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From the * Universität Hohenheim, Institut
für Tierhaltung und Tierzüchtung, Fachgebiet Tierhaltung und
Leistungsphysiologie (470A), Stuttgart, Germany; and the
Justus-Liebig Universität Gießen,
Institut für Veterinäranatomie, -histologie und -embryologie,
Gießen, Germany.
| Correspondence to: Rolf Claus, Universität Hohenheim, Institut für Tierhaltung und Tierzüchtung, Fachgebiet Tierhaltung und Leistungsphysiologie (470A), Garbenstr. 17, 70599 Stuttgart, Germany (e-mail: thsekret{at}uni-hohenheim.de). |
| Received for publication April 26, 2006; accepted for publication July 24, 2006. |
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Abstract |
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(ER). The specificity of
ER staining was confirmed by RT-PCR and Western blot. Immunization
decreased LH and testosterone to minimal concentrations in immunized and
E217ß-infused immunized boars, whereas follicle-stimulating
hormone (FSH) was not significantly altered. Estradiol decreased to base
levels after immunization. Infusion increased E2-17ß in
peripheral blood plasma of the immunized boars to physiological levels. Except
for A-spermatogonia, all spermatogenic cells decreased after immunization by
about 60%. After estradiol infusion, cell counts increased again and were
intermediate between control and immunized boars. Mitosis of spermatogonia was
reduced by nearly 50% due to immunization but was partly restored by
E2-17ß infusion. Expression of ER was localized in
spermatogonia, suggesting stimulation of mitosis, which was further confirmed
due to its predominant occurrence in stage I of the seminiferous epithelial
cycle (main stage of cell division). Apoptosis was minimal in boars but
elevated in the other 2 groups. Data showed that estrogens in physiological
concentrations supported mitosis but were not sufficient to normalize sperm
production because apoptosis was still high.
Key words: Estrogen receptor, spermatogenic stage, apoptosis, mitosis
and
ß (ER, ERß) both in Sertoli cells and in several types of
spermatogenic cells, so that a role of estrogens for spermatid adhesion to
Sertoli cells was suggested (Miura et al,
1999; Ebling et al,
2000; Pelletier et al,
2000; Hess,
2003). In a few species, high amounts of estrogens are synthesized in the Leydig cells and are measurable in high concentrations in peripheral blood plasma, as shown for the stallion (Raeside, 1969; Setchell and Cox, 1986; Claus et al, 1992; Lemazurier et al, 2001) and the boar (Velle, 1958). In the latter species, peripheral concentrations may reach nearly 280 pg/mL blood plasma (Claus and Hoffmann, 1980). In the mature boar, aromatase activity occurs exclusively in the Leydig cells (Fra˛cek et al, 2001; Mutembei et al, 2005). Sertoli cell expression of aromatase is limited to a narrow period of fetal development (Claus et al, unpublished data). Substitution experiments with castrated boars demonstrated that the synergistic action of androgens and estrogens is necessary to ensure normal function of accessory sex glands, male libido, and sexual behavior (Joshi and Raeside, 1973; Parrot and Booth, 1984; Booth, 1988), and also the establishment of the anabolic potential (van Weerden and Grandadam, 1976).
In the boar, high amounts of total estrogens also occur in the fluid of
spermatogenic tubules. They are primarily represented by 17ß-estradiol
(E2-17ß), which may reach concentrations of 41 ng/mL in this
fluid (Claus et al, 1985). A
high proportion of E2-17ß is transferred into the semen, so
that the total amounts per ejaculate may reach 110 ng. These amounts influence
female reproductive functions such as sperm transport and ovulation (Claus,
1989,
1990). The high tubular
concentrations of estrogens additionally suggest an effect on spermatogenesis.
An individual function of estrogens and a possible synergism with androgens
remain to be clarified. Two types of estrogen receptors are well described,
estrogen receptors and ß (ER, ERß). In the boar the
presence of the receptor types in different cells within the testes was
investigated by immunocytochemistry and the ERß demonstrated in Sertoli
cells (Mutembei et al, 2005),
germ cells (Rago et al, 2004;
Mutembei et al, 2005), and
Leydig cells (Mutembei et al,
2005). ER immunoreactivity was shown in germ cells
(Rago et al, 2004;
Mutembei et al, 2005) and
Leydig cells (Rago et al,
2004; Mutembei et al,
2005). A more detailed study, however, using in situ hybridization
combined with laser-assisted cell picking revealed a clear separation between
expression of ER and ERß between cell types, so that ER
expression was found to be limited to spermatogonia and spermatocytes, whereas
ERß expression occurred only in Sertoli cells
(Lekhkota et al, 2005). It
appears, therefore, that E2-17ß may be directly involved in
the regulation of mitotic activity of early stages of spermatogenesis, but a
possible effect cannot be separated from an effect of androgens so far. Active
immunization of boars with potent antigens against gonadotropin-releasing
hormone (GnRH) leads to an inhibition of luteinizing hormone (LH), so that
androgen and estrogen formation in the testes drops
(Metz et al, 2002).
Follicle-stimulating hormone (FSH) concentrations are not influenced by active
immunization, so that blood plasma concentrations do not differ significantly
between normal and immunized boars (Awoniyi
et al, 1988; Wagner and Claus,
2004). In consequence, immunization allows us to study
spermatogenesis in the absence of steroids and additionally to perform
substitution experiments to clarify individual or combined effects of
androgens and estrogens.
So far the effects of immunization on spermatogenesis have been compared with normal control boars (Wagner and Claus, 2004). It was found that steroid withdrawal leads to a reduction of the mitotic rate of spermatogonia. In addition, the expression of the glucocorticoid receptor in spermatogonia was increased so that apoptosis was elevated in spermatogonia and spermatocytes. In consequence, reduced mitosis and increased apoptosis led to a decrease of spermatogenic activity in the absence of testicular steroids (Wagner and Claus, 2004).
Based on these effects, the present paper reports the consequences of a
continuous infusion with E2-17ß on ER-mediated effects
on spermatogenesis in immunized boars.
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Material and Methods |
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All steps of the animal experiments, including cannulation, immunization, infusion, sampling of blood, and killing, were approved by the local animal welfare commission.
Animals, Housing, and Sampling
German landrace boars from the university herd were kept individually in
stalls (1.9 x 2.8 m). The animals were fed a diet with 10 MJ
metabolizable energy/kg feed and 13.9% crude protein at an amount of 3 kg per
day. The average weight of the boars increased from 82 ± 3.4 kg (mean
± SEM: 20 weeks of age) to 140 ± 5.3 kg (32 weeks) during the
experiment. As a vaccine, portions of 2 mL Improvac (CSL Limited ACN,
Victoria, Australia) were injected as recommended by the supplier.
Blood samples were collected over the 7-week period daily at 0800 hours
prior to feeding and used for radioimmunological determination of
E2-17ß in plasma extracts with a highly specific antiserum
which had been raised in rabbits against E2-17ß-6-CMO-BSA
(Claus et al, 1983). This
antiserum revealed cross reactivities only with E2-17 (0.4%)
and with estrone (0.43%). The sensitivity was 11.5 pg/mL. The intra-assay
coefficient of variation was 18%, for a concentration of 86 pg/mL. The
interassay variation varied depending on the concentration between 10% and
16%.
Testosterone was also determined radioimmunologically (Bubenik et al, 1982). The antiserum against testosterone had been raised against testosterone-3-CMO-BSA and revealed cross-reactivity only with 5a-dihydrotestosterone (28.5%). The sensitivity was 0.04 ng/mL plasma. The intra-assay coefficient of variation varied between 3.9% and 6.7% depending on the concentration and between 7% and 18% for the interassay variation coefficient. The latter figure refers to the immunized group, where concentrations were close to the detection limit.
For LH and FSH determination, window sampling was performed 5 times over the experimental period. For each window, blood was drawn every 20 minutes for 12 hours (0800 till 2000 hours). Determinations of LH and FSH were performed by RIA as published earlier (Claus et al, 1990), with the exception that in case of LH the first antibody was preincubated with the sample before adding the tracer. LH (AFP: 11043B) and FSH (AFP: 10640B) for iodination were obtained from Dr. Parlow (NIDDK, Torrance, Calif). From the same source the species-specific antisera (LH: AFP 15103194; FSH: AFP 2062096Rb) had been obtained. The LH antiserum could be used at a final dilution of 1:666 000. The FSH antiserum was used at a dilution of 1:200 000.
The reliability criteria were as follows: For LH the sensitivity was 25 pg/mL blood plasma. The intra-assay coefficient of variation was 7.9% and the interassay coefficient 8%. For FSH the sensitivity was 36 pg/mL blood plasma. The intra-assay coefficient of variation was 11% and the interassay coefficient 12%.
The pigs were killed by intravenous infusion of 0.2 mL Eutha 77/kg body weight (Essex Pharma, Munich, Germany). Testes were collected, the epididymides removed, and the weight determined. Samples for histology and immunocytochemistry were taken from the testes within = minutes after killing and fixed either in Bouin solution or 4% formaldehyde. For confirmation of the specificity of apoptosis staining, additional samples were fixed in 2.5% glutaraldehyde for later analysis by electron microscopy.
Histological Evaluations
All evaluations are based on = animals for control and immunized boars and
on 6 animals for the E217ß-infused immunized boars.
The Bouin-fixed samples were used to characterize the spermatogenic activity by counting the following spermatogenic cells: A-spermatogonia, B-spermatogonia, primary spermatocytes, round spermatids, and elongated spermatids. Because complete spermatogenesis in the boar can be separated into 8 stages (Swierstra, 1968), 5 representative round tubules for each of stages I–V and VIII were localized in the sections. Because stages VI and VII cannot be differentiated with certainty by light microscopy, the resulting data were combined (stage VI/VII). The cells were counted so that the quantitative data for each cell type are based on 40 tubules in each boar. Details of the histological procedure were given earlier (Wagner and Claus, 2004).
The immunocytochemical staining of Ki67 (mitosis) was performed in
formaldehyde-fixed tissue as described
(Mentschel et al, 2001).
Apoptosis was determined also in formaldehyde-fixed samples using the TUNEL
reaction as modified by Gavrieli et al
(1992). The determination of
the estrogen receptor (ER) was based on a polyclonal antibody
raised in rabbits against a synthetic 67 kda N-terminal epitope of the human
estrogen receptor protein (Ab 17, Lab Vision Corporation, Fremont, Calif). It
was shown by the supplier that it also detects the porcine ER. It was
used at a dilution of 1:150. Counterstaining was performed by hematoxylin.
Staining of the ER was also extended to slices from paraffinembedded
testis samples from control boars and immunized boars. For immunocytochemical
evaluation of testis, generally 2 slides with 3 tissue cross sections were
scored for each animal. Each immunocytochemical parameter was counted in 100
tubules per slide and is given as "positive cells per tubule." The
tubules were selected randomly, and counting was performed by 2 independent
observers. Mitosis (Ki67-stained cells) and the ER expression were
additionally referred to the stages of the seminiferous epithelial cycle.
Western Blot Analysis of the ER
To confirm the specificity of the antiserum for the ER, Western blot
analysis was carried out in porcine testicular tissue that had been stored at
–80°C. For protein extraction the tissue was transferred into
ice-cold lysis buffer (2 mL/g tissue; 10 mmol Tris-HCl, pH 7.5; 20 mmol sodium
molybdate; 10 mmol dithiothreitol; 10% glycerol; 0.05% Triton X-100; 1 mmol
EDTA; 2 mmol PMSF) containing protease inhibitors (Aprotinin, 10 µg/mL;
Leupetin, 10 µg/mL; Pepstatin A, 10 µg/mL; AppliChem, Darmstadt,
Germany). Tissue was homogenized with a bead mill (Mikrodismembrator U; B.
Braun, Melsungen, Germany) for 20 seconds at 2000 rpm, centrifuged (10 min,
9500 g, 4°C) and the protein content in the supernatant determined
(Bradford, 1976).
Equal amounts of protein were boiled in loading buffer (60 mmol Tris, pH 6.8; 15% SDS, 35% glycerol, 25% ß-mercaptoethanol; 0.1% bromphenol blue) for 5 minutes. Electrophoresis (50 µg protein/lane) was performed with a 12% SDS-polyacrylamide separating gel and a 5% stacking gel. For calibration a prestained protein ladder (10–180 kDa; MBI Fermentas, St Leon-Rot, Germany) was additionally loaded onto the gel. Proteins were transferred to polyvinylidene difluoride (PVDF) membranes (0.45 µm, Hybond-P; Amersham, Freiburg, Germany) using the Semi-Phor semidry blotting chamber (Hoefer Scientific Instruments, San Francisco, Calif). For blocking, the membranes were incubated in PBS containing 0.1% Tween 20 (PBS-T) and 5% skim milk powder at 4°C overnight.
The ER antibody was then added and incubated for 2.5 hours at room
temperature at a dilution of 1:2000. After washing, the second biotinylated
antibody (sheep against rabbit-IgG) was added and incubated for 2 hours at a
dilution of 1:2000. After washing, the
streptavidine-biotin-horseradishperoxidase-complex (ABC-complex; DAKO,
Hamburg, Germany) was added at a dilution of 1:5000 and incubated for 1 hour
at room temperature. The visualization was carried out with a luminol-based
detection reagent (250 mmol luminol; 80.4 mmol p-coumaric acid 1M Tris/HCl).
The chemiluminescent signal was documented on an X-ray film. The Western blot
analysis was repeated twice.
Confirmation of ER by RT-PCR
The occurrence of the ER mRNA was additionally confirmed by RT-PCR.
Total cellular RNA was isolated from testicular tissue using TRIzol Reagent
(Life Technologies, Karlsruhe, Germany). RNA samples were evaluated for purity
and integrity using absorbance (A260/A280) and r-RNA (28s/18s) ratios,
respectively. Primers for ER were based on a porcine sequence (GenBank
accession number AF035775). The following primers were used: forward primer
5'-GCT CCT GTT TGC TCC TAA C–'9, reverse primer 5'-GAC
ACG GTG GAT ATG GTC–'9. PCR protocols were optimized for
amplification within the exponential portion of the curves, as described for
other genes in our laboratory. PCR was performed in 50-µL reactions
containing 1 µL of reverse transcription mixture, Taq DNA Polymerase, 2 mol
MgCl2,10 x PCR-buffer, 10 mmol dNTPs, and 10 pmol/µL
primers. Reactions were incubated for 3 minutes at 95°C, followed by
cycles of 1 minute at 94°C, 2 minutes at 56°C, 1 minute 30 seconds at
72°C. The number of cycles was 35. After cycles were completed, reactions
were incubated at 72°C for 10 minutes, which was followed by cooling to
4°C. A set of negative controls was processed in a similar manner, except
that reverse transcriptase was replaced by water in reverse transcription
reactions. Products were separated by electrophoresis in 2% agarose gel,
visualized by ethidium bromide staining, and documented on Polaroid T 57 film.
The RT-PCR procedure was repeated 10 times. For Western blot and RT-PCR
different sample preparations were used.
Statistical Evaluation
All data represent the arithmetic means ± SEM from 5 boars in the
control boars and the immunized boars and from 6 boars in the
E2-17ß infused immunized group.
For the testosterone concentrations, each boar is represented by 22 blood samples; estradiol concentrations represent 50 blood samples from each boar. The concentrations of LH and FSH represent the arithmetic means ± SEM from 2 windows, with 37 samples from 5 boars each. The histological data are given as arithmetic means ± SEM of at least 40 tubules from each boar. The immunocytochemical evaluation refers to the arithmetic means ± SEM of 100 counted tubules per animal.
All data were tested for normal distribution by the Kolmogorov-Smirnov test.
They were further analyzed using the mixed model analysis of the Statistical Package for the Social Sciences (version 11; SPSS, Chicago, Ill).
The following model was used:
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Yijk = mean count of kth animal of ith group within jth replicate
µ = general effect
i = main effect of ith group
ßj = main effect of jth replicate
( ß)ij = group x replicate interaction
eijk = residual error
In this design, the animal was assumed to be chosen at random.
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Results |
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TopAbstractMaterial and MethodsResultsDiscussionReferences |
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Hormone Concentrations
Concentrations of LH, FSH, testosterone, and estradiol are summarized in
Table 1. LH concentrations
decreased considerably, from 130 to 27 pg/mL respectively. Infusion of
estradiol had no effect on LH concentrations in immunized boars.
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In contrast, FSH concentrations were not influenced neither by GnRH-immunization nor the additional supply of estradiol. Testosterone in the control boars revealed mean concentrations of 4 ng/mL. The variation is mainly explained by differences in the concentrations between individuals (range of mean levels: 1.3–5.4 ng/mL). Immunization effectively decreased testosterone concentrations, but as expected, they were not influenced by E2-17ß infusion.
The mean E2-17ß concentrations were 106 pg/mL in the control boars, with an age-dependent increasing tendency along the 50-day sampling period. E2-17ß values differed considerably between individual boars, so that the range of mean estradiol concentration was 54–284 pg/mL.
Immunization decreased E2-17ß concentrations from 106 to 12 pg/mL (range: <12 pg/mL to 36 pg/mL). The infusion of estradiol led to an immediate rise in plasma. The infusion of high amounts of E2-17ß led to a mean level of 512.4 ± 49.1 pg/mL. After lowering the amount of E2-17ß, the concentration decreased to a mean concentration of 234 pg/mL, which was 1.6-fold higher than the control boars.
Germ Cells
A comparison of the frequency of the individual spermatogenic cell types is
given in Table 2. For
B-spermatogonia, primary spermatocytes, and round and elongated spermatids,
the absence of testicular steroids in the immunized boars led to a significant
decrease of cell counts compared to control boars. In the estrogen-infused
boars the frequency of these cells increased again, but the counts were
intermediate between control boars and immunized boars. In consequence, all
counts in the E2-17ß-infused pigs differed significantly when
compared to both the control and the immunized boars.
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A-spermatogonia, however, did not differ significantly between controls and immunized boars. E2-17ß-infused immunized boars had the lowest number of A-spermatogonia, which differed significantly compared to both control and immunized boars. Thus it appears that cell counts had shifted from A- to B-spermatogonia due to estradiol infusion.
Results From Immunocytochemistry, RT-PCR, and Western Blot
An example for the ER staining is shown for an immunized boar in
Figure 1. As can be seen,
ER-staining is clearly separated from the neighboring cells and
specifically occurs in spermatogonia. The specificity was additionally
confirmed by Western blot and RT-PCR. Western blot analysis confirming the
specificity of the antiserum had been performed by the supplier with human
breast carcinoma cells and is additionally verified for testicular tissue in
Figure 2. The bands of the
expected size confirmed the applicability of the antiserum for boar testicular
tissue.
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mRNA (Figure
3).
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-staining in the 3 groups of boars revealed that positive stained
cells only occur within the tubules, but not in interstitial cells and Sertoli
cells. Within the tubules, positive staining was primarily observed for A- and
B-spermatogonia and only occasionally in more advanced spermatogenic cells.
The occurrence of ER in spermatogonia suggests a role for mitosis, so
the relationships between ER-expression and mitosis were analyzed for
boars and referred to the stages of the seminiferous epithelial cycle, as
shown in Figure 4. ER
was mainly detected in stage I. The number of Ki-67-positive cells which
indicate the initiation of mitotic activity is included in
Figure 4 and reveals highest
cell counts in stage VIII. Because the stage-dependent pattern of
ER–staining did not differ between the other 2 groups, further
quantification of ER-positive cells was limited to stage I of the
seminiferous epithelial cycle. These data and the number of cells undergoing
mitosis and apoptosis are summarized for the 3 groups in
Table 3. In
Figure 5 an example of
apoptosis is revealed by electron microscopy and confirms the occurrence of
apoptosis in early stages of spermatogenesis.
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In the estradiol-infused immunized boars, the numbers of mitotic cells was
intermediate compared to control boars and immunized boars
(Table 3). In consequence,
mitosis in E2-17ß-infused immunized boars again differs
significantly from the other 2 groups. No significant difference was
detectable when comparing apoptosis in immunized and estradiol-infused boars,
whereas apoptosis in the control boars was significantly lower. No significant
differences exist in the expression of the ER between the 3 groups,
although mean positive cell counts are more than 2-fold in the
E2-17ß-infused boars compared to the immunized boars.
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Discussion |
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TopAbstractMaterial and MethodsResultsDiscussionReferences |
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An alternative approach had been chosen by knockout experiments in mice inhibiting either the aromatase activity, and thus aromatization of androgens to estrogens (Fisher et al, 1998; Robertson et al, 1999), or alternatively the estrogen receptor (Lubahn et al, 1993; Krege et al, 1998; for review see O'Donnell et al, 2001). In both cases possible effects of estrogens on different traits of male reproductive physiology had to be studied in the presence of androgens. More recently E2-17ß formation was inhibited with an aromatase inhibitor, but effects on spermatogenesis were not investigated (At-Taras et al, 2005).
In our study the expression of ER in boar testis was additionally
confirmed by RT-PCR and by Western blot analysis in all 3 groups. In Western
blot the preparations from the nonimmunized control boars obviously led to
additional bands which may be attributed to a different endocrine background
(eg, due to the presence of androgens). The underlying substances were not
further identified. In addition, we could demonstrate previously that
ER expression is limited to early stages of germ cell development
(Lekhota et al, 2005). ER expression in the noninfused immunocastrates
shows that it is partly independent from the presence of the ligand. A
tendency for elevated receptor density in E2-17ß-infused boars
may be due to an additional ligand-dependent expression. The ER could
be exclusively localized in A- and B-spermatogonia. Staining, however, was
focused on spermatogonia in stage I of the seminiferous epithelial cycle,
which represents the main stage of spermatogonial division
(Frankenhuis et al, 1982;
Garcia-Gil et al, 2002).
Stages VI/VII and VIII also belong to the mitotic stages, but mainly represent
the G1 and S-phase, whereas actual division (M-phase) occurs in stage I
(Guraya, 1987) where also
ER was detected in the present study. A mitosis-supporting effect of
estrogens for spermatogenesis is not surprising, because their promotion of
cell division is well known for a variety of other tissues, including
estrogen-dependent tumors (Altucci et al,
1997; Zhang et al,
1998). In such tissues it was also found that estrogens promote
cells to finish the G1-phase and to enter the M-phase
(Leung and Potter, 1987;
Zhang et al, 1998). A role of
estrogens for mitosis of spermatogonia was also assumed for the stallion
(Sipahutar et al, 2003) and
for rodents and the eel (O'Donnell et al,
2001; Miura et al,
1999).
By the use of subcutaneous E2-17ß Silastic implants it was shown in GnRH-deficient mice that the treatment led to an increase of the tubular volume (Ebling et al, 2000), as also shown in the hamster (Pak et al, 2002). In the latter study the effect was attributed to a spermatogenesis-initiating effect of estrogens.
Quantitative analysis revealed that counts of the individual spermatogenic cells other than A-spermatogonia were intermediate in the E2-17ß-infused immunized boars, compared to both boars with significantly higher and immunized boars with significantly lower cell counts.
These data thus additionally support a role of E2-17ß in
spermatogenesis in the boar. This does not mean necessarily that overall
production of sperm is improved in E2-17ß-infused immunized
boars compared to immunized boars, because counteracting apoptosis in the
following spermatogenic cells was not significantly different between these 2
groups. In control boars, however, apoptosis was markedly reduced, suggesting
an additional role of androgens. It was not possible to compare sperm counts
in ejaculates, because semen collection attempts were unsuccessful both in
immunized and E2-17ß-infused immunized boars. Taken together,
the data show that in the boar ER is localized only in spermatogonia.
Infusion of E2-17ß led to an increase of mitosis in the
tubules of GnRH-immunized boars, indicating that estrogens are necessary for
germ cell renewal in the testis of boars.
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Acknowledgments |
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