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From the * Department of Morphofisiological
Science, DCM-Bloco H-79, University of State of Paraná (UEM),
Paraná, Brazil; Laboratory of Cellular
Biology, Department of Morphology, Institute of Biological Science, Federal
University of Minas Gerais, Belo Horizonte, MG, Brazil;
Department of Anatomy, Institute of
Bioscience, State University of São Paulo, Botucatu, SP, Brazil;
Department of Morphology and Pathology,
Laboratory of Anatomy, UFSCAR, São Carlos, SP, Brazil.
| Correspondence to: Tânia Mara Segatelli, Department of Morphofisiological Science, DCM-Bloco H-79, University of State of Paraná (UEM), Av Colombo, 5790, Maringá, Paraná, Brazil (e-mail: tmsegatelli{at}uem.br). |
| Received for publication May 13, 2004; accepted for publication June 20, 2004. |
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Abstract |
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Key words: Testis, spermatogenesis, morphometry, sperm production
Estimates for the duration of the seminiferous epithelium cycle have been obtained utilizing several methods (Roosen-Runge, 1951; Clermont and Perey, 1957; Clermont and Antar, 1973; Rosiepen et al, 1994; Dolbeare, 1995; Smithwick et al, 1996a,1996b; Rosiepen et al, 1997; Weinbauer et al, 1998; França et al, 1999). However, tritiated thymidine, a very specific precursor for DNA, is classically utilized as a germ cell marker in order to determine the duration of spermatogenesis (Swierstra and Foote, 1965; Swierstra, 1968; Barr, 1973; Neves et al, 2002; França and Godinho, 2003).
Accurate morphometric information can provide answers to important questions about the spermatogenic process and also is valuable for correlations with physiological and biochemical findings (Wing and Christensen, 1982; Gaytan et al, 1986; França and Russell, 1998). There are numerous quantitative studies in the literature related to germ, Sertoli, and Leydig cells in mammals, and most quantitative investigations of spermatogenesis require identification of the stages of the seminiferous epithelium cycle and the knowledge of its duration (Berndtson, 1977; França and Russell, 1998; França and Godinho, 2003). Also, because each Sertoli cell is able to support only a limited number of germ cells, in a species-specific manner, the number of Sertoli cells that are established before puberty in mammals and Sertoli cell efficiency are the best indicators of spermatogenic efficiency (daily sperm production per gram of testis) (Hess et al, 1993; Johnson, 1995; França and Russell, 1998).
The gerbil (Meriones unguiculatus) is a native rodent in the arid regions of Mongolia and China and represents an interesting experimental model in biomedical research, being easily bred in laboratory conditions. Although there are some reports in the literature regarding the descriptions of the testicular postnatal development and benign testicular hyperplasia (Ninomiya and Nakamura, 1987a,b) and ultrastructural investigation of acrosome formation and development during spermatogenesis (Segatelli et al, 2000, 2002), little is known about the reproductive biology in male gerbils (Schwentker, 1963; Rich, 1968; Williams, 1974). The objectives of the present study were to perform a detailed quantitative investigation of the testis and to determine the duration of spermatogenesis and the spermatogenic efficiency in the gerbil.
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Materials and Methods |
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Thymidine Injections and Tissue Preparation
To estimate the duration of spermatogenesis, the animals received
intraperitoneal injections of tritiated thymidine (thymidine
[methyl-3H], specific activity 82.0 Ci/mmol; Amersham, Life
Science, England). The injections of 100 µCi of 3H-thymidine
were performed using a hypodermic needle. Two animals were utilized for each
time interval considered (approximately 1 hour, 7, 14, and 21 days) after
thymidine injections. Before sacrifice, the animals were anesthetized with
ether and perfusedfixed with Karnovsky fluid
(Sprando, 1990).
Historesin-embedded testis fragments were sectioned at 3-µm thickness and
placed on glass slides. To perform autoradiographic analysis, the histological
slides were dipped in autoradiography emulsion (Kodak NTB-2, Eastman Kodak
Company, NY) at 45°C. After drying for approximately 1 hour at 25°C,
sections were placed in sealed black boxes containing silica gel as a drying
agent and stored in a refrigerator at 4°C for approximately 30 days.
Subsequently, testis sections were developed in Kodak D-19 solution at
15°C according to Bundy
(1995) and stained with
toluidine blue. Analyses of these sections were performed by light microscopy.
Cells were considered labeled when 4–5 or more grains were present over
the nucleus in the presence of low-to-moderate background
(Neves et al, 2002;
França and Godinho,
2003).
Stages Characterization and Relative Frequency
Stages of seminiferous epithelium cycle were characterized based on the
development of the acrosomic system and morphology of the developing spermatid
nucleus. This method provides 12 stages of the seminiferous epithelium cycle
and both stage characterization and relative frequencies were described
previously for the gerbil by Segatelli et al
(2000;
2002). The relative stage
frequencies were determined according to Hess et al
(1990), utilizing the analysis
of 200 seminiferous tubule cross-sections in 12 animals, at 400x
magnification. The duration of the seminiferous epithelium cycle was estimated
based on the stage frequencies and considering the most advanced germ cell
type labeled at each time period investigated after thymidine injection.
Morphometric Analysis of the Testis
The 8 animals utilized in this part of the study were also anesthetized
with ether and perfused-fixed with Karnovsky fluid
(Sprando, 1990). After this
procedure, testes were trimmed out from the epididymis and weighed, and the
percentage occupied by the tunica albuginea was determined. Because the testis
mediastinum in the gerbil is very small, its percentage was not determined in
the present study. Testis samples were embedded in Historesin®, sectioned
at 4-µm thickness, placed on glass slides, and stained with toluidine blue
and Schiff-hematoxylin periodic acid. The tubular diameter of the seminiferous
tubule and the height of the seminiferous epithelium were measured at
100x magnification using an ocular micrometer calibrated with a stage
micrometer. Thirty tubular profiles that were round or nearly round were
chosen randomly and measured for each animal. The epithelium height was
obtained in the same tubule sections utilized to determine tubular diameter.
The volume densities of various testicular components were determined by light
microscopy using a 441-intersection grid placed in the ocular of the light
microscope. Twenty fields chosen randomly (8820 points) were scored for each
animal at 400x magnification. Points were classified as one of the
following: seminiferous tubule (comprising tunica propria, epithelium, and
lumen), Leydig cell, blood and lymphatic vessels, connective tissue, and
others (including fibers and cells of connective tissue). The volume of each
component of the testis was determined as the product of the volume density
and testis volume. To obtain a more precise measure of the testis parenchyma
volume, 3.15% of testicular capsule was excluded from testis weight. The total
length of seminiferous tubule per testis and per gram of testis, expressed in
meters, was obtained by dividing the seminiferous tubule volume by the squared
radius of the tubule times the pi value (
R2)
(Johnson and Neaves, 1981; França and Godinho,
2003)
Cell Counts and Cell Numbers
All germ cell nuclei and Sertoli cell nucleoli present at stage VII of the
seminiferous epithelium cycle (SEC) were counted in 10 round or nearly-round
seminiferous tubule cross-sections, chosen at random, for each animal. These
counts were corrected for section thickness and nucleus or nucleolus diameter
according to Abercrombie
(1946), modified by Amann
(1962). The following germ
cells were counted: type A1 spermatogonia, leptotene primary spermatocytes,
pachytene primary spermatocytes, and round spermatids. The following ratios
were obtained from the corrected counts:
The total number of Sertoli cells was determined from the corrected counts of Sertoli cell nucleoli per seminiferous tubule cross-section and the total length of seminiferous tubules according to the method described by Hochereau-de-Riviers and Lincoln (1978). The daily sperm production (DSP) per testis and per gram of testis were obtained according to the following formula developed by França (1992): DSP = (total number of Sertoli cells per testis) x (the ratio of round spermatids to Sertoli cells at stage VII) x (stage VII relative frequency [%])/(stage VII duration [days]).
The individual volume of Leydig cell was obtained from the nucleus volume
and the proportion between nucleus and cytoplasm. Thirty spherical Leydig cell
nuclei showing evident nucleolus were measured for each animal. Leydig cell
nuclear volume obtained by the formula (4/3)R3
(R = nuclear diameter/2) was expressed in µm3. To
calculate the proportion between nucleus and cytoplasm, a 441-point square
lattice was placed over the sectioned material at 400x magnification.
One thousand points over Leydig cells were counted for each animal. The number
of Leydig cells per testis and per gram of testis were estimated from the
Leydig cell individual volume and the volume occupied by Leydig cells in the
testis parenchyma.
Statistical Analysis
All data are presented as the mean ± SEM, and these data were
analyzed by descriptive statistic.
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Results |
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Duration of Spermatogenesis
The most advanced labeled germ cell type present at several times after the
3H-thymidine injections (approximately 1 hour, 7, 14, and 21 days)
are shown in Table 1 and
Figure 2. Approximately 1 hour
after injection, the most advanced labeled germ cell type observed was
leptotene primary spermatocytes present at stage VII
(Figure 3a). However, it should
be stated that all XII stages characterized presented labeled germ cell nuclei
near the basal lamina; these nuclei had the morphological characteristics of
type A, intermediate, and type B spermatogonia. The most advanced labeled
cells found 7 days after injection were pachytene spermatocytes, observed at
stage III (Figure 3b). Meiotic
figures from the first meiotic divisions and located at the middle of stage
XII were observed as the most advanced labeled germ cell 14 days after
injection (Figure 3c), whereas
21 days after injection, round spermatids present at stage VI were the most
advanced cell labeled (Figure
3d). Based on these labeling patterns and the stage frequencies,
the mean duration of the seminiferous epithelium cycle was estimated to be
10.55 ± 1.0 days. As can be observed in
Table 1 and
Figure 2, this value was
obtained based on labeling of leptotene spermatocytes and intermediate
labeling points for the various time intervals considered. The duration of
each stage of the seminiferous epithelium cycle was determined taking into
account the cycle duration and the percentage of occurrence of each stage
(Figure 2). Thus, stage I was
the longest stage (1.46 days), whereas the shortest was stage V (0.42 days).
Considering that approximately 4.5 cycles are necessary for the
spermatogenesis process to be completed, the total duration of spermatogenesis
in the gerbil was estimated as being 47.47 days.
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Morphometry of the Testis
The mean testis weight found for the gerbil was approximately 0.54 g,
providing a gonadosomatic index (testes mass divided by body weight) of
approximately 0.72% (Table 2).
The volume density of tubular and interstitial compartments was approximately
92% and 8%, respectively, and Leydig cells occupied 3% of the testis
parenchyma or almost 40% of the interstitial space
(Table 2). The mean tubular
diameter and epithelium height were 253 and 100 µm, respectively. Based on
the volume of the testis parenchyma (testis weight minus tunica albuginea
weight) and the volume occupied by the seminiferous tubules in the testis and
the tubular diameter, approximately 10 and 18 m of seminiferous tubules were
found, respectively, per testis and per testis gram
(Table 2).
Table 3 shows the corrected
number of germ cells and Sertoli cell nucleoli found per seminiferous tubule
cross-section at stage VII of the seminiferous epithelium cycle, whereas the
cell ratios obtained from these corrected counts are shown in
Table 4. Approximately 12
pachytene primary spermatocytes and 34 round spermatids were found per each
type A1 spermatogonia. The meiotic index, measured as the number of round
spermatids produced per pachytene spermatocytes, was 2.8, indicating that 30%
of cell loss occurs during meiosis. The Sertoli cell efficiency at stage VII,
estimated from the total number of germ cells and the number of round
spermatids per Sertoli cell was 21 and 12.6, respectively
(Table 4). The total number of
Sertoli cells per testis was 15 million, whereas the daily sperm production
per testis and per gram of testis (spermatogenic efficiency) was 18 and 33
million, respectively (Table
5). Leydig cell and nuclear volumes were approximately 328 and
1148 µm3, respectively. Coincidentally, the values found for the
number of Leydig cell per testis and per gram of testis was strikingly similar
to those found for Sertoli cells (Table
6).
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Discussion |
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In mammals, the duration of spermatogenesis is considered to be species specific and, although strain or breed differences can be found among members of the same species (Russell et al, 1990a), apparently the spermatogenic cycle length cannot be altered by any natural factor or experimental manipulation (Clermont, 1972; Berndtson and Desjardins, 1974; Amann and Schanbacher, 1983). In approximately half of the mammalian species already investigated, the duration of each spermatogenic cycle is situated in an interval of 9–12 days. In this way, the cycle length found for the gerbil is situated exactly in the middle of this predominant range. The same trend is observed when the gerbil is compared with other rodent species investigated. In these species, the duration of each spermatogenic cycle varies from 6.7 days for bank vole (Grocock and Clarke, 1976) to 17 days for the Chinese hamster (Oud and de Rooij, 1977). Although the duration of spermatogenesis is not necessarily the same for species closely related (Russell et al, 1990a; França and Russell, 1998; Paula et al, 1999), it is considered that the cycle duration is not phylogenetically determined in mammals (Neves et al, 2002). However, recent work has shown that this very important aspect of spermatogenesis is controlled by the germ cell genotype (França et al, 1998).
Compared with most mammalian species already investigated
(Kenagy and Trombulak, 1986), the gonadosomatic index in the gerbil is high and similar to other laboratory
rodent species investigated, such as rats and mice. Also, considering the
values found for the seminiferous tubule compartment in most mammalian species
(
70% to 90%) (Russell et al,
1990b; França and
Russell, 1998), the volume density observed for this parameter is
very high in the gerbil. However, the opposite is observed for Leydig cells
(França and Russell,
1998; França and
Godinho, 2003), the percentage of which is similar to that found
for rats (Russell and França,
1995; Rocha et al,
1999). The length of seminiferous tubules per gram of testis is
related to the tubular diameter and the seminiferous tubule volume. In
general, there are 10–15 m of tubules per gram of testis in mammals
(Wing and Christensen, 1982;
Neaves and Johnson, 1985; Sinha-Hikim et al, 1988;
França and Russell,
1998; França and
Godinho, 2003). Due to the high seminiferous tubule occupancy and
the fact that mean value found for the tubular diameter in the gerbil is
situated around the mean range observed for mammals
(Roosen-Runge, 1977;
Setchell et al, 1994;
França and Russell,
1998), approximately 18 m of tubules per gram of testis parenchyma
was found in this species.
Germ cell loss (apoptosis) plays an important role in the seminiferous epithelium homeostasis by limiting the number of sperm produced. This occurs mainly during meiosis, through the elimination of germ cells that are defective or carry DNA mutations, and during the spermatogonial phase in a process named cell-density regulation (Roosen-Runge, 1973; Huckins, 1978; Sinha-Hikim et al, 1985; de Rooij and Lok, 1987; França and Russell, 1998; de Rooij and Russell, 2000; Young and Nelson, 2001; Weinbauer et al, 2001). Overall, germ cell apoptosis results in the loss of up to 75% of the potential number of mature spermatozoa that can be produced by one differentiated type A1 spermatogonia (de Rooij and Lok, 1987; Bartke, 1995; Johnson, 1995; França and Russell, 1998; Lee et al, 1997, 1999; Sinha-Hikim and Swerdloff, 1999; de Rooij and Russell, 2000).
Germ cell loss can be estimated comparing the ratios between germ cells counted during specific steps of spermatogenesis (Johnson, 1991). We did not perform an investigation specifically related to the kinetics of spermatogonia in the gerbil. However, the results found for the ratio of primary spermatocytes to type A spermatogonia, investigated at stage VII, suggest that at least 5 generations of differentiated spermatogonia are present in this species. The ratio of spermatids to primary spermatocytes show that 30% of germ cell loss occurred in the gerbil during meiosis. This value is similar to that observed for most mammalian species investigated (Roosen-Runge, 1973; França and Russell, 1998). Also, assuming that 5 generations of differentiated spermatogonia are present in the gerbil, the ratio of round spermatids to type A1 spermatogonia observed in this species indicate that 75% of germ cell loss took place during spermatogenesis.
The Sertoli cell number established during the prepubertal period in mammals determines the final testicular size and the number of sperm produced in sexually mature animals (Orth et al, 1988; Hess et al, 1993). This occurs because each Sertoli cell can support only a limited number of germ cells, with this support capacity being both variable and species-specific (Berndtson and Desjardins, 1974; Russell and Peterson, 1984; França and Russell, 1998). Approximately 28 million Sertoli cells were found per testis gram in the gerbil. This value is situated in the range (20–40 million) observed for most mammalian species studied up to date (Russell et al, 1990b; França and Russell, 1998). Because the number of Sertoli cells is stable in the adult animal and throughout the different stages of the cycle, these cells are used as a reference point to quantify and functionally evaluate the spermatogenic process (França and Russell, 1998; França and Godinho, 2003). The noticeable flexibility among species in the number of spermatids supported by a single Sertoli cell shows that, in general, species in which the ratio of spermatids to Sertoli cells is higher also have higher spermatogenic efficiency (daily sperm production per gram of testis) (Russell and Peterson, 1984; Sharpe, 1994; França and Russell, 1998). According to Russell and Peterson (1984), the ability to accommodate more germ cells may be dependent on the size of the Sertoli cells and also to a certain degree on the size and/or shape of the elongated spermatids. In the gerbil, almost 13 spermatids were found for each Sertoli cell. Compared with other mammalian species and even to other rodent species, this ratio is relatively high (Wing and Christensen, 1982; Russell and Peterson, 1984; Sharpe, 1994; França and Russell, 1998; Rocha et al, 1999).
Very little is known about the mechanisms responsible for the regulation of the Leydig cell size and the number of Leydig cells per testis and a dramatic variation in these parameters, and for the organization of these cells in the interstitial compartment, is found in the literature for different mammalian species (Fawcett et al, 1973; Russell, 1996; França and Russell, 1998; França et al, 2000). However, it is already established that Leydig cell volume and Leydig cell capacity to secrete testosterone is positively correlated with the quantity of smooth endoplasmic reticulum (Ewing et al, 1979; Zirkin et al, 1980). A cross-talk between the seminiferous epithelium and the Leydig cells is strongly suggested in the literature (Habert et al, 2001; Russell et al, 2001; Neves et al, 2002). As found for some species investigated during sexual maturity (Neves et al, 2002; França and Godinho, 2003) and around the pubertal period (França et al, 2000), perhaps it might be more than a coincidence that the number of Leydig and Sertoli cells per testis are strikingly similar in the gerbils investigated in the present study.
Several parameters, such as the number of germ cells supported by each Sertoli cell, the volume density of seminiferous tubules, the number of spermatogonial generations, the number of Sertoli cells per gram of testis, are positively correlated with the spermatogenic efficiency (Russell and Peterson, 1984; França and Russell, 1998; Johnson et al, 2000). On the other hand, this parameter shows a negative correlation with the volume density of Sertoli cells in the seminiferous epithelium and the length of spermatogenic cycle. It means that species showing long cycle length and, paradoxically, bigger Sertoli cells are those with lower spermatogenic efficiency (Russell et al, 1990b). Therefore, probably because the volume density of the seminiferous epithelium and the support capacity (efficiency) of Sertoli cells are very high in the gerbil, this species presents high spermatogenic efficiency compared with other mammalian species, including rodents, already investigated (Russell and Peterson, 1984; Russell et al, 1990b; França and Russell, 1998; Johnson et al, 2000).
In summary, in the present investigation, we obtained several fundamental basic data regarding the testis structure and function in the gerbil. These data might provide the basis for future research involving the reproductive biology in this species that is an important experimental model in biomedical research.
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Footnotes |
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