Amann and Schanbacher1983; França & Russell, Reference França, Russell, Regadera and Martinez-Garcia1998; Leal & França, Reference Leal and França2006; Hess & França, Reference Hess, França and Cheng2007). Because the nuclear volume of pachytene primary spermatocytes grows markedly during meiotic prophase (França & Russell, Reference França, Russell, Regadera and Martinez-Garcia1998; Neves et al, Reference Neves, Chiarini-Garcia and França2002), the size of their nuclei was used to determine more precisely the location of the most advanced labelled germ cell, particularly when these cells were present in stages showing high frequency.
Cell counts and cell numbers
All germ cells nuclei and SCs nucleoli present at stage VI (near spermiation) of the cycle were counted in ten randomly chosen round or nearly round seminiferous tubule cross-sections for each animal. These counts were corrected for section thickness (4 µm) and nuclear or nucleolar diameter according to Abercrombie (Reference Abercrombie1946), as modified by Amann & Almquist (Reference Amann and Almquist1962). For this purpose, 10 nuclei or nucleoli diameter were measured per animal for each cell type analyzed. Cell ratios were obtained from the corrected counts obtained. The total number of SCs was determined from the corrected counts of Sertoli cell nucleoli per seminiferous tubule cross-sections and the total length of ST (Hochereau-de Reviers & Lincoln, Reference Hochereau-de Reviers and Lincoln1978; Leal & França, Reference Leal and França2006; Costa et al., Reference Costa, Leal, Silva, Cássia, Ferreira, Guimarães and França2010). Daily sperm production (DSP) per testis and per gram of testis was determined based on the formula:
{\fontsize{7}{9}\selectfont{
$$\begin{eqnarray*}
{\rm DSP} = {\rm total\,number\,of\,SCs\,per\,testis}\\
&&\times \,{\rm ratio\,of\,round\,spermatids\,to\,Sertoli\,cells\,at\,stage\,VI}\\
&&\times \,{\rm stage\,VI\,relative\,frequency\,(\%)/stage\,VI\,duration}\left( {\rm days} \right)
\end{eqnarray*}$$}}
(Leal & França, Reference Leal and França2006; Auharek et al., Reference Auharek, Avelar, Lara, Sharpe and França2011, Cordeiro-Junior et al., Reference Cordeiro-Junior, Costa, Talamoni and França2010).
The individual volume of Leydig cells was obtained from the nucleus volume and the proportion between the nucleus and cytoplasm (Leal & França, Reference Leal and França2006; Auharek et al., Reference Auharek, Avelar, Lara, Sharpe and França2011). As the Leydig cell nucleus in this species is spherical, nucleus volume was calculated from the mean nucleus diameter and, for this purpose, 30 nuclei with an evident nucleolus was measured per animal. Leydig cell nuclear volume was expressed in µm3 and obtained by the formula 4/3πr3, where r = nuclear diameter/2. To calculate the proportion between nucleus and cytoplasm, a 441-point square lattice was placed over the sectioned material at ×1000 magnification and 1000 points over Leydig cells were counted for each animal. Subsequently, the total number of Leydig cells per testis was estimated from the Leydig cell individual volume and the volume density occupied by Leydig cells in the testis parenchyma.
Results
Biometric data and testis stereology
Biometric and testis morphometric data are shown in Table 1. The obtained testis weight was 1.63 ± 0.2 g, while the gonadosomatic index (GSI; testes mass divided by body weight) was 1.15 ± 0.2%. The ST occupied nearly 96% of the testis parenchyma, whereas Leydig cells comprised 1.5 ± 0.4%. Mean seminiferous tubule diameter and epithelium height were 168 ± 7 and 65 ± 2 µm, respectively. Based on the volume of testis parenchyma and the volume occupied by ST in the testis and the tubular diameter, the total length of seminiferous tubules per testis was 66 ± 8 m.
Table 1 Biometric and morphometric data in P. guyannensis
Stages of the seminiferous epithelium cycle and relative frequencies
Based on the development of the acrosomic system and the morphology of the developing spermatid nucleus, 12 stages of the seminiferous epithelium cycle were characterized. These stages (shown in Fig. 1) are briefly described below.
Figure 1 Stages of the seminiferous epithelium cycle and its frequencies. (a) Stages I–XII of the seminiferous epithelium cycle in P. guyannensis based on the acrosomic system. The individual germ cell nuclei shown in the right column represent the germ cells found in each particular stage. A: type A differentiated spermatogonia; In: Intermediate spermatogonia; B: type B spermatogonia; Pl: preleptotene spermatocytes; L: leptotene spermatocytes; Z: zygotene spermatocytes; P: pachytene spermatocytes; D: diplotene spermatocytes; Meiosis: meiotic figures; R: round spermatids; E: elongating/elongated spermatids; SC: Sertoli cells. (b) Frequencies (mean percentage ± SEM) of the 12 stages of the cycle in P. guyannensis.
Stage I
Two generations of spermatids were observed in this stage: early round spermatids and elongated spermatids. The newly formed round spermatids were smaller than secondary spermatocytes and were characterized by the lack of proacrosomal granules, although a juxtanuclear Golgi apparatus was evident at ×1000 magnification. Elongated spermatid bundles are more packed. As occurs with all spermatogonial cells, type A differentiated spermatogonia are in contact with the basal membrane.
Stage II
Early round spermatids with two small proacrosomal vesicles were present. At the end of this stage, these proacrosomal vesicles have coalesced to form a single acrosomal vesicle in contact with the nucleus and containing an acrosomal granule. Intermediate spermatogonia are also present in the basal compartment.
Stage III
This stage begins as the acrosomal vesicle flattens over the surface of the nucleus. The elongated spermatids moved toward the seminiferous tubule lumen. Type B spermatogonia were also observed.
Stage IV
The acrosome spreads over the surface of the nucleus. The elongated spermatids bundles moved deep inside the seminiferous epithelium. As observed in the previous stage, type B spermatogonia were in contact with the basement membrane.
Stage V
Elongated spermatid bundles were more dissociated and moved toward the lumen. Type B spermatogonia in transition to preleptotene spermatocyte were present at the base of the seminiferous epithelium.
Stage VI
Elongated spermatids with a spatulated head were being released/spermiated toward the tubule lumen. Two generations of primary spermatocytes were present: preleptotene spermatocytes lining the basal membrane; and pachytene spermatocytes positioned between round spermatids and preleptotene spermatocytes.
Stage VII
Only one spermatid generation, with round nuclei and forming several layers within the upper part of the seminiferous epithelium, was present in this stage. The acrosome continued spreading over the nucleus. Preleptotene spermatocytes were in contact with the basement membrane.
Stage VIII
Together with the acrosome, the spermatid nuclei begun to elongate, becoming ovoid in shape. Preleptotene spermatocytes in transition to leptotene were observed.
Stage IX
The acrosome followed the nuclear elongation of the spermatids. Elongated spermatids were with their heads oriented toward the SC nuclei. Leptotene spermatocytes were present in the base of the seminiferous epithelium.
Stage X
A ventral angle was formed in the elongated heads of the spermatids. These elongated spermatids formed bundles in the seminiferous epithelium. The pachytene spermatocytes were in transition to the diplotene phase of the meiotic prophase, while the leptotene spermatocytes changed to zygotene.
Stage XI
Elongation of spermatids was complete at this stage. Two generations of primary spermatocytes were present: zygotene and diplotene.
Stage XII
The presence of meiotic figures related to the first and second meiotic divisions was the main characteristic of this stage. Secondary spermatocytes and newly formed round spermatids were also observed. The zygotene spermatocytes were in the transition to pachytene.
Sertoli cells and undifferentiated type A spermatogonia were observed in all twelve stages characterized. The mean frequencies of the stages of the seminiferous epithelium cycle are displayed in Fig. 1. Stages VI and VII had the highest frequencies (19.6 and 17.2% respectively), whereas stages II–IV were less frequent (~2 to ~4%). The frequencies of pre-meiotic (stages VII–XI), meiotic (stage XII) and post-meiotic (stages I–VI) phases of the cycle were ~49, ~10 and ~41%, respectively.
Length of the seminiferous epithelium cycle
The most advanced labelled germ cell types observed following BrdU injection are shown in Fig. 2. Approximately 1 h after injection, the most advanced germ cells labelled were identified as preleptotene spermatocytes or cells in the transition from preleptotene to leptotene spermatocytes. Based on the mean pachytene nucleus diameter in both animals investigated in this aspect, these spermatocytes were present at the middle part of stage VII. At 20 days after BrdU injection, the most advanced germ cells labelled were elongated spermatids present in stage III. Based on the most advanced germ cell labelled at each time period and the stage frequencies, the mean duration of one seminiferous epithelium cycle was estimated to be 7.48 ± 0.01 days, whereas the total duration of spermatogenesis, considering that 4.5 cycles are necessary for the spermatogenic process to be completed, was estimated to be 33.6 ± 0.06 days.
Figure 2 Most advanced germ cells labelled after BrdU injection and the germ cell composition of each stage of the seminiferous epithelium cycle. (a) One hour after injection, preleptotene/leptotene spermatocytes (arrows) at the middle part of stage VII are labelled. (a′) Negative control. (b) Twenty days after injection, elongated spermatids (arrows) at the stage III are labelled. (b′) Negative control. (c) Diagram showing germ cell composition of each stage of the seminiferous epithelium cycle in P. guyannensis. Letters within each column indicate the germ cell type present in each stage. A: type A spermatogonia; In: intermediate spermatogonia; B: type B spermatogonia; Pl: preleptotene; L: leptotene; Z: zygotene; P: pachytene; D: diplotene; R: round spermatids; and E: elongating/elongated spermatid. Scale bars: 10 µm
Cell counts and cell numbers
The cell counts, ratios and sperm production are shown in Table 2. The meiotic index (number of round spermatids produced per primary pachytene spermatocyte) was 2.7 ± 0.1. Sertoli cell efficiency (number of round spermatids per Sertoli cell) was approximately 8 ± 1. The number of SCs per testis and per gram of testis were 112 × 106 ± 12 × 106 and 78 × 106 ± 11 × 106 cells, respectively. The DSP per testis and per gram of testis (spermatogenic efficiency) was approximately 121 × 106 ± 24 × 106 and 78 × 106 ± 9 × 106, respectively. It means that, in total, around 240 × 106 sperm were produced daily.
Table 2 Cell counts, cell ratios and sperm production
Leydig cell nuclear volume and Leydig cell individual size were 183 ± 4 and 746 ± 51 µm³, whereas their number per testis and per gram of testis were respectively 28.3 × 106 ± 6.7 × 106 and 19 × 106 ± 4.6 × 106 cells (Table 3).
Table 3 Leydig cell morphometry
Discussion
The Proechimys guyannensis is a neotropical rodent that is a natural host of infectious parasites and is currently used in biomedical research, particularly due to its resistance to epilepsy. Comprehensive studies on testicular structure and function have been performed on less than 2% of living mammalian species (Nowak, Reference Nowak1999; Almeida et al., Reference Almeida, Leal and França2006; Leal & França, Reference Leal and França2009; Cordeiro-Junior et al., Reference Cordeiro-Junior, Costa, Talamoni and França2010; Costa et al., Reference Costa, Leal, Silva, Cássia, Ferreira, Guimarães and França2010), and to our knowledge, there are few reports related to the reproductive biology of this species. Due to the very high ST volume density (%) and number of SCs per testis gram and the very short duration of spermatogenesis, the spermatogenic efficiency (DSP per gram of testis) obtained for P. guyannensis is one of the highest obtained for the mammalian species investigated to date. This DSP is similar to that observed for Trinomys moojeni (Cordeiro-Junior et al., Reference Cordeiro-Junior, Costa, Talamoni and França2010), which is another species of the same family herein investigated. Because there are very few data available for the members of this family, the discussion below will be focused mainly on the data available for the main investigated laboratory rodents (rats, mice and hamsters) and T. moojeni (please see Table 4).
Table 4 Comparative parameters related to biometry, testis stereology and spermatogenesis in some well investigated rodent species
a Cordeiro-Junior et al., Reference Cordeiro-Junior, Costa, Talamoni and França2010.
b Clermont & Trott, Reference Clermont and Trott1969; Avelar et al., Reference Avelar, Leal, França, Levy, Taskén and Hansson2004; Auharek et al., Reference Auharek, Avelar, Lara, Sharpe and França2011.
c Clermont & Harvey, Reference Clermont and Harvey1965; Rocha et al., Reference Rocha, Debeljuk and França1999; França, Reference França2007.
d Sinha Hikim et al., Reference Sinha-Hikim, Bartke and Russell1988; Van Haaster & De Rooij, Reference Van Haaster and De Rooij1993.
e Combined stages frequencies after spermiation and prior to metaphase.
f Meiotic division I through meiosis II.
g Combined stages frequencies after completion of meiosis until spermiation.
h Measured as the number of round spermatids produced per pachytene primary spermatocyte (presumptive germ cell loss during meiosis in parenthesis).
Based on the development of the acrosomic system, 12 stages of the seminiferous epithelium cycle were characterized in P. guyannensis and only one stage per seminiferous tubule cross-section was observed. This segmental arrangement of the stages is peculiar of rodents and is present in most mammals so far investigated (Leblond & Clermont, Reference Leblond and Clermont1952; Sharpe, Reference Sharpe, Knobil and Neill1994; França et al., Reference França, Avelar and Almeida2005). Several studies developed in our laboratory indicates that pre-meiotic and post-meiotic stage frequencies are phylogenetically determined (França & Russell, Reference França, Russell, Regadera and Martinez-Garcia1998; Neves et al., Reference Neves, Chiarini-Garcia and França2002; França et al., Reference França, Avelar and Almeida2005; Almeida et al., Reference Almeida, Leal and França2006; Costa et al., Reference Costa, Chiarini-Garcia, Morato, Alvarenga and França2008; Silva et al., Reference Silva, Costa, Andrade and França2010). In these studies, two clear patterns are observed for these frequencies in rodents: species in which the pre-meiotic frequency is about one-quarter of the entire spermatogenic cycle and species that show an equilibrium between the combined pre-meiotic and post-meiotic frequencies. In this aspect, similar to other rodents phylogenetically closely related (Paula et al., Reference Paula, Chiarini-Garcia and França1999; Leal & França, Reference Leal and França2009; Costa et al., Reference Costa, Leal, Silva, Cássia, Ferreira, Guimarães and França2010), P. guyannensis and T. moojeni belong to the latter pattern, whereas mice, rats and hamsters belong to the first pattern (Table 4). Conversely, the duration of spermatogenesis is considered to be species specific (Clermont, Reference Clermont1972; Amann & Schanbacher, Reference Amann and Schanbacher1983) and controlled by the germ cell genotype (França et al., Reference França, Ogawa, Avarbock, Brinster and Russell1998). Particularly when compared with other rodent species investigated (Russell et al, Reference Russell, Ettlin, Sinha-Hikim and Clegg1990; França & Russell, Reference França, Russell, Regadera and Martinez-Garcia1998; França et al., Reference França, Avelar and Almeida2005; Hess & França, Reference Hess, França and Cheng2007; Costa et al., Reference Costa, Leal, Silva, Cássia, Ferreira, Guimarães and França2010), the duration of one spermatogenic cycle (7.5 days) and the total duration of spermatogenesis (33.6 days) found in the present study is one of the shortest so far obtained.
In the literature, it is considered that total number of SCs per testis is established before puberty (Orth et al, Reference Orth, Gunsalus and Lamperti1988; Sharpe et al, Reference Sharpe, Fraser, Brougham, McKinnell, Morris, Kelnar, Wallace and Walker2003; França et al, Reference França, Avelar and Almeida2005; Holsberger & Cooke, Reference Holsberger and Cooke2005) and that the number of this key somatic cell per seminiferous tubule cross-section is stable along the different stages of the seminiferous epithelium cycle. Therefore, these cells are used as a reference to quantify and evaluate spermatogenesis (França & Russell, Reference França, Russell, Regadera and Martinez-Garcia1998; Johnson et al, Reference Johnson, Varner, Roberts, Smith, Keillor and Scrutchfield2000; França & Hess, Reference França, Hess, Skinner and Griswold2005). Specifically, the number of germ cells supported by each SC (Sertoli cell efficiency) is considered species specific and positively correlates with spermatogenic efficiency (França & Russell, Reference França, Russell, Regadera and Martinez-Garcia1998; Johnson et al, Reference Johnson, Varner, Roberts, Smith, Keillor and Scrutchfield2000; França & Hess, Reference França, Hess, Skinner and Griswold2005; França et al, Reference França, Avelar and Almeida2005; Hess & França, Reference Hess, França and Cheng2007). Although not very different from the values observed for mice, rats and hamsters (Sinha-Hikim et al, Reference Sinha-Hikim, Bartke and Russell1988; Van Haaster & De Rooij, Reference Van Haaster and De Rooij1993; França & Russell, Reference França, Russell, Regadera and Martinez-Garcia1998; Johnson et al, Reference Johnson, Varner, Roberts, Smith, Keillor and Scrutchfield2000; França & Hess, Reference França, Hess, Skinner and Griswold2005; França et al, Reference França, Avelar and Almeida2005; França, Reference França2007), the result found for SC efficiency in P. guyannensis (7.9) is much lower than that observed (~15) for the other investigated species from the Echimyidae family (T. moojeni; Cordeiro-Junior et al., Reference Cordeiro-Junior, Costa, Talamoni and França2010). However, because the number of SCs per testis gram obtained in the present study is strikingly high and the duration of spermatogenesis is ~15% faster, a similar spermatogenic efficiency was observed for P. guyannensis when compared to T. moojeni (Cordeiro-Junior et al., Reference Cordeiro-Junior, Costa, Talamoni and França2010). For most mammalian species already investigated, including laboratory rodents such as mice, rats and hamsters, this efficiency ranges from 20 × 106 to 50 × 106 (Sharpe, Reference Sharpe, Knobil and Neill1994; Johnson et al., Reference Johnson, Varner, Roberts, Smith, Keillor and Scrutchfield2000; Hess & França, Reference Hess, França and Cheng2007; Sousa et al., Reference Sousa, Campos-Junior, Costa and França2014). Therefore, the spermatogenic efficiency found for P. guyannensis can be included in the top level for mammals (Table 4).
In comparison with most mammalian species investigated (Kenagy & Trombulak, Reference Kenagy and Trombulak1986; França, Reference França2007; Leal & França, Reference Leal and França2008; Cordeiro-Junior et al., Reference Cordeiro-Junior, Costa, Talamoni and França2010; Costa et al., Reference Costa, Leal, Silva, Cássia, Ferreira, Guimarães and França2010), the gonadosomatic index (GSI) in P. guyannensis is relatively high. In the literature, it is considered that species with higher GSI are usually promiscuous (Kenagy & Trombulak, Reference Kenagy and Trombulak1986; Short, Reference Short1997; Costa et al., Reference Costa, Leal, Silva, Cássia, Ferreira, Guimarães and França2010). Besides that, only two to three pups are observed per gestation in this species (Madureira et al., Reference Madureira, Passos, Resende, Souza, Almeida and Zanini2014). These aspects deserve further investigation in P. guyannensis. Because the ST volume density observed in the present study is strikingly high (~96%), it could be expect that the Leydig cell volume occupancy would be very low. However, due to the relatively small cell size, the obtained number of this steroidogenic cell per testis gram is similar to many rodents so far investigated, such as agouti and paca (Costa et al., Reference Costa, Leal, Silva, Cássia, Ferreira, Guimarães and França2010) and rats (Clermont & Harvey, Reference Clermont and Harvey1965; Rocha et al, Reference Rocha, Debeljuk and França1999; França, Reference França2007), and even higher than the values found for T. moojeni (Table 4).
In summary, due to the very high seminiferous tubule volume density and number of SCs per testis gram, as well as to the short duration of spermatogenesis, the spermatogenic efficiency found in the present study for P. guyannensis is strikingly high. However, surprisingly, the number of pups per litter cited in the literature for this rodent is very small. Therefore, besides its importance in biomedical research, this species may represent an interesting model for investigating reproductive strategies in mammals.
Acknowledgements
Financial support from the Brazilian National Council for Scientific and Technological Development (CNPq), the National Council for the Improvement of Higher Education Personnel (CAPES), and the Foundation for Research Support of Minas Gerais (FAPEMIG) is gratefully acknowledged. Technical help from Mara L. Santos is also highly appreciated.
Declaration of conflicting interests
None.
