Tissue & Cell, 1999 31 (3) 372–379 © 1999 Harcourt Publishers Ltd Article no. tice.1999.0043
Tissue&Cell
Exposure to constant light during testis development increases daily sperm production in adult Wistar rats D. C. M. Rocha1, L. Debeljuk2, L. R. França1
Abstract. Testis histometry and daily sperm production (DSP) were evaluated in adult (160-day-old) Wistar rats exposed to constant light for the first 25 days after birth, and compared with control animals which were exposed to a 12 h-light–12 h-dark light regimen. Significantly greater (P < 0.05) numbers of Sertoli cell nucleoli and round spermatids per cross-section of seminiferous tubule were found in animals exposed to constant light. In addition, epididymis weight, DSP per testis and per gram of testis, as well as Leydig cell compartment volume, were significantly increased in treated animals. Although there was a clear trend toward an increased Sertoli cell population per testis in animals exposed to constant light, this difference was not statistically significant (P < 0.05). The number of round spermatids as expressed per Sertoli cell was the same in both groups. Surprisingly, the diameter and volume of round spermatid nucleus at stages I and VII of the cycle of seminiferous epithelium were significantly lower (P < 0.05) in treated animals. In conclusion, constant illumination during neonatal testis development increased sperm production and Leydig cell compartment volume in adult rats probably through a mechanism involving elevated follicle stimulating hormone and luteinizing hormone during the prepubertal period. To our knowledge, this is the first study showing that altering the light regimen can affect sperm production in non-seasonal breeders.
Keywords: Pineal gland, photoperiod, morphometry, daily sperm production, rats
Introduction Sertoli cell proliferation in rats occurs from day 16 postconception until virtually the 16th day postnatally (Steinberger & Steinberger, 1971; Orth, 1982), and is stimulated by follicle stimulating hormone (FSH) (Orth, 1982; Almirón & Chemes, 1988). Since this cell has a relatively fixed support capacity of germ cells, its population ultimately dictates testis size and sperm production (Orth et al., 1988; Hess et al., 1993; Franca et al., 1995). Melatonin, secreted by the pineal gland, plays an important role in the reproductive function of seasonal mammals 1 Laboratory of Cellular Biology, Department of Morphology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil 31270–901 2Department of Physiology, Southern Illinois University, Carbondale, IL, 62901–6512, USA
Received 6 April 1999 Accepted 11 May 1999 Correspondence to: Dr Luiz Renato de França Tel.: 55 31 4992816; Fax.: 55 31 4992780; E-mail
[email protected]
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(Reiter, 1991). This hormone cannot be considered a pro- or antigonadothropic substance, since its action differs among, and within, species depending on the time, length and, to a lesser extent, quantity of melatonin secretion or administration. Melatonin cannot be easily compared to other classical hormones because its action is very likely widespread and does not necessarily require the presence of receptors (Cagnacci & Volpe, 1996). It is well known that exposure to constant light reduces the ability of the rat pineal gland to synthesize melatonin (Wurtman et al., 1964), probably due to the rhythm of sympathetic nerves innervating the pineal gland (Brownstein & Axelrod, 1974). In hamsters and rats, pineal maturation and the establishment of a melatonin rhythm takes place around 15 days of age (Tamarkin et al., 1980). In newborn rats that are physiologically blind, the rhythm of N-acetyltransferase, the key enzyme for melatonin synthesis, is not yet synchronized by the environmental lighting, but rather by circadian changes in the mother’s pattern of rearing the newborn rats (Deguchi, 1977; Jarrige
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Table 1
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Biometric and morphometric data and testosterone levels.
Parameters Body Weight (g) Testis Weight (g) Gonadosomatic Index (%) Epididymal Weight (g) Seminiferous Tubule Diameter (µm) Epithelium Height (µm) Total Length of Seminiferous Tubule (m) Seminal Vesicle Weight (g)1 Testosterone (ng/mL)
Control (n = 8)
Treated (n = 7)
414 ± 9 1.57 ± 0.04 0.76 ± 0.02 0.56 ± 0.09 354 ± 5 111 ± 2 13.5 ± 0.5 1.15 ± 0.04 1.86 ± 0.3
411 ± 10a 1.72 ± 0.07a 0.84 ± 0.04a 0.61 ± 0.02* 362 ± 9 114 ± 2 14.0 ± 0.8 1.03 ± 0.05 0.86 ± 0.2b
Values are the mean ± SE. *statistically significant (P < 0.05). an = 8; bn = 3; 1includes coagulating gland.
Table 2
Volume density (Vv%)1 and volume of testis compartments (mL).
Parameters 2
Testis Volume Seminiferous Tubule Volume Seminiferous Epithelium Lumen Tunica Propria Intertubular Volume Lymphatic Space Leydig Cell Blood Vessels Others
Control (n = 8)
Treated (n = 7)
1.473 ± 0.04 1.321 ± 0.03 (89.7) 1.126 ± 0.01 (76.5) 0.158 ± 0.01 (10.7) 0.038 ± 0.002 (2.6) 0.153 ± 0.01 (10.3) 0.046 ± 0.01 (3.1) 0.048 ± 0.004 (3.3) 0.053 ± 0.003 (3.6) 0.005 ± 0.003 (0.3)
1.603 ± 0.08 1.437 ± 0.08 (89.5) 1.222 ± 0.06 (76.2) 0.175 ± 0.02 (10.8) 0.040 ± 0.003 (2.5) 0.167 ± 0.01 (10.5) 0.046 ± 0.01 (2.9) 0.060 ± 0.003 (3.9)* 0.056 ± 0.003 (3.5) 0.005 ± 0.002 (0.3)
Values are the mean ± SE. *statistically significant (P < 0.05). 1Vv% in parenthesis. 2Excludes 6.5% of testis capsule.
et al., 1992). Maternally-derived melatonin is transported via milk and reaches all tissues in the neonate (Reppert & Klein, 1978). Melatonin effect is age-and time-dependent (Vanecek & Vollrath, 1990). Although adult rats are only marginally photoperiodic, immature rats are markedly influenced by photoperiod and melatonin (Lang et al., 1983; Vanecek & Illnerova, 1985). The concentration of melatonin receptor sites in the pituitary is high in 20-day-old fetuses but gradually decreases in the course of postnatal life (Vanecek, 1988). Although rat Leydig cells are capable of synthesizing melatonin (Tijmes et al., 1996), the main source of this hormone is the pineal gland (Reiter, 1980). Unless olfactory bulbs are removed (Nelson et al., 1982), the reproductive system of the adult rat is not sensitive to melatonin (Lang et al., 1983). However, in developing rats it was shown that photoperiodically-induced changes in the melatonin synthesis affected the growth of rat testes (Vanecek & Illnerova, 1985; Jarrige et al., 1992), and testicular testosterone and androstenedione concentration (Jarrige et al., 1990; 1992). Also, melatonin is considered to be an important factor for the timing of sexual maturation (Lang et al., 1984). Pinealectomy in rats has been shown to advance the onset of puberty (Relkin, 1971), while daily melatonin injections delay sexual maturation in the young male (Lang et al., 1983) by supressing the pubertal peaks of pituitary GnRH receptors number and also the plasma levels of testosterone, luteinizing hormone (LH) (Lang et al., 1984) and FSH (Debeljuk et al., 1970; Lang et al., 1984).
Many reports are available in the literature demonstrating the effects of melatonin in pubertal testis development (Reiter, 1991). However, no morphometric evaluation of the testis was performed in these experiments. The aim of the present work is to verify whether exposure to constant light during postnatal testis development is able to increase Sertoli cell population and sperm production in adult rats.
Materials and methods Animals and experimental design Two groups of eight randomly-selected newborn Wistar rats were utilized. Control rats were exposed to a 12 h-light–12 h-dark photoperiod. Treated animals and their respective mothers were placed in a room under a 24 h light regimen (80 w) from 0–25 days of age and then exposed to 12L–12D photoperiod in the same room as controls. The temperature and humidity were approximately 22°C and 70%, respectively. Food pellets and tap water were provided ad libitum. Tissue preparation Fifteen min before the initiation of perfusion for tissue fixation, rats were injected intraperitoneally with heparin at a dose of 130 IU/kg of body weight. The anesthetized rats were perfused through the left ventricle with Bouin’s fixative. Two animals from each group were perfused with 4% glutaraldehyde in cacodylate buffer 0.1 M, pH 7.4. Testis
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Fig. 1 Number of round spermatids nuclei per seminiferous tubule cross section at stage VII of the seminiferous epithelium cycle. Mean cell numbers (±SEM) were increased significantly (*) in treated animals (P < 0.05). Fig. 2 Number of Sertoli cell nucleoli per seminiferous tubule cross section at stage VII of the seminiferous epithelium cycle. Mean cell numbers (±SEM) were increased significantly (*) in treated animals (P < 0.05). Fig. 3 Ratio of round spermatids nucleoli per Sertoli cell nucleolus at stages VII of the cycle. No significant difference was observed between the means (±SEM) of treated and control animals (P > 0.05).
tissue was embedded in plastic (glycol methacrylate) and 4 µm thick sections were stained with toluidine blue and analyzed. Testosterone assay Immediately before sacrifice, blood samples were taken and centrifuged. In each serum sample testosterone levels were
determined using a solid-phase radioimmunoassay system kit (ICN Pharmaceuticals, Costa Mesa, CA, USA). Morphometry of the testis The tubular diameter and the height of seminiferous tubule epithelium were measured at X 160 magnification using an ocular micrometer calibrated with a stage micrometer.
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Fig. 4 Round spermatid nuclear diameter at stages I and VII of the seminiferous epithelium cycle. Mean nucleus diameter (±SEM) were significantly lower (*) in treated animals (P < 0.05). Fig. 5 Round spermatid nuclear volume at stages I and VII of the seminiferous epithelium cycle. Mean nucleus volume (±SEM) were significantly lower (*) in treated animals (P < 0.05). Fig. 6 Pachytene spermatocyte nuclear volume at stage VII of the seminiferous epithelium cycle. No significant difference was observed between the means (±SEM) of treated and control animals (P > 0.05).
Twenty tubular profiles that were round or nearly round were measured, and a mean was determined for each animal. The epithelium height was obtained in the same tubules utilized to determine tubular diameter. Basic morphometric data on testis composition were obtained using point counting by
systematic placement of a 441-point square lattice over sectioned material at X 400 magnification. Approximately 6600 points were counted for each animal. The volume of each component of the testis was determined as the product of the volume density and testis volume. For subsequent
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morphometric calculations, the specific gravity of testis tissue was considered to be 1.0. To obtain a more precise measure of testis volume, 6.5% of the testis capsule was excluded from testis weight (Russell & França, 1995). The total length of the seminiferous tubules, expressed in meters, was obtained by dividing seminiferous tubule volume by πR2. To estimate the volume of round spermatid nucleus, the diameters of thirty nuclei were blindly measured for each animal at stage VII of the cycle. As stage VII is present at a high relative frequency (18%), measured nuclei were sampled equally at the beginning, middle, and at the end of stage VII. Except for the ‘cap’ region, all nuclear profiles were measured. The volume of round spermatid nucleus at stage I of the cycle, and the volume of pachytene primary spermatocyte nucleus at stage VII were also determined from thirty nuclear diameter measurements for each animal. Individual nuclear volumes were expressed in µm3 using the following formula: 4/3πR3. Cell counts and cell numbers Round spermatid nuclei and Sertoli cells nuclei and nucleoli were counted in ten round seminiferous tubule cross sections at stage VII of the cycle. Except for Sertoli cell nuclei these counts were corrected for section thickness and nucleus size according to Abercrombie (1946), modified by Amann (1962). From these corrected counts the ratio of round spermatids to Sertoli cells was obtained. The total number of Sertoli cells was determined from the corrected counts of Sertoli cell nucleoli per cross-section of seminiferous tubule and the total length of seminiferous tubules according to the method of Hochereau-de-Reviers and Lincoln (1978). The daily sperm production per testis and per gram of testis was obtained according to the formula developed by França (1992) as follows: DSP = Total number of Sertoli cell per testis x the ratio of round spermatids to Sertoli cells at stage VII x stage VII relative frequency (%)/Stage VII duration (days) Statistical analysis All data are presented as the mean ± SEM. Student’s t-test was used to determine significant differences (P < 0.05) between means from control and treated animals.
Results Basic biometric and morphometric data are shown in Table 1. Although all testicular parameters were numerically larger in rats exposed to constant illumination, none of them was statistically significant. Nevertheless, the epididymal weight was significantly higher (P < 0.05) in treated animals. Testosterone levels are shown in Table 1. Treated rats had 54% less serum testosterone (P > 0.05). However, only three samples were available for animals exposed to constant light. Table 2 shows the volume density and
volume of the testis compartments. Except for the volume of the Leydig cell compartment, which was 25% larger (P < 0.05), all other parameters were only marginally higher in rats exposed to constant light. The number of nuclei of round spermatids and Sertoli cells nucleoli per seminiferous tubule cross sections at stage VII of the cycle were about 20% higher (P < 0.05) in treated animals (Figs 1 & 2). Nevertheless, the ratio of round spermatids to Sertoli cell was the same in both control and treated group (Fig. 3). There were 25% more (P < 0.05) Sertoli cell nuclei per cross-section of seminiferous tubule in rats under constant light (20.3 ± 0.7 vs 26.7 ± 0.6). Surprisingly, the diameter of nuclei of round spermatid and their volume at stage I and VII of the seminiferous epithelium cycle were significantly lower (P < 0.05) in rats exposed to constant illumination (Figs 4 & 5). These findings were not observed in diameter measurements of nuclei from pachytene primary spermatocyte and their volume at stage VII (Fig. 6). Although the total number of Sertoli cells per testis and daily sperm production were numerically higher (22%) in treated rats (Figs 7 & 8), only the latter parameter attained statistically significant difference (P < 0.05). The efficiency of sperm production, measured as DSP per gram of testis, was also higher (13%; P < 0.05) in treated animals (Fig. 9).
Discussion To our knowledge, this is the first investigation to perform a quantitative study of testicular cells and compartments in adult rats exposed to constant light during the neonatal period. With this approach we found that treated rats produced 22% more sperm than animals exposed to a 12L–12D light regimen. This finding is also corroborated by heavier epididymis observed for the same animals. According to Russell & Peterson (1984) spermatid-Sertoli cell ratio is a prime factor determining Sertoli cell efficiency. Therefore, increased sperm production in treated rats, which have testis weight only 10% larger than controls, resulted mainly from a higher number of Sertoli cells that maintained the same support capacity of round spermatids. The literature is conflicting regarding the effect of both photoperiod and pinealectomy on reproductive function in rats. Wallen and Turek (1981) showed that daylength has little effect on neuroendocrine-gonadal function in male albino rats. From this result the authors suggested that some components of the photoperiodic system involving the pineal gland have been preserved, being manifested only in certain experimental conditions. Some of these components may be playing an important role in our present experiment. Unexpectedly, we found that the diameter of round spermatid nuclei and volume at stage I and VII of the spermatogenic cycle were significantly decreased in rats exposed to constant light. Round spermatids nuclei were measured three times, the last series of measurements made blindly, the same
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Fig. 7
Number of Sertoli cells per testis. No significant difference was observed between the means (±SEM) of treated and control animals (P > 0.05).
Fig. 8 Daily sperm production per testis. Mean round spermatid numbers (±SEM) were significantly higher (*) in animals exposed to constant light (P < 0.05). Fig. 9 Daily sperm production per gram of testis (±SEM). The efficiency of spermatogenesis was significantly increased (*) in animals exposed to constant light (P < 0.05).
results with statistical significance were found for all sets of measurements. Changes in the volume of pachytene primary spermatocytes nuclei were not observed at stage VII. Perfusion and the kind of fixatives utilized (Bouin’s fixative and glutaraldehyde) did not appear to influence this finding, since treated and control animals were perfused in a comparable manner and the means found for nucleus diameter and
volume did not differ when Bouin’s fixative was considered (six animals for each group). A similar result was found for glutaraldehyde fixed tissues. To our knowledge there is no data in the literature showing that any experimental condition is capable of altering germ cell volume at a similar phase of their development during spermatogenic process. In fact, this is not considered to occur when germ cells counts are being
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obtained and compared in many different experimental approaches. The volume density of seminiferous epithelium was around 76% in both treated and control groups. Since spermatids occupy approximately 55% of the seminiferous epithelium during the spermatogenic cycle (Russell & França, 1995), a 10% reduction in nucleus volume, assuming that all cell size is affected, explains in part why the efficiency of spermatogenesis was around 13% higher in treated rats. There are enormous varieties of pathways through which solutes may cross plasma membranes and a number of factors that may influence cell composition (Macknight et al., 1994). Alterations in cell volume occur during various physiological and pathophysiological conditions (Lang et al., 1990), because, in a process named homeostasis, cells attempt to minimise changes in composition when faced with perturbations in their environment (Macknight et al., 1994). The fact that only spermatids in treated animals had their nucleus decreased in volume probably indicates that some regulatory control of nucleus size was affected during spermiogenesis. Normally, the volume of nuclei in round spermatids increases 50% from step 1 to step 7 and dramatically decreases thereafter, reaching about 2% of its maximal size (França et al., 1995). Recently it was demonstrated that a water channel protein (Aquaporin 7), which causes cell volume reduction, is expressed in the cytoplasm of spermatids at late steps (Ishibashi et al., 1997). Studies investigating the regulatory mechanisms of cell size in rats during spermiogenesis might shed light on this subject. Nevertheless, whatever caused the decrease in the volume of round spermatids nuclei probably involves a mechanism that occurred during the constant light period and lasted the entire animal’s life, since treated rats were sacrificed more than four months after being exposed to constant light. In the present work, we found that the volume of Leydig cell compartment was 25% larger in treated rats. However, it was not established if this increase was in cell size or cell population. Postnatal proliferation of the Leydig cells is stimulated by IGF-I and factors secreted by both Sertoli cells and testicular macrophages, and, to a lesser degree by LH (Ge et al., 1996). Melatonin significantly inhibits basal and LHRHstimulated cAMP in 5 to 14-day-old rats (Vanecek & Vollrath, 1990). Thus, inhibiting the pineal gland through constant light during postnatal development of the testis probably increased LH and FSH. It may not be a coincidence that the numerical increase observed in the volume of Leydig cell compartment and in the total number of Sertoli cells per testis in treated animals were of the same magnitude. On the other hand, serum testosterone levels in rats exposed to constant light were 54% lower than in the controls. It is well established that spermatogenesis is dependent on androgens. However, according to Sharpe et al. (1988), the testicular content of testosterone required for the quantitative maintenance of spermatogenesis in the adult rat is between 24% and 46% of the normal levels. It is also well demonstrated that FSH is required for the maintenance of spermatogenesis in adult rats (Russell et al., 1993; Meachem et al., 1998). In the present study, germ cell apoptosis was rarely observed in treated or in control rats.
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