The Overexpression of the Insl3 in Female Mice Causes Descent of the Ovaries
Ibrahim M. Adham,
Gerd Steding,
Tarvo Thamm,
Erika E. Büllesbach,
Christian Schwabe,
Ilona Paprotta and
Wolfgang Engel
Institute of Human Genetics (I.M.A., T.T., I. P., W.E.), University
of Göttingen, D-37073 Göttingen, Germany; Department of
Embryology (G.S.), University of Göttingen, D-37075
Göttingen, Germany; and Department of Biochemistry and Molecular
Biology (E.E.B., C.S.), Medical University of South Carolina,
Charleston, South Carolina 29425
Address all correspondence and requests for reprints to: Dr. Ibrahim M. Adham, Institute of Human Genetics, University of Göttingen, Heinrich-Düker-Straße 12, 37073 Göttingen, Germany. E-mail
iadham{at}gwdg.de
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ABSTRACT
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Testicular descent in mice is dependent upon proper outgrowth of
the gubernaculum primordia under the influence of the insulin-like 3
gene product (Insl3). Deletion of this gene prevents gubernaculum
growth and causes bilateral cryptorchidism. In vitro
experiments have led to the suggestion that Insl3 and androgens
together induce outgrowth of the gubernacular primordia. The
experiments reported here were designed specifically to determine
whether in vivo the Insl3-mediated gubernaculum
development is independent of androgens. To that effect transgenic male
and female mice were generated that overexpressed Insl3 in the pancreas
during fetal and postnatal life. Expression of the transgenic allele in
the Insl3-deficient mice rescued the cryptorchidism in
male mutant, indicating that the islet ß-cells efficiently processed
the Insl3 gene product to the functional hormone. All transgenic
females displayed bilateral inguinal hernia. The processus vaginalis
developed containing intestinal loops. The Müllerian derivatives
gave rise to oviduct, uterus, and upper vagina, and Wolffian duct
derivatives were missing, indicating the absence of the androgen- and
anti-Müllerian hormone-mediated activities in transgenic females.
The ovaries descended into a position over the bladder and attached
to the abdominal wall via the well developed cranial suspensory
ligament and the gubernaculum. Administration of
dihydrotestosterone during prenatal development suppressed formation of
the cranial suspensory ligament and thereby allowed the descent of the
ovaries into the processus vaginalis. These results suggest that
Insl3-mediated activity induces gubernaculum development and precludes
a role of androgen in this process. Furthermore, the transgenic females
exhibit reduced fertility, which is due to fetal mortality during
midgestation.
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INTRODUCTION
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CRYPTORCHIDISM IS THE most common disorder
of sexual differentiation in humans, affecting approximately 35% of
male infants. Complications of undescended testes include infertility
and increased risk of testicular malignancy (1).
In sexually undifferentiated embryos the gonads are located on the
ventrolateral aspect of the kidney and attached to the abdominal wall
by the cranial suspensory ligament (CSL) and the gubernaculum, which
are derived from the cranial and caudal part of the gonadal mesentery,
respectively. The different positions of testes and ovaries are the
result of the differential development of both ligaments in males and
females. Regression of the CSL and growth of the gubernaculum cause
descent of the testis into the inguinal region of the abdominal cavity.
In the female, in contrast, development of the CSL and impairment of
the gubernaculum growth result in localization of the ovary lateral to
the kidneys (reviewed in Ref. 2). Gonadal hormones control
the development of both ligaments. Regression of the CSL in females
after prenatal exposure to androgens, and continued development of the
gubernaculum in males after prenatal treatment with antiandrogen,
demonstrates that androgens prevent the development of the CSL
primordia (3, 4, 5). Localization of ARs in the CSL and
development of the CSL in mice with testicular feminization, which have
a nonfunctional AR, further demonstrate the sensitivity of the CSL to
androgens (6, 7). Impairment of gubernaculum growth in
Insl3-deficient male mice leaves no doubt about the
importance of this factor in gonadal positioning (8, 9).
In the double mutant mice that lack the androgen- and Insl3-mediated
activities, the ovaries are positioned as in wild-type females, which
provides strong evidence for the essential role of both hormones in
sex-specific positioning of the gonads (8, 10).
The development of the gubernaculum during the transabdominal descent,
which occurs in mice between embryonic d 15.5 (E15.5) and E17.5, is
characterized by rapid proliferation of the mesenchymal cells and by
differentiation of the outer cellular layers into myoblasts (11, 12). During the inguinoscrotal descent of the testis, which
occurs in mice during the first 3 wk of postnatal development, the
mesenchymal cells of the gubernacular bulb gradually disappear while
the muscular layer invaginates and grows caudally into the space
of the degradated mesenchymal core of the gubernaculum bulb. The
invagination of the muscular layer in the direction of the developed
scrotum forms the processus vaginalis, which is also called the
cremastric sac (13). Contraction of the inverted cremaster
muscle and the intraabdominal pressure move the testes into the
scrotum. Because the processus vaginalis in rodents does not develop
into a narrow inguinal canal, as seen in humans, the testis can freely
pass from the scrotum to the abdominal cavity.
The Insl3, a member of the insulin superfamily, is synthesized as
preproprotein in the Leydig cells during all phases of life and, after
birth, in the theca and granulosa cells of the ovary (14, 15). Although the structure of circulating Insl3 is unknown, the
presence of certain conserved amino acids of the A and B chains at the
N and C termini of the pro-Insl3 suggests that the mode of in
vivo processing of pro-Insl3 and the resulting structure of Insl3
is similar to that of insulin and relaxin (16). It is
known that the processing of the proinsulin occurs in the trans-Golgi
by a regulated secretory pathway (17, 18), whereas in
hepatocytes, which have only a constitutive pathway of protein
secretion, proinsulin processing to insulin is extremely inefficient
(19, 20). To assess the efficiency of the pancreas in
processing the pro-Insl3 to the biologically active Insl3 hormone and
to determine the ability of the Insl3 to stimulate gubernaculum
development in female embryos, we overexpressed Insl3 in the
pancreatic islets during pre- and postnatal development. The gene was
targeted using the upstream regulatory sequences of rat insulin II
(Insl2) gene (21). The transgenic females
displayed inguinal hernia, and their ovaries had descended into the
inguinal region. Using histological and scanning electron
microscopic analyses, we investigated development of the
gubernaculum during fetal life. To determine whether the pancreatic
Insl3 would rescue cryptorchidism observed in the
Insl3-deficient males, we introduced the transgenic allele
into the Insl3-/- mice.
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RESULTS
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Generation of Insl2-Insl3 Transgenic Mice
The rat insulin II promoter-mouse Insl3 hybrid gene
(Insl2-Insl3) (Fig. 1
) was
designed to direct the overexpression of the Insl3 gene in
pancreatic ß-cells during the pre- and postnatal development of male
and female transgenic mice. The fusion gene was microinjected into the
pronuclei of fertilized FVB/N mouse eggs. One female and four males
were found to carry the Insl2-Insl3 transgene, as detected
by Southern analysis using the Insl2 promoter as probes
(data not shown). Transgenic mice were maintained as hemizygotes and
mated with either wild-type FVB/N or with transgenic hemizygotes for
further analysis. The phenotypes associated with the insertion of the
Insl2-Insl3 fusion gene, as described below, were identical
in all transgenic offspring of five founders. Therefore, one (L4) of
the five transgenic lines was further analyzed in detail.

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Figure 1. Schematic Representation of the
Insl2-Insl3 Fusion Gene
The Insl2-Insl3 fusion gene consists of 570 bp of the
promoter and 22 bp of the 5'-noncoding region of the rat insulin II
gene (solid box), and the entire mouse
Insl3 gene extending from 2 bp of the 5'-uncoding region
up to 24 bp of the 3'-flanking region. Numbered boxes
indicate location of exons of the Insl3; thin
lines show intron and 3'-flanking sequence. Translation
initiation (ATG) and termination (TAA) codons and relevant restriction
sites used for the transgene construct are indicated. B,
BamH; X, XbaI; N, NotI;
Xh, XhoI; *, Disruption of the restriction site after
cloning.
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Northern Blot and Immunohistochemical Analyses
To determine whether the Insl2-Insl3 fusion gene was
transcriptionally active, a Northern blot containing total RNA
extracted from different tissues of wild-type and hemizygous
Insl2-Insl3 mice was hybridized with the Insl3
cDNA probe. As shown in Fig. 2
, the
transgenic mice expressed the mouse Insl3 in testis and
pancreas but not in other tissues such as spleen, brain, liver, and
heart. In the wild type, the expression of the Insl3 was
restricted to the testis as described by Zimmermann et al.
(14). The expression of the Insl3 in the
pancreas did not affect the level of endogenous gene expression in the
testis, which was not significantly different in the transgenic
compared with wild-type animals. We examined wild-type and transgenic
mouse pancreas for presence of the Insl3. Thin sections of the pancreas
were assayed with polyclonal antimouse Insl3 antibody. The islets of
the transgenic mice were densely stained for the Insl3 (Fig. 3
) whereas those from the wild-type mice
were not. On the other hand, the pancreatic exocrine cells showed no
detectable staining for Insl3 in either the transgenic or nontransgenic
mice. These results indicate that the transgenic allele was expressed
and correctly translated only in the islet cells of the pancreas.

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Figure 2. Analysis of Insl3 Expression in
Adult Tissues of Wild-Type and Transgenic Mice
Total RNA (10 µg) extracted from various tissues of adult wild-type
(WT) and transgenic (TG) mice was subjected to Northern blot
hybridization using the Insl3 cDNA as a probe. Integrity
of the RNA samples was confirmed by hybridization with human elongation
factor-2 cDNA (EF-2). T, Testis; S, spleen; B, brain; L, liver; H,
heart; P, pancreas.
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Figure 3. Immunohistochemical Detection of Insl3 in Mouse
Pancreas
Sections of the pancreas of a nontransgenic control mouse (A) and
of a transgenic mouse (B) were incubated with the antiserum against
Insl3. Immunoreactive cells were restricted in pancreatic islets of the
transgenic mouse. Scale bar, 20 µm.
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Transabdominal Descent of the Ovary and Development of the
Processus Vaginalis in Transgenic Females
Female founder and all female progenies from the four founder
males display bilateral inguinal hernias (Fig. 4C
), which are different in appearance
from the scrotal sac of the wild-type male (Fig. 4A
). The inguinal
hernia becomes more prominent in the transgenic female after the third
postnatal week. Removal of the abdominal skin reveals the development
of the processus vaginalis in the hemizygous females (Fig. 4F
). The
development of the processus vaginalis in transgenic females occurred
on both sides and seems more laterally directed than in the males,
which show caudally directed outgrowth (Fig. 4D
). The inguinal hernia
contains peritoneal contents with intestinal loops. In all transgenic
females, the Müllerian derivatives are well developed and form
the oviducts, uterus, and upper vagina, whereas Wolffian duct
derivatives were absent (Fig. 4H
), indicating the absence of androgens-
and anti-Müllerian hormone-mediated activity in transgenic
females. The ovaries moved into a position over the bladder and
attached to the abdominal wall via the well developed CSLs and the
gubernacula (Fig. 4K
). The descent of the ovaries into the inguinal
region interfered with the formation of a distinct V-shaped uterus in
transgenic females, as observed in wild-type females (Fig. 4G
). The
development of both ligaments in transgenic females prevents the
descent of ovaries into the developed processus vaginalis. To further
determine whether the regression of the CSL under the influence of
androgen will cause the descent of the ovaries in the processus
vaginalis, pregnant females were treated with dihydrotestosterone
during gestation d 12.517.5. The ovaries of treated transgenic mice
descended further into the processus vaginalis (Fig. 4
, I and L).

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Figure 4. Development of Processus Vaginalis and Ovarian
Position
AC, Inguinal region of a 3-month-old wild-type male (A), wild-type
female (B), and transgenic female (C) shows the scrotum (s) of male (A)
and the inguinal hernia (ih) of transgenic female (C). DF,
Development of the processus vaginalis (pv) in a 3-month-old wild-type
male (D) and in a 3-month-old transgenic female (F). In contrast, the
processus vaginalis is not developed in a 3-month-old wild-type female
(E). G, Dissected abdominal region of a 6-wk-old wild-type female shows
the position of the ovaries (o) adjacent to the kidneys (k) and the
distinct V-shaped uterine horns (u). H, Genital tract of a 6-wk-old
transgenic female reveals the localization of the ovaries over the
bladder (b). I, Genital tract of a prenatal dihydrotestosterone-treated
transgenic female shows the descent of the ovaries into the inguinal
hernia. JL, Schematic representation reveals the differential
development of the cranial suspensory ligament (csl) and Gubernaculum
(g) in wild-type female (J), transgenic female (K), and prenatal
dihydrotestosterone-treated transgenic female (L).
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The transabdominal migration of the testis is mediated by the
outgrowing gubernaculum bulb, in particular the differentiated outer
layers of muscle cells. In contrast to wild-type female fetuses,
developmental impairment of the gubernaculum leads to retention of the
ovaries near the dorsocaudal region of the kidneys. To determine
whether the observed phenotype in the transgenic females is due to the
gubernaculum development during fetal life, we examined the
gubernaculum development in transgenic female fetuses by scanning
electron microscopic and histological analyses and compared them
with those of wild-type males and females (Fig. 5
). The gubernaculum in E17.5 is extended
between the gonad and the inguinal region and subdivided in cranial and
caudal parts, which have been named gubernacular cord and -bulb,
respectively. In the wild-type male and the transgenic female, the
gubernacular cord is shortened, whereas the bulb is enlarged (Fig. 5
, A
and C) and differentiated into mesenchyme in the center and myoblast in
circumferential layers (Fig. 5
, D and F). In wild-type females, the
gubernacular bulb is poorly developed (Fig. 5B
) and contains only loose
mesenchymal cells (Fig. 5E
). As a consequence of gubernaculum
development, testes and ovaries in wild-type males and transgenic
females are localized at a lower position in the abdomen. These data
demonstrate that the Insl3 induces development of the gubernaculum in
males as well as in transgenic females.

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Figure 5. Gubernaculum Development in E17.5 Transgenic Female
AC, Scanning electron microscopy of the reproductive tract at
E17.5 revealing a outgrowth of the gubernaculum in wild-type male (A)
and transgenic female (C), and impaired development of the gubernaculum
in wild-type female (B). DF. Histological analysis of inguinal
abdomen at E17.5 shows a well developed gubernaculum bulb in wild-type
male (D) and transgenic female (F), as indicated by differentiation
into mesenchymal core surrounded by muscular outer layers; whereas in
wild-type female (E) the gubernacular bulb is undifferentiated. b,
Bladder; c, mesenchymal core; gb, gubernaculum bulb; gc, gubernaculum
cord; m, myogenic outer layer; o, ovary; ov, oviduct; t, testis; v, vas
deferens; Scale bar, A, 500 µm; B, 1,000 µm; C, 200
µm; DF, 100 µm.
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Fertility of Transgenic Females
Breeding of the hemizygous male and female transgenic animals with
wild-type mice revealed fertile males, whereas fertility in females was
reduced. The litter size of transgenic females was significantly
smaller (4.3 ± 1.9) than that of the wild-type females (9.6
± 1.7) (Table 1
). The percentage of
hemizygous and wild-type pups did not differ from the expected ratio
(54% transgenics, 46% wild-type, n = 88). Thus, the genotype of
the mother, and not that of her pups, determined litter size. The cause
of reduced fertility was investigated by observing the follicle
development and embryo survival during gestation. Histological analysis
of ovaries from 3-month-old females showed normal folliculogenesis and
corpora lutea formation (data not shown). Females mated to wild-type
males were killed at various times, and the number of live embryos was
counted. At 8.5 d of gestation the number of living embryos of
transgenic females was the same as of wild types, whereas on d 10.5,
12.5, and 14.5 (Table 2
) only
approximately half of the embryos were surviving in the uteri of
transgenic females. We have previously shown that the expression of the
Insl3 in ovaries of wild-type mice decreased to undetectable
levels between d 8.5 and 17.5 of pregnancy (14). To
determine whether the fetal death in the transgenic females is a
consequence of the overexpression of the Insl3 in the
pancreas during midgestation, the total RNA was extracted from the
pancreas at various days of midpregnancy and subjected to blot
hybridization. The levels of Insl3 transcripts in the
pancreas did not significantly change during midgestation (Fig. 6A
). These results show that fetal death
in midgestation in transgenic females is the cause for their reduced
fertility as opposed to defective oogenesis or implantation. It is
possible that the high levels of Insl3 seen in transgenic pancreas are
causing the fetal death in midgestation.

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Figure 6. Insl3 Transgene Expression in
Females and Males During Pregnancy and During Postnatal Development
A, Northern blot with total RNA isolated from pancreas of transgenic
mice at different stages of midpregnancy. B, Northern blot with total
RNA extracted from pancreas of transgenic males at different stages of
postnatal development. Integrity and variation of loaded RNA samples
were checked by rehybridization with a probe for a human elongation
factor-2 (EF-2).
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Transgenic Insl2-Insl3 Rescued the Cryptorchid
Phenotype of the Insl3-Deficient Male Mice
The increase of the Insl3 expression in wild-type
testis after postnatal d 25 suggests that the Insl3 may play a
functional role in male fertility (14). In that context
the expression pattern of the transgenic allele was determined by
Northern blot analysis with pancreatic RNA of 10-, 20-, 30-, and
60-d-old transgenic males. The levels of expression of the
Insl2-Insl3 gene did not change during postnatal development
(Fig. 6B
).
The efficacy of the transgenic allele as regards descent of testes in
the Insl3-deficient male was tested in the
Insl3-/- male transgenic for
Insl2-Insl3. Anatomical analyses of fetuses at E17.5 and of
adult Insl2-Insl3:
Insl3-/- males revealed normal
descent of testes to the inguinal region at E17.5 and into the scrotum
of adult mice (data not shown). Breeding of these adult males with
wild-type females resulted in pregnancies with normal litter size. From
these results we conclude that pancreatic Insl3 rescues cryptorchidism
resulting from the deletion of the testicular endogenous
Insl3 gene. Furthermore, the transient increase in the level
of the Insl3 expression during postnatal development does
not affect male fertility.
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DISCUSSION
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Cryptorchidism in the Insl3-deficient male is a
consequence of impaired development of the gubernaculum during prenatal
life (8, 9). Synergistic effects of other testicular
factors in the induction of gubernaculum development could not be
excluded by these experiments. Coculture of gubernaculum explants
in vitro with testes from
Insl3+/- and
Insl3-/- or with synthetic androgen
(R1881) demonstrated that both Insl3 and androgen induce the
gubernaculum development in vitro (12). The
evidence reported in this paper shows clearly that in vivo
Insl3-mediated activity is responsible for induction and development of
the gubernaculum independent of androgen action. This conclusion is
supported by results with transgenic mice overexpressing active Insl3
in the pancreas during prenatal and postnatal development of female and
males. Furthermore, development of the gubernaculum in female mice,
which lack androgen-mediated activities during fetal life, excludes the
potential role of androgens in this developmental process.
Tissue-specific expression of the transgene has been achieved by using
the well characterized rat insulin II gene regulatory
sequences, which have been used for starting the ß-cell-specific
expression of a reporter gene from E12 in an in vivo model
system (22). The rationale for choosing the rat insulin II
promoter to direct the expression of the Insl3 is 2-fold: 1)
Insulin appears in the developing mouse pancreas at E12.5 (23, 24), which means that the pancreatic Insl3 will be produced in
male and female embryos when gubernaculum development occurs. In
contrast, endogenous Insl3 is only expressed in the male
embryos; 2) The enzymatic machinery in ß-cells that converts
proinsulin to insulin might efficiently process the pro-Insl3 to
bioactive Insl3. This proved to be correct according to our
results.
Using this strategy, we have generated five independent transgenic
lines. The expression of the Insl3 in the pancreatic islets
has been demonstrated by mRNA and protein analysis. Functional
complementation with the transgene rescued cryptorchidism in the
Insl3-/- male, which leaves no doubt
about the importance of Insl3 and the competence of the secretory
pathway in islet ß-cells to process the pro-Insl3 to a functional
hormone.
The asymmetry of the gonadal positions observed in the true
hermaphrodite, where the testes are descended on one side while on the
contralateral side there are undescended ovaries or ovotestes, suggests
that the Insl3 reaches the gubernaculum by exocrine transport and acts
locally. However, the induction of gubernacular development in
transgenic females and Insl2-Insl3:
Insl3-/- males by pancreatic Insl3
clearly shows that Insl3 exerts biological activity via an endocrine
pathway. Further support for that proposition comes from the work of
Büllesbach et al. (25), who showed also
that the human INSL3 circulates at a high level in postpubertal
males.
The testicular descent in mice is essentially a two-step process. In
the first step or intraabdominal descent, which occurs between E15.5
and E17.5, the development of the gubernaculum and regression of the
CSL cause the testis to migrate into the inguinal region. In the second
step, the inguinoscrotal descent, which occurs in the first 3 wk after
birth, the evagination of the outer myoblast layer of the gubernaculum
and the caudal extension into the developed scrotum create the
extraabdominal space into which the testes descend. Several experiments
suggest that the inguino-scrotal descent of testes is mediated by
androgens (26, 27). The development of the processus
vaginalis in the transgenic female may rule out participation of the
androgen-mediated activity in the evagination of the gubernaculum and
the formation of the processus vaginalis. Thus, our results are
consistent with the presence of an inguinal hernia in all human males
with complete androgen-insensitivity syndrome (28, 29).
Because all transgenic lines show a similar disruption of fertility in
females, it is unlikely to be caused by integration of the transgenic
allele into an unrelated gene. The follicle development in the
transgenic mice, as monitored by histological analysis, seems to be
unaffected. The pregnancies of these animals are normal until d 8.5,
but between gestation d 10.5 and 14.5 approximately half of the embryos
die. In wild-type mice, the level of Insl3 expression in
ovaries remains fairly constant between d 0.5 and 8.5, falls to a
barely detectable level between gestation d 8.517.5, and increases
again on d 18.5. It seems likely that overexpression of the pancreatic
Insl3 during midpregnancy explains the fetal death in the transgenics.
Loss of fetuses in the transgenic females mimics a disease phenotype
mouse model with a mutation in the 5a-reductase type 1 gene
(Srd5a1). In that case the elevation of estrogens in the
Srd5a1-/- mice during midgestation
causes fetal death (30). It would be of interest to
determine which tissues are targeted in the transgenic female and the
mechanism by which the overexpression of the Insl3 exerts
this lethal effect on embryos.
The development of the processus vaginalis in the
Insl2-Insl3 transgenic females resembles, to some extent,
infant girls with congenital inguinal hernias. Inguinal hernias are
common in preterm and low-birth-weight infants with a male-to-female
ratio of 8:1 (31, 32, 33). The clinical definition of the
inguinal hernia in a girl is a bulge or swelling in the inguinal
region, which becomes more prominent with increased intraabdominal
pressure such as crying or straining. The developed processus vaginalis
in patients contains peritoneal contents such as intestinal loops and,
in some cases, ovaries (34). The phenotypic similarity
between the transgenic females and infant girls with congenital
inguinal hernia led us to suggest that misexpression of
Insl3 during prenatal development or the overexpression of
the Insl3 in the mother during midgestation may be the cause
of the development of the gubernaculum and, consequently, the
development of the processus vaginalis in the affected girls.
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MATERIALS AND METHODS
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Generation of Transgenic Mice
The BamHI/XbaI fragment of pRIP1-Tag
(21), containing the rat insulin II promoter from -695 to
+8, was subcloned into the EcoRI site of pZero vector
(Invitrogen, San Diego, CA) to give the vector pZ-RIP. A
genomic Insl3 fragment with an artificial NotI at
the 5'-end and a XhoI site at the 3'-end was amplified by
PCR, using mouse genomic DNA and primers that spanned nucleotides -2
to +1,329 of the mouse Insl3 gene (14). The PCR
fragment was digested with NotI/XboI and
subcloned into the NotI/XhoI-digested pZ-RIP
vector to create pZ-RIP-Insl3 (Fig. 1
). The nucleotide sequence of the
amplified Insl3 fragment was confirmed by DNA
sequencing.
The rat insulin II promoter-mouse Insl3 hybrid gene
(Insl2-Insl3) was separated from the pZ-RTP-Insl3
construct by SpeI/ApaI, purified by agarose gel
electrophoresis, and microinjected into fertilized FVB/N eggs to
generate transgenic mice. Mice were genotyped for the presence of the
transgene by Southern hybridization analysis and by PCR designed to
amplify a region spanning the junction between the rat Insl2
promoter and the mouse Insl3 gene. Oligonucleotide primer
sequences were Insl2F, 5'-TTTGGACTATAAAGCTAGTGG; Insl3R,
5'-AGTTTGAATCCAGCCTGGTCC. Thermal cycling was carried out for 35
cycles, denaturation at 94 C for 30 sec, annealing at 54 C for 30 sec,
and extension at 72 C for 45 sec. Southern hybridization analysis of
tail-derived genomic DNA digested with SstI was performed
according to standard methods using the 570-bp
EcoRI/SsII of the rat Insl2 promoter
as probe. Transgenic lines were maintained on the genetic background of
FVB/N. All animal experiments were carried out using protocols approved
by the Medical Faculty of the University of Göttingen and
Research Advisory Committee.
Androgen-Treated Mice
Four-week-old transgenic females were mated, and the presence of
a vaginal plug was considered d 0.5. Pregnant females were injected
daily sc beginning on d 12.5 through d 16.5 with 0.5 mg 5
-dihydrotestosterone propionate (Steraloids, Hamilton, NH) in 0.1 ml
olive oil (n = 3), or with oil only (n = 3). Female offspring
were killed at the age of 4 wk, and the internal genitalia were exposed
by macroscopic dissection and photographed.
RNA Analysis
Different tissues were dissected out from 3-month-old wild-type
and transgenic mice or from the pancreas of 10-, 20-, 30-, and 60-d-old
males, and 6.5-, 8.5-, 10.5-, 12.5-, and 14.5-d pregnant
females. Total RNA was prepared using the RNA now Kit (ITC
Biotechnologies, Heidelberg, Germany) according to the manufacturers
recommendations. Total RNA (10 µg) was electrophoresed on a
1% formaldehyde gel and transferred onto a nylon membrane. The
membrane was hybridized with 32P-labeled
Insl3 cDNA fragment (14). RNA integrity was
checked by rehybridization of blots with a cDNA probe for human
elongation factor-2 (35).
Scanning Electron Microscopy and Histological Analysis
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 dried to the critical point
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).
For histological analysis, embryos (E17.5 dpc) were
collected in PBS, fixed in Bouins fixative, embedded in paraffin,
sectioned at 10 µm, and stained with hematoxylin-eosin.
Immunohistochemistry
Pancreas from wild-type and Insl2-Insl3 transgenic
mice were fixed in 10% formalin overnight and transferred to 70%
ethanol before paraffin embedding. Sections (7 µm) were dewaxed in
xylene and sequentially rehydrated. Thereafter, tissue sections were
preincubated for 1 h with 5% normal goat serum in 0.05% Triton
X-100/PBS and incubated overnight at 4 C with 1:500 rabbit anti-Insl3
antiserum, washed in PBS (three times), and then incubated with
alkaline phosphatase-conjugated goat antirabbit antibody (1:500)
(Sigma) for 30 min at room temperature. After washing in
PBS (three times) for 5 min, the immunoreactivity was revealed by
incubating the sections with a solution containing feast red
TR/naphthol AS-Mx phosphate tablets (Sigma). Sections were
counterstained for 7 min with hematoxylin.
Rabbit Antimouse Insl3 Antibodies
Mouse Insl3 was synthesized according to the cDNA sequence
(14). It consisted of a 26-amino acid residue A chain and
a 31-residue B chain linked by an insulin-like disulfide bonding
pattern. The mouse Insl3 chains were synthesized by solid-phase
chemistry and combined by sequential, site-directed disulfide bond
formation as described for human Insl3 (36). Two rabbits
received injections of 50 µg mouse Insl3 (sc) each in monthly
intervals. Antibodies were produced in the Antibody Facility at the
Medical University of South Carolina using approved protocols. Blood
collections were made about 2 wk after booster injections, and the
serum titer was determined by RIA using
125I-desaminotyrosyl mouse Insl3 as tracer and
goat-antirabbit IgG-conjugated cellulose as second antibody. The
antibody does not recognize insulin or relaxin.
Generation of Insl2-Insl3:Insl3-/-
Mice
Mating Insl2-Insl3 hemizygous males with homozygous
females for the Insl3-null mutation produced
Insl2-Insl3:Insl3-/- mice. Offspring of
this cross, which were both hemizygous for the Insl2-Insl3
transgene and heterozygous for the Insl3-null gene, were then crossed
to produce mice homozygous for the disrupted Insl3 gene and
hemizygous for the Insl2-Insl3 transgene
(Insl2-Insl3:Insl3-/-).
The wild-type and Insl3-null allele was identified as previously
described (8).
Statistical Analysis
The t test was used to determine the significance of
differences in litter size, and the
2 test was
used as an indicator of significance of genotype recognition.
 |
ACKNOWLEDGMENTS
|
---|
We would like to thank S. Schmidt, H. Riedesel, and S. Wolf for
assistance with the generation and breeding of transgenic mice; K.
Falk-Stietenroth, H.-G. Sydow, U. Sancken, and A. Winkler for
histological preparations and statistical, and secretarial help; and D.
Hanahan, for providing the pRIP1-Tag.
 |
FOOTNOTES
|
---|
This work was supported by a grant from the Deutsche
Forschungsgemeinschaft (through SFB 271) to I.M.A. and W.E., and NIH
Grant 1-RO1-HD40406-1 to C.S.
Abbreviations: CSL, Cranial suspensory ligament; E15.5,
embryonic d 15.5.
Received for publication May 15, 2001.
Accepted for publication September 28, 2001.
 |
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