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Department of Obstetrics and Gynecology, Baylor College of Medicine, Houston, Texas 77030
Address all correspondence and requests for reprints to: Dr. Alexander I. Agoulnik, Department of Obstetrics and Gynecology, 6550 Fannin Street, Baylor College of Medicine, Houston, Texas 77030. E-mail: agoulnik{at}bcm.tmc.edu.
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Introduction |
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Recently, two G protein-coupled receptors (GPRCs), LGR7 and LGR8 (originally called GREAT), have been identified as the putative receptors for RLN (15). Insulin-like factor 3 (INSL3), a specific testicular hormone, has been recognized as the cognate ligand for the LGR8 receptor (16, 17). INSL3 does not activate LGR7 in vitro, and mice deficient for Insl3 or Lgr8 have a distinct cryptorchid phenotype that does not overlap with the phenotype of RLN-deficient mice (18, 19, 20, 21). A novel member of the RLN family, RLN-3 peptide (also called INSL7), mainly expressed in the brain and testis, was initially identified as another ligand for LGR7 (22). However, the INSL7/LGR7 relationship in vivo is not quite clear because INSL7 activates and binds to two other GPCRs, GPCR135 (23) and GPCR142 (24), which are unrelated to LGR7/LGR8.
A recent study demonstrated that female mice with deletion of the Lgr7 receptor exhibit features similar to Rln1 knockout mice (25). Female mutant mice had normal fertility and litter size, but some females were incapable of delivering their pups. Additionally, as in Rln1 knockout mice, the mothers were unable to feed their pups due to poorly developed nipples. However, only a minority of male Lgr7-deficient mice in the first two generations of breeding exhibited abnormalities in spermatogenesis and decreased fertility that were described in Rln1 knockout mice (12). To further ascertain whether there is receptor redundancy in RLN signaling in vivo, we produced mice deficient for the Lgr7 gene as well as mice with deletion of both putative RLN receptors, Lgr7 and Lgr8.
Lgr7-deficient females obtained in our experiments also have severely underdeveloped nipples and are unable to feed milk to their progeny. Additionally, pulmonary fibrosis was observed as early as 1-month-old age in both male and female mutants. However, no significant abnormalities were detected in the testes or the prostate. Female mice with deletion of both Lgr7 and Lgr8 were fertile with normal litter sizes. Double mutant males do not exhibit any gross abnormalities in the testes and prostate, indicating that RLN does not signal through the Lgr8 pathway in vivo. Using Lgr7–/– mice with transgenic overexpression of Insl3, we have shown that overstimulation of the Insl3/Lgr8 pathway fails to compensate for the deletion of Lgr7, suggesting that these signaling pathways are target specific in vivo.
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Materials and Methods |
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Lgr7 gene targeting and production of mutant mice
The 4-kb fragment containing parts of the exons 15 and 17 was obtained by PCR from genomic DNA of AB2.1 ES cells with primers mLgr7ex15F/Sal, 5'-(cccgtcgac)AACCTCTTGGCAAGCATCAT-3', and mLgr7ex17R/Sal, 5'-(cccgtcgac)AAGATGGGAATCCAGCACAG-3'. An insertional targeting vector was constructed by ligation of the PCR fragment into SalI site of the pNTR-LacZ/PGKneo/loxP vector, kindly provided by Dr. R. Behringer (M. D. Anderson Cancer Center, Houston, TX). The resulted plasmid was linearized by restriction with PshAI restriction enzyme (Fig. 1A
). We electroporated targeting construct DNA into AB2.1 ES cells and selected recombinant clones with G418. Analysis of the DNA from ES clones was performed by PCR with screening primers derived from the vector backbone (T3) and from the part of the exon 17 that was not included into the targeting vector: mLgr7ex17R, 5'-TCTTTCACTGCTTCAGGTGG-3'. During homologous integration, the targeting vector was inserted into exon 17 and created a duplication of the 4-kb genomic fragment used in the targeting construct. The internal ribosomal entry site (IRES)-LacZ cassette was therefore introduced into Lgr7 gene in correct/sense orientation. The PCR with screening primers produced 4.2-kb fragment from DNA of the cells with homologous integration of the targeting construct (Fig. 1B). We injected the recombinant clone into C57BL/6J blastocysts and reimplanted these into pseudopregnant female mice using standard procedures. Chimeric males were bred to C57BL/6J females to produce mice heterozygous for targeted allele.
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). The Lgr7ko mutant allele derived from 129 ES cells produced smaller PCR fragment than the one derived from the wild-type C57BL/6 allele. Double mutant mice with mutations of two receptors (Lgr7 and Lgr8) were produced by breeding Lgr7ko mutants with crsp/crsp and Lgr8 (Greatko) mice (20, 21). The Insl3 transgenic mice (26) were obtained from Dr. I. Adham (University of Göttingen, Göttingen, Germany) and were bred to generate Lgr7ko/Lgr7ko Tg(Ins2-Insl3) animals. Details of the genetic typing for Tg(Ins2-Insl3) (26) were described previously.
RT-PCR analysis
Total RNA from mouse tissues was extracted with the TRIzol reagent (Invitrogen Life Technologies, Rockville, MD). First-strand cDNA was synthesized using the oligo(dt) primer and RETROscript kit (Ambion, Austin, TX). The following primers were used for the analysis of the Lgr7 expression: mLgr7ex15F, 5'-CAACACGGATGGGATTTCA-3'; mLgr7ex18R, 5'-GTTGTGCCAGAGTTGATGGA-3'; and for the analysis of Lgr8: Lgr8/6F, 5'-ACTGCATTACCTCTCTCAGGC-3'; Lgr8/1R, 5'-TGGAATCTCTATCCTTTCCAGG-3'. RT-PCR with primers from ubiquitously expressed porphobilinogen deaminase gene (PbgdF, 5'-GGGAACCAGCTCTCTGAGG-3', and PbgdR, 5'-TCTGGGTGCAAAATCTGG-3') were used to assess the quality of cDNA pools.
Specimen collection and analysis
For evaluation of nipples and mammary glands, the two most inferior nipples and mammary glands were dissected from 5 Lgr7–/– and wild-type virgin female mice at 6 months of age. The same two nipples and mammary glands were dissected from lactating Lgr7–/– and wild-type mice 2 d post delivery (n = 7). The nipple size was measured by multiplying the length with the thickness of the nipple in mm for all specimens. Male and female reproductive tissues from the age-matched Lgr7–/– and wild-type males and females were taken from six to 10 mice for each group. The organs were examined for signs of any gross abnormalities. The uteri and ovaries were dissected out and weighed separately from nonpregnant females, n = 8 for Lgr7–/– and n = 10 for wild-type (age between 5 and 6 months). Lungs from 1-month-old (n = 5) and 6-month-old (n = 5) Lgr7–/– and wild-type mice were examined morphologically as well as histopathologically. Quantification of collagen content was performed on five wild-type and four Lgr7–/– mice at 6 months of age.
Mouse tissues were fixed in 10% buffered formalin, embedded into paraffin, sectioned, and stained with hematoxylin and eosin to observe general morphology of tissues. Masson’s trichrome staining (Sigma Diagnostics, St. Louis, MO) was used to examine collagen fiber accumulation in tissues samples according to manufacturer’s directions. For histological analysis of LacZ expression, 6 x 6-mm samples of adult mouse tissues or whole mount embryos (embryonic d 15.5) were processed for X-gal staining. Briefly, after fixation for 60 min [0.1 M sodium phosphate (pH 7.3), 0.2% glutaraldehyde, 2% formaldehyde], samples were rinsed for 90 min in 0.02% Nonidet P-40, 2 mM MgCl2, and 0.01% sodium deoxycholate and stained with X-gal staining solution (1 mg/ml) at room temperature overnight as previously described (27). Tissue was processed through increasing concentrations of ethanol, dehydrated, and paraffin wax-embedded, and 7-µm sections were cut and counterstained with eosin.
Analysis of histological sections
Serial sections of mouse tissues from five to 10 age-matched animals of different genotypes were used for the various histological analyses. The exact number and age of animals of each genotype are indicated in Results. The means and variations within each group were calculated based on the values obtained for each animal. The parameters used for the different tissues were as follows:
Spermatogenesis.
All stages of spermatogenesis were studied in the seminiferous tubules in 10 random high power fields in each section. Five sections were studied in both Lgr7–/– (n = 8–10, 3–6 months) and Lgr7+/+ (n = 8–10, 3–6 months) animals.
Prostate.
Five hematoxylin and eosin (H&E) sections of prostatic tissue (from dorsal, ventral, and lateral lobes) were examined for each group, Lgr7–/– (n = 6) and Lgr7+/+ (n = 6). Ten random high-power fields were studied in each section, and both glandular as well as stromal elements were examined.
Uterus.
Three H&E sections of the uterine horns were evaluated at three levels (base of uterus, mid-uterus, and proximal to the oviduct). For each genotype, three to five animals were examined. Ten random high-power fields were studied. The myometrium and endometrium as well as stromal components were examined in a blinded fashion for structural abnormalities.
Lungs.
Evaluation of histopathology was performed on lungs that were not lavaged in a blinded fashion. Serial sections were stained with either H&E or Masson’s trichrome stain. Three sections were examined for every animal in each group at 1 and 6 months of age, respectively. Alveolar thickness and fibrosis were evaluated in 10 random fields at three levels in each lung for each animal. The extent of fibrosis was divided into four categories: normal lung, minimal fibrous thickening of alveoli or bronchioles, moderate thickening without damage to lung architecture, and severe fibrosis with alteration of lung architecture, each corresponding to a numerical score of 0, 1, 2, and 3 by two investigators blinded to the genotype of the animal examined. In all cases, scoring was divided into fields including only lung parenchyma and in those surrounding the vascular bundle as well as the bronchioles. Both male and female mice in both genotypes were analyzed, the number of animals in each group is indicated (see Table 3).
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Statistical analysis
The Student’s t test and ANOVA were used to assess significance of differences among the different groups. P < 0.05 was considered significant.
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). To analyze the consequences of the genetic targeting, the expression of Lgr7 was analyzed at the RNA level (Fig. 1D) using RT-PCR in two organs with high level of Lgr7 expression, namely the brain and testis. We detected Lgr7 expression in the wild-type testis and brain mRNA, using a primer pair derived from the distal part of exon 15 (before targeting vector insertion) and from exon 17 (after vector insertion) (Fig. 1A). RT-PCR failed to amplify the expected cDNA fragment from Lgr7ko/Lgr7ko mice (Fig. 1D), indicating the absence of correctly spliced Lgr7 transcripts. All heterozygous animals had a wild-type phenotype and normal fertility. Intercrossing between heterozygous parents produced homozygous animals in the expected ratios, indicating their full viability (data not shown). The homozygous mutants described below were on mixed 129xC57BL/6 background. The Lgr7ko/Lgr7ko homozygous mice did not exhibit any visible anatomical or behavioral abnormalities, except underdeveloped nipples of mammary glands in females as was described previously for RLN-deficient females (8) (see below). Contrary to previous reports concerning RLN knockout mice (12), no differences in the mean body weight of Lgr7ko/Lgr7ko male mice at 2 months of age (18.5 g ± 1.56, n = 6) were detected compared with heterozygous or the wild-type littermates (19.04 g ± 1.81, n = 5; P > 0.05).
Lgr7 expression in mouse tissues
Expression of Lgr7 was analyzed first by RT-PCR in RNAs isolated from a variety of adult wild-type organs. As shown in Fig. 2, we detected presence of the Lgr7 mRNA in heart, aorta, testis, bladder, and uterus. The highest level of expression was detected in brain and different organs of cardiovascular and reproductive systems. Despite effects of Lgr7 deletion on the lungs (see below), the expression of Lgr7 in this organ was only marginal and detected only after Southern blot hybridization of the resulted RT-PCR fragments with the Lgr7 probe (data not shown). We have established the expression pattern of the second putative receptor for RLN, Lgr8. In addition to previously described sites (brain, testis, gubernaculum), we have detected Lgr8 transcripts in other organs. Both receptors were expressed in several organs including brain, aorta, bladder, testis, prostate, seminal vesicles, epididymis, uterus, and ovary, indicating that their functions might overlap. Different splice variants of both receptors have been described previously (21, 29), and it is possible that some of these transcripts are tissue specific.
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. In the developing embryo, the expression of Lgr7 was most prominent in the genital papilla (Fig. 3A).
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Previously, it has been shown that RLN-deficient and Lgr7-deficient females have a prolonged parturition time (8, 25). In our colony, five mutant females of 56 (9%) were unable to deliver pups within 10 h and had to be killed. In two of these animals, dead pups were found entrapped within the birth canal. In all cases, the majority of pups were dead. None of the wild-type females demonstrated any abnormalities with delayed parturition.
We compared weight of the uteri and ovaries in adult nonpregnant 6-month-old mutant (n = 8) and wild-type females (n = 10) and found no significant differences between the two groups (Table 1). Histological analysis did not reveal any structural abnormality in the Lgr7–/– uteri (Fig. 4). Contrary to the results obtained with RLN-deficient mice, we have not detected any abnormalities in the testes or prostate in the Lgr7ko/Lgr7ko males. The mean weight of the testes was the same in wild-type and mutant homozygous males (Table 1). Histopathologic analysis of the testes and prostate glands of 1-month and at 6-month age males did not reveal any abnormalities associated with Lgr7 deficiency (Fig. 4).
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). Although the difference in nipple size was more striking between the two groups during pregnancy and lactation, this effect was observed even in nonpregnant females (Table 2). The Lgr7–/– females exhibited normal nursing behavior but were unable to feed milk to their pups. The latter died within 24 h with no milk in their stomachs unless fostered with a lactating female.
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). These abnormalities were present in varying degrees only in the mutant animals, but not in any of the wild-type littermate controls (n = 5) (Fig. 6). Morphometric analysis of both collagen deposition as well as fibrosis surrounding the vascular bundles and the bronchioles revealed a significant increase in the Lgr7–/– at 1 month in both sexes (Table 3). However, there does not appear to be significant collagen deposition in the lung parenchyma in either the wild-type or the Lgr7–/– animals (data not shown), as was reported in the Rln1ko mice (10). At 6 months, lungs of male mice showed the same difference in peribronchiolar and perivascular fibrosis, but the parenchyma appeared to be unaffected (Table 3). Quantification of the mean hydroxyproline content of lungs from Lgr7–/– (95.9 ± 31.3 µg hydroxyproline/mg lung, n = 4) and wild-type (106.9 ± 31.8 µg hydroxyproline/mg lung, n = 4) mice at 6 months of age did not reveal any significant differences in the two groups, likely due to focal areas of collagen deposition in the mutant lungs (P > 0.05).
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, no histological abnormalities were noted in the prostates in the double mutant mice.
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). Some of the Lgr7-deficient females derived from this cross were able to feed their pups despite having reduced nipple size (two of six). Morphometric analysis did not reveal a significant difference between the groups in the amount of collagen deposition or fibrosis either in the lung parenchyma or in the perivascular and peribronchial regions (Table 3). No additional gross abnormalities were noted in any other organs in either male or female double mutants.
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). In two other double mutant animals (Lgr7ko/Lgr7ko, Lgr8–/Lgr8–), the right testis was absent unilaterally, which, most likely, represented an end result of the testicular torsion. No testicular torsion or necrosis was detected in cryptorchid Lgr8–/Lgr8– males with wild-type allele of Lgr7 (n = 7) derived from the same crosses. Analysis of the testicular phenotype in Lgr8–/Lgr8– (20, 21) or our Lgr7ko/Lgr7ko colonies (more than 60 males analyzed) never revealed any such abnormality.
Phenotype of mice deficient for Lgr7 with overexpression of Insl3
Because the expression of Lgr7 and Lgr8 overlaps in several tissues and both receptors use the Gs/adenylate cyclase/cAMP signaling pathway, one might expect that Insl3 overstimulation could compensate for the deletion of Lgr7. To address this question, we created mice with transgenic overexpression of Insl3 and deficiency for Lgr7. Female mice of this genotype demonstrated differentiation of gubernaculae and descent of the ovaries in the low intraabdominal position as was reported for the Insl3 transgenics (26). The Lgr7ko/Lgr7ko females with Insl3 transgene had smaller nipples than their Lgr7ko/Lgr7+ littermates (data not shown), suggesting that the overstimulation of the Insl3/Lgr8 pathway failed to rescue the Lgr7-deficient phenotype. Majority of the females were unable to effectively deliver milk to their offspring. However, about a third of the Lgr7ko/Lgr7ko females (four of 11) (with or without transgene) were able to feed their pups. Analysis of the phenotype of female Lgr7-deficient mice with Insl3 transgene did not exhibit any rescue of the Lgr7–/– phenotype, indicating a nonoverlapping nature of RLN and Insl3 signaling in vivo in the female mice.
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Discussion |
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The phenotypic characteristics of the female Lgr7 knockout mice described here show a number of abnormal characteristics previously described in RLN-deficient mice and confirm recently published findings by Krajnc-Franken et al. (25). Male and female Lgr7–/– mice do not exhibit any gross anatomic or behavioral abnormalities and have normal fertility. Mutant Lgr7–/– and Rln1–/– females both have severely underdeveloped nipples and are unable to feed milk to their young. Interestingly, our data indicate that Lgr7–/– females had significantly smaller nipples even in the nonpregnant state. Given that circulating RLN levels in virgin mice are not detectable, it is possible that the effect seen here is due to the local paracrine action of RLN on the nipples. Lgr7-deficient female mice demonstrate negligible growth in the nipples during pregnancy and lactation as previously seen in Rln1-deficient females (8), indicating that signaling through Lgr7 plays a key role in RLN-mediated growth and development of the nipples in mice before and during pregnancy.
In crosses with Lgr8–/– and Tg(Insl3) mice, we noticed that some of the Lgr7–/– females with or without Lgr8 or Tg(Insl3) were able to feed their pups despite significantly reduced nipples. These animals had a different genetic background (C57BL/6 x 129xFVB intercrosses) compared with the original Lgr7–/– colony (C57BL/6 x 129). Thus, other genetic factors might influence the RLN signaling in target tissues. Similar genetic influences on male fertility phenotype have been previously suggested in Lgr7ko mice (25).
As in RLN-deficient mice, 9% of the Lgr7–/– females were unable to effectively deliver their pups. In some of these animals, the dead pups were entrapped within the birth canal, implicating abnormal parturition as a possible mechanism. We have established that Lgr7 is highly expressed in the uterus. Interestingly, the highest level of ß-Gal activity in mutant females with knock-in Lgr7ko allele and thus Lgr7 expression was detected in the myometrium, which is consistent with a known function of RLN in uterine contractility (4). However, histopathologic analysis of the myometrium of the mutant animals did not reveal structural or architectural abnormalities, confirming the findings described by Krajnc-Franken et al. (25). However, a functional abnormality of the myometrium due to deletion of Lgr7 cannot be ruled out. Another explanation for the abnormal parturition seen in some of the mutant females could be due to defective cervical ripening. Studies from RLN-deficient mice showed increased collagen levels in the cervix during pregnancy (9). 5
Reductase knockout mice also exhibit a phenotype of prolonged parturition thought to be due to defective cervical ripening (30). Because these mice have normal levels of RLN in the circulation compared with wild-type mice, it is thought that aberrant expression of the RLN receptor or a downstream signaling target could be the underlying defect. Nevertheless, additional studies with histopathological analysis and quantification of collagen content in the cervix of Lgr7ko mice at different stages of pregnancy will be needed to ascertain this hypothesis for a fact.
One of the striking phenotypes associated with RLN deficiency is an age-related progression of pulmonary fibrosis (10). The Lgr7ko mice obtained in our experiments demonstrate abnormal connective tissue remodeling in the lungs in varying degrees. The most severe changes were seen surrounding the bronchioles and the blood vessels as early as 1 month of age in both male and female mice. The lung parenchyma did not show significant collagen accumulation. The focal nature of fibrosis and collagen deposition may account for the fact that no significant difference was detected in the amount of total collagen in the Lgr7–/– lungs compared with wild-type at 6 months of age. However, the age-related progression of collagen accumulation as described with Rln1–/– mice (10) was not observed in Lgr7 mutant male mice. These data suggest that RLN signaling through Lgr7 appears to play some role in connective tissue remodeling in the lungs. The earlier but nonprogressive nature of the effects seen in Lgr7-deficient lungs compared with Rln1 mutants may be the result of subtle variations in genetic background or the compensatory effect of other ligands such as Rln3/Insl7 on Lgr7 receptor. Additionally, the double mutant mice bred on an FVB genetic background did not exhibit the same changes as described in the Lgr7ko animals of 129xC57BL/6 origin, again indicating a possible influence of the genetic background on the penetrance of the Lgr7ko mutation.
Comparison of the phenotypic abnormalities in Rln1 and Lgr7-deficient mice demonstrated several additional differences. The Rln1–/– males had smaller testes, with varying degrees of defects in spermatogenesis, and underdeveloped prostate. Extracellular matrix of the testis and prostate was noticeably abnormal, which correlated with an increase in the rate of cell apoptosis (12). Lgr7ko males did not exhibit any abnormalities in fertility or histopathology of the prostate in our experiments. It has been previously shown that RLN can activate Lgr8 receptor in vitro (15); and thus, Lgr8 may be responsible for the phenotypic differences between Rln1 or Lgr7-deficient mutants. To examine whether some of these effects could be due to the redundancy of RLN receptors in vivo, we created mice deficient for both Lgr7 and Lgr8 receptors. Mice with the deletion of both receptors did not exhibit any abnormalities in prostate development. Due to a cryptorchid phenotype of the Lgr8 mutants, it was difficult to compare the effect of ablation of both receptors on spermatogenesis. Nevertheless, the size of the cryptorchid testis and their histology were similar in double Lgr7–/– Lgr8–/– and single Lgr8–/– mutant males. These findings indicate that the RLN does not interact with Lgr8 in vivo.
In two of the adult double mutant males right testis was absent and in one of the males we found right testicular torsion. No such abnormalities were detected previously in any of Lgr8-deficient males (20, 21), or in Lgr7ko mice (our data and Ref.25). Of note, RLN-deficient mice do not exhibit a similar phenotype. Although these observations are preliminary and need further investigation, it seems logical to suggest that two independent factors, weakened testicular support due to abnormal connective tissue remodeling and high mobility of intraabdominal cryptorchid testis could contribute to the torsion of the vas deferens and testicular artery, leading to testicular necrosis seen only in double mutant animals.
As stated earlier, LGR7 and LGR8 respond to ligand stimulation through a cAMP-dependent pathway (15). At the same time, little is known about the downstream signaling targets of these receptors. Our data show that the overstimulation of Lgr8 by transgenic expression of its ligand Insl3 does not compensate for the lack of Lgr7. The Insl3 transgenic Lgr7–/– mice maintain the small nipple phenotype of Lgr7–/– mice. Conversely, deletion of Lgr7 did not affect gonadal descent in the Insl3 transgenics. Thus, not only is there a specificity of ligand-receptor pairing for Lgr7 and Lgr8 in target tissues, but there also appear to be distinct downstream signaling pathways.
Recently, Krajnc-Franken et al. (25) described mice with an independently obtained Lgr7 mutation. These mice exhibited the same reduction of nipple size and parturition defects as in our experiments, although the abnormalities of the lung phenotype were not described (25). A minority of the mutant males exhibited disrupted spermatogenesis and reduced fertility. This phenotype appears to be transitory, disappearing in older males from the F2 generation and in F3 mice. As noted above, in our experiments too, we noticed a great variability of all mutant characteristics in mice with different genetic backgrounds. It is possible that a more careful analysis and production of different Lgr7 mutant lines on certain permissive backgrounds will reveal additional phenotypic effects of ablation of the RLN receptor.
In summary, the data presented here demonstrate specificity of hormone-receptor pairing in the RLN peptide family of hormones. Analysis of the phenotypes of Lgr7, Rln1, and Lgr8-deficient mice, Tg(Insl3) mice, as well as animals with different combinations of these mutations, indicates that the signaling pathways for Lgr7 and Lgr8 in target tissues are distinct and nonoverlapping in vivo.
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Acknowledgments |
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Footnotes |
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Abbreviations: GPCR, G protein-coupled receptor; H&E, hematoxylin and eosin; INSL3, insulin-like factor 3; LGR, leucine-rich repeat-containing GPCR; Pgbd, porphobilinogen deaminase gene; RLN, relaxin.
Received April 21, 2004.
Accepted for publication July 6, 2004.
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-reductase type 1 knockout mice is due to impaired cervical ripening. Mol Endocrinol 13:981–992This article has been cited by other articles:
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B. M. C. McGowan, S. A. Stanley, K. L. Smith, N. E. White, M. M. Connolly, E. L. Thompson, J. V. Gardiner, K. G. Murphy, M. A. Ghatei, and S. R. Bloom Central Relaxin-3 Administration Causes Hyperphagia in Male Wistar Rats Endocrinology, August 1, 2005; 146(8): 3295 - 3300. [Abstract] [Full Text] [PDF]
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