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From the * School of Biotechnology, Yeungnam
University, Gyeongsan, Republic of Korea; the
Department of Biochemistry and Molecular
Biology, School of Medicine, Kyunghee University, Seoul, Republic of Korea;
the School of Agricultural Biotechnology,
Seoul National University, Seoul, Republic of Korea; the
Department of Animal Science, Chungbuk National
University, Cheongju, Republic of Korea; the ||
Department of Agricultural Sciences, Korea
National Open University, Seoul, Republic of Korea; the ¶
Hanwoo Experimental Station, National Institute
of Animal Sciences, Pyeongchang, Republic of Korea; and the #
Department of Biochemistry and Molecular Biology and
Center of Anti-aging, School of Medicine, Eulji University, Daejeon, Republic
of Korea.
| Correspondence to: Dr Ki-Ho Lee, Department of Biochemistry and Molecular Biology, College of Medicine, Eulji University, 143-5 Yongdoo-dong, Joong-goo, Daejeon, Republic of Korea (301-110) (e-mail: kiholee{at}eulji.ac.kr). |
| Received for publication January 29, 2008; accepted for publication August 27, 2008. |
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Abstract |
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Key words: Leydig cells, male reproductive tract, steroid hormones, estradiol, nandrolone
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.
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-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). |
Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Total RNA and Protein Isolation
Total RNA was isolated according to the instructions provided with TRIzol
RNA extraction solution (Invitrogen, Carlsbad, California). In brief,
50–100 mg of fresh testis tissue was homogenized in 1 mL of extraction
buffer using a Polytron homogenizer (Fisher Scientific, Pittsburgh,
Pennsylvania), followed by chloroform and isopropanol total RNA precipitation.
The isolated RNA pellets were dissolved in RNA storage buffer (Ambion, Austin,
Texas) and stored at –80°C until used for the reverse transcription
(RT) reaction. The purity and yield of the total RNA were determined by an
ultraviolet (UV) spectrophotometer (Eppendorf, New York, New York), and the
qualities of the total RNAs were checked by gel electrophoresis prior to the
RT reaction.
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RT and Real-time PCR
RT was carried out according to the instructions in the ImProm-II RT system
(Promega, Madison, Wisconsin). Briefly, 1 µg of total RNA was
reverse-transcribed in a total volume of 20 µL using oligo-dT primer. The
RT reaction was performed at 25°C for 5 min, 42°C for 1 hour, and
70°C for 15 minutes. One microliter of cDNA was used as a template for
real-time PCR in a 25 µL reaction mixture, including 0.75 U of GoTaq DNA
polymerase (Promega), 5 µL of 5x buffer, 0.2 mM of
deoxyribonucleotide triphosphate (Promega), 2.5 µL of 3000x SYBR
Green I (BMA, Rockland, Maine), and 10 pmol of each primer. Oligonucleotide
primers for real-time PCR were prepared either by using Primer 3 software
(Whitehead Institute/MIT Center for Genomes Research, Cambridge,
Massachusetts;
http://www.bioneer.co.kr/cgi-bin/primer/primer3.cgi)
or utilizing published information. Information and sequences of primers of
steroidogenic enzymes tested in the present study are summarized in
Table 1. The PCR program
employed an initial step of 95°C for 5 minutes for predenaturation,
followed by denaturation at 94°C, annealing, and extension at 72°C
using the PTC-200 Chromo 4 real-time system (Bio-Rad Laboratories). The final
extension was carried out for 10 minutes at 72°C. No RNA, no cDNA
template, and no primer controls were included for PCR control purposes. The
PCR products were visualized on 1.2% agarose gel and photo-captured under UV
light using an image documentation system (Vilber Loumat,
Marne-la-Vallée, France). Cyclophilin (PPIA) was included as
an internal PCR control. For quantification of real-time PCR results, the
relative standard curve method was used to obtain quantitative values. Each
sample was replicated 3 or 4 times, and the normalized mean value to
PPIA was used for final comparison.
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Immunohistochemistry
The male reproductive tract was fixed in Bouin fixative for 18–24
hours. The testes were separated from other parts of the reproductive tract.
The testes were dehydrated in a serial of ethanol, cleared in xylene, and
infiltrated with paraffin. Paraffin-embedded testes were sectioned at
thicknesses of 4–5 µm. For immunohistochemistry, tissue sections were
deparaffinized in xylene and rehydrated in a series of ethanol. After
microwaving in 0.01 M citrate buffer, pH 6.0, for 10 minutes for antigen
retrieval, tissue sections were placed in 0.3%
H2O2/methanol for 15 minutes to inactivate endogenous
peroxidase. After washing in PBS, tissue sections were incubated in 10% normal
goat (Chemicon International, Temecula, California) or rabbit serum (Jackson
ImmunoResearch Laboratories Inc, West Grove, Pennsylvania), for 30 minutes at
room temperature to block nonspecific binding. Diluted primary antibodies were
applied on the tissue sections and incubated in a humidified chamber at
4°C overnight. The dilutions of the primary antibodies were selected after
a series of multiple preliminary trials for each antibody. We used dilutions
of 1:1000 for CYP19 (polyclonal rabbit anti-CYP19; a generous gift from Dr
Nobuhiro Harada, Fujita Health University, Japan), 1:400 for CYP11A1 (AB1244;
Chemicon), 1:2000 for HSD17B4 (monoclonal mouse anti-HSD17B4; a kind gift from
Dr Gabriele Möller, GSF-Research Center for Environment and Health,
Neuherberg, Germany), 1:500 for CBR1 (ab4148; Abcam Ltd, Cambridge, United
Kingdom), 1:500 for CYP17A (polyclonal rabbit anti-CYP17A; a generous gift
from Dr Anita Payne, Stanford University, Stanford, California), 1:200 for
ALR2 (polyclonal rabbit anti-ALR2; a gracious gift from Dr Motoko Takahashi,
Saga University, Saga, Japan), and 1:500 for HSD3B (polyclonal rabbit
anti-HSD3B; a benevolent gift from Dr Ian Mason, University of Edinburgh,
Edinburgh, United Kingdom). Excess primary antibodies were washed off the
tissue sections using PBS. Tissue sections were then incubated with
biotinylated goat anti-rabbit IgG (DAKO Corporation, Carpinteria, California)
for CYP19, CYP11A1, CYP17A, ALR2, and HSD3B, biotinylated goat anti-mouse IgG
(DAKO) for HSD17B4, or biotinylated rabbit anti-goat IgG secondary antibody
(DAKO) for CRB1 in a humidified chamber at room temperature for 1 hour.
Unbound secondary antibodies were washed off with PBS, and elite avidin-biotin
peroxidase (Vector Laboratories, Burlingame, California) was placed on slides
in a humidified chamber at room temperature for 30 minutes. After three
5-minute washes in PBS, the tissue sections were treated with a mixture of
3,3'-diaminobenzidine (Sigma, St Louis, Missouri), 0.05 M Tris-HCl
buffer, and 5% hydrogen peroxide to detect the peroxidase. The tissue sections
were then counterstained with hematoxylin, followed by dehydration in ethanol
and mounting. For negative controls, tissue sections were treated with normal
rabbit, mouse (Chemicon), or goat serum at the same dilutions in the place of
primary antibodies. Immunostaining was evaluated with digitalized images
captured with an Olympus-CoolSNAP cf color/OL camera (Olympus America,
Melville, New York) using RSImage version 1.1 software (Roper Scientific,
Duluth, Georgia). The photographic images were processed in Photo-Shop
software (Adobe Systems, San Jose, California).
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Data Presentation and Statistical Analysis
Data for mRNA and protein abundance were expressed relative to 0 weeks of
age as arbitrary units. In the figures, data are presented as mean ±
SD. A lack of bars indicates an insignificant SD. Comparison of mean
differences among neonatal and prepubertal ages were made using 1-way analysis
of variance, followed by Tukey's test, using SPSS software (SPSS Inc, Chicago,
Illinois). In all cases, results were considered significant if P
< .05.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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50 kDa) at 1 week and 3
weeks of age were not significantly changed
(Figure 1B). However, the
abundance of CYP19 was significantly lower at 4 months of age than at 0 weeks
of age (Figure 1B).
Immunohistochemical analysis showed an exclusive localization of CYP19 in
Leydig cells of the testis, regardless of the postnatal ages
(Figure 1C;
Table 2). Sex cords (SCs) in
the neonatal testis and seminiferous tubules (STs) in the prepubertal testis
were devoid of CYP19 staining (Figure
1C). Strong immunopositive staining of CYP19 in Leydig cells was
found at all neonatal ages (Figure
1C). However, the immunoreactivity of CYP19 was visibly reduced in
Leydig cells at 4 months of age (Figure
1C; Table 2).
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Differential Expression and Immunolocalization of CYP11A1 mRNA and Protein
The expression level of CYP11A1 mRNA was significantly increased
at 1 week of age, compared with the expression level at 0 weeks of age
(Figure 2A). The abundance of
CYP11A1 mRNA remained significantly high at 2 and 3 weeks of age
(Figure 2A), but the expression
of CYP11A1 mRNA was significantly reduced at 4 months of age
(Figure 2A). In contrast to the
mRNA expression pattern, the highest level of CYP11A1 protein (52
kDa) was found at 0 weeks of age, followed by significantly decreased levels
of CYP11A1 at 1, 2, and 3 weeks of age
(Figure 2B). At 4 months of
age, the testes possessed the lowest level of CYP11A1
(Figure 2B). Restricted
immunoreactivity of CYP11A1 was found in Leydig cells
(Figure 2C;
Table 2). No positive
immunostaining of CYP11A1 was observed in SCs during the neonatal period
(Figure 2C). However, at 4
months of age, CYP11A1 was immunolocalized in some germ cells, including
secondary spermatocytes and round spermatids, as well as Leydig cells
(Figure 2C;
Table 2).
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30 kDa) at 2
and 3 weeks of age (Figure 3B).
As in CBR1 mRNA, the expression of CBR1 protein in the testis was
significantly reduced at 4 months of age
(Figure 3B). Regardless of age,
the strong immunoreactivity of CBR1 was exclusively localized in Leydig cells,
but not in SCs or Sertoli or germ cells in STs
(Figure 3C;
Table 2).
Expression and Immunohistochemical Localization of HSD17B4 Transcript and Protein
The expression and immunohistochemical localization of HSD17B4
mRNA and protein are shown in Figure
4. The level of HSD17B4 mRNA was increased with neonatal
age, followed by a significant decrease at 4 months of age
(Figure 4A). The highest mRNA
expression of HSD17B4 was found at 3 weeks of age
(Figure 4A). Western blot
analysis showed a single band of HSD17B4 protein (32 kDa) in the testis
(Figure 4B). Interestingly, the
highest level of HSD17B4 protein was found at 0 weeks of age, followed by a
significant decrease at 1 week of age
(Figure 4B). However, the
amounts of HSD17B4 protein at 2 and 3 weeks of age were not significantly
different from the level at 0 weeks of age
(Figure 4B). As seen in mRNA
expression, the lowest expression of HSD17B4 protein was found at 4 months of
age (Figure 4B). During the
neonatal period, HSD17B4 expression was localized in Leydig cells, as
determined by immunohistochemistry (Figure
4C; Table 2).
However, at 4 months of age, the Leydig cells were devoid of HSD17B4
(Figure 4C), and the
blood-testis barrier (BTB) along the Sertoli cells was strongly immunostained
for HSD17B4 (Figure 4C;
Table 2). Neither Sertoli cells
nor germ cells were immunopositive for HSD17B4 at 4 months of age
(Figure 4C;
Table 2).
Expression and Localization of CYP17A Transcript and Protein
The expression of CYP17A mRNA was significantly increased at 1, 2,
and 3 weeks of age, compared with that at 0 weeks of age
(Figure 5A). A significant
decrease in the CYP17A mRNA level was detected in the testis at 4
months of age (Figure 5A). The
expression pattern of CYP17A protein (50 kDa) during the neonatal period
differed from that of mRNA expression
(Figure 5B). A significant
reduction of the CYP17A protein level was found at 2 weeks of age, whereas the
levels of CYP17A protein at 1 and 3 weeks of age were not significantly
different from the CYP17A protein level at 0 weeks of age
(Figure 5B). A significant
decrease in the CYP17A protein level was also detected at 4 months of age
(Figure 5B).
Immunohistochemical analysis showed strong immunoreactivity of CYP17A in
Leydig cells of the testis at all ages
(Figure 5C;
Table 2). The Sertoli cells and
germ cells were immunonegative for CYP17A
(Figure 5C;
Table 2).
Changes of Expression and Localization of HSD3B mRNA and Protein
The expression of HSD3B mRNA was significantly increased at 1 week
of age (Figure 6A). The
abundance of HSD3B mRNA at 2 and 3 weeks of age was significantly
decreased compared with that at 1 week of age
(Figure 6A). A significant
decrease of HSD3B mRNA expression was seen at 4 months of age
(Figure 6A). Western blot
analysis showed that the level of HSD3B protein (45 kDa) was the highest
at 0 weeks of age (Figure 6B).
A significant decrease of HSD3B level was found at 1 week of age, followed by
further significant decrease at 3 weeks of age
(Figure 6B). The lowest level
of HSD3B protein in the boar testis was detected at 4 months of age
(Figure 6B).
Immunohistochemistry revealed the localization of HSD3B in Leydig cells and
SCs at neonatal ages (Figure
6C; Table 2).
Strong immunoreactivity of HSD3B was detected in SCs at all neonatal ages,
whereas the intensity of the positive reaction of HSD3B in Leydig cells varied
to some extent with age (Figure
6C; Table 2). At 4
months of age, the immunoreactivity of HSD3B became visibly weaker and was
found in Leydig cells and Sertoli cells, as well as the BTB
(Figure 6C;
Table 2).
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36 kDa) level until 2 weeks of age
(Figure 7B). A significant
decrease of the protein level was found at 3 weeks of age, and the lowest
level of ALR2 protein was observed at 4 months of age
(Figure 7B).
Immunohistochemical analysis showed a strong positive reaction of ALR2 in
Leydig cells and SCs at all neonatal ages
(Figure 7C;
Table 2). At 4 months of age,
the immunoreactivity of ALR2 in Leydig cells was visibly reduced, whereas a
strong immunostaining of ALR2 was found in Sertoli cells
(Figure 7C;
Table 2).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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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
17-hydroxyprogesterone to 17,20β-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
3- 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 20-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.
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Footnotes |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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