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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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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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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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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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)
enabled us to use positive and negative selection (19).
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)
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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). 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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, 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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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 Denhardt’s 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 10–15 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
manufacturer’s 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
Bouin’s 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 Locke’s 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.
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ACKNOWLEDGMENTS |
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, 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 |
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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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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