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Urogenital Research Unit, State University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil
1 Biochemistry, INRA2006-EA2608, University, Esplanade de la Paix, 14032, CAEN-Cedex, France
(Requests for offprints should be addressed to S Carreau; Email: serge.carreau{at}unicaen.fr)
(C da Fonte Ramos is in US postdoctoral position at Stanford University, Department of Urology, California, USA).
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Abstract |
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(ER) receptors expression in the pups at weaning. From parturition until weaning, Wistar rats were separated into three groups: (C) control group, with free access to a standard laboratory diet containing 23% protein; protein-energy restricted (PER) group, with free access to an isoenergy and protein-restricted diet containing 8% protein; and energy-restricted (ER) group, receiving standard laboratory diet in restricted quantities, which were calculated according to the mean ingestion of the PER group. All pups were killed at weaning, corresponding to 21 days post partum. Compared with the C group, body weights (C = 48 ± 2.3 g; PER = 20 ± 1.3 g; ER = 25.4 ± 0.9 g; P < 0.01) and testicular weights (C = 0.15 ± 0.02 g, PER = 0.05 ± 0.01 g, ER = 0.06 ± 0.02 g, P < 0.001) of both PER and ER groups were lower. However, there was no significant difference in the testicular/body weight ratio in PER and ER groups compared with the C group. The testosterone serum concentration (ng/ml) was significantly higher in the PER group compared with ER and C groups (C = 0.09 ± 0.012; PER = 0.45 ± 0.04; ER = 0.15 ± 0.03, P < 0.01). Testicular testosterone concentration (C = 2.1 ± 0.43; PER = 6.5 ± 0.7; ER = 13 ± 2.3, P < 0.01) was increased in treated groups when compared with controls. The estradiol serum concentration (pg/ml) was lower in both dietary groups (C = 74 ± 4.6; PER = 49 ± 3.2; ER = 60 ± 5.5, P < 0.01). The amounts of aromatase mRNA and ER transcripts were significantly lower (P < 0.05) in PER and ER groups; conversely AR (both mRNA and protein) was significantly enhanced (P < 0.05) in treated animals. The nutritional state in early phases of development is important since we have demonstrated here that the maternal malnutrition during lactation leads to alterations in estradiol and testosterone serum concentrations, testicular testosterone concentration, AR and ER expression together with a decrease of aromatase expression. All together, these changes of steroid status may be deleterious for future germ cell development and reproductive function of these male pups submitted to early malnutrition. |
Introduction |
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Androgen (AR) and estrogen (ER, ERß) receptors are members of the steroid/thyroid hormone receptor gene superfamily and, like other steroid receptors, are mainly nuclear and function as ligand-dependent transcription factors (Lindzey et al. 1994, Zhou et al. 1994). Using testicular cell cultures, Cardone et al.(1998) showed that both estrogen and androgen may autoregulate the expression of their own receptor mRNAs, yet estrogen can downregulate AR mRNA.
Androgens can promote downregulation of AR mRNA concentrations (Quarmby et al. 1990, Krongrad et al. 1991, Hackenberg et al. 1992, Wolf et al. 1993), although androgenic upregulation of receptor mRNA has been observed in few tissues (Gonzalez-Cadavid et al. 1993, Nastiuk & Clayton 1994, Kerr et al. 1995). The removal of endogenous androgens by ethane dimethane sulfonate treatment of adult rats, resulted in an obviousdecrease in the AR immunoexpression, but replacement of testosterone for 4 h restored the expression of this receptor (Turner et al. 2001). The physiological significance of AR autoregulation has not been established; however, cellular AR concentration correlates relatively well with the extent of androgen responsiveness, suggesting that autoregulation of AR may influence hormonal sensitivity (Dai et al. 1996). There is also evidence showing that estradiol regulates ER gene expression in a dose-dependent manner (Tena-Sempere et al. 2000).
Undernutrition is known to have a wide variety of effects on endocrine systems (Rocha de Melo & Guedes 1997, Ramos et al. 2000, Cónsole et al. 2001). Regarding the reproductive system, it has been shown that food restriction can inhibit both the maintenance and onset of reproductive capability (Desjardins & Lopez 1983). However, long-term caloric restriction can transiently suppress the decreases of steroidogenic activities that are characteristics of aging (Chen et al. 2005).
In adult rats, food restriction can reduce the body weight (Chik et al. 1989), as well as testes, epididymis, and prostate weights (Grewal et al. 1971). The serum concentration of luteinizing hormone (LH), follicle stimulating hormone (FSH), and testosterone are also decreased by undernutrition (Grewal et al. 1971, Howland 1975, Herbert 1980, Chik et al. 1987, 1989, Dong et al. 1994, Young et al. 2000). It was shown that malnutrition led to gonadotroph morphological alterations that are typical of those found in cells whose secretory activities are suppressed (Herbert 1980).
Despite the fact that undernutrition-related reproductive suppression in rats is a well-documented phenomenon, we do not have preceding knowledge until now, on how under-nutrition affects aromatase and steroid receptor (AR and ERs) expressions in testicular tissues of male rats whose mothers were subjected to undernutrition during lactation, as well as on estradiol and testosterone serum concentrations. Therefore, the present study was designed to identify underlying mechanisms related to potential reproductive problems associated with undernutrition during the neonatal period.
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Materials and Methods |
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Wistar rats were kept in a room with controlled temperature (25 ± 1 °C) and an artificial dark–light cycle (lights on from 0700 to 1900 h). Virgin female rats of 3 months of age were caged with one male rat at a proportion of 2:1. After mating, determined by the presence of a vaginal plug, each female was placed in an individual cage with free access to water and food until delivery. The handling of the animals was approved by the Animal Care and Use Committee of the Biology Institute of State University of Rio de Janeiro, which based their analysis on the Guide for the Care and Use of Laboratory Animals (Bayne 1996), and the study design was approved by the local Ethical Committee for the care and use of laboratory animals.
Experimental design
Six pregnant Wistar rats were separated after delivery into three groups: (C) control group – with free access to a standard laboratory diet containing 23% protein; protein-energy restricted (PER) group – with free access to an isoenergy and protein-restricted diet containing 8% protein; and energy-restricted (ER) group – receiving standard laboratory diet in restricted quantities, which were calculated, every day, according to the mean ingestion of the PER group and correspond to 60% of that consumed by the control group. The PER group, in spite of having free access to diet, consume about 60% of that consumed by the control group (Passos et al. 2000). In this way, the amount of food consumed in both ER and PER groups was almost the same and was measured every day (data not shown).
Within 24 h of birth, excess pups were removed, so that only six pups were kept per dam, because it has been shown that this procedure maximizes lactation performance (Fishbeck & Rasmussen 1987). At weaning (21 days post partum), seven male pups of each group were slaughtered under thiopental anesthesia (0.10 ml/100 g body weight), always in the morning. The blood was collected by cardiac puncture and the serum kept at –20 °C for subsequent determination of hormonal parameters. The testes were excised, dissected, weighted then divided in two parts: one was kept at –70 °C for subsequent measurements of aromatase, estrogen, and AR transcripts by RT-PCR and proteins by western blotting as well as endogenous testosterone by RIA. The other part of the testes was paraffin-embedded, sectioned at 5 µm thickness and processed by routine histological analyses. Some samples were stained with hematoxylin and eosin to check the integrity of the tissue, while others were used for AR and ER immunohistochemistry.
Nutritional diet
The low-protein diet was prepared in our laboratory and its composition is shown in Table 1
. Vitamins and mineral mixtures were formulated to meet the American Institute of Nutrition AIN-93G recommendation for rodent diets (Reeves et al. 1993).
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Total RNA from testicular tissue was extracted using the guanidium thiocyanate–phenol–chloroform method (Chomczynski & Sacchi 1987). Testes were homogenized in a lysis buffer (1 M Tris, 4 M guanidium thiocyanate, 0.5% sarcosyl, 1% ß-mercaptoethanol). Then, RNA was extracted by a phenol/chloroform/isoamylic alcohol solution and precipitated by isopropyl alcohol. After washing with 75% ethanol, the RNA was dried and dissolved with diethyl pyrocarbonate-treated water. The quality of RNA samples was verified by determination of the ratio 260 nm/280 nm and electrophoresis on a 1% agarose gel. The samples were stored at –80 °C until utilization.
Semi-quantitative RT-PCR
Total RNA (2 µg) was reverse transcribed into cDNA in a total volume of 40 µl. RNA was incubated at 42 °C for 1 h with 200 IU Moloney murine leukemia virus reverse transcriptase (M-MLV-RT), 0.5 mM dNTP, 0.2 µg oligo-dT and 20 IU Rnasin. Then, cDNA coding for aromatase, AR, estrogen receptors and ß and ribosomal protein L19 were amplified by PCR with specific primers (Table 2). The reactions were performed in a final volume of 50 µl from 4 µl cDNA with 1.5 IU Taq polymerase, 0.2 mM dNTP, 1.5 mM MgCl2 and 25 pmoles of the forward and reverse primers. Primers were obtained from Invitrogen and all others products used for RT-PCR are from Promega. In order to quantify aromatase, ER and AR transcripts, we have determined the optimal number of amplification cycles, for each gene. After an initial step of denaturation at 95 °C for 5 min, a variable number of cycles of amplification: 30 s at 95 °C, 30 s at 60 °C, 45 s at 72 °C were performed (Table 2), followed by a final extension at 72 °C for 7 min. We have chosen L19 transcripts which do not vary among all the samples in order to correct the difference in the quantities of total RNA used for reverse transcription (Tena-Sempere et al. 2002). All cDNA fragments amplified were run on a 2% agarose gel stained with ethidium bromide, visualized under u.v. transillumination and analyzed with NIH software (http://rsb.info.nih.gov/nih-image). For each cDNA amplified, we have verified their identity by sequencing (GENOME express, Meylan, France) and alignment with the published sequences (Table 2).
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Each testis was homogenized in 500 µl TEG buffer (50 mM Tris pH 7.4, 1.5 mM EDTA, 50 mM NaCl, glycerol 10%, 5 mM DTT, 10 µg/ml leupeptin). The homogenatewas centrifuged at 100 000 g for 2 h at 4 °C. The proteins extracted from the supernatants were measured by the Bradford method (Bradford 1976) and subsequent quantification of estrogen and AR was determined by western blot analysis. Total protein (70 µg) was loaded in each lane of 8% PAGE. The proteins were transferred to nitrocellulose membrane, and the detection of specific proteins was done with AR-specific antibody (rabbit polyclonal 200 µg/ml, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and ER-specific antibody (mouse monoclonal 200 µg/ml, Santa Cruz Biotechnology). Horseradish peroxidase IgG was used as a secondary antibody followed by autoradiography using enhanced chemic luminescence (ECL) detection reagents supplied by Amersham (Braunschweig, Germany). Bands were scanned in a computer and their relative intensities determined by densitometry using Scion Image v. 4.0.2 (Scion Corporation, Frederick, USA). The molecular weights of AR and ER have been determined using a molecular weight standard kit.
Steroid determinations
The serum and intratesticular hormonal concentrations were determined by using specific RIA (ICN Pharmaceuticals, Inc, CA, USA). The intra- and inter-assay variation coefficients were 4.6 and 7.5% for testosterone and 6.4 and 5.9% for estradiol. The intratesticular testosterone concentration was performed after homogenization of the testes in 10 ml TEG buffer and was expressed as ng/ml per g of tissue.
AR and ER immunohistochemistry
Deparaffined sections were hydrated, treated with trypsin solution for 15 min at 37 °C for antigen retrieval, and then treated with 3% hydrogen peroxide solution in methanol for 10 min to block endogenous peroxidase activity. These steps were followed by washing the sections in PBS and subsequently incubated 10 min at room temperature with 10% goat serum to block unspecific binding. The sections were then incubated overnight at 4 °C with specific primary antibody (mentioned above) diluted in PBS with 1% BSA. Sections were then washed in PBS and incubated at room temperature for 30 min with specific secondary antibody (Zymed Laboratories Inc., San Francisco, CA, USA). Sections were washed in PBS, then revealed by treating with liquid diaminobenzidine (Zymed Laboratories), and then counterstained with hematoxylin. The negative controls were processed by replacing the primary antibody with PBS and no indication of staining was observed.
Statistical analysis
All results are means ± S.E.M. Statistical analysis was performed by one-way ANOVA followed by Student–Newman–Keuls test (Sokal & Rohlf 1995). Values of P < 0.05 were considered significant.
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Results |
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The body weight of pups whose mothers were subjected to a protein-energy (PER) or ER diet during lactation was significantly lower (PER = 58%; ER = 46%) than that of the controls at the end of lactation. The absolute weights of the testes at the end of lactation were significantly decreased in both groups on dietary treatment (65% in the PER and 60% in the ER group). On the other hand, the testicular weight in relation to the body weight showed only a slight reduction in PER and ER groups compared with the C group (Fig. 1).
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Compared with the control group, the serum testosterone concentration was significantly increased in the PER group (390%). Serum testosterone concentration in the ER group was slightly, but not significantly, higher than in controls (Fig. 2A). The testicular testosterone concentration was significantly increased in both PER and ER groups compared with controls (210% in the PER and 520% in the ER group, P < 0.01). The animals of the ER group also showed a significant (P < 0.01) increase in testicular testosterone concentration when compared with the animals of PER group (Fig. 2B).
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). Aromatase expression
We performed semi-quantitative RT-PCR in order to determine whether aromatase gene expression in testis is affected by a protein-energy or an ER diet during lactation (Fig. 3). We have observed a significant decrease of more than 70% of the aromatase/L19 ratio in testes of PER and ER groups compared with the control group.
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The concentration of mRNAs recorded as the AR/L19 ratio was significantly increased in the two groups (111% in PER and 120% in ER groups; Fig. 4A). The AR protein expression (Fig. 4B) was also significantly enhanced in both groups (558% in the PER and 120% in the ER groups). The animals of the PER group showed a significant increase (198%) in the androgen testicular receptor protein concentration when compared with the animals of ER group.
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The values of the ER/L19 ratio are shown in Fig. 5A and it is clear that the amount of transcripts is lower in PER and ER groups compared with control group; however, this diminution was only significant in the PER group. The concentrations of ERß transcripts were unchanged despite the treatments (data not shown). Conversely, the estrogen receptor- protein concentration was significantly increased (Fig. 5B) in both PER and ER groups (PER = 617%; ER = 393%).
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H&E sections are shown in Fig. 6A (400 x magnification) and 6B (1000 x magnification). AR immunoreactive staining was strong in most Leydig cells and peritubular myoid cells. Sertoli cells and germ cells (spermatogonia) were also stained (Fig. 7A). ER staining was present in Leydig cells, peritubular myoid cells, Sertoli, and germ cells (Fig. 7B). Despite some difference in the intensity of staining, the testes of all the three groups studied showed staining in the same cells.
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Discussion |
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The reduction in body and testes weights observed in this study is in agreement with past results (Nelson et al. 1992, Ramos et al. 1997, Young et al. 2000, Léonhard et al. 2003). In spite of the significant reduction in body and testes weights, the difference in the testicular/body weight ratio is not significant in either malnourished groups compared with the control group. The reduction in body and organs weights is independent of the animal’s age, nutrient deficit, and duration of the malnutrition (Young et al. 2000). Recently, we showed that both PER and ER groups exhibited a significant reduction in the total, luminal, and epithelial areas and in the epithelial height evaluated by stereological methods. These structural alterations could contribute to some differences observed in this study and probably have an impact on the capacity of the testes to synthesize and/or metabolize steroids (Ramos et al. 2006).
Our past results show that the offspring of mothers that were subjected to protein or energy malnutrition during lactation present an increase in leptin serum concentration (Teixeira et al. 2002) that could be related to the low weight gain of the animals. Conversely, leptin is also a potent stimulator of the LH and FSH production (Caprio et al. 2001, Ogura et al. 2001). Our results did not agree with others’ data showing that food restriction leads to low serum testosterone concentration (Desjardins & Lopez 1983, Nelson et al. 1992, Young et al. 2000), probably related to differences in the experimental model used, since the age of animals, time, and kind of malnutrition are important factors that could influence the animals in different ways.
In agreement with the literature, we showed in this paper that AR is present in Leydig cells, peritubular myoid cells and Sertoli cells (Sar et al. 1990, Bremner et al. 1994, Zhou et al. 1996, Pelletier et al. 2000, Zhu et al. 2000). The presence of AR in germ cells is controversial and it was shown only in spermatogonia of prepubertal animals (Zhou et al. 1996) what is in agreement with the results observed in this paper.
In relation to ER, our data are in agreement with the literature showing that ER immunolocalization is detected in Leydig cells (Saunders et al. 1998, Bilinska et al. 2001, Zhou et al. 2002) and in the peritubular myoid cells (Zhou et al. 2002). Sertoli and germ cells were also stained (Pelletier et al. 2000). Malnutrition during lactation did not alter the localization of either androgen and estrogen receptors.
In males, testosterone synthesized by Leydig cells is the primary endocrine and/or paracrine factor involved in the regulation of testicular development, initiation of spermatogenesis and formation of secondary sexual characteristics. The testicular testosterone concentration is a determinant factor for spermatogenesis (Wright & Frankel 1979), and the spermatozoa production is related to testosterone concentration (Awoniyi et al. 1989, Mc Lachlan et al. 1994). The increase in the testicular testosterone concentration observed in both dietary treated groups could be a result of an adaptive mechanism of the Leydig cells as reported elsewhere in other experimental models (Boujrad et al. 1995) and/or is the consequence of an abnormal excretion of testosterone.
The increment observed in the serum testosterone concentration in the PER group suggests that this hormone is being normally synthesized and secreted into the circulation. On the other hand, the ER group presented normal concentration of serum testosterone, in spite of an increase in testicular testosterone concentration. In this last group, the synthesis is not correlated with the secretory process, therefore is in favor of an abnormal secretory pathway.
The present results show that independent of the alteration in the synthesis and secretion of testosterone, the androgen receptor (mRNA and protein concentrations) is altered in the same manner as serum testosterone, i.e. a higher increase in the PER group, what could suggest a receptor upregulation. However, it is known that dihydrotestosterone has four times more affinity for the androgen receptor than testosterone (Tapanaien et al. 1984); therefore, we cannot discard the possibility of an autoregulation by this hormone, and indeed 5 reductase activity increases at that age (Tapanaien et al. 1984).
The aromatase gene expression is controlled by androgens likely via specific responsive elements leading to either an enhancement (Genissel & Carreau 2001, Lambard et al. 2005) or a decrement of the amounts of specific transcripts (Douglas et al. 2006). As these animals present low serum estradiol concentration, it is obvious that maternal malnutrition leads to a reduction in the synthesis of aromatase enzyme, which is indeed observed as there is a decrease of more than 65% of the aromatase expression in both groups. This reduction of aromatase expression is correlated with an increase of endogenous testosterone in PER and ER pups. It should also be stressed that the impact of caloric restriction (ER) is more dramatic than that of the reduced protein intake (PER) for most of the parameters determined and especially for blood testosterone and androgen receptor concentrations as also observed in adult rats under the same diet (Marcelly de Souza Santos et al. 2004)
Tena-Sempere et al.(2000) reported that neonatal exposure to estrogens differentially alters testicular expression of and ß ER mRNAs. ER mRNA concentrations, as well as those of AR, were significantly decreased, whereas relative and total expression concentrations of ERß mRNA increased during postnatal/prepubertal development after neonatal estrogen exposure, a phenomenon that was not mimicked by luteinizing hormone-releasing hormone antagonist treatment. The authors suggest that the effect of estrogen on the expression concentrations of AR and ER mRNAs probably involves a direct action on the developing testis and cannot be attributed to estrogen-induced suppression of gonadotropin secretion during the neonatal period. If estrogens can indeed have a direct action on the developing testis, we can assume that, in our malnutrition experimental model, the increment in the ER and AR protein concentration expression in both malnourished groups could be a consequence of a lack of the estrogen action on the developing testis since a decrease of the concentration of aromatase transcripts has been observed in both PER and ER groups.
The importance of aromatase which is present in most of the testicular cells and the widespread distribution of ERs in male reproductive tract is obvious (Carreau et al. 2003); thus, any changes induced by diet which will reduce the amount of estrogens will likely affect testicular development, and thus spermatogenesis as shown elsewhere (Bilinska et al. 2004).
We can surmise that an adequate nutritional state in early life is important for normal maturation of the male gonad and steroidogenic pathway on the one hand, and in the expression of steroid receptors on the other hand. Gene expression and protein synthesis are therefore under several hormonal controls which depend on numerous factors including protein and energy as demonstrated in the diet studies herein. The reduced protein intake is less deleterious than the caloric restriction, but both affect the testes of male pups, which could lead to abnormal testicular development and may delay the onset of spermatogenesis for which a correct androgens/estrogens ratio is absolutely required.
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Acknowledgements |
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Received in final form 30 November 2006
Accepted 1 December 2006
Made available online as an Accepted Preprint 11 December 2006
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