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Testis stereology, seminiferous epithelium cycle length, and daily sperm production in the ocelot (Leopardus pardalis)
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Theriogenology 73 (2010) 157–167 www.theriojournal.com
Testis stereology, seminiferous epithelium cycle length, and daily sperm production in the ocelot (Leopardus pardalis) R.C. Silva a,b, G.M.J. Costa a, L.M. Andrade c, L.R. Franc¸a a,* a
Laboratory of Cellular Biology, Department of Morphology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil b Department of Basic Sciences, Federal University of Jequitinhonha and Mucuri Valleys, Diamantina, MG, Brazil c Associac¸a˜o Bichos Gerais, Belo Horizonte, MG, Brazil Received 12 June 2009; received in revised form 14 August 2009; accepted 17 August 2009
Abstract Similar to most wild felids, the ocelot (Leopardus pardalis) is an endangered species. However, knowledge regarding reproductive biology of the ocelot is very limited. Germ cell transplantation is an effective technique for investigating spermatogenesis and stem cell biology in mammals, and the morphologic characterization of germ cells and knowledge of cycle length are potential tools for tracking the development of transplanted germ cells. Our goal was to investigate basic aspects related to testis structure, particularly spermatogenesis, in the ocelot. Four adult males were used. After unilateral orchiectomy, testis samples were routinely prepared for histologic, stereologic, and autoradiographic analyses. Testis weight and the gonadosomatic index were 11 0.6 g and 0.16 0.01%, respectively, whereas the volume density of seminiferous tubules and Leydig cells was 83.2 1.6% and 9.8 1.5%. Based on the acrosomic system, eight stages of spermatogenesis were characterized, and germ cell morphology was very similar to that of domestic cats. Each spermatogenic cycle lasted 12.5 0.4 d, and the entire spermatogenic process lasted 56.3 1.9 d. Individual Leydig cell volume was 2522 mm3, whereas the number of Leydig and Sertoli cells per gram of testis was 38 5 106 and 46 3 106. Approximately 4.5 spermatids were found per Sertoli cell, whereas daily sperm production per gram of testis was 18.3 1 106, slightly higher than values reported for other felids. The knowledge obtained in this study could be very useful to the preservation of the ocelot using domestic cat testes to generate and propagate the ocelot genome. # 2010 Elsevier Inc. All rights reserved. Keywords: Ocelot (Leopardus pardalis); Spermatogenic cycle length; Spermatogenic efficiency; Stereology; Testis
1. Introduction The ocelot (Leopardus pardalis) is a medium-size cat (7 to 15.8 kg) that occupies a wide variety of habitats from rainforests to dry scrubland [1]. Similar to most wild felids, the ocelot is currently listed as an
* Corresponding author. Tel.: +55 31 34092816; fax: +55 31 34092780. E-mail address: [email protected] (L.R. Franc¸a). 0093-691X/$ – see front matter # 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2009.08.009
endangered species by the Convention on International Trade of Endangered Species of Wild Fauna and Flora (CITES) [2] and by other sources [3,4]. In Brazil, Leopardus pardalis populations outside the Amazon Basin are listed as vulnerable [5]. Similar to other species, the loss of genetic diversity poses a serious threat to the conservation of endangered wild felids [6]. Although knowledge of reproductive parameters is crucial for the preservation of any endangered species, there is little information regarding testis function in the ocelot [4,7].
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Characterization of the stages of spermatogenesis and estimation of the spermatogenic cycle length are fundamental to determining spermatogenic efficiency and to performing comparative studies among species. The total duration of spermatogenesis requires approximately 4.5 cycles, lasts from 30 to 75 d in mammals [8,9], is generally considered constant for a species [10], and is under the control of the germ cell genotype [11]. Germ cell transplantation is a recently developed technique that has considerable potential for studies involving biotechnology, transgenics, and preservation of the genetic stock of valuable animals or endangered species [12–15]. In this regard, germ cell transplantation studies are currently under way in our laboratory, using the domestic cat as a recipient; one of our objectives is to use this species to preserve the genetic stock of wild felids, including the ocelot. Consequently, detailed knowledge regarding spermatogenesis and length of the spermatogenic cycle are very important. As there are few reports in the literature regarding the reproductive biology of the ocelot [4,7], the main objectives of the current study were to perform a detailed, comprehensive histologic and stereologic investigation of the testis, as well as determine the duration of spermatogenesis and spermatogenic and Sertoli cell efficiency in the sexually mature ocelot. 2. Materials and methods 2.1. Animals Four ocelots (2.5 to 3.5 yr old) weighing 13.8 1 kg were used. These animals were from the Brigitte Johnson animal facility, located in the city of Belo Horizonte (198550 1500 S; 438560 1600 W), state of Minas Gerais, Brazil. As no significant seasonal changes have been found for sperm production in ocelots kept in captivity [7], in the current study, testis samples were collected during Brazilian winter (July). The animals were contained and kept under general anesthesia using a combination of xylazine hydrochloride (Vetbrands, Sespo Indu´stria e Come´rcio Ltda, Jacareı´, SP, Brazil) and ketamine (Ko¨nig, Ko¨nig do Brasil Ltda, Santana de Parnaı´ba, SP, Brazil), using 1.1 and 10 mg per kg body weight, respectively. After unilateral orchiectomy, the testis was separated from the epididymis, weighed, and cut longitudinally (with a razor blade) into small slabs, which were fixed by immersion in 4% buffered glutaraldehyde for 12 to 24 h. Tissue samples (2 to 3 mm thick) were routinely processed and embedded in plastic (glycol methacrylate) for histologic, stereologic, and autoradiographic evaluation. To investigate their morphology, sperm were
collected from the cauda epididymidis, and the smears were routinely prepared for histologic evaluation and comparisons with smears available in our laboratory for the jaguar and domestic cat. All surgical procedures were performed by a veterinarian and followed approved guidelines for the ethical treatment of animals. 2.2. Intratesticular thymidine administration and tissue preparation Prior to orchiectomy, an intratesticular administration (75 mCi/testis) of tritiated thymidine (thymidine [methyl-3H], specific activity 82.0 Ci/mmol; Amersham, Life Science, Buckinghamshire, England)—a specific marker for cells that are synthesizing DNA—was performed close to the cauda epididymidis in two animals to estimate the duration of spermatogenesis. For each animal, two time intervals (1 h and 24 d; and 1 h and 28 d) were considered after the thymidine administration. Tissue samples (2 to 3 mm thick) were collected near the thymidine administration site, routinely fixed, and embedded as described above. For autoradiographic analysis, unstained testis sections (4 mm) were dipped in an autoradiographic emulsion (Kodak NTB-2; Eastman Kodak Company, Rochester, NY, USA) at 43 to 45 8C. After drying for approximately 1 h at 25 8C, the testis sections were placed in sealed black boxes and stored in a refrigerator (4 8C) for approximately 4 wk. Subsequently, testis sections were developed in Kodak D-19 solution at 15 8C [16] and stained with toluidine blue. Analyses of these sections were performed under light microscopy to detect the most advanced germ cell type labeled at the three time periods after thymidine administration. Cells were considered labeled when at least four or five grains were present over the nucleus on a low to moderate background. 2.3. Testis stereology Volume densities of the testicular tissue components were determined under light microscopy, using a 441intersection grid placed in the ocular of the light microscope. Fifteen randomly chosen fields (6615 points) were scored per testis for each animal at 400 magnification. Tubule diameter and seminiferous tubule epithelium height were measured at 100 magnification, using an ocular micrometer calibrated with a stage micrometer. Thirty round or nearly round tubule profiles were chosen randomly and measured for each animal. Epithelium height was obtained in the same tubules used to determine the tubule diameter. The total length of seminiferous tubule (meters) was obtained by
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dividing seminiferous tubule volume by the squared radius of the tubule multiplied by p [17]. 2.4. Stages of the seminiferous epithelium cycle Stages of the seminiferous epithelium cycle were characterized based on the development of the acrosomic system and morphology of the developing spermatid nucleus [18,19]. Similar to the jaguar and domestic cat [20], this method provided eight stages of the seminiferous epithelium cycle for the ocelot. The relative stage frequencies were determined by evaluating 150 seminiferous tubule cross sections per animal at 400 magnification. The measurement of the angle of the acrosome on the nuclear surface was obtained from 15 germ cells (per animal and per cycle stage) at 1000 magnification. 2.5. Length of the seminiferous epithelium cycle The histologic sections used were those that were of better quality and had more tubule cross sections. The duration of the spermatogenic cycle was estimated in two animals, based on stage frequencies and the most advanced germ cell type labeled at different times after thymidine administration. 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 [21]. As the volume of the nucleus of pachytene primary spermatocytes grows markedly during meiotic prophase and these cells are present in nearly all stages of the cycle [8,22], the size of their nuclei was used as a reference point to more precisely determine the location of the most advanced labeled germ cell. 2.6. Cell counts and cell numbers All germ cell nuclei and Sertoli cell nucleoli present in Stage V of the cycle 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, based on the method described by Abercrombie [23] and modified by Amann and Almquist [24]. For this purpose, 10 nuclei or nucleoli diameters were measured per animal for each cell type analyzed. Cell ratios were obtained from the corrected counts obtained in Stage V. 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 the seminiferous tubules [25]. Daily sperm production
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(DSP) per testis and per gram of testis (spermatogenic efficiency) was obtained according to the following formula: DSP = total number of Sertoli cells per testis the ratio of round spermatids to Sertoli cells in Stage V Stage V relative frequency (%)/Stage V duration (d) [26]. Individual volume of the Leydig cells was obtained from the nucleus volume and proportion occupied by the nucleus and cytoplasm. As the Leydig cell nucleus was spherical, nucleus volume was calculated from the mean nuclear diameter. For this purpose, 30 nuclei with an evident nucleolus were measured for each animal. Leydig cell nuclear volume was expressed in mm3 and obtained from the formula 4/ 3pR3, in which R = nuclear diameter/2. To calculate the relative proportions occupied by the nucleus and cytoplasm, a 441-point square lattice was placed over the sectioned material at 400 magnification, and 1000 points over Leydig cells were counted for each animal. The total number of Leydig cells per testis was estimated from the individual Leydig cell volume and the volume occupied by Leydig cells in the testis parenchyma. 3. Results 3.1. Biometric data and testis volume density All data are presented in Table 1. Mean (SEM) testis weight for the adult ocelot was 11 0.6 g, providing a gonadosomatic index (testes mass divided by body weight) of 0.16 0.01%. Volume density of seminiferous tubules and Leydig cells was 83.2 1.6% and 9.8 1.5%, respectively. Therefore, Leydig cells occupied nearly 60% of the intertubular compartment. Mean tubular diameter and epithelium height were Table 1 Biometric and morphometric data in the ocelot (mean SEM). Parameters Body weight (kg) Testis weight (g) Gonadosomatic index (%) Testis parenchyma volume density (%) Tubular compartment Tunica propria Seminiferous epithelium Lumen Intertubular compartment Leydig cell Others Tubular diameter (mm) Seminiferous epithelium height (mm) Tubular length per gram of testis (m) Total tubular length per testis (m)
13.8 1 11 0.6 0.16 0.01 83.2 1.6 4.7 0.2 69.8 2 8.7 1.5 16.8 1.6 9.8 1.5 5.2 1.2 252 3 86 3 16.7 0.4 150 13
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252 3 and 86 3 mm. The mean percentage found for the tunica albuginea was approximately 20 0.02% (data not shown). Based on the volume of the testis parenchyma (testis weight minus tunica albuginea weight) and the volume occupied by seminiferous tubules in the testis and tubule diameter, there were 16.7 0.4 and 150 13 m of seminiferous tubules per testis gram and per testis (Table 1). The morphology observed for the sperm obtained from the cauda epididymidis in the ocelot was very similar to the morphology previously determined for the jaguar and domestic cat (data not shown).
spermatids. Because the proacrosomal granules cannot be seen under a light microscope, the newly formed spermatids were characterized by their lack of distinguishing features. However, a juxtanuclear Golgi apparatus was evident. Some elongated spermatid bundles were located deep within the epithelium; many were closer to the epithelium base than to the lumen. Type A spermatogonia and intermediate spermatogonia nuclei were found at the base of the tubule. Young pachytene spermatocytes were located between round spermatids and the basal lamina.
3.2. Stages of the seminiferous epithelium cycle and relative stage frequencies
3.2.2. Stage II Early round spermatids usually had two small acrosomal vesicles in which only occasional proacrosomal granules were present. At the end of this stage, the small proacrosomal vesicles coalesced to form one large acrosomal vesicle containing a single acrosomal granule, and the acrosomal vesicle made contact with the nucleus. The elongated spermatid bundles had moved much closer toward the lumen of the seminiferous tubule. Pachytene spermatocyte nuclei were larger than in Stage I and slightly more distant from the basal lamina. Type B and Type A spermatogonia were observed in this stage.
The eight stages of the cycle characterized based on the acrosomic system (Fig. 1) were very similar to those previously described for the jaguar and domestic cat [20] and are described later. To better characterize these stages, the angle formed by the acrosome in relation to the spermatid was measured. 3.2.1. Stage I Two generations of spermatids were present, including early round spermatids and elongated
Fig. 1. Stages I to VIII of the seminiferous epithelium cycle in the ocelot, based on the development of the acrosome in spermatids: A, Type A spermatogonia; B, Type B spermatogonia; Pl, preleptotene spermatocyte; L, leptotene spermatocyte; Z, zygotene spermatocyte; P, pachytene spermatocyte; D, diplotene spermatocyte; M, meiotic figure; R, round spermatids; E, elongating/elongated spermatids; SC, Sertoli cells; Rb, residual bodies; 28, secondary spermatocyte. The inserts present in the top right corner of each panel represent the development of the spermatid acrosome.
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3.2.3. Stage III The acrosome spread slightly over the nucleus during this stage, and the acrosomal vesicle remained round. The acrosome vesicle subtended at an angle on the nuclear surface of 55 4.3 degrees (range, 42 degrees to 70 degrees). Elongated spermatid bundles had dissociated, and spermatid nuclei were located very close to the tubule lumen. Type A spermatogonia, pachytene spermatocytes with larger nuclei, round spermatids, and Type B spermatogonia were present in this stage; type B spermatogonia nuclei were characterized by a round to ovoid shape and the presence of a large amount of heterochromatin. 3.2.4. Stage IV An extensive acrosomal vesicle was seen in spermatids in this stage. The acrosome vesicle extended over the nucleus and began to flatten at the point of contact with the nucleus. The acrosome vesicle subtended at an angle on the nucleus of approximately 80 6 degrees (range, 60 degrees to 97 degrees). The main characteristic of this stage was the location of elongated spermatids just being released at the lumen of the seminiferous tubule. Residual bodies were observed just below elongated spermatids. Preleptotene spermatocytes, originated from Type B spermatogonia, were in contact with the basal lamina and represented the other germ cell type present in this stage. Overall, the nucleus morphology of round spermatids, pachytene spermatocytes, and Type A spermatogonia was similar to that of the previous stage. 3.2.5. Stage V The nuclei of spermatids were still round, and the acrosome vesicle subtended over the nucleus at an angle of approximately 100 10.2 degrees (73 degrees to 140 degrees). Only one spermatid generation was present and formed several layers within the upper part of the seminiferous epithelium. Occasional Type A spermatogonia and two generations of primary spermatocytes were present: leptotene, whose nuclei were located closer to the basal lamina; and pachytene, sandwiched between round spermatids and leptotene spermatocytes. 3.2.6. Stage VI The ratio between the longest or longitudinal axis and the shortest axis (transversal line passing across the nucleus at the equatorial zone) was approximately 1.43 0.1. Spermatid nuclei began elongation, and the chromatin in young elongated spermatids was more condensed than that in the previous stage. Primary
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spermatocytes were in the transition from leptotene to zygotene, and pachytene spermatocyte nuclei were noticeably larger than in Stage V. Type A spermatogonia were also present. 3.2.7. Stage VII Elongation of spermatids was completed during this stage. The ratio between the longest and shortest axis of the nucleus was 2.54 0.3. Condensation of the nucleus (as reflected by staining intensity) was occurring during the latter phase of this stage. Elongated spermatids first formed bundles, with their heads oriented toward the Sertoli cell nuclei, usually located at the base of the tubule. Young primary spermatocytes had the characteristics of zygotene cells. At the end of this stage, pachytene spermatocytes transitioned to the diplotene phase of the meiotic prophase. The nuclei of Type A spermatogonia were more frequent and similar in appearance to those seen in the previous stages. 3.2.8. Stage VIII In comparison with the previous stage, nuclei in elongated spermatids had a similar shape. Judging by staining affinity, condensation of these cells was still occurring. The main feature of this stage was the presence of meiotic figures of the first and second divisions; secondary spermatocytes and early round spermatids were also observed. Zygotene spermatocytes were present in this stage and were in transition to pachytene spermatocytes. Elongated spermatid bundles were located within Sertoli cell crypts at approximately the middle of the seminiferous epithelium. The nuclei of Type A spermatogonia were present in greater numbers. The mean percentage of each of the eight stages of the seminiferous epithelium cycle characterized for the ocelot is shown in Fig. 1. Stage II was less frequent (5.2%), whereas Stages IV, V, and VII were more frequent (18% to 21%). The frequencies of premeiotic (Stages V to VII), meiotic (Stage VIII), and postmeiotic (Stages I to IV) stages of the cycle were 51.1%, 8.9%, and 40%, respectively. 3.3. Length of the seminiferous epithelium cycle The most advanced labeled germ cell type found at different time periods after thymidine administration is shown in Table 2 and Figs. 2 and 3. Approximately 1 h after administration, the most advanced labeled germ cells were identified as either preleptotene spermatocytes or cells in the transition from preleptotene to leptotene (Figs. 2A and 3). These cells were present at
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Table 2 The length (d) of seminiferous epithelium cycle in the ocelot (mean SEM). Animal
Time after administration
Most advanced germ cell type labeled
1
1h 27.92 d* 1h 23.97 d*
Pl/L E Pl/L R Mean duration of the cycle
2
Stage of the cycle VI I VI I based on Pl/L = 12.5 0.4
Number of cycles traversed
Cycle length based on labeling in leptotene
— 2.32 — 1.86 d.
— 12.04 — 12.88
Pl/L, preleptotene/leptotene primary spermatocytes; E, elongate spermatids, R, round spermatids. * Total time after thymidine injection minus 1 h.
the beginning of Stage VI. Based on the mean pachytene nucleus diameter, these cells traversed approximately 20% of this stage in both animals and were located in the basal compartment. At 24 d, these cells were round spermatids present in Stage V that went through 70% of this stage. At 28 d, these cells were elongated spermatids in Stage I that had traversed 40% of this stage. Based on the most advanced labeled germ cell types observed at each time period after thymidine administration and stage frequencies, the mean duration of the seminiferous epithelium cycle for the two animals investigated was estimated to be 12.5 0.4 d (Table 2). The duration of the various stages of the cycle was determined taking into account the cycle length and the percentage of occurrence of each stage. The shortest stage was Stage II (0.65 d) and the longest was Stage V (2.66 d; Fig. 3). As approximately 4.5 cycles are necessary for the spermatogenic process to be completed, the total length of spermatogenesis was estimated as 56.3 1.9 d. 3.4. Testis stereology The data related to testis stereology in the ocelot are shown in Tables 3 and 4. The meiotic index (measured
as the number of round spermatids produced per pachytene primary spermatocyte) was 2.9 0.1. Therefore, 30% cell loss occurred during the meiotic prophase. Sertoli cell efficiency in the ocelot (estimated from the number of round spermatids per each Sertoli cell) was 4.5 0.1. The number of Sertoli cells per gram of testis was 46 3 106, whereas this figure per testis was 419 54 106. Regarding spermatogenic efficiency, the daily sperm production per gram of testis and per testis was approximately 18.3 1 and 163 21 106, respectively. The mean nuclear volume and size of Leydig cells (Table 4) was 421 16 and 2522 172 mm3, whereas their number per gram of testis and per testis was 38 5 106 and 300 20 106. 4. Discussion Little information is available in the literature regarding the reproductive biology of the family Felidae, which includes some of the most successful predators on earth and currently the most threatened [27]. The current study is the first to report a more comprehensive stereologic and functional investigation of the testis in the ocelot (Leopardus pardalis) and, to
Fig. 2. The most advanced-labeled germ cells found (arrows) at various intervals after intratesticular administration of tritiated thymidine in the ocelot; (A) 1 h after injection, preleptotene/leptotene spermatocytes at the beginning of Stage VI; (B) at 24 d, these cells were round spermatids present at the beginning of the last quarter of Stage V; (C) at 28 d, these cells were elongated spermatids in the middle of Stage I.
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Fig. 3. This diagrammatic figure illustrates the eight stages of the seminiferous epithelium cycle characterized in the ocelot based on the acrosomic system. The vertical columns (designated by roman numerals) depict cell associations. The developmental progression of a cell is followed horizontally until the right-hand border of the diagram. The cell progression continues on the left of the diagram, one row up. The cycle of the diagram ends with the completion of spermiation. The following symbols are used to designate specific germ cell types: In, Type intermediate spermatogonia; B, Type B spermatogonia; Pl, preleptotene; L, leptotene; Z, zygotene; P, pachytene; D, diplotene; M/28, secondary spermatocyte. Arabic numbers are used to designate the steps of the spermiogenic phase. Frequencies (%) and duration (d) of each stage of the seminiferous epithelium cycle are also shown. The most advanced germ cell types labeled in the eight stages of the cycle at three time period intervals (1 h, 24 h, and 28 d) after thymidine administration are also depicted.
our knowledge, following studies on the jaguar (Panthera onca) [20], it is the second comprehensive study related to the basic histologic and quantitative aspects of the testis in wild felids. In the current study, the testis samples were taken during Southern Hemisphere winter (July). In a study developed in Brazil, with adult captive male ocelots, Morais and colleagues [7] observed that although serum testosterone concentrations were significantly higher in the spring, a non-significant sperm production peak was noted in summer. However, in their study, the investigated species (ocelot, margay, and tigrinas) were capable of reproducing year-round. In comparison with
data obtained in our laboratory for the domestic cat and jaguar (Table 5), the gonadosomatic index for the ocelot was twofold and threefold higher, respectively, thereby suggesting that the ocelot may have a different reproductive strategy and/or invest more in sperm production. Although exhibiting lower Sertoli cell efficiency (number of spermatids per Sertoli cell) and relatively long spermatogenic cycle length, due to having approximately 50% more Sertoli cells per testis gram the ocelot exhibited slightly more spermatogenic efficiency (daily sperm production per gram of testis) in comparison with data published on the domestic cat and jaguar. It is noteworthy that the Sertoli cell efficiency
Table 3 Cell counts, cell ratios, and sperm production in the ocelot (mean SEM).
Table 4 Leydig cell morphometry in the ocelot (mean SEM).
Parameters
Parameters
Round spermatids:pachytene spermatocyte Round spermatids:Sertoli cell nucleoli Sertoli cell number per gram of testis (106) Sertoli cell number per testis (106) Daily sperm production per gram of testis (106) Daily sperm production per testis (106)
2.9 0.1 4.5 0.1 46 3 419 54 18.3 1 163 21
Nuclear diameter (mm) Leydig cell volume (mm3) Nucleus volume (mm3) Cytoplasm volume (mm3) Leydig cell number per gram of testis (106) Leydig cell number per testis (106)
9.3 0.1 2522 172 421 16 2101 165 38 5 300 20
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Table 5 Comparative parameters related to testis morphometry and spermatogenic events in the sexually mature ocelot, domestic cat, and jaguar. Parameters
Ocelot
Domestic cat*
Jaguary
Body weight (kg) Testis weight (g) Gonadosomatic index (%) Testis parenchyma volume density (%) Seminiferous tubule Leydig cell Leydig cell volume (mm3) Leydig cell number per gram of testis (106) Sertoli cell number per gram of testis (106) Round spermatids per Sertoli cell Spermatogenic cycle length (d) Spermatogenic efficiency (106)
13.8 1 11 0.6 0.16 0.01
3.1 0.2 1.17 0.07 0.078 0.007
77 3 17.7 2.2 0.05 0.01
83.2 1.6 9.8 1.5 2522 172 38 5 46 3 4.5 0.1 12.5 0.4 18.3 1
88.2 1.2 6 0.6 2044 150 30 2.9 32 3.9 5.1 0.6 10.4 0.3 15.7 1.6
74.7 3.8 16.7 1.6 1602 180 107 12 29 4 7.9 0.8 12.8 0.01 16.9 1.2
* y
Ref. [30], related to data obtained for the domestic cat. Ref. [20], related to data obtained for the jaguar.
found for the ocelot was one of the lowest reported for mammals thus far [9]. Nevertheless, the spermatogenic cycle length and the total duration of spermatogenesis in the ocelot were very similar to the jaguar and longer than values found for the domestic cat (Table 5), as well as for most mammalian species studied [9]. In mammals, the stages of the seminiferous epithelium cycle may be characterized based on changes in the shape of the spermatid nucleus, the occurrence of meiotic divisions, and the arrangement of spermatids within the germinal epithelium [28–30], as well as based on the development of the acrosomic system and morphology of developing spermatids [18–
20]. Eight stages of spermatogenesis were characterized for the ocelot based on the acrosomic system, and germ cell morphology was very similar to that described for the domestic cat [30] and jaguar [20]. Sperm morphology was also similar when the ocelot was compared with the domestic cat. Therefore, sperm from these two species cannot readily be distinguished through the use of morphology. There is strong evidence that premeiotic and postmeiotic stage frequencies may be phylogenetically determined among members of the same mammalian family [8,22,31]. Conversely, spermatogenic cycle length is generally considered constant for a given
Fig. 4. Frequencies (mean SEM) of eight stages of the spermatogenic cycle, characterized based on the development of the acrosome in the spermatids in the ocelot (current study; n = 4), domestic cat (n = 25), and jaguar (n = 4) [20]. Note that the frequencies of several stages were different in these three felid species.
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Fig. 5. Comparative frequencies (mean SEM) of premeiotic (Stages V to VII), meiotic (Stage VIII), and postmeiotic (Stages I to IV) stages of the cycle, characterized based on the acrosomic system in the ocelot (current study; n = 4), domestic cat (n = 25), and jaguar (n = 4) [20]. In comparison with the jaguar, these frequencies were similar for the ocelot and domestic cat, particularly for postmeiotic frequencies.
species [10] and is under the control of germ cell genotype [11]. Recent studies using molecular genetic assessment found that the phylogenetic divergence between the domestic cat and ocelot and between the domestic cat and jaguar is about 2 106 and 5 106 yr, respectively [32,33,27]. Therefore, the combined premeiotic and postmeiotic stage frequencies observed for the ocelot were similar to those found for domestic cats (Figs. 4 and 5). However, the spermatogenic cycle length and total duration of spermatogenesis were similar to values obtained for the jaguar (Table 5). Similar to the domestic cat [30], the tunica albuginea in the ocelot testis was very thick, and its volume density was much greater than that of most mammalian species investigated [8], including the jaguar [20]. The tubule diameter and seminiferous epithelium height found for the ocelot was similar to that of most mammalian species investigated [34] and not very different from values observed for the domestic cat [30] and jaguar [20]. However, the seminiferous tubules in the domestic cat occupy nearly 90% of the testis parenchyma, whereas these values are 83% and 75% in the ocelot and jaguar, respectively (Table 5). The tubule length per gram of testis was nearly 15% bigger in the domestic cat. The Leydig cell volume density in the ocelot (Table 4) was higher than that observed for most mammals [9], including the values obtained for the domestic cat [30]. Nevertheless, whereas the Leydig cell size was in the upper range for mammals, the number of this type of cell per gram of testis was similar to that described for the domestic cat and approximately one third of that described for the jaguar (Table 5).
Germ cell apoptosis normally occurs during spermatogenesis in all mammals investigated, particularly during the spermatogonial phase (density-dependent regulation) and during meiosis, probably due to chromosome damage [8,9,35]. Similar to the few felids investigated in this aspect [20,30] and to most mammalian species investigated thus far [8,9,35], 30% of germ cell loss occurred during meiosis in the ocelot. However, unlike the domestic cat [30] but similar to the jaguar [20], no missing generation of germ cells was observed in the ocelots examined in the current study. With the exception of the domestic cat and a few other small cat species, nearly every one of the 37 felid species is considered endangered or threatened [36]. Considering the importance of the propagation of animals in captivity for the preservation of endangered species, reproduction of wild cats needs to become an integral component of conservation efforts [37]. There are currently several assisted-reproduction techniques and biotechnologies that could potentially be used to preserve endangered wild species [3,4,38,39], such as germ cell transplantation using fresh, frozen, and cultured germ cells [6,40,41], testis grafting [42], and cell aggregates [43]. For this purpose, we are currently developing methods for using the domestic cat as a recipient for spermatogonial stem cell transplantation with the aim of preserving and propagating male germ plasm from endangered wild felid species, including the ocelot. In the current investigation we obtained substantial basic data related to testis function in the ocelot,
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including characterization of stages of the seminiferous epithelium cycle, the total duration of spermatogenesis, and Sertoli cell and spermatogenic efficiencies. Besides providing baseline information for comparative studies among felines and other mammalian species, the data generated in this study may be useful to the preservation of the ocelot (Leopardus pardalis) using domestic cat testes to generate and propagate the ocelot genome, considering that these two felid species are phylogenetically closely related. Acknowledgments Financial support from the Brazilian National Council for Research (CNPq) and from the Minas Gerais State Foundation (FAPEMIG) is gratefully acknowledged. The scholarships awarded to Robson Campos Silva from CAPES and to Guilherme Mattos Jardim Costa from the Minas Gerais State Foundation (FAPEMIG) and CNPq are greatly appreciated. Technical help from Adriano Moreira and Mara L. Santos is also greatly appreciated. References [1] Eizirik E, Bonatto SL, Johnson WE, Crawshaw Jr PG, Vie´ JC. Brousset DM, et al. Phylogeographic patterns and evolution of the mitochondrial DNA control region in two neotropical cats (Mammalia, Felidae). J Mol Evol 1998;47:613–24. [2] CITES [Convention on International Trade of Endangered Species of Wild Fauna and Flora]. Appendices I, II and III, CITES Secretariat, 2009. Available at: www.cites.org. [3] Pelican KM, Wildt DE, Pukazhenthi B, Howard J. Ovarian control for assisted reproduction in the domestic cat and wild felids. Theriogenology 2006;66:37–48. [4] Baudi DLK, Jewgenow K, Pukazhenthi BS, Spercoski KM, Santos AS, Reghelin ALS, et al. Influence of cooling rate on the ability of frozen–thawed sperm to bind to heterologous zona pellucida, as assessed by competitive in vitro binding assays in the ocelot (Leopardus pardalis) and tigrina (Leopardus tigrinus). Theriogenology 2008;69:204–11. [5] Machado ABM, Drummond GM, Paglia AP. Livro vermelho da fauna brasileira ameac¸ada de extinc¸a˜o, Ministe´rio do Meio Ambiente. Fundac¸a˜o Biodiversitas 2008;785–6. [6] Kim Y, Selvaraj V, Dobrinski I, Lee H, Mcentee MC, Travis AJ. Recipient preparation and mixed germ cell isolation for spermatogonial stem cell transplantation in domestic cats. J Androl 2006;27:248–56. [7] Morais RN, Mucciolo RG, Gomes ML, Lacerda O, Moraes W, Moreira N, et al. Seasonal analysis of semen characteristics, serum testosterone and fecal androgens in the ocelot (Leopardus pardalis), margay (L. wiedii) and tigrina (L. tigrinus). Theriogenology 2002;57:2027–41. [8] Franc¸a LR, Russell LD. The testis of domestic animals. In: Martı´nez F, Regadera J, editors. Male Reproduction, A Multidisciplinary Overview. Churchill Livingstone; 1998. p. 197–219.
[9] Hess RA, Franc¸a LR. Spermatogenesis. Cycle of the seminiferous epithelium. In: Cheng CY, editor. Molecular Mechanisms in Spermatogenesis. Landes Bioscience; 2007. p. 1–15. [10] Clermont Y. Kinetics of spermatogenesis in mammals: seminiferous epithelium cycle and spermatogonial renewal. Physiol Rev 1972;52:198–236. [11] Franc¸a LR, Ogawa T, Avarbock MR, Brinster RL, Russell LD. Germ cell genotype controls cell cycle during spermatogenesis in the rat. Biol Reprod 1998;59:1371–7. [12] Dobrinski I. Germ cell transplantation and testis tissue xenografting in domestic animals. Anim Reprod Sci 2005;89:137– 45. [13] Khaira H, McLean D, Ohl DA, Smith GD. Spermatogonial stem cell isolation, storage, and transplantation. J Androl 2005;26:442– 50. [14] Brinster RL. Male germline stem cells: from mice to men. Science 2007;316:404–5. [15] Dobrinski I, Travis AJ. Germ cell transplantation for the propagation of companion animals, non-domestic and endangered species. Reprod Fertil Dev 2007;6:732–9. [16] Bundy DC. Photographic emulsions and processing. In: Stumpf WE, Solomon HF, editors. Autoradiography and Correlative Imaging. Academic Press; 1995. p. 49–57. [17] Johnson L, Neaves WB. Age-related changes in the Leydig cell population, seminiferous tubules and sperm production in stallions. Biol Reprod 1981;24:703–12. [18] Leblond CP, Clermont Y. Definition of the stages of the cycle of the seminiferous epithelium in the rat. Ann N Y Acad Sci 1952;55:548–84. [19] Russell LD, Ettlin RA, Sinha-Hikim AP, Clegg ED. Histological and Histopathological Evaluation of the Testis. Cache River Press; 1990. [20] Costa GMJ, Chiarini-Garcia H, Morato RG, Alvarenga RLLS, Franc¸a LR. Duration of spermatogenesis and daily sperm production in the jaguar (Panthera onca). Theriogenology 2008;70:1136– 46. [21] Amann RP, Schanbacher BD. Physiology of male reproduction. J Anim Sci 1983;57:380–403. [22] Neves ES, Chiarini-Garcia H, Franc¸a LR. Comparative testis morphometry and seminiferous epithelium cycle length in donkeys and mules. Biol Reprod 2002;67:247–55. [23] Abercrombie M. Estimation of nuclear populations from microtome sections. Anat Rec 1946;94:238–48. [24] Amann RP, Almquist JO. Reproductive capacity of dairy bulls. VIII. Direct and indirect measurement of testicular sperm production. J Dairy Sci 1962;45:774–81. [25] Hochereau DE, Reviers MT, Lincoln GA. Seasonal variation in the histology of the testis of the red deer. Cervus elephus J Reprod Fertil 1978;54:209–13. [26] Franc¸a LR. Daily sperm production in Piau boars estimated from Sertoli cell population and Sertoli cell index. In: Dieleman SJ, editor. Proceedings of the 12th International Congress on Animal Reproduction and Artificial Insemination. Elsevier Science Ltd; v. 4. 1992. p. 1716–8. [27] O’Brien SJ, Johnson W, Driscoll C, Pontius J, Pecon-Slattery J, Menotti-Raymond M. State of cat genomics. Trends Genet 2008;24:268–79. [28] Courot M, Hochereau-de Reviers MT, Ortavant R. Spermatogenesis. In: Johnson AD, Gomes WR, Vandemark NL, editors. The Testis. Academic Press; 1970. p. 339–432. [29] Berndtson WE. Methods for quantifying mammalian spermatogenesis: a review. J Anim Sci 1977;44:818–83.
R.C. Silva et al. / Theriogenology 73 (2010) 157–167 [30] Franc¸a LR, Godinho CL. Testis morphometry, seminiferous epithelium cycle length, and daily sperm production in domestic cats (Felis catus). Biol Reprod 2003;68:1554–61. [31] Almeida FF, Leal MC, Franc¸a LR. Testis morphometry, duration of spermatogenesis, and spermatogenic efficiency in the wild boar (Sus scrofa scrofa). Biol Reprod 2006;75:792–9. [32] Johnson WE, Eizirik E, Pecon-Slattery J, Murphy WJ, Antunes A, Teeling E, et al. The late Miocene radiation of modern Felidae: a genetic assessment. Science 2006;311:73–7. [33] Driscoll CA, Menotti-Raymond M, Roca AL, Hupe K, Johnson WE, Geffen E, et al. The near eastern origin of cat domestication. Science 2007;317:519–23. [34] Setchell BP. Possible physiological bases for contraceptive techniques in the male. Hum Reprod 1994;97:381–7. [35] Roosen-Runge EC. Germinal-cell loss in normal metazoan spermatogenesis. J Reprod Fertil 1973;35:339–48. [36] O’Brien SJ, Johnson WE. Big cat genomics. Annu Rev Genomics Hum Genet 2005;6:407–29. [37] Paz RCR, Eduardo Dias EA, Adania CH, Barnabe VH, Barnabe RC. Ovarian response to repeated administration of alternating exogenous gonadotropin regimens in the ocelot (Leopardus
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Testis stereology, seminiferous epithelium cycle length, and daily sperm production in the ocelot (Leopardus pardalis) R.C. Silva a,b, G.M.J. Costa a, L.M. Andrade c, L.R. Franc¸a a,* a
Laboratory of Cellular Biology, Department of Morphology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil b Department of Basic Sciences, Federal University of Jequitinhonha and Mucuri Valleys, Diamantina, MG, Brazil c Associac¸a˜o Bichos Gerais, Belo Horizonte, MG, Brazil Received 12 June 2009; received in revised form 14 August 2009; accepted 17 August 2009
Abstract Similar to most wild felids, the ocelot (Leopardus pardalis) is an endangered species. However, knowledge regarding reproductive biology of the ocelot is very limited. Germ cell transplantation is an effective technique for investigating spermatogenesis and stem cell biology in mammals, and the morphologic characterization of germ cells and knowledge of cycle length are potential tools for tracking the development of transplanted germ cells. Our goal was to investigate basic aspects related to testis structure, particularly spermatogenesis, in the ocelot. Four adult males were used. After unilateral orchiectomy, testis samples were routinely prepared for histologic, stereologic, and autoradiographic analyses. Testis weight and the gonadosomatic index were 11 0.6 g and 0.16 0.01%, respectively, whereas the volume density of seminiferous tubules and Leydig cells was 83.2 1.6% and 9.8 1.5%. Based on the acrosomic system, eight stages of spermatogenesis were characterized, and germ cell morphology was very similar to that of domestic cats. Each spermatogenic cycle lasted 12.5 0.4 d, and the entire spermatogenic process lasted 56.3 1.9 d. Individual Leydig cell volume was 2522 mm3, whereas the number of Leydig and Sertoli cells per gram of testis was 38 5 106 and 46 3 106. Approximately 4.5 spermatids were found per Sertoli cell, whereas daily sperm production per gram of testis was 18.3 1 106, slightly higher than values reported for other felids. The knowledge obtained in this study could be very useful to the preservation of the ocelot using domestic cat testes to generate and propagate the ocelot genome. # 2010 Elsevier Inc. All rights reserved. Keywords: Ocelot (Leopardus pardalis); Spermatogenic cycle length; Spermatogenic efficiency; Stereology; Testis
1. Introduction The ocelot (Leopardus pardalis) is a medium-size cat (7 to 15.8 kg) that occupies a wide variety of habitats from rainforests to dry scrubland [1]. Similar to most wild felids, the ocelot is currently listed as an
* Corresponding author. Tel.: +55 31 34092816; fax: +55 31 34092780. E-mail address: [email protected] (L.R. Franc¸a). 0093-691X/$ – see front matter # 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2009.08.009
endangered species by the Convention on International Trade of Endangered Species of Wild Fauna and Flora (CITES) [2] and by other sources [3,4]. In Brazil, Leopardus pardalis populations outside the Amazon Basin are listed as vulnerable [5]. Similar to other species, the loss of genetic diversity poses a serious threat to the conservation of endangered wild felids [6]. Although knowledge of reproductive parameters is crucial for the preservation of any endangered species, there is little information regarding testis function in the ocelot [4,7].
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Characterization of the stages of spermatogenesis and estimation of the spermatogenic cycle length are fundamental to determining spermatogenic efficiency and to performing comparative studies among species. The total duration of spermatogenesis requires approximately 4.5 cycles, lasts from 30 to 75 d in mammals [8,9], is generally considered constant for a species [10], and is under the control of the germ cell genotype [11]. Germ cell transplantation is a recently developed technique that has considerable potential for studies involving biotechnology, transgenics, and preservation of the genetic stock of valuable animals or endangered species [12–15]. In this regard, germ cell transplantation studies are currently under way in our laboratory, using the domestic cat as a recipient; one of our objectives is to use this species to preserve the genetic stock of wild felids, including the ocelot. Consequently, detailed knowledge regarding spermatogenesis and length of the spermatogenic cycle are very important. As there are few reports in the literature regarding the reproductive biology of the ocelot [4,7], the main objectives of the current study were to perform a detailed, comprehensive histologic and stereologic investigation of the testis, as well as determine the duration of spermatogenesis and spermatogenic and Sertoli cell efficiency in the sexually mature ocelot. 2. Materials and methods 2.1. Animals Four ocelots (2.5 to 3.5 yr old) weighing 13.8 1 kg were used. These animals were from the Brigitte Johnson animal facility, located in the city of Belo Horizonte (198550 1500 S; 438560 1600 W), state of Minas Gerais, Brazil. As no significant seasonal changes have been found for sperm production in ocelots kept in captivity [7], in the current study, testis samples were collected during Brazilian winter (July). The animals were contained and kept under general anesthesia using a combination of xylazine hydrochloride (Vetbrands, Sespo Indu´stria e Come´rcio Ltda, Jacareı´, SP, Brazil) and ketamine (Ko¨nig, Ko¨nig do Brasil Ltda, Santana de Parnaı´ba, SP, Brazil), using 1.1 and 10 mg per kg body weight, respectively. After unilateral orchiectomy, the testis was separated from the epididymis, weighed, and cut longitudinally (with a razor blade) into small slabs, which were fixed by immersion in 4% buffered glutaraldehyde for 12 to 24 h. Tissue samples (2 to 3 mm thick) were routinely processed and embedded in plastic (glycol methacrylate) for histologic, stereologic, and autoradiographic evaluation. To investigate their morphology, sperm were
collected from the cauda epididymidis, and the smears were routinely prepared for histologic evaluation and comparisons with smears available in our laboratory for the jaguar and domestic cat. All surgical procedures were performed by a veterinarian and followed approved guidelines for the ethical treatment of animals. 2.2. Intratesticular thymidine administration and tissue preparation Prior to orchiectomy, an intratesticular administration (75 mCi/testis) of tritiated thymidine (thymidine [methyl-3H], specific activity 82.0 Ci/mmol; Amersham, Life Science, Buckinghamshire, England)—a specific marker for cells that are synthesizing DNA—was performed close to the cauda epididymidis in two animals to estimate the duration of spermatogenesis. For each animal, two time intervals (1 h and 24 d; and 1 h and 28 d) were considered after the thymidine administration. Tissue samples (2 to 3 mm thick) were collected near the thymidine administration site, routinely fixed, and embedded as described above. For autoradiographic analysis, unstained testis sections (4 mm) were dipped in an autoradiographic emulsion (Kodak NTB-2; Eastman Kodak Company, Rochester, NY, USA) at 43 to 45 8C. After drying for approximately 1 h at 25 8C, the testis sections were placed in sealed black boxes and stored in a refrigerator (4 8C) for approximately 4 wk. Subsequently, testis sections were developed in Kodak D-19 solution at 15 8C [16] and stained with toluidine blue. Analyses of these sections were performed under light microscopy to detect the most advanced germ cell type labeled at the three time periods after thymidine administration. Cells were considered labeled when at least four or five grains were present over the nucleus on a low to moderate background. 2.3. Testis stereology Volume densities of the testicular tissue components were determined under light microscopy, using a 441intersection grid placed in the ocular of the light microscope. Fifteen randomly chosen fields (6615 points) were scored per testis for each animal at 400 magnification. Tubule diameter and seminiferous tubule epithelium height were measured at 100 magnification, using an ocular micrometer calibrated with a stage micrometer. Thirty round or nearly round tubule profiles were chosen randomly and measured for each animal. Epithelium height was obtained in the same tubules used to determine the tubule diameter. The total length of seminiferous tubule (meters) was obtained by
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dividing seminiferous tubule volume by the squared radius of the tubule multiplied by p [17]. 2.4. Stages of the seminiferous epithelium cycle Stages of the seminiferous epithelium cycle were characterized based on the development of the acrosomic system and morphology of the developing spermatid nucleus [18,19]. Similar to the jaguar and domestic cat [20], this method provided eight stages of the seminiferous epithelium cycle for the ocelot. The relative stage frequencies were determined by evaluating 150 seminiferous tubule cross sections per animal at 400 magnification. The measurement of the angle of the acrosome on the nuclear surface was obtained from 15 germ cells (per animal and per cycle stage) at 1000 magnification. 2.5. Length of the seminiferous epithelium cycle The histologic sections used were those that were of better quality and had more tubule cross sections. The duration of the spermatogenic cycle was estimated in two animals, based on stage frequencies and the most advanced germ cell type labeled at different times after thymidine administration. 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 [21]. As the volume of the nucleus of pachytene primary spermatocytes grows markedly during meiotic prophase and these cells are present in nearly all stages of the cycle [8,22], the size of their nuclei was used as a reference point to more precisely determine the location of the most advanced labeled germ cell. 2.6. Cell counts and cell numbers All germ cell nuclei and Sertoli cell nucleoli present in Stage V of the cycle 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, based on the method described by Abercrombie [23] and modified by Amann and Almquist [24]. For this purpose, 10 nuclei or nucleoli diameters were measured per animal for each cell type analyzed. Cell ratios were obtained from the corrected counts obtained in Stage V. 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 the seminiferous tubules [25]. Daily sperm production
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(DSP) per testis and per gram of testis (spermatogenic efficiency) was obtained according to the following formula: DSP = total number of Sertoli cells per testis the ratio of round spermatids to Sertoli cells in Stage V Stage V relative frequency (%)/Stage V duration (d) [26]. Individual volume of the Leydig cells was obtained from the nucleus volume and proportion occupied by the nucleus and cytoplasm. As the Leydig cell nucleus was spherical, nucleus volume was calculated from the mean nuclear diameter. For this purpose, 30 nuclei with an evident nucleolus were measured for each animal. Leydig cell nuclear volume was expressed in mm3 and obtained from the formula 4/ 3pR3, in which R = nuclear diameter/2. To calculate the relative proportions occupied by the nucleus and cytoplasm, a 441-point square lattice was placed over the sectioned material at 400 magnification, and 1000 points over Leydig cells were counted for each animal. The total number of Leydig cells per testis was estimated from the individual Leydig cell volume and the volume occupied by Leydig cells in the testis parenchyma. 3. Results 3.1. Biometric data and testis volume density All data are presented in Table 1. Mean (SEM) testis weight for the adult ocelot was 11 0.6 g, providing a gonadosomatic index (testes mass divided by body weight) of 0.16 0.01%. Volume density of seminiferous tubules and Leydig cells was 83.2 1.6% and 9.8 1.5%, respectively. Therefore, Leydig cells occupied nearly 60% of the intertubular compartment. Mean tubular diameter and epithelium height were Table 1 Biometric and morphometric data in the ocelot (mean SEM). Parameters Body weight (kg) Testis weight (g) Gonadosomatic index (%) Testis parenchyma volume density (%) Tubular compartment Tunica propria Seminiferous epithelium Lumen Intertubular compartment Leydig cell Others Tubular diameter (mm) Seminiferous epithelium height (mm) Tubular length per gram of testis (m) Total tubular length per testis (m)
13.8 1 11 0.6 0.16 0.01 83.2 1.6 4.7 0.2 69.8 2 8.7 1.5 16.8 1.6 9.8 1.5 5.2 1.2 252 3 86 3 16.7 0.4 150 13
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252 3 and 86 3 mm. The mean percentage found for the tunica albuginea was approximately 20 0.02% (data not shown). Based on the volume of the testis parenchyma (testis weight minus tunica albuginea weight) and the volume occupied by seminiferous tubules in the testis and tubule diameter, there were 16.7 0.4 and 150 13 m of seminiferous tubules per testis gram and per testis (Table 1). The morphology observed for the sperm obtained from the cauda epididymidis in the ocelot was very similar to the morphology previously determined for the jaguar and domestic cat (data not shown).
spermatids. Because the proacrosomal granules cannot be seen under a light microscope, the newly formed spermatids were characterized by their lack of distinguishing features. However, a juxtanuclear Golgi apparatus was evident. Some elongated spermatid bundles were located deep within the epithelium; many were closer to the epithelium base than to the lumen. Type A spermatogonia and intermediate spermatogonia nuclei were found at the base of the tubule. Young pachytene spermatocytes were located between round spermatids and the basal lamina.
3.2. Stages of the seminiferous epithelium cycle and relative stage frequencies
3.2.2. Stage II Early round spermatids usually had two small acrosomal vesicles in which only occasional proacrosomal granules were present. At the end of this stage, the small proacrosomal vesicles coalesced to form one large acrosomal vesicle containing a single acrosomal granule, and the acrosomal vesicle made contact with the nucleus. The elongated spermatid bundles had moved much closer toward the lumen of the seminiferous tubule. Pachytene spermatocyte nuclei were larger than in Stage I and slightly more distant from the basal lamina. Type B and Type A spermatogonia were observed in this stage.
The eight stages of the cycle characterized based on the acrosomic system (Fig. 1) were very similar to those previously described for the jaguar and domestic cat [20] and are described later. To better characterize these stages, the angle formed by the acrosome in relation to the spermatid was measured. 3.2.1. Stage I Two generations of spermatids were present, including early round spermatids and elongated
Fig. 1. Stages I to VIII of the seminiferous epithelium cycle in the ocelot, based on the development of the acrosome in spermatids: A, Type A spermatogonia; B, Type B spermatogonia; Pl, preleptotene spermatocyte; L, leptotene spermatocyte; Z, zygotene spermatocyte; P, pachytene spermatocyte; D, diplotene spermatocyte; M, meiotic figure; R, round spermatids; E, elongating/elongated spermatids; SC, Sertoli cells; Rb, residual bodies; 28, secondary spermatocyte. The inserts present in the top right corner of each panel represent the development of the spermatid acrosome.
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3.2.3. Stage III The acrosome spread slightly over the nucleus during this stage, and the acrosomal vesicle remained round. The acrosome vesicle subtended at an angle on the nuclear surface of 55 4.3 degrees (range, 42 degrees to 70 degrees). Elongated spermatid bundles had dissociated, and spermatid nuclei were located very close to the tubule lumen. Type A spermatogonia, pachytene spermatocytes with larger nuclei, round spermatids, and Type B spermatogonia were present in this stage; type B spermatogonia nuclei were characterized by a round to ovoid shape and the presence of a large amount of heterochromatin. 3.2.4. Stage IV An extensive acrosomal vesicle was seen in spermatids in this stage. The acrosome vesicle extended over the nucleus and began to flatten at the point of contact with the nucleus. The acrosome vesicle subtended at an angle on the nucleus of approximately 80 6 degrees (range, 60 degrees to 97 degrees). The main characteristic of this stage was the location of elongated spermatids just being released at the lumen of the seminiferous tubule. Residual bodies were observed just below elongated spermatids. Preleptotene spermatocytes, originated from Type B spermatogonia, were in contact with the basal lamina and represented the other germ cell type present in this stage. Overall, the nucleus morphology of round spermatids, pachytene spermatocytes, and Type A spermatogonia was similar to that of the previous stage. 3.2.5. Stage V The nuclei of spermatids were still round, and the acrosome vesicle subtended over the nucleus at an angle of approximately 100 10.2 degrees (73 degrees to 140 degrees). Only one spermatid generation was present and formed several layers within the upper part of the seminiferous epithelium. Occasional Type A spermatogonia and two generations of primary spermatocytes were present: leptotene, whose nuclei were located closer to the basal lamina; and pachytene, sandwiched between round spermatids and leptotene spermatocytes. 3.2.6. Stage VI The ratio between the longest or longitudinal axis and the shortest axis (transversal line passing across the nucleus at the equatorial zone) was approximately 1.43 0.1. Spermatid nuclei began elongation, and the chromatin in young elongated spermatids was more condensed than that in the previous stage. Primary
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spermatocytes were in the transition from leptotene to zygotene, and pachytene spermatocyte nuclei were noticeably larger than in Stage V. Type A spermatogonia were also present. 3.2.7. Stage VII Elongation of spermatids was completed during this stage. The ratio between the longest and shortest axis of the nucleus was 2.54 0.3. Condensation of the nucleus (as reflected by staining intensity) was occurring during the latter phase of this stage. Elongated spermatids first formed bundles, with their heads oriented toward the Sertoli cell nuclei, usually located at the base of the tubule. Young primary spermatocytes had the characteristics of zygotene cells. At the end of this stage, pachytene spermatocytes transitioned to the diplotene phase of the meiotic prophase. The nuclei of Type A spermatogonia were more frequent and similar in appearance to those seen in the previous stages. 3.2.8. Stage VIII In comparison with the previous stage, nuclei in elongated spermatids had a similar shape. Judging by staining affinity, condensation of these cells was still occurring. The main feature of this stage was the presence of meiotic figures of the first and second divisions; secondary spermatocytes and early round spermatids were also observed. Zygotene spermatocytes were present in this stage and were in transition to pachytene spermatocytes. Elongated spermatid bundles were located within Sertoli cell crypts at approximately the middle of the seminiferous epithelium. The nuclei of Type A spermatogonia were present in greater numbers. The mean percentage of each of the eight stages of the seminiferous epithelium cycle characterized for the ocelot is shown in Fig. 1. Stage II was less frequent (5.2%), whereas Stages IV, V, and VII were more frequent (18% to 21%). The frequencies of premeiotic (Stages V to VII), meiotic (Stage VIII), and postmeiotic (Stages I to IV) stages of the cycle were 51.1%, 8.9%, and 40%, respectively. 3.3. Length of the seminiferous epithelium cycle The most advanced labeled germ cell type found at different time periods after thymidine administration is shown in Table 2 and Figs. 2 and 3. Approximately 1 h after administration, the most advanced labeled germ cells were identified as either preleptotene spermatocytes or cells in the transition from preleptotene to leptotene (Figs. 2A and 3). These cells were present at
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Table 2 The length (d) of seminiferous epithelium cycle in the ocelot (mean SEM). Animal
Time after administration
Most advanced germ cell type labeled
1
1h 27.92 d* 1h 23.97 d*
Pl/L E Pl/L R Mean duration of the cycle
2
Stage of the cycle VI I VI I based on Pl/L = 12.5 0.4
Number of cycles traversed
Cycle length based on labeling in leptotene
— 2.32 — 1.86 d.
— 12.04 — 12.88
Pl/L, preleptotene/leptotene primary spermatocytes; E, elongate spermatids, R, round spermatids. * Total time after thymidine injection minus 1 h.
the beginning of Stage VI. Based on the mean pachytene nucleus diameter, these cells traversed approximately 20% of this stage in both animals and were located in the basal compartment. At 24 d, these cells were round spermatids present in Stage V that went through 70% of this stage. At 28 d, these cells were elongated spermatids in Stage I that had traversed 40% of this stage. Based on the most advanced labeled germ cell types observed at each time period after thymidine administration and stage frequencies, the mean duration of the seminiferous epithelium cycle for the two animals investigated was estimated to be 12.5 0.4 d (Table 2). The duration of the various stages of the cycle was determined taking into account the cycle length and the percentage of occurrence of each stage. The shortest stage was Stage II (0.65 d) and the longest was Stage V (2.66 d; Fig. 3). As approximately 4.5 cycles are necessary for the spermatogenic process to be completed, the total length of spermatogenesis was estimated as 56.3 1.9 d. 3.4. Testis stereology The data related to testis stereology in the ocelot are shown in Tables 3 and 4. The meiotic index (measured
as the number of round spermatids produced per pachytene primary spermatocyte) was 2.9 0.1. Therefore, 30% cell loss occurred during the meiotic prophase. Sertoli cell efficiency in the ocelot (estimated from the number of round spermatids per each Sertoli cell) was 4.5 0.1. The number of Sertoli cells per gram of testis was 46 3 106, whereas this figure per testis was 419 54 106. Regarding spermatogenic efficiency, the daily sperm production per gram of testis and per testis was approximately 18.3 1 and 163 21 106, respectively. The mean nuclear volume and size of Leydig cells (Table 4) was 421 16 and 2522 172 mm3, whereas their number per gram of testis and per testis was 38 5 106 and 300 20 106. 4. Discussion Little information is available in the literature regarding the reproductive biology of the family Felidae, which includes some of the most successful predators on earth and currently the most threatened [27]. The current study is the first to report a more comprehensive stereologic and functional investigation of the testis in the ocelot (Leopardus pardalis) and, to
Fig. 2. The most advanced-labeled germ cells found (arrows) at various intervals after intratesticular administration of tritiated thymidine in the ocelot; (A) 1 h after injection, preleptotene/leptotene spermatocytes at the beginning of Stage VI; (B) at 24 d, these cells were round spermatids present at the beginning of the last quarter of Stage V; (C) at 28 d, these cells were elongated spermatids in the middle of Stage I.
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Fig. 3. This diagrammatic figure illustrates the eight stages of the seminiferous epithelium cycle characterized in the ocelot based on the acrosomic system. The vertical columns (designated by roman numerals) depict cell associations. The developmental progression of a cell is followed horizontally until the right-hand border of the diagram. The cell progression continues on the left of the diagram, one row up. The cycle of the diagram ends with the completion of spermiation. The following symbols are used to designate specific germ cell types: In, Type intermediate spermatogonia; B, Type B spermatogonia; Pl, preleptotene; L, leptotene; Z, zygotene; P, pachytene; D, diplotene; M/28, secondary spermatocyte. Arabic numbers are used to designate the steps of the spermiogenic phase. Frequencies (%) and duration (d) of each stage of the seminiferous epithelium cycle are also shown. The most advanced germ cell types labeled in the eight stages of the cycle at three time period intervals (1 h, 24 h, and 28 d) after thymidine administration are also depicted.
our knowledge, following studies on the jaguar (Panthera onca) [20], it is the second comprehensive study related to the basic histologic and quantitative aspects of the testis in wild felids. In the current study, the testis samples were taken during Southern Hemisphere winter (July). In a study developed in Brazil, with adult captive male ocelots, Morais and colleagues [7] observed that although serum testosterone concentrations were significantly higher in the spring, a non-significant sperm production peak was noted in summer. However, in their study, the investigated species (ocelot, margay, and tigrinas) were capable of reproducing year-round. In comparison with
data obtained in our laboratory for the domestic cat and jaguar (Table 5), the gonadosomatic index for the ocelot was twofold and threefold higher, respectively, thereby suggesting that the ocelot may have a different reproductive strategy and/or invest more in sperm production. Although exhibiting lower Sertoli cell efficiency (number of spermatids per Sertoli cell) and relatively long spermatogenic cycle length, due to having approximately 50% more Sertoli cells per testis gram the ocelot exhibited slightly more spermatogenic efficiency (daily sperm production per gram of testis) in comparison with data published on the domestic cat and jaguar. It is noteworthy that the Sertoli cell efficiency
Table 3 Cell counts, cell ratios, and sperm production in the ocelot (mean SEM).
Table 4 Leydig cell morphometry in the ocelot (mean SEM).
Parameters
Parameters
Round spermatids:pachytene spermatocyte Round spermatids:Sertoli cell nucleoli Sertoli cell number per gram of testis (106) Sertoli cell number per testis (106) Daily sperm production per gram of testis (106) Daily sperm production per testis (106)
2.9 0.1 4.5 0.1 46 3 419 54 18.3 1 163 21
Nuclear diameter (mm) Leydig cell volume (mm3) Nucleus volume (mm3) Cytoplasm volume (mm3) Leydig cell number per gram of testis (106) Leydig cell number per testis (106)
9.3 0.1 2522 172 421 16 2101 165 38 5 300 20
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Table 5 Comparative parameters related to testis morphometry and spermatogenic events in the sexually mature ocelot, domestic cat, and jaguar. Parameters
Ocelot
Domestic cat*
Jaguary
Body weight (kg) Testis weight (g) Gonadosomatic index (%) Testis parenchyma volume density (%) Seminiferous tubule Leydig cell Leydig cell volume (mm3) Leydig cell number per gram of testis (106) Sertoli cell number per gram of testis (106) Round spermatids per Sertoli cell Spermatogenic cycle length (d) Spermatogenic efficiency (106)
13.8 1 11 0.6 0.16 0.01
3.1 0.2 1.17 0.07 0.078 0.007
77 3 17.7 2.2 0.05 0.01
83.2 1.6 9.8 1.5 2522 172 38 5 46 3 4.5 0.1 12.5 0.4 18.3 1
88.2 1.2 6 0.6 2044 150 30 2.9 32 3.9 5.1 0.6 10.4 0.3 15.7 1.6
74.7 3.8 16.7 1.6 1602 180 107 12 29 4 7.9 0.8 12.8 0.01 16.9 1.2
* y
Ref. [30], related to data obtained for the domestic cat. Ref. [20], related to data obtained for the jaguar.
found for the ocelot was one of the lowest reported for mammals thus far [9]. Nevertheless, the spermatogenic cycle length and the total duration of spermatogenesis in the ocelot were very similar to the jaguar and longer than values found for the domestic cat (Table 5), as well as for most mammalian species studied [9]. In mammals, the stages of the seminiferous epithelium cycle may be characterized based on changes in the shape of the spermatid nucleus, the occurrence of meiotic divisions, and the arrangement of spermatids within the germinal epithelium [28–30], as well as based on the development of the acrosomic system and morphology of developing spermatids [18–
20]. Eight stages of spermatogenesis were characterized for the ocelot based on the acrosomic system, and germ cell morphology was very similar to that described for the domestic cat [30] and jaguar [20]. Sperm morphology was also similar when the ocelot was compared with the domestic cat. Therefore, sperm from these two species cannot readily be distinguished through the use of morphology. There is strong evidence that premeiotic and postmeiotic stage frequencies may be phylogenetically determined among members of the same mammalian family [8,22,31]. Conversely, spermatogenic cycle length is generally considered constant for a given
Fig. 4. Frequencies (mean SEM) of eight stages of the spermatogenic cycle, characterized based on the development of the acrosome in the spermatids in the ocelot (current study; n = 4), domestic cat (n = 25), and jaguar (n = 4) [20]. Note that the frequencies of several stages were different in these three felid species.
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Fig. 5. Comparative frequencies (mean SEM) of premeiotic (Stages V to VII), meiotic (Stage VIII), and postmeiotic (Stages I to IV) stages of the cycle, characterized based on the acrosomic system in the ocelot (current study; n = 4), domestic cat (n = 25), and jaguar (n = 4) [20]. In comparison with the jaguar, these frequencies were similar for the ocelot and domestic cat, particularly for postmeiotic frequencies.
species [10] and is under the control of germ cell genotype [11]. Recent studies using molecular genetic assessment found that the phylogenetic divergence between the domestic cat and ocelot and between the domestic cat and jaguar is about 2 106 and 5 106 yr, respectively [32,33,27]. Therefore, the combined premeiotic and postmeiotic stage frequencies observed for the ocelot were similar to those found for domestic cats (Figs. 4 and 5). However, the spermatogenic cycle length and total duration of spermatogenesis were similar to values obtained for the jaguar (Table 5). Similar to the domestic cat [30], the tunica albuginea in the ocelot testis was very thick, and its volume density was much greater than that of most mammalian species investigated [8], including the jaguar [20]. The tubule diameter and seminiferous epithelium height found for the ocelot was similar to that of most mammalian species investigated [34] and not very different from values observed for the domestic cat [30] and jaguar [20]. However, the seminiferous tubules in the domestic cat occupy nearly 90% of the testis parenchyma, whereas these values are 83% and 75% in the ocelot and jaguar, respectively (Table 5). The tubule length per gram of testis was nearly 15% bigger in the domestic cat. The Leydig cell volume density in the ocelot (Table 4) was higher than that observed for most mammals [9], including the values obtained for the domestic cat [30]. Nevertheless, whereas the Leydig cell size was in the upper range for mammals, the number of this type of cell per gram of testis was similar to that described for the domestic cat and approximately one third of that described for the jaguar (Table 5).
Germ cell apoptosis normally occurs during spermatogenesis in all mammals investigated, particularly during the spermatogonial phase (density-dependent regulation) and during meiosis, probably due to chromosome damage [8,9,35]. Similar to the few felids investigated in this aspect [20,30] and to most mammalian species investigated thus far [8,9,35], 30% of germ cell loss occurred during meiosis in the ocelot. However, unlike the domestic cat [30] but similar to the jaguar [20], no missing generation of germ cells was observed in the ocelots examined in the current study. With the exception of the domestic cat and a few other small cat species, nearly every one of the 37 felid species is considered endangered or threatened [36]. Considering the importance of the propagation of animals in captivity for the preservation of endangered species, reproduction of wild cats needs to become an integral component of conservation efforts [37]. There are currently several assisted-reproduction techniques and biotechnologies that could potentially be used to preserve endangered wild species [3,4,38,39], such as germ cell transplantation using fresh, frozen, and cultured germ cells [6,40,41], testis grafting [42], and cell aggregates [43]. For this purpose, we are currently developing methods for using the domestic cat as a recipient for spermatogonial stem cell transplantation with the aim of preserving and propagating male germ plasm from endangered wild felid species, including the ocelot. In the current investigation we obtained substantial basic data related to testis function in the ocelot,
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including characterization of stages of the seminiferous epithelium cycle, the total duration of spermatogenesis, and Sertoli cell and spermatogenic efficiencies. Besides providing baseline information for comparative studies among felines and other mammalian species, the data generated in this study may be useful to the preservation of the ocelot (Leopardus pardalis) using domestic cat testes to generate and propagate the ocelot genome, considering that these two felid species are phylogenetically closely related. Acknowledgments Financial support from the Brazilian National Council for Research (CNPq) and from the Minas Gerais State Foundation (FAPEMIG) is gratefully acknowledged. The scholarships awarded to Robson Campos Silva from CAPES and to Guilherme Mattos Jardim Costa from the Minas Gerais State Foundation (FAPEMIG) and CNPq are greatly appreciated. Technical help from Adriano Moreira and Mara L. Santos is also greatly appreciated. References [1] Eizirik E, Bonatto SL, Johnson WE, Crawshaw Jr PG, Vie´ JC. Brousset DM, et al. Phylogeographic patterns and evolution of the mitochondrial DNA control region in two neotropical cats (Mammalia, Felidae). J Mol Evol 1998;47:613–24. [2] CITES [Convention on International Trade of Endangered Species of Wild Fauna and Flora]. Appendices I, II and III, CITES Secretariat, 2009. Available at: www.cites.org. [3] Pelican KM, Wildt DE, Pukazhenthi B, Howard J. Ovarian control for assisted reproduction in the domestic cat and wild felids. Theriogenology 2006;66:37–48. [4] Baudi DLK, Jewgenow K, Pukazhenthi BS, Spercoski KM, Santos AS, Reghelin ALS, et al. Influence of cooling rate on the ability of frozen–thawed sperm to bind to heterologous zona pellucida, as assessed by competitive in vitro binding assays in the ocelot (Leopardus pardalis) and tigrina (Leopardus tigrinus). Theriogenology 2008;69:204–11. [5] Machado ABM, Drummond GM, Paglia AP. Livro vermelho da fauna brasileira ameac¸ada de extinc¸a˜o, Ministe´rio do Meio Ambiente. Fundac¸a˜o Biodiversitas 2008;785–6. [6] Kim Y, Selvaraj V, Dobrinski I, Lee H, Mcentee MC, Travis AJ. Recipient preparation and mixed germ cell isolation for spermatogonial stem cell transplantation in domestic cats. J Androl 2006;27:248–56. [7] Morais RN, Mucciolo RG, Gomes ML, Lacerda O, Moraes W, Moreira N, et al. Seasonal analysis of semen characteristics, serum testosterone and fecal androgens in the ocelot (Leopardus pardalis), margay (L. wiedii) and tigrina (L. tigrinus). Theriogenology 2002;57:2027–41. [8] Franc¸a LR, Russell LD. The testis of domestic animals. In: Martı´nez F, Regadera J, editors. Male Reproduction, A Multidisciplinary Overview. Churchill Livingstone; 1998. p. 197–219.
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