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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
CRYPTORCHIDISM IS THE most common disorder of sexual differentiation in humans, affecting approximately 3–5% 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Generation of Insl2-Insl3 Transgenic Mice
The rat insulin II promoter-mouse Insl3 hybrid gene (Insl2-Insl3) (Fig. 1Go) 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.

 
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. 2Go, 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. 3Go) 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.

 
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. 4CGo), which are different in appearance from the scrotal sac of the wild-type male (Fig. 4AGo). 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. 4FGo). 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. 4DGo). 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. 4HGo), 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. 4KGo). 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. 4GGo). 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 12.5–17.5. The ovaries of treated transgenic mice descended further into the processus vaginalis (Fig. 4Go, I and L).



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Figure 4. Development of Processus Vaginalis and Ovarian Position

A–C, 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). D–F, 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. J–L, 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).

 
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. 5Go). 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. 5Go, A and C) and differentiated into mesenchyme in the center and myoblast in circumferential layers (Fig. 5Go, D and F). In wild-type females, the gubernacular bulb is poorly developed (Fig. 5BGo) and contains only loose mesenchymal cells (Fig. 5EGo). 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

A–C, 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). D–F. 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; D–F, 100 µm.

 
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 1Go). 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 2Go) 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. 6AGo). 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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Table 1. Reduced Fertility of Transgenic Female

 

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Table 2. Effect of Overexpression of the Insl3 on Embryo Survival

 


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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).

 
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. 6BGo).

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.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 8.5–17.5, 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go). 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 {alpha}-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 manufacturer’s 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 Locke’s 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 Bouin’s 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 {chi}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.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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