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ABSTRACT: The early neonatal development of boars is characterized by significant testicular production of androgens and estrogens, including an anabolic steroid hormone, 19-nortestosterone. The present study was conducted to determine the expression and presence of steroidogenic and steroid hormone metabolism—related enzymes in the testes of neonatal and 4-month-old prepubertal pigs. Quantitative analyses with real-time polymerase chain reaction and Western blotting were utilized to reveal mRNA and protein expression, respectively. The localization of the molecules in the testes was determined by immunohistochemistry. mRNA expressions of the molecules tested were mostly significantly increased between 1 and 3 weeks of age and decreased at 4 months of age, compared with those at 0 weeks of age. The protein levels of cytochrome P450 aromatase and carbonyl reductase 1 were significantly increased between 1 and 3 weeks of age and decreased at 4 months of age. However, protein expression patterns of other molecules differed from those of mRNA expression, which implied the existence of posttranscriptional gene regulation. Immunohistochemical analysis revealed that all of the molecules were present in Leydig cells of the pig testis, regardless of age, except cytochrome P450 side chain cleavage in germ cells and 17β-hydroxysteroid dehydrogenase 4 on the blood-testis barrier at 4 months of age. Aldose reductase and 3β-hydroxysteroid dehydrogenase were localized in both Leydig and Sertoli cells. We postulate that marked rises in the expression of steroidogenic enzymes in the pig testis during early neonatal development could be associated with peak production of 19-nortestosterone, thus eventually leading to the early growth of male pigs.
Aremarkable feature of the domestic boar (Sus scrofa) is high circulating estrogen concentrations (Claus and Hoffman, 1980; Setchell et al, 1983). Estrogen concentrations peak during neonatal development, between 1 and 3 weeks of age, and transiently decrease and remain at low level until pubertal development (Ford, 1983). Changes in the serum levels of free androgens and conjugated steroids also show similar patterns to those of estrogens during postnatal development of male pigs (Colenbrander et al, 1978; Schwarzenberger et al, 1993). Along with androgens and estrogens, 19-nortestosterone (17β-hydroxy-19-nor-4-androsten-3-one, also known as nandrolone) is normally found at high levels in pig serum during early neonatal development (Schwarzenberger et al, 1993). Particular attention is paid to nandrolone because of its high anabolic activity (Kuhn, 2002). Because the production of steroid hormone requires actions of a number of steroidogenic enzymes, it is suggested that there is a strong association between elevated steroid production and enhanced expression of steroidogenic enzymes during the early neonatal period.
Dynamic morphologic and histochemical changes in the pig testis appear during the early neonatal period. Increases of Sertoli cell proliferation and Leydig cell volume occur during the first month after birth (França et al, 2000). In addition, the majority of testicular volume is made up of Leydig cells in the early neonatal pig, predominantly between 2 and 3 weeks of age (van Straaten and Wensing, 1978). In mammal testes, syntheses of androgens and estrogens occur mostly in Leydig cells, and require a number of steroidogenic enzymes. In fact, the expression and presence of steroidogenic enzymes in the domestic pig testis are well documented (Sasano et al, 1989; Hall, 1991; Clark et al, 1996; Conley et al, 1996; Conley and Bird, 1997; Moran et al, 2002). A number of investigations have demonstrated that the expressions and activities of steroidogenic enzymes in pig testis are dependent on a variety of extragonadal and intragonadal factors (Chuzel et al, 1996; Clark et al, 1996; Lejeune et al, 1998; Moran et al, 2002). Estrogens are synthesized from the aromatization of androgens through the action of cytochrome P450 aromatase (CYP19). Differential expression of CYP19 has been found during different stages of pig development. In fetal pig testis, CYP19 is present in Leydig cells and/or gonocytes (Conley et al, 1996; Parma et al, 1999; Haeussler et al, 2007), whereas the expression of CYP19 is exclusively limited to Leydig cells of immature and mature pigs (Fraczek et al, 2001; Mutembei et al, 2005). During early neonatal development, an increase of CYP19 activity has been detected between 1 and 7 days after birth (Moran et al, 2002). However, the ontogeny of CYP19 expression in the pig testis during early neonatal development has not yet been determined, in spite of the peak production of estrogen during the first month after birth (Schwarzenberger et al, 1993). Differential expressions of other steroidogenic enzymes in the pig testis during fetal and postnatal development have also been reported (Conley et al, 1994; Moran et al, 2002; Haeussler et al, 2007). However, a detailed examination of the expression of these steroidogenic enzymes during early neonatal development is needed, because of the significant production of steroid hormones in pigs during the neonatal period (Schwarzenberger et al, 1993).
As noted above, nandrolone is a potent anabolic steroid that is found at high levels in male pig serum, particularly during early neonatal development and after puberty (Schwarzenberger et al, 1993; Choi et al, 2007). Endogenous production of nandrolone is also detected in mares (Sterk et al, 1998) and some ruminants, including goat, cow, and sheep (Mayer et al, 1992; De Brabander et al, 1994; Sterk et al, 1998). The mechanism of nandrolone synthesis in the pig testis has not been revealed in detail. Kao et al (2000) showed that the porcine CYP19 is capable of converting testosterone into nandrolone via demethylation. In addition, Corbin et al (1999) demonstrated the catalytic activity of the porcine CYP19 on the formation of nandrolone using testosterone as a substrate. These findings imply that the presence of a high serum level of nandrolone in the male pig during early neonatal development would be associated with the expression of CYP19, as well as other steroidogenic enzymes, in the pig testis.
Comprehensive evaluation of differential gene expression in the pig testis during postnatal development has not been studied. Our recent, unpublished cDNA microarray data have shown dramatic expressional changes of a variety of molecules in pig testis between 2 weeks of age and prepuberty. Based on preliminary data and other investigations, we hypothesized that peak production of steroid hormones in the male pig during early neonatal development would relate to increases of gene expression of steroidogenic enzymes in the pig testis. To test this hypothesis, based on our cDNA microarray results, we selected a total of 7 genes that are involved in the synthesis and metabolism of steroid hormones in the pig testis. These molecules were CYP19, cytochrome P450 side chain cleavage (CYP11A1), 17β-hydroxysteroid dehydrogenase 4 (HSD17B4), 3β-hydroxysteroid dehydrogenase (HSD3B), carbonyl reductase 1 (CBR1), aldose reductase (ALR2), and 17α-hydroxylase (CYP17A). In the present study, we first attempted to evaluate the differential expression of mRNA and protein by real-time polymerase chain reaction (PCR) and Western blot analyses, respectively. In addition, immunohistochemical analysis was performed to localize the molecules in the pig testis at different neonatal ages (0, 1, 2, and 3 weeks of age). We also included the testis at 4 months of age for comparison in the present study, because steroid hormones were present at the basal level in circulating blood at this age (Schwarzenberger et al, 1993).
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This study examined the expression and localization of the enzymes involved in steroidogenesis and metabolism of steroid hormone in the early neonatal and prepubertal pig testes. Quantitative real-time PCR and Western blotting analyses were used to determine the expressions of the mRNA and proteins of enzymes, respectively. In addition, the localization of these molecules in the pig testes was evaluated by immunohistochemistry. Criteria used to select the molecules tested in the present study were based on our unpublished DNA microarray analysis, which showed differential expression of pig testicular genes between 2 weeks of age and prepuberty. To our knowledge, this is the first time that the expression and localization patterns of a number of enzymes related to the synthesis and metabolism of steroid hormones in the pig testis have been investigated during early neonatal development. Significant increases of the mRNA levels of all of the molecules examined were clearly observed between 1 and 3 weeks of age. Interestingly, except in the cases of CYP19 and CBR1, changes of protein abundance during early neonatal development were not consistent with the patterns of mRNA expression. However, the expression pattern of mRNA in the prepubertal pig testis at 4 months of age was similar to the protein expression pattern. Thus, these results suggest the existence of posttranscriptional regulatory mechanisms on the expression of steroidogenic enzymes in the pig testis during early neonatal development. Results of immunohistochemical analysis are summarized in Table 2. Immunohistochemistry revealed the presence of steroidogenic enzymes in Leydig cells, and Sertoli cells in the cases of ALR2 and HSD3B, during neonatal development. However, as seen in CYP11A1 and HSD17B4, differential localization of molecular expression was found in the prepubertal pig testis, indicating a change and/or addition to the functional roles of these molecules in the pig testis during postnatal development.
The synthesis and metabolism of steroid hormones require a variety of steroidogenic enzymes. The boars have extraordinarily high plasma and testicular levels of estrogens, compared with the males of other species and females of the same species (Claus and Hoffman, 1980; Setchell et al, 1983; Schwarzenberger et al, 1993). During postnatal development, the first peak of estrogen concentration occurs during the first month after birth, followed by transient decreases until the second peak, which occurs after puberty (Christenson et al, 1984; Schwarzenberger et al, 1993). The production of estrogens is catalyzed by the action of CYP19, which results in irreversible conversion of androgens to estrogens (Carreau and Levallet, 1997). Thus, a surge of estrogen production in the pig testis during early neonatal development would result in an increase of CYP19 expression. Indeed, the present study showed marked increases of CYP19 mRNA and protein levels in the pig testis at 2 weeks of age, in parallel with our previous finding (Choi et al, 2007). A slight but significant increase of the CYP19 protein level was observed at 1 and 3 weeks of age. In addition, the present study demonstrated the exclusive localization of CYP19 in Leydig cells, in agreement with the findings of other investigators (Conley et al, 1996; Mutembei et al, 2005; Haeussler et al, 2007). In spite of dramatic increases of plasma estrogen concentrations in the first few weeks (Schwarzenberger et al, 1993), increases of CYP19 mRNA and protein levels were unexpectedly low, 1.7 times or less. A similar finding for CYP19 activity was found in the neonatal pig testes (Moran et al, 2002). However, because the interstitial volume, density, and cytoplasmic volume of Leydig cell in pig testis increase greatly between birth and 1 month of age (van Straaten and Wensing, 1978; França et al, 2000), it is reasonable to consider that overall CYP19 level and activity in the pig testes during early neonatal development would be greater than observed in the findings from the present study, as well as previous studies (Moran et al, 2002). Thus, it is speculated that such increase of CYP19 activity during the first 2 weeks after the birth would strongly correlate with a significant secretion of nandrolone from the neonatal pig testis.
Interconversion of 17-ketosteroids with the corresponding 17β-hydroxysteroids requires the action of HSD17B. Of a number of HSD17B isoforms, HSD17B4 is responsible for the inactivation of estradiol and androstene-3β, 17β-diol into estrone and dehydroepiandrosterone, respectively (Labrie et al, 1997). In the pig testis, HSD17B4 is localized in Leydig cells and predominantly directs the oxidation of estradiol to estrone (De Launoit and Adamski, 1999). High plasma estrone concentrations have been measured in boars during neonatal development (Claus and Hoffmann, 1980; Ford, 1983). In the present study, the lowest level of HSD17B4 mRNA in the pig testis during the early neonatal period was detected at 0 weeks of age, whereas the highest level of HSD17B4 protein was found at the same age. These data indicate the existence of posttranscriptional regulation of HSD17B4 expression. Even though the present study showed lower levels of HSD17B4 protein between 1 and 3 weeks of age compared with that at 0 weeks of age, it is speculated that overall HSD17B4 protein levels would remarkably increase because of apparent increases of Leydig cell volume and density during the neonatal period (van Straaten and Wensing, 1978; França et al, 2000). An unexpected finding was the alternation in HSD17B4 localization in the pig testis at 4 months of age. A strong immunoreactivity of HSD17B4 was localized on the BTB at 4 months of age, whereas only Leydig cells were immunopositive for HSD17B4 during early neonatal development. The role of HSD17B4 on the BTB is not currently known. Booth (1983) showed the stimulatory effect of estrone on the development of male characteristics in the boar. Entry of steroid hormones such as testosterone and dehydroepiandrosterone into rete testis fluid through the BTB has been demonstrated in rats (Cooper and Waites, 1975). Rete testis fluid in the boar testis contains a significant concentration of estrogens (Setchell et al, 1983). Thus, it is presumed that HSD17B4 on the BTB would play a role in the accumulation of estrone in rete testis fluid through the active conversion of estradiol synthesized from Leydig cells. Another possible role of HSD17B4 on the BTB would be a stimulatory effect on spermatogenesis and/or Sertoli cell proliferation by estrone. Additional investigation should be conducted to resolve the role of HSD17B4 on BTB in the pig testis.
The synthesis of androgens from a cholesterol precursor requires a number of steroidogenic enzymes, including CYP11A1, CYP17A, and HSD3B. The CYP11A1 is the rate-limiting enzyme for steroidogenesis and converts cholesterol into pregnenolone, which is then metabolized into progesterone by the action of HSD3B. The CYP17A is a pivotal enzyme that converts pregnenolone or progesterone to 17-hydroxypregnenolone or 17-hydroxyprogesterone, respectively. These 2 intermediates serve as precursors for androstenedione that is further converted into testosterone by the action of HSD17B. The expression and localization of these 3 enzymes in the pig testis have been demonstrated from the findings of other studies (Suzuki et al, 1992; Clark et al, 1996; Moran et al, 2002; Weng et al, 2005). Androstenone, dehydroepiandrosterone, and testosterone are types of androgens that are found in pig plasma at relatively high levels (Sinclair et al, 2001). The initial peaks in plasma androgen concentrations are seen within the first month after birth during postnatal development (Sinclair et al, 2001), which implies a requirement for marked increases of gene expression for CYP11A1, CYP17A, and HSD3B. In fact, the present study showed significant increases of mRNA levels of these enzymes between 1 and 3 weeks of age. However, protein levels during early neonatal development were lower or equivalent to those at 0 weeks of age. The discordance between the mRNA and protein expressions of these enzymes implies the existence of posttranscriptional modulation on gene expression during early neonatal development. In addition, we could not rule out the possibility of posttranslational regulation, leading to the enhancement of enzyme activities during neonatal development. Immunohistochemical analysis revealed the primary localization of CYP11A1, CYP17A, and HSD3B in Leydig cells, regardless of age. Interestingly, we also found a positive immunoreaction of CYP11A1 in germ cells of the prepubertal pig testis. Moreover, positive immunoreactivity of HSD3B was found not only in Leydig cells, but also in Sertoli cells in the neonatal testis and Sertoli cells and BTB in the prepubertal testis. Similar observations were made for CYP11A1 in the bear testis (Tsubota et al, 1993) and for HSD3B in the monkey testis (Liang et al, 1999). Such differential testicular expression would indicate distinguishable roles of steroidogenic enzymes in the pig testes during postnatal development. To our knowledge, the present study is the first report to demonstrate the differential localization of CYP11A1 and HSD3B in the domestic pig testis. Further examinations are needed to determine the functional roles of steroidogenic enzymes in different cell types of the pig testis.
In the present study, we examined the expression and presence of 2 metabolic enzymes, ALR2 and CBR1. ALR2 is a member of the aldo-keto reductase superfamily, whereas CBR1 is a member of the short-chain dehydrogenase/reductase superfamily (Hoffmann and Maser, 2007). Both of these enzymes share a common characteristic: NADPH-dependent reduction. Porcine testicular CBR catalyzes the reduction of ketones on androgens and progesterone (Tanaka et al, 1992). The CBR1 is expressed and localized only in Leydig cells of the neonatal pig testis (Kobayashi et al, 2002), and this is in agreement with our present finding. The expression of CBR1 mRNA and protein during early neonatal development increases according to age, and shows a transient decrease at 4 months of age. Similar findings on CBR1 mRNA expression and activity in the neonatal pig testes have been demonstrated in previous studies (Ohno et al, 1992; Tanaka et al, 1992). It is believed that CBR1 is responsible for the conversion of 17α-hydroxyprogesterone to 17α,20β-dihydroxy-4-pregnen-3-one, which is present in the neonatal pig testis (Ghosh et al, 2001). The CBR has 2 distinct activities, 20β-HSD (Tanaka et al, 1992) and 3α- and 3β-HSDs (Ohno et al, 1992), thus implying a diverse role in the metabolism of steroid hormones. Thus, it is speculated that multifunctional actions of CBR1 in the pig testis would play an important role in metabolic reactions of steroid hormones synthesized in Leydig cells, eventually leading to adequate testicular function during early neonatal development. The expression and localization of ALR2 in the domestic boar testis have not yet been determined. It has been demonstrated that progesterone is a substrate for the reducing activity of ALR2 with 20α-HSD activity (Warren et al, 1993). The present study demonstrates immunolocalization of ALR2 in Leydig and Sertoli cells of the pig testes. In the rat testis, ALR2 is exclusively present in Sertoli and spermatogenic cells (Kobayashi et al, 2002), suggesting species-specific cellular expression of ALR2 in the testis. The functional role of ALR2 in the pig testis is not understood at this point. However, significant increases of mRNA and protein levels during early neonatal development indicate that ALR2 would be involved in the metabolism of steroid hormones in pig testes following exposure to high concentrations of steroid hormones. In fact, Kobayashi et al (2002) suggested a potential role of ALR2 on the reduction of steroid hormones in the rat testis. Detailed information for a role of ALR2 in the pig testis should be addressed in future studies.
A number of investigations have shown a correlation between the remarkable increase of the Leydig cell number and size and steroidogenic activity in the pig testis during the first month after birth (França et al, 2000; Herrera et al, 1983; Schwarzenberger et al, 1993; van Straaten and Wensing, 1978). In addition, van Straaten and Wensing (1977) reported that a marked increase of the volume percentage of the Leydig cells in the pig testis reaches the highest value at 3 weeks of age after the birth. These findings imply that a proportional increase of the Leydig cells relative to the testicular interstitium and STs would contribute to enhanced expression of steroidogenic enzymes in the pig testis during the early neonatal period. In pigs, the total body weight shows an almost 10-fold increase, with maximal growth in the skeletal muscle, during the first month of birth (Sarkar et al, 1977). As stated earlier, nandrolone, an androgen having 10 times higher anabolic activity than testosterone, is found at high concentrations in pigs during early neonatal development (Schwarzenberger et al, 1993). Thus, it is believed that anabolic steroid hormones synthesized from the pig testis may play a role in early postnatal development of pigs. In conclusion, the present study demonstrates that differential gene and protein expressions of various steroidogenic and steroid metabolism—related enzymes in the neonatal pig testes would contribute to the significant increases of plasma and testicular steroid hormone concentrations during early neonatal development, eventually leading to overall growth of the pig.