Targeted Disruption of the Insl3 Gene Causes Bilateral Cryptorchidism
Stephan Zimmermann,
Gerd Steding,
Judith M. A. Emmen,
Albert O. Brinkmann,
Karim Nayernia,
Adolf F. Holstein,
Wolfgang Engel and
Ibrahim M. Adham
Institut für Humangenetik der Universität
Göttingen (S.Z., K.N., W.E., I.M.A.) D-37073
Göttingen, Germany
Abteilung Embryologie der
Universität Göttingen (G.S.) D-37075 Göttingen,
Germany
Endocrinology and Reproduction (J.M.A.E., A.O.B.)
Erasmus University Rotterdam N-3000 DR Rotterdam, The
Netherlands
Abteilung für Mikroskopische Anatomie
(A.E.H.) Universität-Krankenhaus Eppendorf D-20251
Hamburg, Germany
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ABSTRACT
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The sexual dimorphic position of the gonads in mammals
is dependent on differential development of two ligaments, the cranial
suspensory ligament (CSL) and the gubernaculum. During male
embryogenesis, outgrowth of the gubernaculum and regression of the CSL
result in transabdominal descent of the testes, whereas in the female,
development of the CSL in conjunction with failure of the gubernaculum
development holds the ovaries in a position lateral to the kidneys.
Several lines of evidence suggest that regression of the CSL and
induction of gubernaculum development are mediated by testosterone and
a yet unidentified testicular factor, respectively. The
Insl3 gene (orginally designated Ley I-L), a
member of the insulin-like superfamily, is specifically expressed in
Leydig cells of the fetal and postnatal testis and in theca cells of
the postnatal ovary. Here we show that male mice homozygous for a
targeted deletion of the Insl3 locus exhibit bilateral
cryptorchidism with free moving testes and genital ducts. These
malformations are due to failure of gubernaculum development during
embryogenesis. In double-mutant male mice for Insl3 and
androgen receptor genes, testes are positioned adjacent to
the kidneys and steadied in the abdomen by the CSL. These findings
demonstrate, that the Insl3 induces gubernaculum development in an
androgen-independent way, while androgen-mediated regression of the CSL
occurs independently from Insl3.
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INTRODUCTION
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Since the appearance of Josts theory that male sexual
differentiation in eutherian mammals is regulated by two fetal
testicular hormones (1), several lines of evidence have demonstrated
the role of anti-Müllerian hormone, also known as
Müllerian-inhibiting substance (MIS), from the Sertoli cells for
the regression of Müllerian ducts and of androgen from the Leydig
cells for the differentiation of Wolffian ducts into vas deferens,
epididymis, and accessory glands. In female fetuses, the absence of MIS
and androgen leads to the development of the Müllerian ducts
derivatives and the passive regression of the Wolffian ducts. However,
the molecular mechanism underlying the sexual dimorphic position of the
gonads in mammals was not included in Josts theory and has received
limited attention, although the respective positions of ovary
and testis are of utmost importance for fertility. Over the last
century, numerous theories have been proposed to explain the process of
testis descent. Controversies between these theories are often centered
upon the targeted structures and factors that are involved in this
process (2, 3).
The genital mesentery of the internal genital tract is a
retroperitoneal structure that connects the gonads and genital ducts to
the abdominal wall. The differential development of two parts of the
genital mesentery, the cranial suspensory ligament (CSL) and the caudal
genital ligament, also called gubernaculum, during male and female
development has been determined and proposed to be responsible for a
sexual dimorphic position of testis and ovary (2, 3). In mammals, the
process of testis descent has been divided into two functional phases
(3). During the first or transabdominal phase, occurring between days
15.5 and 17.5 postcoitum (dpc) in murine development, the development
of the gubernaculum and regression of the CSL result in the
transabdominal movement of the testis into the inguinal region. In the
female embryo, development of the CSL and developmental impairment of
the gubernaculum keep the ovary near the kidney (Fig. 1
). During the second or inguinoscrotal phase
of testis descent, occurring in the mouse between postnatal weeks 2 and
3, the testis descends from the inguinal region to the scrotum
while the gubernaculum is inverting or regressing.

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Figure 1. Schematic Representation of Differential
Development of the Cranial Suspensory Ligament (CSL) and the
Gubernaculum during Sexual Differentiation
At embryonic day 13.5, position of the developed gonad is identical in
male and female fetuses and attached to the abdominal wall by the CSL
and the gubernaculum. Between embryonic days 15.517.5, development of
the gubernaculum and regression of the CSL in the male embryo result in
the transabdominal descent of the testis into the inguinal region. In
the female embryo, further development of the CSL and developmental
impairment of the gubernaculum lead to sustain the ovary near the
kidney.
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To date, a substantial amount of data has accumulated to indicate the
relevance of testicular factors in the differential growth of both
ligaments during male embryogenesis. The prevention of outgrowth of the
fetal CSL is an androgen-dependent process. Prenatal exposure of
females to androgen prevent development of the CSL (4). The persistence
of the CSL in completely androgen-insensitive male mice that contain a
mutation in the gene encoding the androgen receptor (Ar),
and in male rats that were exposed to antiandrogen during fetal life,
further supports the role of androgen in suppression of the CSL
development (5, 6, 7). Development of the male-like gubernaculum in bovine
freemartin, a female fetus exposed to the blood of a male twin by
chorioallantoic anastomosis, and in female rabbit fetus that has been
grafted with a fetal testis, demonstrated the participation of fetal
testicular factors in gubernacular development (8, 9, 10). Because a
proportion of human males with persistent Müllerian duct syndrome
(PMDS) and human males with complete androgen insensitivity syndrome
have undescended testes, a potential role of both MIS and androgen in
testicular descent was suggested (3, 11). However, recent data showed a
normal testicular descent in the homozygous MIS and
MIS type II receptor mutant mice, and the outgrowth of the
gubernaculum in male mice with testicular feminization rule out the
direct action of MIS and androgen in induction of the gubernacular
outgrowth during the transabdominal descent of the testis (5, 12, 13).
These data suggested a role of a third fetal testicular factor in
gubernacular development (10, 14, 15).
We have previously characterized a novel member of the insulin-like
hormone superfamily, Insl3, which is specifically expressed
in Leydig cells of the fetal and adult testis and in the theca cells of
the postnatal ovary (16, 17). The Insl3 gene is expressed at
high levels in the adult testis and at much lower levels in the adult
ovary. Analyses of Insl3 transcripts in testis and ovary
throughout the pre- and postnatal life of the mouse revealed a sexual
dimorphic pattern of Insl3 expression during development. No
Insl3 transcripts were detected in female embryos of any
stage, whereas in male embryos transcripts were first detected at 13.5
dpc. After birth, the level of Insl3 transcription in testis
remains constant during the first 3 weeks, increases at the time at
which the first wave of round spermatids undergoes spermiogenesis, and
reaches the highest level in adult testis (18). These results led us to
suggest that the Insl3 factor plays an essential role in
differentiation and maintenance of the male phenotype and
spermatogenesis (16, 18). In the female, expression of Insl3
is first detected in the ovary at day 6 after birth. This, taken
together with the distinct expression pattern of Insl3
during the estrous cycle and pregnancy, implies a functional role of
Insl3 during follicular development (18).
To determine the role of Insl3 in sexual differentiation and
gametogenesis, we have generated mice containing a targeted disruption
of the Insl3 gene. Morphological abnormalities were only
observed in male Insl3-/- mice,
which exhibited bilateral cryptorchid testes located high in the
abdomen. To investigate the role of Insl3 in the process of the testis
descent, we have histologically analyzed gubernacular development
during transabdominal descent of the testis in the wild-type and the
Insl3 mutant males. To address the question of whether
androgen and Insl3 function independently in the development of CSL and
gubernaculum, we have generated double-mutant male mice in which the
action of both factors is eliminated. Finally, we have surgically
descended the testes of the
Insl3-/- mice in the inguinal
canal to determine the role of Insl3 for male germ cell
development.
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RESULTS
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Generation of Insl3-Deficient Mice
To elucidate the potential role of the Insl3 gene, we
deleted the gene in mice through homologous recombination. A
replacement targeting vector was designed to delete the two exons
encoding the Insl3 factor and replaced them with the neomycin
phosphotransferase (neo) gene under the control of the
phosphoglycerate kinase promoter. Introduction of a negative selection
marker, the herpes simplex virus thymidine kinase (tk) gene,
at the 3'-end of the construct (Fig. 2A
)
enabled us to use positive and negative selection (19).

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Figure 2. Targeted Disruption of Insl3 Gene,
and RNA and DNA Analysis of Generated Mice
A, Structure of the wild-type allele, targeting vector, and targeted
allele are shown together with the relevant restriction sites. A 1.8-kb
XhoI-SstI fragment containing both exons
of the gene was replaced by a pgk-neo selection cassette
(NEO). TK, Thymidine kinase cassette; B,
BamHI; S, SstI; Sa, SalI;
X, XhoI. B, Southern blotting of
BamHI-digested DNA from F2 mice, hybridized
with a 3'-external probe, revealing a 10-kb wild-type and a 14-kb
mutated fragment. C, Northern blotting of testicular RNA from
Insl3+/+,
Insl3+/-, and
Insl3-/- adult mice,
hybridized with the mouse Insl3 cDNA probe, revealing a
0.9-kb mRNA prominent in Insl3+/+, reduced
in Insl3+/-, and absent in
Insl3-/- testes.
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MP1 ES cells were transfected with the targeting vector and selected
for homologous recombination events (20). Drug-resistant clones were
selected, and DNA was isolated and screened by Southern blot analysis
using an external probe (data not shown). Three recombinant clones had
undergone homologous recombination. One clone produced germ
line-transmitting chimeras after aggregation with morula derived from
CD1 females. These chimeras were bred with CD1 and 129/Sv females to
establish the Insl3-deleted allele on a CD1 x 129/Sv
hybrid and a 129/Sv inbred genetic background. Southern blot analysis
on DNA isolated from tail biopsies was used to determine the genotype
of the offspring. Hybridization with the 3'-external probe (Fig. 2A
)
visualized a 10-kb BamHI fragment in the case of a wild-type
allele and a 14- kb fragment for a targeted allele (Fig. 2B
). Both male
and female mice heterozygous for the Insl3 mutation appeared
normal and fertile. Heterozygous animals were mated, and approximately
25% (76 of 302) of the offspring were homozygous for the null allele.
Northern blot analysis of RNA derived from testes of these mice
revealed that the Insl3-/- mice
failed to produce detectable Insl3 mRNA (Fig. 2C
). These
results confirm that the introduced mutation results in a complete loss
of Insl3 mRNA in testis of
Insl3-/- mice. The phenotypes
associated with the homozygous mutation that are described below were
on a mixed (CD1 x 129/Sv) genetic background, but were not
different from that on an inbred (129/Sv) genetic background.
Insl3 Homozygous Mutant Male Mice Are Sterile and Have
Bilateral Cryptorchidism
The pattern of Insl3 expression in ovaries at various
stages of the estrous cycle and during pregnancy showed a correlation
with follicular development (18). However, homozygous mutant females
underwent normal estrous cycles, as indicated by the cytology of
vaginal smears, and after mating with wild-type or heterozygous male
mice, they became pregnant and produced litters of normal size
[9.1 ± 0.6 (n = 18) vs. 9.8 ± 0.9 (n
= 14) in control females]. Normal folliculogenesis was observed in the
ovaries of the Insl3-deficient females (data not shown),
suggesting that the Insl3 factor is not essential for female germ cell
development or folliculogenesis.
Morphological abnormalities were only observed in male
Insl3-/- mice, which were
infertile despite normal sexual behavior toward female mice and
production of copulation plugs. Anatomical examination of the male
Insl3-/- mice revealed that the
Wolffian duct derivatives had differentiated normally into vas
deferens, epididymis, and accessory glands and no Müllerian duct
derivatives were present (Fig. 3
, A and C).
However, all Insl3-/- males
exhibited bilateral cryptorchid testes located high in the abdomen
(Fig. 3
, A and C). The testicular arteries originated in the
abdominal aorta below the renal arteries and ran just below the kidneys
in an ovarian vasculature-like fashion. No tight attachment of the
testis and epididymis to the inguinal region was found. Therefore,
gubernacular development could be affected in these mutant mice.
Torsion of the vas deferens and testicular artery and localization of
the right testis in the contralateral position did occur in some
Insl3-/- mice, presumably due to
the absence of tight attachment of the testes to the inguinal region in
combination with regression of the CSL.

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Figure 3. Testicular Position in
Insl3-/-, Ar/Y,
and Ar/Y
Insl3-/- Male Mice
A, Dissected abdominal region of a 4-week-old wild-type mouse shows the
testes (t), which were already descended into the scrotal sac, adjacent
to the bladder (b). B, Genital tract of a 4-week-old wild-type female
shows the position of ovaries (o) adjacent to the kidneys (k), uterine
horns (u), and ovarian arteries (oa). C, Free moving genital tract in
the abdomen of 4-week-old Insl3-deficient male. The
Wolffian duct derivatives are normally differentiated into epididymis
(e), vas deferens (v), and accessory glands (not shown). Note the
torsion (arrowhead) of the vas deferens and testicular
artery (ta). D, Testes of 3-week-old Ar/Y mouse located
above the bladder and attached to the abdominal wall with cranial
suspensory ligament and gubernaculum. E, Testes of 4-week-old
Ar/Y Insl3-/-
male situated adjacent to the kidneys in a comparable position as
ovaries in wild-type mouse (B).
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Roles of Androgen and Insl3 in Development of the CSL and the
Gubernaculum Are Independent
The structural abnormalities contributing to cryptorchidism in
Insl3-/- male mice are different
from those observed in male mice with testicular feminization. The
development of CSL and gubernaculum in Ar/Y mice disrupts
normal testis descent. Consequently, testes of these mice are located
at an intermediate position of ovaries and testes in wild-type mice
(Fig. 3D
). To address the question of whether androgen and Insl3
function independently in the development of both ligaments, we have
generated double- mutant male mice in which the action of both factors
is eliminated. Testes in Ar/Y
Insl3-/- mice are completely
undescended (Fig. 3E
), and tight attachment of the testes to the
inguinal region is absent. The male external and internal genitalia are
not virilized, and Müllerian and Wolffian duct derivatives are
absent. In contrast to Insl3-/-
mutant mice, the testes are situated adjacent to the kidneys in a
comparable position as ovaries in wild-type mice (Fig. 3
, B and E) and
are attached to the dorsal abdominal wall via well developed CSLs.
Insl3 Is Required for Normal Development of the
Gubernaculum during Transabdominal Descent of the Testis
The transabdominal descent of the testis coincides with the
regression of the CSL, the shortening of the gubernacular cord, and the
outgrowth of the gubernacular bulb including the differentiation of its
outer mesenchymal layer into myoblasts (4, 21, 22). Analysis of E17.5
wild-type males by scanning electron microscopy reveals that the
gubernaculum shows swelling (Fig. 4A
),
whereas the gubernaculum of the
Insl3-/- male and control female
displays a small bulb and an elongated cord (Fig. 4
, B and C). To
investigate whether the cryptorchidism found in the
Insl3-/- male mice may result from
an affected development of the gubernaculum, we have analyzed
transverse sections from fetuses at stages before (E15.5) and during
(E17.5) the transabdominal descent of the testes. At E15.5, the
gubernacular bulb of wild-type males and females and
Insl3-/- males is similar in size
and contains loose mesenchymal cells (data not shown). At E17.5, the
gubernacular bulb in wild-type males is enlarged and well developed
into mesenchyme in the center and myoblasts in circumferential layers
(Fig. 4D
). In contrast, the gubernacular bulb in the
Insl3-/- males and in the
wild-type females is poorly developed, as indicated by the lack of
structural organization into outer and inner layers (Fig. 4
, E and F).
These observations suggest that Insl3 stimulates gubernacular
development in male mice.

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Figure 4. Impaired Gubernacular Development in E17.5
Insl3-/- Male
AC, Scanning electron microscopy of the reproductive tract revealing
a swelled gubernaculum in control male (wild-type or
Insl3+/-) (A), and thin, elongated
gubernaculum in Insl3-/- male
(B) and control female (C). DE, Histological analysis of inguinal
abdomen at E17.5 shows a well developed gubernacular bulb in control
male (D), as indicated by marked differentiation into a mesenchymal
core surrounded by muscular outer layers; whereas in
Insl3-/- male (E) and control
female (F) the gubernacular bulb is undifferentiated. bl, Bladder; bw,
abdominal body wall; c, mesenchymal core; gb, gubernacular bulb; gc,
gubernacular cord; m, myogenic outer layer; o, ovary; t, testis; u,
uterus; v, vas deferens. Scale bar: DF, 100 µm.
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Insl3 Is Not Essential in Male Germ Cell Development
Histological analyses of the testes of 3-month-old
Insl3-/- mice revealed abnormal
spermatogenesis (Fig. 5
, A and B). All seminiferous
tubules showed a reduced number of spermatogonia, karyolysis of most of
the primary spermatocytes, and vacuolization of Sertoli cell cytoplasm.
Most notably, there was a complete absence of postmeiotic cells such as
spermatids and spermatozoa. Electron microscopy documented a normal
appearance of Leydig cells (data not shown). Furthermore, the testes of
Insl3-/- mice at 5 days of age
showed an intact tubular structure with normal development of Sertoli
and spermatogenic cells (Fig. 6
, A and B).
Clear signs of germ cell depletion were observed at 2 weeks of age when
the first wave of spermatogenic cells undergoes meiotic divisions (Fig. 6
, C and D). The fact that there is an increase in the testicular
expression level of the Insl3 after the third week of
postnatal development raises the question of whether germ cell
depletion reflects a primary or a secondary defect. The cryptorchid
testes of 3-week-old Insl3-/- mice
were surgically displaced and fixed in the inguinal canal. Three months
after the operation, mating of four
Insl3-/- male mice with wild-type
females failed to produce any offspring, and no spermatozoa were
detected in uteri of the females possessing vaginal plugs. Histological
examinations of the surgical descended testes of the
Insl3-/- mice revealed occurrence
of normal spermatogenesis in most seminiferous tubules (Fig. 5C
) and
the presence of sperm in the epididymis (data not shown). Taken
together, these observations demonstrate that Insl3 is not essential
for male germ cell development. The germ cell depletion in abdominal
testes of Insl3-/- mice might be
attributed to a higher testis temperature, which is known to affect
spermatogenesis.

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Figure 5. Spermatogenesis in Cryptorchid and Surgically
Descended Testes of Insl3-/-
Mice
A, Histology of a descended testis from a 3-month-old adult control
shows normal spermatogenesis. B, Section through a seminiferous tubule
of cryptorchid testis from a 3-month-old
Insl3-/- male revealing
karyolysis of pachytene spermatocytes and the absence of spermatids or
spermatozoa. Sertoli cells contain lipid-filled or empty vacuoles
(arrows). C, Three months after operation, the descended
testis from an Insl3-/- male
shows normal spermatogenesis and presence of mature spermatids in most
of the seminiferous tubules. Magnification: AC, x490.
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Figure 6. Testes Histology of Wild-Type and
Insl3-/- Mice at 5 and 15 Days Postpartum
A and B, Histology of a testis from a wild-type (A) and an
Insl3-/- mouse (B) at 5 days
of age showing the presence of immature Sertoli cells and
spermatogonia. C and D, Section through a testis from a wild-type (C)
and an Insl3-/- mouse (D) at
postnatal day 15 showing a few degenerated spermatocytes with condensed
and darkly stained nuclei (arrow) in the center of a
seminiferous tubule of
Insl3-/- testis.
Magnification: AD, x800.
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DISCUSSION
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To address the biological role of Insl3 in sexual differentiation
and fertility, we generated mice lacking the Insl3 gene. All
Insl3-/- male mice have bilateral
cryptorchid testes and presumably, therefore, are infertile, whereas
the Insl3-/- female mice are
fertile. This striking phenotype was displayed in all
Insl3-/- male mice regardless of
genetic background. Thus, Insl3 plays an essential role in the process
of testis descent.
Transabdominal descent of the testis from the posterior abdominal wall
to the inguinal region occurs in the fetal mouse as a result of
outgrowth of the gubernaculum and regression of the CSL (2, 3). Lack of
gubernaculum development and localization of the testis adjacent to the
kidney in E17.5 mutant males demonstrate that arrest of the testis
descent in the Insl3-/- mice takes
place during the transabdominal phase. Furthermore, a successful
initiation of the early stages of transabdominal descent is evidenced
by proliferation of the gubernacular bulb and the differentiation of
its outer mesenchymal layer into myoblasts (22). Histological analysis
of a E17.5 male mutant showed the lack of structural organization of
the gubernacular bulb into an outer layer of myoblasts and an inner
mesenchymal layer in both E17.5 male mutants and control females. These
observations and the absence of Insl3 gene expression in
female mice during fetal life suggest that Insl3 stimulates the
outgrowth and differentiation of the primordium of the gubernaculum in
male mice. Whether the Insl3 exerts its role in gubernacular
development by direct signaling, through activation of downstream genes
that are required for mesenchymal cell proliferation and development,
remains to be determined.
The involvement of a third testicular hormone in testis descent has
been described by several research groups (10, 14, 15). In an in
vitro analysis of testicular hormone action on pig fetal
gubernaculum, MIS, inhibin, or androgen could not stimulate the
proliferation of gubernacular cells (14). Normal outgrowth of the
gubernaculum in Ar/Y mice and full descended testes in the
homozygous MIS and MIS type II receptor mutant
mice (5, 12, 13) support the idea that neither androgen nor MIS but,
rather, a third testicular factor is involved in prenatal development
of the gubernaculum. Both androgen and MIS are still potentially
involved in postnatal regression/inversion of the gubernaculum during
the inguinoscrotal phase (23). We hypothesize that the Insl3 factor is
the as-yet-unidentified testicular factor, which is specifically
involved in gubernacular development. Full virilization of the male
external genitalia, normal differentiation of the Wollfian duct
derivatives into vas deferens, epididymis, and accessory glands, and
absence of Müllerian duct derivatives in
Insl3-deficient mice are a strong indication that failure of
gubernacular development in Insl3 mutant male mice is not
due to absence of androgen- and MIS-mediated activities during fetal
life.
The ovary-like position of the testes in the Ar/Y
Insl3-/- double-mutant mice,
which, similar to wild-type females, lack androgen- and Insl3-mediated
activities during prenatal development, demonstrates that the
testicular factors androgen and Insl3 are essential for the
establishment of the sexual dimorphic position of the gonads via
regulation of CSL regression and gubernacular development,
respectively. Normal regression of the CSL in the male Insl3
mutants indicates that the action of androgen on CSL regression does
not require Insl3. Furthermore, the development of the gubernaculum in
male Tfm/Y mice, which lack androgen-mediated activity,
demonstrates that the function of Insl3 in gubernacular development is
independent from androgen.
Although the pattern of Insl3 expression during postnatal
development of testis and ovary showed a correlation with
spermatogenesis and folliculogenesis (18), normal spermatogenesis and
follicle development were observed in the surgically descended testes
of Insl3-/- mice and in ovaries of
Insl3-deficient mice, respectively. These results suggest
that Insl3 is not essential for germ cell development. The germ cell
depletion in abdominal testis of
Insl3-/- mice might be attributed
to the higher testis temperature, which is known to affect
spermatogenesis (24). The infertility of the
Insl3-/- male mice with surgically descended
testis may be due to anatomical alteration of the reproductive organs
during the operation, which mechanically obstructed the transfer of the
sperm along their normal pathway from the epididymis to the uteri of
the female mice, which had a vaginal plug.
The insulin-like family ligands are structurally related to each other
and mediate many of the biological effects on cellular metabolism,
growth, and differentation through binding and activation of their
receptors, which are also structurally very similar (25, 26). It is
known that insulin can bind to the insulin-like growth factor-1
receptor (IGF-1R), and the insulin-like growth factor-I and II (IGF-I
and -II) to the insulin receptor (IR), albeit with lower affinities.
The result of targeted mutagenesis of genes encoding members of
insulin-like family ligands and receptors exhibit a growth deficiency
in mouse embryos carrying a null mutation of the gene encoding IGF-I
and II and IGF-1R, while mice homozygous for a null allele of the
insulin-1 and -2 and insulin receptor are born with apparently normal
intrauterine growth but die within hours after birth as a result of
diabetic ketoacidosis (27, 28, 29, 30, 31). The striking phenotype of the
Insl3 mutant mice described suggests that the action of
Insl3 on gubernacular development is specific and that other members of
the insulin-like family do not compensate for the lack of the Insl3
during fetal development of male
Insl3-/- mice. However, it remains
to be investigated whether the action of Insl3 on gubernacular
development is mediated through an interaction with its own receptor,
which has not yet been identified, or through cross-talk with other
members of the insulin-like receptor family located in the gubernacular
primordia.
Cryptorchidism is the most common disorder of sexual differentiation in
human males, with an incidence of 3.4% in the term newborn, which
decreases to 0.8% at 1 yr of age. Severe complications of
cryptorchidism are infertility and an increased risk for testicular
malignancy (32). The complex process of testicular descent involves a
series of hormonal and mechanical factors. Since the INSL3
gene is also present in human genome (33), INSL3 could be one of these
factors, and mutations in the gene encoding INSL3 could be a new
etiology of cryptorchidism in humans.
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MATERIALS AND METHODS
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Construction of the Targeting Vector
The Insl3-targeting vector was constructed by using
the plasmid pPNT (provided by Dr. R. Mulligan). A 7.5-kb
SstI fragment containing a 3'- flanking region of the
Insl3 gene was isolated and ligated with the
XbaI/EcoRI-digested pPNT vector after filling the
end with Klenow enzyme (clone Insl3/1) (18, 34). Finally,
the 2.0-kb SalI/XhoI fragment (Sa/I
site from polylinker of phage clone) containing a 5'-flanking region of
the Insl3 gene was isolated and inserted in the
XhoI-digested clone Insl3/1 by blunt end
ligation. The resulting 16.5-kb targeting vector was linearized with
NotI before electroporation.
ES Cell Culture, Generation of Chimeric Mice, and
Screening
The ES cell line MPI (provided by Dr. P. Gruss) was cultured as
described previously (35). Confluent plates were washed in PBS buffer
and trypsinized, and the cells were suspended in the same buffer at
2 x 107/ml. Aliquots of this cell suspension were
mixed with 30 µg of linearized targeted vector and electroporated at
250 V and 500 µF using a Gene Pulser apparatus (Bio-Rad Laboratories, Inc., Richmond, CA). Cells were plated into
nonselective medium in the presence of G418-resistant embryonic mouse
fibroblasts. Selection was applied 36 h later using medium
containing G418 at 350 µg/ml and gancyclovir at 2 µM.
After 10 days of selection, individual drug-resistant clones were
picked into 24-well trays. Three days later, individual recombinant ES
clones were replicated into 24-well trays for freezing and isolation of
DNA.
Genomic DNA was extracted from ES cells, digested with
BamHI, electrophoresed, and blotted onto Hybond
N+ membranes (Amersham, Arlington Heights,
IL). The blots were hybridized with 32P-labeled 1.3-kb
SalI/BamHI fragment (Fig. 2A
). To confirm a
correct homologous recombination event of the targeted Insl3
gene and absence of additional random integration of targeted
construct, a neomycin fragment was used to probe Southern blots.
Hybridization was carried out at 65 C overnight in the following
solution: 5x SSC/5x Denhardts solution, 0.1% SDS, and 100 µg/ml
denatured salmon sperm DNA. Filters were washed twice at 65 C to final
stringency at 0.2x SSC/0.1% SDS.
Chimeric mice from ES cells carrying the disrupted Insl3
allele were generated by aggregating 1015 compact ES cells with
2.5-day-old embryos of the CD1 mouse strain as described previously
(36). Chimeric animals obtained were mated to CD1 or 129/Sv partners,
and F1 agouti offspring were genotyped by Southern blot
analysis. Heterozygous animals were crossed to obtain homozygous mice,
which were genotyped by Southern and PCR analyses. PCR was performed
according to standard protocols to discriminate wild-type and mutant
alleles in the DNA from the mouse tails and from the head of embryos.
Primer sequences were as follows: 1 (Insl3 sense),
5'-CCGCACCTGGGAGAGGACTTC; 2 (Insl3 antisense),
5'-GTTATCCACGCTTGTCCAACC; 3 (Pgk antisense),
5'-TTCCATTGCTCAGCGGTG CTG. Thermal cycling was carried out for 30
cycles, denaturation at 94 C for 1 min, annealing at 58 C for 1 min,
and extension at 72 C for 1 min. Animal studies were conducted in
accordance with The Endocrine Society Guidelines for the Care and Use
of Experimental Animals.
RNA Analysis
Total RNA was extracted from testes of 12-week-old mice using
the RNA now Kit (ITC Biotechnologies) according to the
manufacturers recommendation. The RNA was size fractionated by
electrophoresis on a 1% agarose gel containing formaldehyde,
transferred to a nylon membrane, and hybridized with
32P-labeled Insl3 cDNA fragment under the same
conditions as used for Southern blot hybridization (18).
Generation of Ar/Y
Insl3-/-
Mutant Mice
To generate Ar/Y
Insl3-/- double-mutant mice,
females Ta Ar/++, which have tabby
variegated coats owing to X chromosome inactivation, were mated with
Insl3+/- males. Females Ta
Ar/++ Insl3+/- in the progeny
were then crossed with Insl3+/-
males. Ta Ar/Y Insl3-/-
mice, which were phenotypic females with tabby coat, were identified by
a Insl3- and a Zfy-specific PCR-based assay
(37).
Histological Analysis
Embryos (15.5 and 17.5 dpc) were collected in PBS, fixed in
Bouins fixative, embedded in paraffin, sectioned at 6 µm, and
stained with hematoxylin-eosin. Testes from 5- and 15-day-old and
12-week-old mice were fixed with 5% glutaraldehyde in 0.2
M phosphate buffer, postfixed with 2% osmium tetroxide,
and embedded in epoxy (Epon) resin. Sections at 1 µm were stained
with 1% toluidine blue/pyronine.
Scanning Electron Microscopy
After material was preserved for genotyping, the abdominal
cavity of the E17.5 was opened, and the gastrointestinal tract and the
urinary bladder were removed. After fixation by immersion in 1.5%
glutaraldehyde in Lockes solution for 12 h and dehydration in a
graded ethanol series, the embryos were critical point dried using
ethanol as the transitional and CO2 as the exchange fluid.
The dried specimens were mounted with conducting silver and spattered
with gold-palladium to a layer of about 40 nm. Specimens were examined
and photographed in a DSM 960 scanning electron microscope (Carl Zeiss, Thornwood, NY).
Surgical Transplantation of the Cryptorchid Testis into the
Inguinal Canal (Orchiopexy)
After anesthesia of 3-week-old
Insl3-/- males, the abdominal
cavity was opened by a 4-mm long transversal incision immediately below
the umbilicus. The testicular artery was cut, and the testes were
mobilized, brought down, and steadied into the inguinal canal by
suturing their capsule to peritoneum. These testes retained sufficient
vascularity from collateral blood flow through the deferential
artery.
 |
ACKNOWLEDGMENTS
|
---|
The authors are indebted to P. van der Schoot for advice with
the phenotypic analyses of the Insl3 mutant mice and help with the
histological techniques, respectively. We would like to thank R.
Shamsadin, K. Sand, H. Oberwinkler, and S. Wolf for assistance with the
generation of knock-out mice; H.-G. Sydow, U. Sancken, and A. Winkler
for scanning microscopy preparation, preparing Fig. 1
, and secretarial
help; and P. Gruss for providing MPI II ES cells. We also thank J.
A. Grootegoed and S. Bohlander for discussion, review, and
comments.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Ibrahim M. Adham, Institut für Humangenetik der Universität, Gosslerstrasse 12d, 37073 Göttingen, Germany. E-mail: iadham{at}gwdg.de
This work was supported by a grant from the Deutsche
Forschungsgemeinschaft (through SFB 271) to I.M.A.
Received for publication November 17, 1998.
Revision received January 29, 1999.
Accepted for publication February 2, 1999.
 |
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