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Biology of Reproduction 67, 247-255 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Comparative Testis Morphometry and Seminiferous Epithelium Cycle Length in Donkeys and Mules1

Elizabeth S. Nevesa, Hélio Chiarini-Garciaa, and Luiz R. França2,a

a Laboratory of Cellular Biology, Department of Morphology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil 31270-901


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The mule (Equus mulus mulus) is a sterile hybrid domestic animal that results from the breeding of a male donkey (Equus asinus) to a female horse (Equus caballus). Usually, spermatogenesis in mules does not advance beyond spermatocytes. In the present study, we performed a comparative and more accurate morphometric and functional investigation of the testis in donkeys and mules. Due to the smaller testis size, lower seminiferous tubule volume density, and fewer germ cells, the total length of seminiferous tubules in mules was significantly smaller than in donkeys. However, the percentage of seminiferous tubules containing germ cells (spermatogonia and spermatocytes) in mules was approximately 95%. The total number of Sertoli cells per testis observed in donkeys and mules was very similar. However, the total number of Leydig cells in mules was approximately 70% lower than in donkeys. At least in part, this difference was probably related to the lower number of germ cells present in mule seminiferous tubules. Although spermatogenesis in mules did not advance beyond secondary spermatocytes/newly formed round spermatids, germ cell associations in the seminiferous epithelium and pachytene spermatocytes nuclear volume in donkeys and mules were similar. The duration of spermatogenesis was estimated using intratesticular injections of tritiated thymidine. Each spermatogenic cycle in donkeys lasted 10.5 days. A similar value was found in mules (~10.1 days). Considering that the entire spermatogenic process takes approximately 4.5 cycles to be completed, its total duration in donkeys was estimated to last 47.2 days. The results found for mules suggest that the mechanisms involved in the determination of testis structure and function are probably originated from donkeys. Also, the data found for mules suggest that their seminiferous tubules are able to sustain complete spermatogenesis. In this regard, this species is a potential model for transplants of germ cells originated from donkeys and horses or other large animals.

apoptosis, Leydig cells, Sertoli cells, spermatogenesis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The male mule (Equus mulus mulus, 63 chromosomes) is a sterile domestic animal that results from the breeding of a male donkey (Equus asinus, 62 chromosomes) to a female horse (Equus caballus, 64 chromosomes) [1]. As the number of metacentric and acrocentric autosomal chromosomes in horses and donkeys diverges approximately 35%, the main cause of sterility in mules is probably related to the homologous chromosome pairing failure that occurs during meiosis [2, 3].

Because Sertoli cells are the first cell type known to differentiate in the gonad, they are believed to act as the organizing center of the male gonad [4]. Also, there is strong genetic evidence that the pre-Sertoli cell expresses the testis-determining factor (Sry) [4], which is located in the short arm of the Y chromosome [5, 6]. This aspect makes the mule and donkey an interesting model on which to perform comparative studies related to testis structure and function in both species, mainly when it is considered that the Sry shows great interspecific variability [7].

Spermatogenesis is a cyclic and highly coordinated process in which diploid spermatogonia differentiate into mature haploid spermatozoa. This highly organized process encompasses different cell associations called stages. The sequence of events that occurs from the disappearance of a given cellular association to its reappearance constitutes the cycle of seminiferous epithelium, while the interval required for one complete series of cellular associations to appear at one point within the tubule is called duration of the cycle of the seminiferous epithelium [8].

Spermatogenesis is one of the most productive self-renewing systems in the body, lasting from 30 to 75 days in mammals [9]. Although it is not established yet which genes regulate the duration of spermatogenesis, recent work has demonstrated that the spermatogenic cycle length is under the control of germ cell genotype [10].

The general organization of spermatogenesis is essentially the same in all mammals [11]. However, there are some specific characteristics concerning the types and the number of spermatogonial generations and the morphological characteristics of germ cells present at the various stages of spermatogenesis among different species [9, 12]. The major criteria for stage identification resides in the morphological characteristics of spermatids, in particular, their nucleus and acrosomic system [8, 9, 13]. With this method, the number of stages and the features used for the classification scheme will vary between species and even among different investigators concerning the same species [14]. Another method, the tubular morphology system, is based on the shape and location of spermatid nuclei, presence of meiotic divisions, and overall seminiferous epithelium composition. This method yields 8 stages of the cycle for all species, is less arbitrary, and is a simpler method to characterize the stages of the cycle [15].

Although the basic structure of the testis is highly conserved among vertebrates [4], specific characteristics of the testis structure might be found for a particular species. In this regard, quantitative data can be used to answer important questions about the testis function and to provide a more complete understanding of spermatogenesis [9, 16]. Many quantitative investigations of spermatogenesis require identification of the stages of seminiferous epithelium cycle and the knowledge of the cycle length [15]. There are few reports in the literature concerning the spermatogenic process and testis morphometry in donkeys [17, 18] and testis structure in mules [17]. In these studies, the seminiferous epithelium cycle length and quantitative investigation of Sertoli and Leydig cells were not performed. The present work shows a more accurate morphometric analysis of the testis and the spermatogenic cycle length in donkeys and mules. A possible role of donkey Y chromosome in determining important structural and functional aspects of mule testis is also investigated.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Five sexually mature donkeys (Equus asinus; 4–11 yr of age) and 9 young adult mules (Equus mulus mulus; 2.5–3.5 yr of age) were utilized in the present work. The investigated donkeys belonged to the Pêga breed. They were donated by the Brazilian Pêga Donkey Association and were being utilized as breeders. Although reproduction in this species is not seasonal [19, 20], the animals were orchiectomized during the equine-breeding period, which is from September to February in the southern hemisphere. All mules investigated were offspring of Pêga donkeys, and six of nine were fathered by the same jack. Before surgery, all animals received i.v. injections of 0.8 ml of Sedivet (Boehringer De Angell, U.K.) per 100 kg of body weight. All surgical procedures were performed by a veterinarian and followed approved guidelines for the ethical treatment of animals.

Thymidine Injections and Tissue Preparation

To estimate the duration of seminiferous epithelium cycle, animals received intratesticular injections of tritiated thymidine (thymidine [methyl-3H], specific activity 82.0 Ci/mmol; Amersham Life Science, Little Chalfont, Buckinghamshire, U.K.). The injection of 150 µCi of tritiated thymidine was performed in three different regions (50 µCi in 0.5 ml) of each testis (near the epididymal head, body, and tail) using a hypodermic needle. For each animal, the right or left testis was excised at different time intervals after injections so that each time interval had two different animals. These intervals were 1 h and 7 and 14 days in donkeys and 1 h and 10 days in mules. The latter interval chosen for mules was based on the preliminary estimation of the cycle length found for donkeys, which took into account labeling in preleptoene/leptotene spermatocytes. After the testes and epididymis were removed, the blood was cleared by introducing 0.9% saline with heparin (125 IU/L) via a needle through the testicular artery. Subsequently, they were perfused-fixed by gravity-fed perfusion with 4% buffered glutaraldehyde for 25–30 min. After perfusion, testes were trimmed out from the epididymis and weighed and cut longitudinally by hand with a sharp knife. Tissue samples, measuring 1- to 3-mm thickness, were taken near the tunica albuginea and close to the locations of the thymidine injections. Testis fragments were routinely processed and embedded in plastic (glycol methacrylate). Sections of 4-µm thickness were obtained for light microscopic investigations and were subsequently placed on glass slides and stained with toluidine blue.

To perform autoradiographic analysis, unstained testis sections (4 µm) were dipped on the autoradiography emulsion (Kodak NTB-2; Eastman Kodak Company, Rochester, NY) at 45°C. After drying for approximately 1 h at 25°C, sections were placed in sealed black boxes and stored in a refrigerator at 4°C for 4–6 wk. Subsequently, testis sections were developed in Kodak D-19 solution at 15°C according to Bundy [21] and stained with toluidine blue. Analyses of these sections were performed by light microscopy to detect the most advanced germ cell type labeled at different periods postthymidine injection. Cells were considered labeled when four to five or more grains were present over the nucleus in the presence of low-to-moderate background.

Stages of the Seminiferous Epithelium Cycle in Donkeys and Histological Evaluation of the Seminiferous Tubules in Mules

Stages of the cycle in donkeys were characterized based on the shape and location of spermatid nuclei, presence of meiotic divisions, and overall seminiferous epithelium composition [15, 22, 23]. This method provides 8 stages of the seminiferous epithelium cycle. The relative stage frequencies were determined from the analysis of 400 seminiferous tubule cross-sections per animal (n = 5) at 400x magnification. Both testes were analyzed for each animal. The histological sections utilized were those that presented better quality and more tubular cross-sections.

Although spermatogenesis in mules rarely advances beyond the meiotic phase, the same criteria for stage classification was utilized for this species. However, the stages of the cycle in this species were characterized mainly based on the association of different types of primary spermatocytes and spermatogonia [23, 24]. Seminiferous tubules in mules were also classified according to the presence of lumen and apoptosis and the presence of meiotic figures and/or secondary spermatocytes in the seminiferous epithelium. Two hundred to 400 seminiferous tubule cross-sections were evaluated per animal (n = 9) at 400x magnification. Both testes were analyzed for each animal. Because the presence of germ cells in mule seminiferous tubules was highly variable, stage frequencies were not estimated for this species.

Length of the Seminiferous Epithelium Cycle

The duration of the spermatogenic cycle in donkeys was estimated based on the stage frequencies and the most advanced germ cell type labeled at different periods postthymidine injections. In mules, the duration of the spermatogenic cycle took into account the most advanced germ cell labeled in the interval between the two different periods of injections utilized, according to the criteria utilized by França et al. [10].

Morphometry of the Testis in Donkeys and Mules

The tubular diameter was measured at 400x magnification using an ocular micrometer calibrated with a stage micrometer. At least 30 tubular profiles that were round or nearly round, chosen randomly, were measured for each animal. The volume densities of various testicular tissue components were determined by light microscopy using a 441-intersection grid placed in the ocular of the light microscope. Fifteen fields chosen randomly (6 615 points) were scored for each animal at 400x magnification. Artifacts were rarely seen and were not considered in the total number of points utilized to obtain volume densities. Points were classified as one of the following: seminiferous tubule, tubular lumen, Leydig cell, blood and lymphatic vessels, and connective tissue. The volume of each component of the testis was determined as the product of the volume density and testis volume. For subsequent morphometric calculations, the specific gravity of testis tissue was considered to be 1.0. To obtain a more precise measure of testis volume, 10.8% and 20.4% of the testis capsule plus mediastinum in donkeys and mules [25], respectively, was excluded from the testis weight. The total length of seminiferous tubule (meters) was obtained by dividing seminiferous tubule volume by the squared radius of the tubule times the {pi} value [26].

Pachytene Spermatocyte Nuclear Volume

Primary spermatocyte nuclear volume grows markedly during meiotic prophase [27]. The magnitude of this growth is variable among different species [16]. To investigate comparatively the evolution of nuclear volume in pachytene during spermatogenesis in both donkeys (n = 3) and mules (n = 5), 30 nuclear diameters from early (stage 5), middle (stage 8), and late (stage 2) pachytene primary spermatocytes were measured. These pachytenes were associated with type A spermatogonia (early), type A and B spermatogonia and preleptotene spermatocytes (middle), and type A spermatogonia and zygotene (late). Pachytene nuclear volume was expressed in µm3 and was obtained by the formula (4/3){pi}R3, where R = nuclear diameter/2.

Cell Counts and Cell Numbers

Sertoli cells nucleoli were counted in 20 round or nearly round seminiferous tubule cross-sections, chosen at random, for each animal. These counts were corrected for section thickness and nucleolus diameter according to Abercrombie [28], as modified by Amann [29]. Twenty Sertoli cell nucleoli diameter were measured for each animal. The total number of Sertoli cells per testis in donkeys and mules was determined from the corrected counts of Sertoli cell nucleoli per tubule cross-section and the total length of seminiferous tubules, according to Hochereau-de Reviers and Lincoln [30].

Leydig cell individual volume was obtained from nucleus volume and the proportion between nucleus and cytoplasm. Because the Leydig cell nucleus in donkeys and mules is spherical, its nucleus volume was obtained from the knowledge of the mean nuclear diameter. For this purpose, 30 nuclei showing evident nucleolus had their diameters measured for each animal. Leydig cell nuclear volume was expressed in µm3 and was obtained by the same formula mentioned for pachytene. 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 in donkeys and mules was 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. Student t-test and analysis of correlation and variance (Newman-Keuls test) were done using the program STATISTICA for windows (StatSoft, Inc., Tulsa, OK). All values for volume densities were subjected to arcsine transformation prior to analysis. The significance level considered was P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Biometric Data and Testis Morphometry

In spite of the age difference observed between donkeys and mules utilized in the present work (~8 and 2.8 yr, respectively), animals from both species investigated were adult. As shown in Table 1, their mean body weights were very similar (P > 0.05). However, testis size in donkeys was almost 5-fold higher (P < 0.05) than in mules. Also, the values found for seminiferous tubule volume density, tubular diameter, and total length of seminiferous tubule were significantly higher in donkeys (P < 0.05; Table 1).


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  		TABLE 1. Biometric and morphometric data in donkeys and mules (mean ± SEM)

Histological Evaluation of the Seminiferous Tubules in Mules

The analysis of at least 200 seminiferous tubule cross-sections per animal showed that approximately 70% of the tubules presented patent lumens (Fig. 1, a and c). In approximately 25%, only vacuoles of different sizes and shapes were observed in the epithelium (Fig. 1b). Tubules containing no lumen comprised 5% of the tubular profiles investigated (Fig. 1a). Basically, the same trend was observed when the germ cell type present in the epithelium was analyzed. In this regard, 75% of the tubules presented spermatogonia and spermatocytes (Fig. 1d), 20% showed only spermatogonia (Fig. 1c), while 5% were Sertoli cell-only tubules (Fig. 1b). Also, meiotic figures and/or secondary spermatocytes were observed in 5% of the tubules recorded (Fig. 1e). Early spermatids were rarely seen and were present in only 1 animal investigated. Because all seminiferous tubules in donkeys presented normal spermatogenesis, the abovementioned analysis performed for mules was not done for donkeys.



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  		FIG. 1. Seminiferous tubules/cords in mules. a) Seminiferous tubules (ST) depicting evident lumen (asterisk), a group of seminiferous tubules delimited by arrowheads, and seminiferous cords (SC) with no lumen. b) Seminiferous cords (SC) containing only vacuoles (asterisk) and Sertoli cells (S) are observed. c) Seminiferous tubules with patent lumen and containing only Sertoli cells (S) and spermatogonia (G). d) In other cross-sections of seminiferous tubules (ST), primary spermatocytes (C), apoptotic cells (Ap), type A spermatogonia (G), and Sertoli cells (S) are observed. e) A seminiferous tubule (ST) cross-section shows meiotic figures (M) and secondary spermatocytes (II). Bar = 50 µm (a); bar = 15 µm (b–e)

Approximately 45% of the tubules investigated showed apoptotic cells in the epithelium. In almost 95% of them, these cells were located in the middle and apical regions of the epithelium. Presumably, they were germ cells (spermatocytes).

Stages of the Seminiferous Epithelium Cycle in Donkeys and Mules

The 8 stages of the cycle in donkeys, characterized according to the tubular morphology system, were very similar to those already described for horses [23]. For this reason, they will be only briefly described in the caption of Figure 2.



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  		FIG. 2. Stages 1–8 of the seminiferous epithelium cycle based on the tubular morphology system. a) Stage 1 shows pachytene primary spermatocytes (P), preleptotene spermatocytes (Pl), round spermatids (R), and Sertoli cell (S). b) Stage 2 presents type A spermatogonia (A), leptotene spermatocytes (L), pachytene spermatocytes (P), elongating spermatids (E), and Sertoli cells (S). c) Stage 3 contains type A spermatogonia (A), zygotene spermatocytes (Z), diplotene spermatocytes (D), elongate spermatids (E), and Sertoli cell (S). d) Stage 4 shows type A spermatogonia (A), zygotene spermatocytes (Z), meiotic figure (M), secondary spermatocytes (II), newly formed round spermatids (R), elongate spermatids (E), and Sertoli cells. e) Stage 5 contains type A spermatogonia (A), pachytene spermatocytes (P), round spermatids (R), elongate spermatids (E), and Sertoli cells (S). f) Stage 6 presents type B1 spermatogonia (B1), pachytene spermatocytes (P), round spermatids (R), elongate spermatids (E), and Sertoli cell (S). g) Stage 7 shows type A spermatogonia (A), type B2 spermatogonia (B2), pachytene spermatocytes (P), round spermatids (R), elongate spermatids (E), Sertoli cells (S), and residual bodies (Rb). h) Stage 8 shows type A spermatogonia (A), pachytene spermatocytes (P), round spermatids (R), elongate spermatids (E), Sertoli cells (S), and residual bodies (Rb). Bars = 15 µm.

Although spermatogenesis in mules, investigated in the present study, did not advance beyond the spermatocyte phase, it was possible to characterize 8 different germ cell associations or stages in this species. Except for the presence of spermatids, these cell associations were basically the same as described in donkeys.

The data obtained for nuclear volume of pachytene primary spermatocytes measured at early, middle, and late phases of development were, respectively, as follows: for donkeys, 267 ± 1, 453 ± 2, 511 ± 4 µm3; and for mules, 272 ± 10, 398 ± 21, 483 ± 14 µm3. In both species, significant growth (P < 0.05) of pachytene nucleus volume occurred from early to middle phase and from middle to late phase. However, comparing both species, the size of the pachytene nucleus was similar (P > 0.05) at the different phases analyzed.

Relative Stage Frequencies in Donkeys

The mean percentage of each stage of the seminiferous epithelium cycle in donkeys was as follows: stage 1, 18.1 ± 1.2; stage 2, 9.1 ± 0.3; stage 3, 5.8 ± 1.0; stage 4, 19.3 ± 0.5; stage 5, 8.9 ± 0.5; stage 6, 22.6 ± 1.4; stage 7, 6.3 ± 0.6; and stage 8, 9.9 ± 0.5. As can be noted, stage 6 was the most frequent, while stage 3 presented the lowest frequency. The frequencies of premeiotic (stage 1 to stage 3), meiotic (stage 4), and postmeiotic (stage 5 to stage 8) stages were 33%, 19.3%, and 47.7%, respectively. As mentioned in Materials and Methods, stage frequencies were not obtained for mules.

Seminiferous Epithelium Cycle Length in Donkeys and Mules

The most advanced labeled germ cell types observed at different time periods investigated after thymidine injections in donkeys are shown in Table 2 and Figure 3. One hour after injection, the most advanced labeled germ cells were identified as preleptotene spermatocytes or cells in transition from preleptotene to leptotene. These cells were present at stage 1 and were located in the basal compartment (Fig. 3a). All 8 stages of the seminiferous epithelium cycle showed labeled germ cells that had the typical morphological characteristics of the different spermatogonial cell types present in these stages.


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  		TABLE 2. Length (days) of seminiferous epithelium cycle in donkeys (mean ± SEM)



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  		FIG. 3. Most advanced labeled germ cell type found after different time periods following intratesticular injections of tritiated thymidine. a) In donkeys, 1 h after injection, preleptotene/leptotene spermatocytes (arrows) at stage 1. b) Seven days after injection, pachytene spermatocytes (arrows) at stage 7. c) Fourteen days after injection, secondary spermatocytes (arrows) at stage 4. d) In mules, 1 h after injection, preleptotene/leptotene spermatocytes (arrows) associated with pachytene spermatocytes (P) at stage 1, and e) approximately 10 days after injection, pachytene spermatocytes (arrows) associated with preleptotene (Pl) at stage 1. Bars = 10 µm

The most advanced germ cell type labeled 7 days after thymidine injection was pachytene spermatocyte at stage 7 (Fig. 3b), while labeled meiotic figures and secondary spermatocytes present at stage 4 were observed 14 days after injection (Fig. 3c).

Based on the most advanced labeled germ cell type observed at each time period investigated postthymidine injection in donkeys (Figs. 3 and 4) and the stage frequencies, the mean duration of the seminiferous epithelium cycle was estimated to be 10.5 ± 0.4 days (Table 2). Durations of the various stages of the cycle were determined taking into account the cycle length and the percentage of occurrence of each stage (Fig. 4). The shortest stage was stage 3 (0.61 days), while the longest stage was stage 6 (2.37 days). Considering that approximately 4.5 cycles are necessary for the spermatogenic process to be completed, the total length of spermatogenesis was estimated as being 47.2 days. The approximate duration of the spermatogonial phase was 15.7 days, while the life spans of primary spermatocytes and spermatids in donkeys were 14 and 15.5 days, respectively. The meiotic divisions, corresponding to the duration of stage 4, lasted 2.02 days.



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  		FIG. 4. Diagram showing the most advanced germ cell type labeled at the eight stages of seminiferous epithelium cycle in donkeys at different time periods (1 h and 7 and 14 days) following tritiated thymidine injections. The Roman numerals indicate the spermatogenic cycle. The space given to each stage is proportional to its frequency and duration. A, Type A spermatogonia; B, type B spermatogonia; Pl, preleptotene spermatocytes; L, leptotene; Z, zygotene; P, pachytene; D, diplotene; II, secondary spermatocytes; R, round spermatids; E, elongate spermatids

In mules, the most advanced germ cell types labeled at 1 h and approximately 10 days after injections were preleptotene/leptotene spermatocytes associated with pachytene spermatocytes and pachytene spermatocytes associated with preleptotene/leptotene, respectively (Figs. 3, d–e, and 5). This cell association was characteristic of stage 1 described for donkeys. The mean size of pachytene spermatocyte nuclei present in this cell association at 1 h (440 ± 20 µm3) and 10 days (455 ± 29 µm3) after thymidine injection was very similar (P > 0.05). Based on the labeling pattern found, the duration of the spermatogenic cycle in mules was estimated as being 10.1 days, which is the same value found for donkeys when the cycle length was estimated considering the initial labeling point in preleptotene/leptotene (Table 2).



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  		FIG. 5. Diagram showing the most advanced germ cell type labeled at the eight stages of seminiferous epithelium cycle in mules at the two time periods (1 h and 10 days) following tritiated thymidine injections. The Roman numerals indicate the spermatogenic cycle. A, Type A spermatogonia; B, type B spermatogonia; Pl, preleptotene spermatocytes; L, leptotene; Z, zygotene; P, pachytene; D, diplotene; II, secondary spermatocytes; R, round spermatids; E, elongate spermatids. Apoptosis in the seminiferous epithelium: moderate (light gray), intense (dark gray). Absence of the expected germ cell (stripes)

Leydig and Sertoli Cells

Leydig cell volume density and all parameters related to Leydig cell size were similar in donkeys and mules (P > 0.05). However, the volume occupied by Leydig cells in the testis parenchyma and the number of Leydig cells per testis were, respectively, about 3-fold and 3.5-fold higher in donkeys (P < 0.05; Table 1). Leydig cell nuclear volume showed significant correlation with tubular diameter in donkeys (r = 0.96) and mules (r = 0.78).

Although the nucleolar diameter was approximately 25% bigger (P < 0.05) in donkeys (Table 1), the total number of Sertoli cells per testis in donkeys and mules was virtually the same (P > 0.05).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This is the first study to perform a more accurate and comparative morphometric and functional investigation of the testis in donkeys and mules. The results suggest that common factors, probably originated from donkeys, are involved in the determination of testis structure and regulation of the number of Sertoli cells per testis and the spermatogenic cycle length in both species, while signals resulting from the interaction between Sertoli cells and germ cells could be responsible for proliferation of adult-type Leydig cells.

Due to the smaller testis size, lower seminiferous tubule volume density, and fewer germ cells in mules, the total length of seminiferous tubules in this species was significantly smaller than in donkeys. The percentage of seminiferous tubules containing germ cells (spermatogonia and spermatocytes) in mules investigated in the present study was almost 95%. This figure was 2–3 times higher than the results found in the literature [3, 17]. However, while we analyzed 9 mules, Chandley et al. [3] and Hernández-Jáuregui and Monter [17] investigated only 1 and 3 animals, respectively. Apart from the sample size, this difference found was also probably related to the good quality of tissue fixation and embedding, which allowed us to discriminate precisely spermatogonia from Sertoli cells, and the extensive morphometric evaluation (analysis of thousands of tubule cross-sections) performed in the present work.

Although germ cell apoptosis is a common finding in hybrids, these animals are not necessarily sterile or infertile [31–33]. According to the literature, hybrid sterility might have several causes [32]. The most obvious and observed in mules is autosomal pairing failure [3] that occurs due to differences in parental chromosome numbers [1]. Genic incompatibility between parents [32] and sexual chromosome pairing failure might also cause sterility [34]. In agreement with other studies [3, 17], we rarely observed germ cells more advanced than late pachytene spermatocytes in mule seminiferous tubules. Studies developed in our laboratory [25] and by Hernández-Jáuregui and Monter [17] showed that Leydig cell ultrastructure was apparently normal in mules. This suggests that Leydig cell steroidogenic capacity is probably not affected in this species. Also, Sertoli cell ultrastructure as well as Sertoli cell barrier integrity investigated by transmission electron microscopy and lanthanum were apparently normal in mules [25]. Besides that, epididymal duct epithelium in mules showed no morphological abnormalities [17, 35]. All these findings suggest strongly that sterility in mules is mainly due to chromosome pairing failure [3]. The fact that Sertoli cell nucleolar diameter is decreased in mules, compared with donkeys, is probably related to the functional status of this cell due to the presence of fewer germ cells.

The total number of Sertoli cells per testis was very similar in donkeys and mules and in horses investigated during the breeding season [36, 37]. According to the literature, the Sertoli cell is the major controller of testis development and efficiency of spermatogenesis [4, 38–40]. The total number of Sertoli cells present in the testis is determined during the prepubertal period [39, 41], being regulated mainly by FSH and thyroid hormones [39, 42] and not being dependent on the presence of germ cells [43]. To our knowledge, no data exist in the literature showing what chromosomes or genes might be intrinsically involved in the regulation of Sertoli cell proliferation. However, as it is known that Sry present in the Y chromosome induces an increase in proliferation of the coelomic epithelial cells that give rise to Sertoli cell precursors [4], it could be speculated that the mechanisms involved in Sertoli cell proliferation in mules and donkeys were similar.

The total number of Leydig cells found for donkeys in the present work was similar to adult horses studied during the breeding season and with comparable testis size [26, 44]. Leydig cell proliferation occurs continuously from birth to adulthood in pigs [41] and horses [26], being significantly correlated with testis weight, tubular diameter, total length of seminiferous tubules, and germ cell number per tubule cross-section [41]. Compared with donkeys, the number of Leydig cells in mules investigated in the present study was approximately 70% lower. This difference could be related to the mean age of the mules, which were younger than the donkeys, and the lower number of germ cells present in mules [41]. The significant and positive correlation between Leydig cell nuclear volume and tubular diameter found for donkeys and mules suggests the occurrence of cross-talk between the seminiferous tubules and Leydig cells [39, 45, 46].

The cell composition of the seminiferous tubule epithelium and the germ cells' morphological characteristics at the eight stages of the seminiferous epithelium cycle in donkeys and mules were basically the same as characterized for horses [47]. However, the individual relative frequencies of several stages of the seminiferous epithelium cycle in donkeys studied in the present work were considerably different from the frequencies found for the African donkey (Equus asinus africanus) [18] and horses [47]. In contrast, when the stages were grouped in premeiotic and postmeiotic phases of spermatogenesis, similar percentages were observed for donkeys and horses [18, 47]. These findings support the hypothesis made in recent publications [16, 48, 49], which suggests that this aspect of spermatogenesis might be phylogenetically determined among members of the same mammalian family.

Although strain or breed differences can be found in the literature among members of the same species [9, 50, 51], the length of the spermatogenic cycle has been generally considered to be constant for a given species [50]. Recent studies, using spermatogonial transplants from rats to mice, demonstrated that the spermatogenic cycle length is under the control of the germ cell genotype [10]. To our knowledge, there are no data in the literature regarding the spermatogenic cycle length in sterile hybrids. The spermatogenic cycle length found for donkeys and mules in the present work was very similar. However, the cycle length values obtained in these species were considerably lower than that found in stallions (12.2 days) [47]. Given that the duration of spermatogenesis is not necessarily the same for species closely related [9, 16, 48, 49], it is considered that the cycle length is not phylogenetically determined in mammals. Perhaps hybrids or genetically modified animals could be used as a model to more thoroughly study this important and fascinating aspect of spermatogenesis.

In conclusion, the results found for mules suggest that the mechanisms involved in the determination of testis structure and function probably originated from donkeys. Also, the data found for mules suggest that their seminiferous tubules are able to sustain complete spermatogenesis, making this species a potential model for transplants of germ cells originated from donkeys, horses, or other large animals.


   ACKNOWLEDGMENTS
 
Technical help from Rubens Miranda is highly appreciated.


   FOOTNOTES
 
First decision: 12 December 2001.

1 The scholarship awarded to E.S.N. from the Brazilian Foundation (CAPES) is fully appreciated. Financial support from the Minas Gerais State Foundation (FAPEMIG) and the Brazilian Research Council (CNPq) is gratefully acknowledged. Back

2 Correspondence. FAX: 55 31 34992780; lrfranca{at}icb.ufmg.br Back

Accepted: February 6, 2002.

Received: November 28, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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