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REPRODUCTION-DEVELOPMENT |
Laboratory of Cellular Biology, Department of Morphology, Institute of Biological Sciences, Federal University of Minas Gerais (S.L.P.M., D.A.R.V., L.R.F.), Belo Horizonte MG 31270-901, Brazil; and Graduate Program in Zoology of Vertebrates, Pontifical Catholic University (H.P.G.), Belo Horizonte MG 30535-610, Brazil
Address all correspondence and requests for reprints to: Dr. Luiz Renato França, Laboratory of Cellular Biology, Department of Morphology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte MG 31270-901, Brazil. E-mail: . lrfranca{at}icb.ufmg.br
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Abstract |
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1 g BW and 3.5 cm total in length) were treated for a period of 40 d with different concentrations (100 and 150 ppm) of PTU. The animals were killed and analyzed on d 1, 30, 40, 98, and 208 after the beginning of the treatment. On d 30 and 40 the spermatogenic process was delayed in fish treated with PTU compared with the control group. Also at these periods, treated tilapia had decreased (P < 0.05) body weight and total length. On d 98 body weight and total length had recovered in PTU-treated fish and were similar (P > 0.05) to those of the controls. However, testis weight and gonadosomatic index (testis mass/body weight) were approximately 100% higher (P < 0.05) in treated tilapia. Similarly, the area occupied by seminiferous tubules, the number of Sertoli cells and germ cells per cyst, and the number of Leydig cells per testis were significantly (P < 0.05) greater in treated fish. Nevertheless, nuclear volume and individual Leydig cell volume were significantly lower (P < 0.05) in tilapia receiving PTU treatment. Compared with controls, at 208 d all parameters analyzed presented the same trend as that observed at 98 d. In general, at 98 d the different PTU concentrations used during the treatment period induced similar effects. However, at 208 d the mean values observed for several parameters were significantly higher (P < 0.05) in fish exposed to 150 ppm. Probably due to the higher density of Sertoli cells per cyst in treated tilapia, these cells presented a smaller (P < 0.05) nucleolus and a trend to decrease its support capacity (efficiency). However, the meiotic index (germ cell loss during the two meiotic divisions) was similar (P > 0.05) in the three groups of fish investigated. Remarkably, the results found in tilapia were similar to those found for rats treated with PTU. This suggests strongly that the mechanisms of control of Sertoli cell and Leydig cell proliferation seem to be preserved during vertebrate evolution. |
Introduction |
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The development and function of the germ cells are closely linked to the development of the somatic elements of the testis (3). In this regard, somatic cells are key to the normal functioning of the male reproductive system (4), with the Sertoli cell considered the most important cell to guide germ cell development (5). There is no in vivo instance in fish where germ cells develop without the presence of somatic cells. In well studied laboratory mammals it has been shown that proliferation of Sertoli cells occurs after sex differentiation and before the initiation of spermatogenesis, with early growth of the testis largely due to Sertoli cell proliferation (6). In mammals it has also been shown that differentiated Sertoli cells show no mitotic activity (see review in Ref. 7). Because the Sertoli cell capacity to support germ cells is relatively constant for each species (8), testis size and potential for sperm production are established when the Sertoli cell mitotic period ends (6, 9). Although the role of Sertoli cell number in regulating sperm production is well defined in mammals (10), to our knowledge there is no report in the literature dealing specifically with Sertoli cell proliferation in the teleost.
The thyroid gland plays an essential role in the growth and regulation of the metabolism of vertebrates. There is considerable information that the thyroid gland hormones, T3 and T4, regulate testis maturation, especially in mammals (10, 11). In teleosts, although the role of thyroid hormones is not well established, it is known that T3 and/or T4 are required for normal gonadal maturation (12, 13).
Methods have recently been developed using the goitrogen 6-n-propyl-2-thiouracil (PTU) during testis development to increase testis size and sperm production, with success in laboratory rodents (rat, mouse, and hamster) (10, 14) and chicken (15), among several species investigated. Rats treated with PTU recover the euthyroid state about 2 wk after the end of PTU treatment (16). In this species, transient neonatal hypothyroidism caused by PTU treatment was able to increase testis size, Sertoli cell number, and sperm production up to approximately 80%, 150%, and 140%, respectively (10). As thyroid hormone (T3), which is responsible for Sertoli cell differentiation, is inhibited during the PTU treatment period (10), Sertoli cells undergo an extended period of proliferation (17), "nursing" more germ cells (9). As fertility is not affected (10), the PTU method has great potential for increasing production in economically important species. Although it is already known that PTU treatment induces hypothyroidism in teleosts (18, 19), to our knowledge there are no data in the literature regarding the role of thyroid hormones in Sertoli cell proliferation in this group of vertebrate.
To create a larger testis it is important to treat the animals with PTU during the period of active Sertoli cell proliferation (10). In the present study young fish (Nile tilapia; Oreochromis niloticus) were exposed to two different concentrations of PTU during testis development to investigate the effects of this drug on Sertoli cell proliferation, testis size, and germ cell number. Using this model, the numbers of Sertoli and germ cells were remarkably increased in the adult fish.
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Materials and Methods |
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Nuclear diameter of germ cells and Sertoli cell nucleus size.
To establish the adequate thickness of serial histological sections for counting the number of germ cells and Sertoli cells per cyst in sexually mature animals, testis fragments of 10 sexually mature Nile tilapias (326–368 g BW and 25.6–26.4 cm Lt) were fixed by immersion in 5% glutaraldehyde, 0.1 M phosphate buffer, pH 7.3, and embedded in glycol methacrylate. For histological analysis testis tissue was sectioned at 3-µm thickness in an Reichert-Jung automatic ultramicrotome, stained with 1% toluidine blue, and examined in an Olympus Corp. BX40 light microscope (New Hyde Park, NY). The following mean nuclear diameters, measured at x1000 magnification with an ocular micrometer calibrated with a stage micrometer, were obtained from 20 measurements/cell type/fish: primary spermatogonia, 9.3 ± 0.3 µm; secondary spermatogonia, 6.8 ± 0.2 µm; primary spermatocyte (pachytene), 5.9 ± 0.1 µm; secondary spermatocytes, 4.1 ± 0.1 µm; early spermatids, 2.9 ± 0.06 µm; and late spermatids, 1.8 ± 0.06 µm. The mean Sertoli nucleus length and width were approximately 8.0 and 3.5 µm, respectively. From these data it was established that serial sections, 3 or 2 µm in thickness, were appropriate to count the total number of cells present in cysts of primary spermatocytes, secondary spermatocytes, and early spermatids.
PTU treatment
Based on the preliminary findings, Nile tilapia juveniles (0.95 g BW, 3.5 cm Lt, possibly just before germ cell differentiation) obtained from a local hatchery station were used. After a 5-d period of acclimation to laboratory conditions, the fish were randomly assigned to 3 groups (2 treated and 1 control). Each group, of 100 fish was housed in separate tanks of 250-liter capacity for 40 d. During this period the tank water of the 2 treatment groups was maintained, respectively, with 100 and 150 ppm PTU (Sigma, St. Louis, MO), whereas the control group was not exposed to the drug.
All fish were fed ad libitum a juvenile trout commercial diet. Recirculation and aeration of the water were performed with standard mechanical and biological filters and aerators. Electrical heaters maintained the water temperature between 23 and 26 C. Every 5 d the tank was cleaned, followed by the replacement of 50 liter of water, maintaining the respective concentrations of PTU. Temperature, pH, dissolved oxygen, ammonia, and nitrite were recorded during the experimental period and remained within adequate levels. No fish mortality was recorded in this period. The experiments were conducted according to the institutional animal care protocols and approval.
Posttreatment period
At the end of the PTU treatment period, the fish were transferred to a hatchery station, where they were maintained in separate, approximately 1.5-m3, cages placed in a communal earthen pond. They were fed a commercial diet, and the water parameters routinely measured (temperature, pH, and dissolved oxygen) stayed at adequate levels throughout the experiment. The fish were allowed to grow until 208 d after the beginning of the treatment, when the experiment ended.
Gonad preparation
Fish were killed on d 1, 30, 40, 98, and 208 after the beginning of treatment. On these days, animals were weighed, measured, and had their testes removed. At 98 and 208 d, the killed male tilapias had their testes weighed for calculations of the gonadosomatic index (testes weight/body weight x 100). Fragments of approximately 3 mm in thickness, from several different segment of the testes, were taken transversally, fixed by immersion in 5% glutaraldehyde/0.1 M phosphate buffer, pH 7.3, and embedded in glycol methacrylate. For histological analysis, testis tissue from the mid segment was sectioned at 3-µm thickness in a Reichert-Jung automatic ultramicrotome, whereas serial sections at 3 or 2 µm were used for morphometric analyses of cysts and Leydig cells in sexually mature fish. Sections were stained with 1% toluidine blue and examined in an Olympus Corp. BX40 light microscope.
Number of germ cells and Sertoli cells per cyst, and cell ratios
The total numbers of primary spermatocytes, secondary spermatocytes, and early spermatids per cyst were obtained in 5 cysts per male fish killed at 98 and 208 d. This was accomplished after selecting the respective cysts whose area was entirely encompassed by the serial sections. Cysts of primary spermatocytes (pachytene) and secondary spermatocytes and early spermatids were analyzed in serial sections at 3 and 2 µm thickness, respectively. Approximately 60 µm of tissue were necessary to be serially cut to allow the counting of all cells within an entire cyst of pachytene, whereas for secondary spermatocytes and initial spermatids this figure was 80 µm. The number of Sertoli cell nuclei facing the cyst was simultaneously counted. The nucleolar diameter of 30 Sertoli cells/fish/treatment was also measured at this time. Care was taken not to count one nucleus twice. On d 208, from the total number of fish killed from the control group (29 tilapias) and the 150 ppm group (23 animals), 10 fish/group were randomly selected for morphometric analyses, and 9 fish were used for the 100 ppm group.
To evaluate meiosis and Sertoli cell efficiency, the following ratios between different cell types were determined: secondary spermatocytes per primary spermatocytes, early spermatids per secondary spermatocytes, early spermatids per primary spermatocytes, and primary spermatocytes, secondary spermatocytes, and early spermatids per Sertoli cells.
Volume density of testis parenchyma and area occupied by different testis components
The volume density (percentage) of tubular (seminiferous tubules and respective cysts) and intertubular compartments (Leydig cells, connective tissue cells and fibers, and vessels) were obtained in fish killed at 98 and 208 d. These data were obtained using a 441-point square lattice placed over the testis parenchyma at x400 magnification. Approximately 5300 points were counted for each animal. For this part of the investigation, sections taken from the cranial, midcranial, mid, midcaudal, and caudal region of the testis were evaluated.
The area occupied by seminiferous tubules, intertubular space, hylus, and testis portion of the spermatic duct was also evaluated in tilapias from all three groups killed at 98 and 208 d. This evaluation was performed using an image analysis system (Image Pro Plus for Windows) attached to an Olympus Corp. BX 40 light microscope at x40 magnification using the 2-µm-thick transverse histological sections from the mid segment of the testes. The area encompassing hylus and spermatic duct was estimated by subtracting the area of seminiferous tubules and intertubular space from the total area. These data were expressed in square millimeters.
Leydig cell morphometry
Leydig cell morphometry was also performed on the testis of fish killed at 98 and 208 d. The individual Leydig cell volume (cubic microns) was obtained from the nucleus volume and the proportion (percentage) between nucleus and cytoplasm of this cell. As the Leydig cell nucleus in tilapias is spherical, its nucleus volume was estimated using the mean nuclear diameter. Thirty spherical Leydig cell nuclei presenting evident nucleolus were measured for each fish. Individual nuclear volume was expressed (cubic microns) using the formula 4/3 
R3, where R is nuclear diameter/2. To estimate the proportion between nucleus and cytoplasm, a 441-point square lattice was placed over the sectioned material at x400 magnification. One thousand points over Leydig cells were counted for each animal. The number of Leydig cells per testis was calculated using the data obtained for Leydig cell individual volume and the volume occupied by Leydig cells in the testis.
Statistical analyses
Statistical analyses were performed using Statistica 3.11 for Windows software (StatSoft, Inc., Tulsa, OK). Arcsine transformation was applied to the percentage data before calculations. The results from the three groups were tested using ANOVA (Newman-Keuls test). The significance level considered was P < 0.05. All data are presented as the mean ± SEM.
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Results |
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). A similar trend was observed for total length (Table 1). The histological status of the testis during the treatment period is also shown in Table 1. At the beginning of the treatment (d 1) the gonads in fish from all three experimental groups contained gonocytes/primary spermatogonia and undifferentiated somatic cells (Table 1 and Fig. 1A); most of the somatic cells present were presumably Sertoli cell precursors. On d 30 and 40, spermatogenesis was delayed in both treated groups compared with controls (Table 1 and Fig. 1, B–D). In this regard, although spermatogenesis had advanced up to spermatids or spermatozoa in most control tilapias at 40 d, the predominant germ cell type present in treated tilapias at the same period was spermatogonia.
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and 3). However, both testis size and gonadosomatic index were about 100% higher (P < 0.05) in tilapias exposed to 100 and 150 ppm PTU compared with controls (Tables 2 and 3 and Fig. 2). Although the difference was not significant (P > 0.05; Table 3), compared with animals exposed to 150 ppm the testis weight was approximately 30% larger in tilapias exposed to 100 ppm at 208 d.
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and 3).
No significant differences (P > 0.05) were observed for hylus and testis portion of the spermatic duct area among the three groups investigated at 98 and 208 d after the beginning of PTU treatment (data not shown). However, tilapias from both treated groups presented a noticeable increase in seminiferous tubule and intertubular space area at 98 d (>130%; P < 0.05) and 208 d (75%; P < 0.05; data not shown).
Number of Sertoli cells, spermatocytes, and spermatids per cyst
Sertoli cell number per cysts of spermatocytes and spermatids showed a striking increase in sexually mature treated fish compared with controls (Tables 2 and 3). In this regard, at 98 d the number of this somatic cell was twice as large (P < 0.05) in both treated groups as that in controls (Fig. 3). At 208 d, tilapias treated with 100 and 150 ppm showed approximately 70% and 90% more Sertoli cells per cyst (P < 0.05), respectively (Fig. 3). Except for those associated with spermatid cysts, Sertoli cell number at 208 d was significantly higher (P < 0.05) in the 150 ppm group compared with tilapias exposed to 100 ppm (Table 3). Although at a lower ratio, the number of all germ cell types quantified per cyst at 98 and 208 d was significantly higher (P < 0.05) in both treated groups compared with controls (Tables 2 and 3). At 98 d, the mean increase observed was approximately 55% (Fig. 4), whereas at 208 d this figure was around 30% and 60% for fish exposed to 100 and 150 ppm, respectively (Fig. 4). At 208 d, comparing both treated groups, the number of spermatocytes and spermatids per cyst was higher (P < 0.05) in tilapias exposed to 150 ppm PTU.
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and 3); the differences observed at 208 d were always statistically significant (P < 0.05). Also, compared with fish exposed to 150 ppm PTU, at 208 d the ratio of secondary spermatocytes and spermatids per Sertoli cell was significantly reduced (P < 0.05) in tilapias treated with 100 ppm. Sertoli cell nucleolar diameter was reduced (P < 0.05) in treated fish in a dose-dependent fashion (Tables 2 and 3).
Germ cell loss during meiotic divisions at 98 and 208 d was similar in all experimental groups (P > 0.05). During the first meiotic division, from 20–25% of cell loss occurred, whereas about 20% of germ cells died during the conversion of secondary spermatocytes to spermatids. This means that from the theoretical number of spermatids expected (4 cells) only 2.5 cells were formed, resulting in an overall loss of approximately 40% (Tables 2 and 3).
Leydig cell morphometry
Leydig cell nuclear volume and Leydig cell individual volume were significantly reduced in sexually mature treated tilapias (P < 0.05) compared with controls (Tables 2 and 3). In general, this reduction was higher (P < 0.05) in fish exposed to the highest PTU concentration. On the other hand, the number of Leydig cells per testis showed a striking increase (P < 0.05) in both treated groups at the two different periods investigated (Tables 2 and 3 and Fig. 5).
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Discussion |
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In the present experiment we did not measure thyroid hormone levels during the treatment period. Also, because the thyroid gland in tilapias is a diffused tissue, it was not possible to measure its weight. However, compared with the literature for teleosts and mammals (10, 18), significantly decreased body weight in treated tilapias during the last quarter of PTU treatment showed the effectiveness of the concentrations of PTU used in the present experiment to induce hypothyroidism. Nevertheless, different from laboratory rodents (10), in sexually mature treated tilapias the body weight reached the same size as that of matched controls.
Similar to the results observed for rats, mice, and hamsters (10, 20), testis size and gonadosomatic index were significantly higher in sexually mature tilapias during both periods investigated. Compared with 98 d, testis weight at 208 d after the beginning of treatment was approximately 20-fold higher in all experimental groups investigated. Also, corroborating the results found for rats (9, 21), the volume density of different testis components in treated and control tilapias showed similar values. This indicates that all testis compartments in tilapias grew uniformly and responded positively to the PTU treatment. In rats, the maximum testis size in treated animals was attained between 4 and 5 months after the end of PTU treatment (10). In fish in general, including tilapias, body and testis weights increase for several years. For this reason, it was not the objective of the present work to investigate the long-term effect of PTU treatment on testis size in this species.
Probably due to the hypothyroid condition and the immature status of Sertoli cells (9, 17, 22), spermatogenesis development was delayed in treated fish at 30 and 40 d after the beginning of PTU treatment. Also, in accordance with the results described by Kirby et al. (16) and Cooke et al. (10), 2 wk after the end of the treatment the inhibitory effect of PTU was totally reversed in tilapias, and spermatogenesis in treated animals returned to its normal pace. The duration of spermatogenesis is very short in tilapias, lasting less than 2 wk (23), compared with mammals where this process lasts in general from 40–60 d (24). Thus, the morphometrical and functional evaluation of the testis we performed approximately 60 d after the end of treatment possibly gave us a fairly precise idea regarding the effects of PTU on the number of Sertoli and germ cells per cyst. In fact, most of PTU effects observed on testis function at 208 d after the beginning of treatment, particularly the number of Sertoli cells and germ cells per cyst, showed similar trend compared with those at 98 d. Our findings also show that the augmentation of Sertoli cell number per cyst reflects positively on germ cell number. The addition of more stem cells and Sertoli cells was probably responsible for the higher testis weight observed at 208 d. We did not investigate Sertoli cell kinetics in the present study. Therefore, we do not know whether the increase in Sertoli cell population resulted from mature Sertoli cell division or from Sertoli cell precursors. In mammals, it is believed that mature Sertoli cells do not divide (3, 7).
As observed for mammals (9), our results corroborate that Sertoli cell numbers are the major factor determining the magnitude of sperm production. However, it remains to be established in tilapias whether PTU concentrations different from those used and different windows of treatment during the period when the Sertoli cell is actively proliferating would allow similar results.
The best reflection of the functional efficiency of the Sertoli cell is the number of germ cells supported by this somatic cell. In tilapias, about 100 spermatids/Sertoli cell were observed. Compared with most mammals investigated (7, 8, 25), the number of germ cells per Sertoli cell in tilapias is approximately 10-fold higher, suggesting that the cystic arrangement of spermatogenesis is more efficient compared with the way Sertoli cells are distributed in the seminiferous tubules of mammals. Possibly due to the higher Sertoli cell density per cyst in treated fish, the Sertoli cell presented decreased germ cell support capacity (9, 26) and decreased nucleolar diameter (9).
There is little quantitative information regarding the spermatogenic efficiency in fish. The cystic arrangement of spermatogenesis in tilapias allows a fairly precise morphometrical analysis and a comprehensive understanding of spermatogenesis and the Sertoli/germ cell relationship in this species. Only two cell divisions take place in all vertebrates to form spermatids from primary spermatocytes. Assuming that the spermatogenic cyst in tilapias is formed when the Sertoli cell becomes associated with 1 stem cell (27), the number of primary spermatocytes per cyst found in control and treated tilapias suggests that at least 10 generations of spermatogonia are necessary to form spermatocytes. These data are similar to what is observed in many teleosts and mammals investigated (1, 28, 29). As the number of cells per cyst in treated tilapias is higher than that in controls, it remains to be investigated whether spermatogenesis is more efficient due to the higher number of Sertoli cells per cyst, less germ cell loss during spermatogonial proliferation, or more spermatogonial divisions in treated animals.
It is not established from the literature for mammals whether cell degeneration during spermatogenesis occurs individually or whether the entire clone degenerates (29). The cystic (clonal) arrangement of spermatogenesis in tilapias offers a good experimental model to investigate this question. As the number of the same type of germ cell per cyst varies within the same individual and among different individuals, the results found in the present experiment suggest strongly that cell loss occurs only in a certain portion of the germ cell population originating from a single spermatogonia.
Similar to the results found in rats (21, 30), in treated tilapias Leydig cell volume and nucleus volume decreased significantly; this reduction, mainly for nucleus volume, was more evident in fish exposed to a higher PTU concentration. As testis size increased markedly in these fish, whereas Leydig cell volume density remained similar in treated and control animals, the number of Leydig cells per testis increased remarkably in treated tilapias (21, 30). In neonatal PTU-treated rats, the increase in mesenchymal cell number occurred due to the absence of mesenchymal cell differentiation into Leydig cell precursors (31), which increased the availability of precursors giving rise to Leydig cells at the time of PTU withdrawal. In the present study we did not investigate the effects of PTU on Leydig cell precursors during testis development. However, the remarkable increase in the total number of Leydig cells per testis at 98 and 208 d in both treated groups investigated suggests that thyroid hormones play a very important role in the control of the Leydig cell population; the mechanisms involved were probably similar in fish and mammals.
In conclusion, our data show that similar to the results found for rodents, thyroid hormones are very important for testis development and testis function in Nile tilapias. Also, PTU-induced hypothyroidism offers a potential method for improving spermatogenic efficiency in fish.
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Acknowledgments |
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Footnotes |
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1 Current address: Department of General Biology, Federal University of Viçosa, Viçosa MG 36571-000, Brazil. 
Abbreviations: Lt, Total length; PTU, 6-n-propyl-2-thiouracil.
Received August 6, 2001.
Accepted for publication November 5, 2001.
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