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Department of Endocrinology and Reproduction, Erasmus University Rotterdam (J.M.A.E., A.M., J.A.G., A.O.B.), 3000 DR Rotterdam, The Netherlands; and Institute of Human Genetics, University of Göttingen (I.M.A., W.E.), Göttingen, Germany
Address all correspondence and requests for reprints to: J. M. A. Emmen, Department of Endocrinology and Reproduction, Erasmus University Rotterdam, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands. E-mail: endov{at}endov.fgg.eur.nl
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) (2, 3). During the
transabdominal phase, the male gubernaculum is developing, whereas
outgrowth of the CSL is lacking, resulting in a position of the testis
close to the bladder neck.
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Recently, data became available indicating that insulin-like factor 3 (Insl3), also designated Leydig insulin-like factor or relaxin-like factor, might be an important factor for gubernaculum development in mice (13). Insl3 is specifically expressed in Leydig cells of the pre- and postnatal testis and in thecal cells of the postnatal ovary (14). Insl3 knockout mice, homozygous for a targeted inactivation of the Insl3 gene (Insl3-/- mice), have been generated, and it was observed that male Insl3-/- mice have undescended testes (15). Histological analysis of male Insl3-/- fetuses revealed that the development of the gubernaculum is severely affected. Similar findings were reported by Nef and Parada (16).
Although studies with mutant mouse models have provided more insight into the complicated regulation of gubernaculum development, it remains to be determined whether the targeted factors are directly involved in regulation of gubernaculum development. In the present study, development of the rat gubernaculum during the first phase of testis descent was studied in vivo with respect to cell differentiation and proliferation. Furthermore, an organ culture technique for in vitro culture of the gubernaculum was established to study the effects of different testicular hormones/factors on rat gubernaculum development.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For immunohistochemical analysis of the rat gubernaculum, fetuses were removed from the uterus on E17 and E19 and fixed in 10% neutral buffered formalin for 24 h. After fixation, fetuses were embedded in paraffin and sectioned at 7 µm, and sections were selected for immunohistochemistry.
Cell proliferation in the fetal rat gubernaculum in vivo was assessed using 5'-bromodeoxyuridine (BrdU), a thymidine analog that is incorporated into the DNA during the S phase of the cell cycle. Pregnant rats were injected ip with a single pulse of BrdU (Roche Molecular Biochemicals, Mannheim, Germany) dissolved in saline (100 mg/kg BW) on E17. Two hours after injection, fetuses were removed from the uterus, fixed in 10% neutral buffered formalin, and further processed for immunohistochemical detection of incorporated BrdU.
Organ cultures
For each culture, fetal rat gubernacula and mouse gonads were
isolated. Gubernacula were obtained from E17 male rat fetuses. After
the mothers were killed, the rat fetuses were quickly removed and
placed on ice until dissection. Fetuses were examined under a
dissecting microscope, and gubernacula were obtained from male fetuses
and placed in PBS under sterile conditions. Urogenital ridges were also
removed.
Gonads were obtained from neonatal mice (FVB strain) on postnatal day 6. Additionally, two different mutant mouse models were used: AMH mutant mice (B6 strain) (9) and Insl3 mutant mice (129/SV strain) (15). Homozygous AMH-/- or Insl3-/- female mice were bred with heterozygous AMH+/- or Insl3+/- males, respectively, to obtain homozygous and heterozygous neonates. The macroscopic anatomy of the neonates was determined with a dissection microscope, and homozygous AMH and Insl3 mutant neonates could be reliably distinguished from heterozygous ones based on their phenotypes. Nevertheless, tails were collected for genotyping by PCR. After dissection, testes were decapsulated.
The culture technique used was similar to that used by Cooke et al. (17) for mouse neonatal bulbo-urethral gland. Briefly, tissues were transferred onto CM filters (Millipore Corp., Bedford, MA) and floated on 1 ml medium in four-well plates (Nunc, Roskilde, Denmark). The culture medium used was DMEM/Ham’s F-12 (Life Technologies, Inc., Breda, The Netherlands) supplemented with penicillin (100 IU/ml), streptomycin (100 µg/ml), fungizone (0.6 µg/ml), insulin (10 µg/ml), and transferrin (10 µg/ml; all from Sigma, St. Louis, MO). The medium was supplemented with 2% stripped FCS (Greiner Labortechnik, Kremsmuenster, Austria). In some of the cultures, the synthetic androgen methyltrienolone (R1881; 10-8 M) was added to the medium. The culture dishes were placed in a humidified incubator at 37 C in a 5% CO2 atmosphere, and the medium was changed daily.
Gubernacula were cultured for 5 days with or without added gonads. The gonads were placed in close proximity to the gubernacula on a filter (Millipore Corp.) at the ratio of one gonad to one gubernaculum. After 5 days of culture, some explants were chosen randomly for immunohistochemical analysis. Tissues were fixed in neutral-buffered formalin and embedded in paraffin, and serial sections of 5 µm were cut.
Thymidine incorporation
The proliferation of [3H]thymidine into
DNA was measured on culture day 5. At the end of day 4, 5 µCi/ml
[methyl-3H]thymidine (SA, 48
Ci/mmol; Amersham Pharmacia Biotech, Little Chalfort, UK)
was added to the culture medium. After incubation overnight, the
cultured tissues were removed, rinsed with 10% ice-cold
trichloroacidic acid, and centrifuged. The precipitate was washed twice
with cold 5% trichloroacidic acid and hydrolyzed in 25 µl 1
M NaOH (68 C, 30 min). The radioactivity of the
supernatant was measured using a liquid scintillation counter, and the
incorporation of radioactive thymidine was calculated as
disintegrations per min/gubernaculum.
Immunohistochemistry
Selected sections were mounted on silane-coated slides. After
deparaffinization, sections were treated with 3%
H2O2/methanol solution to
block endogenous peroxidase activity.
To study gubernaculum development in vivo and in vitro, polyclonal antibodies against the rat androgen receptor (SP197) (18) were used as marker for the mesenchymal core, and monoclonal antibodies against sarcomeric myosin (MF20, obtained as hybridoma from the Developmental Studies Hybridoma Bank) (19) were used as a marker for the skeletal muscle outer layer. The procedure used was based upon that described by Janssen et al. (20). Briefly, sections were placed in 10 mM citrate buffer and microwaved three times for 5 min each time at 700 watts. Sections were preincubated with normal goat serum, followed by incubation with the primary polyclonal antibodies SP197 (diluted 1:7000) or monoclonal antibody MF20 (diluted 1:100) overnight at 4 C. The antibodies were detected using biotinylated goat antirabbit antibody (dilution 1:400) or biotinylated goat antimouse antibody (diluted 1:50), respectively, by incubation for 30 min at room temperature, followed by treatment with streptavidin-biotin-peroxidase complex. The peroxidase activity was visualized with 3,3'-diaminobenzidine tetrahydrochloride. Negative controls were included by replacement of the primary antibody by PBS. Cells with nuclear [androgen receptor (AR)] or cytoplasmic (myosin) immunostaining were interpreted as positive. AR expression in the gubernaculum was assessed in qualitative terms by visually determining the average intensity of the positive nuclear staining in this structure.
The BrdU staining procedure has been described previously (21). Briefly, labeling was detected in 7-µm sections, pretreated with 0.1% pronase for 30 min and 2 N HCl for 30 min, followed by 1-h incubation with mouse monoclonal anti-BrdU antibodies (diluted 1:25; Sigma), all at 37 C. Sections were then incubated with peroxidase-conjugated goat antimouse antiserum (diluted 1:100) for 30 min at room temperature. BrdU-labeled nuclei were visualized with 3,3'-diaminobenzidine tetrahydrochloride. Slides were counterstained with hematoxylin.
Controls and statistical analysis
The proliferation index of the rat gubernaculum was estimated by
determining the proportion of BrdU-labeled nuclei per total number of
cells (x100) per fetus. Statistical significance was assessed using
Student’s two-tailed t test.
All culture experiments were performed in triplicate, and the data are expressed relative to [3H]thymidine incorporation induced in gubernaculum cultured with R1881 (10-8 M) in the same experiment. All of the data shown were collected in at least two independent experiments. Statistical analysis was performed using one-way ANOVA supplemented with Hochberg’s GT2 test. All data are presented as the mean ± SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The sexually dimorphic development of
the gubernaculum was further examined by determination of the
proliferation index and by studying the organization of the
gubernacular bulb into mesenchymal cells and myoblasts using two
different cell differentiation markers. On E17, the bulb in both sexes
is organized into a mesenchymal core and an outer muscular layer, but
the male bulb is enlarged compared with the female gubernacular bulb.
The proliferation indexes for the male and female gubernacula on E17
were estimated using the BrdU-labeling technique. BrdU incorporation
was significantly higher in the male gubernacular bulb (15.8 ±
0.5%; n = 4) than in the female bulb (7.2 ± 1.8%; n =
4; P < 0.005). The enlargement or swelling of the male
gubernacular bulb progressed to E19, whereas in the female fetus no
further development occurred (Fig. 2, A and B).
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). However, in the male fetus,
the muscular layer expanded between E17 and E19, and the myoblasts
started to differentiate (Fig. 2, C–E). AR expression was restricted
to the mesenchymal cells of the gubernacular bulb (Fig. 2F). AR
expression in the male gubernaculum was consistently higher than that
in the female gubernaculum at both time points (not shown).
An in vitro model of gubernaculum development
To analyze hormonal control of gubernaculum development, the
possibility of using a culture system was studied. As the in
vivo studies demonstrated that gubernaculum development is
characterized by rapid cell proliferation, it was decided to follow
gubernaculum development in vitro by measurement of the
incorporation of [3H]thymidine into DNA.
Development of the Wolffian duct in vitro is also marked by
rapid proliferation when cultured in presence of a testis or androgen
(22). Therefore, the Wolffian duct was used in each
culture experiment as a control androgen-responsive tissue.
Initial studies indicated that E17 rat gubernacular explants responded
to the presence of testicular tissue in culture by increased
incorporation of [3H]thymidine. To determine
the effects of gonads from AMH-/- and
Insl3-/- mice on gubernaculum development
in vitro, a series of culture experiments with mouse
gubernacula and gonads was carried out. However, in contrast to fetal
rat gubernacular explants, fetal mouse gubernacular explants did not
show any indication of a proliferative response to testicular tissue.
It was therefore decided to perform a coculture of rat gubernaculum
with mouse gonads, outlined in Fig. 3A, which was successful. Maximal differences in
[3H]thymidine uptake per rat gubernaculum
cultured in the presence or absence of mouse testis were observed after
5 days of culture. [3H]Thymidine uptake by the
gubernacular explants was enhanced after 5 days when cultured in the
presence of testis or R1881, the effect of testis being greater than
that of R1881 (Fig. 3B). Ovarian tissue had no effect on gubernacular
growth. As a prenatal mouse testis is much smaller then a prenatal rat
testis, three prenatal mouse testes per rat gubernaculum were necessary
to produce a growth response similar to that produced by one prenatal
rat testis. However, replacing these three fetal mouse testes by one
neonatal mouse testes, dissected on postnatal day 6, induced a growth
response in a E17 rat gubernaculum that compared well with that of an
E17 rat testis.
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, G and H).
Addition of 2% FCS and insulin (10 µg/ml) to the medium enhanced the
viability of gubernaculum and gonadal tissue during culture. On day 0,
the gubernacular bulb was organized into a muscular outer layer and an
inner mesenchymal core (Fig. 2G). By day 5, the entire gubernaculum had
enlarged. The organization into muscular outer layer and mesenchymal
core was preserved in the gubernacular bulb cultured in the presence of
a testis (Fig. 2H).
Immunohistochemistry was performed to study the expression of both
differentiation markers, AR and myosin, in cultured gubernacula (Fig. 2, I–L). Myosin was expressed in the gubernaculum under all culture
conditions, including in gubernacula cultured without hormone or in the
presence of ovarian tissue (Fig. 2, I and J). AR expression was
detected in gubernacular explants cultured in the presence of either
R1881 or testis (Fig. 2, L). The organization of the gubernacular bulb
into an outer muscular layer and an inner AR-positive mesenchymal core,
as seen in vivo, was also observed in gubernacular explants
in vitro, but only when cultured in presence of a testis
(compare Fig. 2, K and L, with Fig. 2, E and F). However, the muscular
layer was thinner and less differentiated compared with in
vivo development (compare Fig. 2K with Fig. 2E). In all other
cultures, myosin-positive cells were found all across the gubernaculum
(Fig. 2I).
Effects of testes from different mutant mice
The culture system described above was used to study the
stimulatory effects of gonads from different mutant mice on
gubernacular explants. Two different mutant mouse models were
available, AMH-/- and
Insl3-/- mice, as well as the heterozygous
animals. Although in vivo analysis of heterozygous and
homozygous AMH and Insl3 mutant male mice did not indicate any defect
in androgen production by the testis, R1881 was added to the culture
medium to compensate for a possible partial deficiency in androgen
production by the testis in vitro. In Fig. 4, the effect of testicular tissue from
the different mutants on [3H]thymidine uptake
by gubernacular explants, cultured in R1881-containing medium, is
shown. AMH-/-, AMH+/-,
and Insl3+/- testicular tissue stimulated
[3H]thymidine incorporation by the gubernacular
explants to the same extent as control testis. In contrast, in the
presence of a Insl3-/- testis,
[3H]thymidine incorporation was significantly
reduced compared with the effect of the control testis
(P < 0.05), to a level similar to that seen with R1881
alone. Heterozygous and homozygous mutant ovarian tissue did not induce
any increase in the proliferative activity of gubernacular explants
compared with the proliferative activity of gubernacular explants
cultured in basal medium (results not shown).
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). Ovarian tissue did not stimulate the
growth of Wolffian duct explants. In contrast to the results obtained
with gubernacular explants, the stimulatory effect of
Insl3-/- testis on Wolffian duct growth
in vitro was not different from that of control or
Insl3+/- testis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Immunohistochemical studies presented herein showed that both male and female rat gubernaculum express myosin, a marker for myoblasts, in vivo. Furthermore, myosin was also expressed in vitro, under all different culture conditions. However, the muscular layer of the gubernaculum in vitro, even after coculture with a testis, was thinner compared with the in vivo situation. Similarly, Radhakrishnan and Donahoe (25) limited their in vitro studies to the muscular layer and did not observe induction of muscle differentiation in cultured rat gubernacular tissue by using whole testis. Studies on in vitro skeletal muscle development showed that only a limited degree of differentiation can take place in vitro, probably due to the lack of innervation, growth factors, and extracellular matrix (26). Although myoblasts can differentiate in the absence of nerves, continuing growth and maturation definitively require innervation (26). Upon isolation, the gubernaculum is separated from the body wall musculature and nerves, which could explain the retarded muscular development in vitro.
Immunostaining of the AR in vivo demonstrated AR-positive cells in the mesenchymal layer of the gubernaculum of both sexes, which is in agreement with published data (18, 27). The level of AR expression increases in the male, but decreases in the female gubernaculum, indicating an initial androgen-independent expression followed by a hormone-dependent expression (28). In agreement with the in vivo situation, the AR was only highly expressed in explants that were cultured in the presence of androgen, either as R1881 added to the medium or in cocultures with testicular tissue.
Generally, gubernacular tissue cultured in vitro showed a more condensed structure than gubernacular tissue in vivo. This could be due to the flattening of the gubernacular explants when cultured on the two-dimensional filter layer. Furthermore, the organization of the gubernaculum into the mesenchymal core and muscular outer layer, as seen in the male gubernaculum development in vivo, was only observed in the gubernacular explants cultured in the presence of a testis. Apparently, testicular factors other then androgens are necessary to maintain the male-specific organization of the gubernaculum during culture.
On E17, the male gubernaculum in vivo was enlarged substantially compared with that in the female, which is in agreement with histological observations reported previously (5, 29, 30). This difference can be explained by a difference in cell proliferation, as determined by BrdU labeling indexes. This is in agreement with observations in the pig fetus, in which gubernaculum outgrowth during the transabdominal phase of testis descent is also characterized by rapid cell proliferation (31, 32, 33). The regulation of proliferation was used as parameter for further analysis of hormonal control of gubernaculum development.
Surprisingly, R1881 added to the culture medium clearly increased [3H]thymidine uptake by gubernacular explants. In two earlier in vitro studies using isolated porcine gubernaculum cells, androgens did not stimulate proliferation (10, 12). As the major difference between rat and pig gubernaculum proper is the presence of a muscular outer layer in the rat (5), the increased proliferation might reflect an increase in the muscular component of the rat gubernaculum. Given the fact that expression of the AR, which is a prerequisite for androgen activity, is detected in the mesenchymal core of both rodent and porcine gubernaculum (27, 34, 35), it is suggested that androgens may act directly on the mesenchymal cells, which, in turn, elicit a growth response on the myogenic cells through paracrine mechanisms. In several androgen-responsive tissues, including prostate, seminal vesicles, and bulbo-urethral glands, testicular androgens act via the AR in the mesenchymal cells (36). In addition, influences of the mesenchyme on the muscular layer and vice versa might be necessary for complete gubernaculum development. Such a mechanism of action might also hold true for the process of gubernaculum outgrowth in the pig. Although the porcine gubernaculum proper only consists of mesenchymal cells, it is in close contact with the cremaster muscle, which is the equivalent of the muscular outer layer of the rat gubernaculum (5). The present in vitro data are in apparent contradiction with the currently available data from in vivo studies that more strongly support a role of androgens in gubernaculum regression then outgrowth (4). However, both Spencer et al. (37) and Cain et al. (38) demonstrated a decrease in gubernacular bulb development upon antiandrogen exposure of male rat fetuses. This indicates the complexity of gubernaculum development in vivo and underlines the usefulness of organ culture for further studies on the role of androgen.
The presence of testicular tissue during the culture period exceeded the stimulatory effect of androgen on [3H]thymidine uptake by gubernacular explants, whereas ovarian tissue did not stimulate [3H]thymidine uptake. This implies that a testicular factor, different from androgens, is also involved in gubernaculum growth. This is in contrast to the Wolffian ducts, which are stimulated to the same extent by testicular tissue as by androgens alone. Two factors that are secreted by the fetal testis at the time of transabdominal testis descent are AMH and Insl3 (14, 39). As neither factor was available for direct studies, coculture experiments were designed in which gonads of AMH and Insl3 mutant mice were tested. R1881 was added to the culture medium to compensate for a possible partial deficiency in androgen production by the testes of AMH and Insl3 mutant testes in vitro. Culturing gubernacula in the presence of Amh-/- testes did not decrease the uptake of [3H]thymidine, compared with the effect of control or Amh+/- testes. This is consistent with the observed lack of a stimulatory effect of AMH on cultured porcine gubernaculum cells (10, 12). It cannot completely be excluded that a small amount of AMH might have been present in the culture medium, as 2% stripped (mixed sex) FCS was used. However, this amount did not cause Müllerian duct regression in a Müllerian duct organ culture assay performed under the same culture conditions as those used for gubernaculum (personal observations).
The effect of an Insl3-/- testes on [3H]thymidine incorporation by the gubernacular explants, cultured in medium containing R1881, was similar to that by explants cultured in medium containing R1881 only. Insl3-/- testes were not competent to further increase the stimulatory effect of androgens present in the culture medium. This result consolidates the putative role of testicular Insl3 in gubernaculum development in vivo and indicates that the primary defect of impaired gubernaculum development observed in Insl3-deficient mice appears to be intrinsic to the testis. In contrast, Insl3 appeared not to be essential for growth of the Wolffian ducts.
Whether Insl3 is secreted by the testis and acts directly on
gubernaculum cells remains to be determined. Recently, however, Insl3
has been demonstrated in the human circulation using antibodies
(40), and specific, high affinity Insl3 receptors have
been identified (41), indicating a possible endocrine role
of Insl3. According to the predicted structure, the biologically active
Insl3 protein consists of a ß-chain (3.3 kDa) and an 
-chain
(13). Fentener van Vlissingen et al.
(10) and Visser et al. (12)
previously demonstrated that low mol wt factors (<30K and <3.5K,
respectively, isolated from fetal pig testis stimulated growth in
cultured gubernaculum cells. As the Insl3 gene has also been identified
in the pig, and porcine Leydig cells express Insl3 messenger RNA
(42), the growth stimulatory effect observed by these
investigators might have been related to Insl3 activity.
In the pig fetus, gubernaculum growth is determined by cell proliferation as well as by changes in the extracellular matrix, resulting in true swelling of the gubernaculum due to water uptake (43). Whether changes in the extracellular matrix are important during transabdominal testis descent in rodents remains to be determined. Interestingly, however, primary structure analysis revealed that Insl3 is closely related to relaxin (44). Furthermore, synthetic human INSL3 peptide augmented the activity of relaxin in the mouse pubic symphysis bioassay, indicating relaxin-like properties of Insl3 (44). Relaxin is a hormone that plays a significant role in promoting the growth and softening of the cervix and loosening the pubic symphysis before parturition by remodeling connective tissue (45, 46). A similar mechanism of Insl3 action in the male fetus during gubernaculum development and testis descent would be an attractive working hypothesis for further research.
Further analysis of gubernaculum development in vivo and in vitro in relation to regulation of Insl3 gene expression might lead to a better understanding of the process of testis descent. A major goal of future studies will be to define downstream genes through which the action of Insl3 on gubernaculum development is mediated.
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Acknowledgments |
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Footnotes |
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Received April 25, 2000.
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