help button home button Endocrine Society Molecular Endocrinology ENDO 08 Sessions Library
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Topilko, P.
Right arrow Articles by Charnay, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Topilko, P.
Right arrow Articles by Charnay, P.
Molecular Endocrinology 12 (1): 107-122
Copyright © 1997 by The Endocrine Society

Multiple Pituitary and Ovarian Defects in Krox-24 (NGFI-A, Egr-1)-Targeted Mice

Piotr Topilko, Sylvie Schneider-Maunoury, Giovanni Levi, Alain Trembleau, Danièle Gourdji, Marc-Antoine Driancourt, Ch. V. Rao and Patrick Charnay

U-368, INSERM (P.T., S.S.-M., G.L., P.C.) Biologie Moléculaire du Développement Ecole Normale Supérieure 75230 Paris, France
Laboratory of Molecular Biology (G.L.) Advanced Biotechnology Center-IST Genova, Italy
URA CNRS 1414 (A.T.) Ecole Normale Supérieure 75230 Paris, France
U-159, INSERM (D.G.) Centre Paul Broca 75014 Paris, France
Institut National de la Recherche Agronomique Physiologie de la Reproduction des Mammifères Domestiques (M.-A.D.) 37380 Nouzilly, France
Laboratory of Molecular Reproductive Biology and Medicine Department of Obstetrics and Gynecology (C.V.R.) University of Louisville Health Science Center Louisville, Kentucky 40292


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The zinc finger transcription factor Krox-24 (NGFI-A, Egr-1) is encoded by an immediate-early serum response gene expressed in various physiological situations and tissues. To investigate its function, we have created a null allele. Mice homozygous for the mutation have a reduced body size, and both males and females are sterile. These phenotypes were related to defects in the anterior pituitary of both sexes and in the ovary. In the pituitary, two cell lineages expressing Krox-24 are differentially affected by the mutation: somatotropes present abnormal cytological features and are reduced in number, consistent with the decreased GH content observed in these animals; in contrast gonadotropes are normal in number, but specifically fail to synthesize the ß-subunit of LH. In the ovary, LH receptor expression is prevented, indicating an involvement of Krox-24 at two levels at least of the pituitary-gonadal axis. Our data, together with the results of a previous report describing another Krox-24 mutant allele, suggest that Krox-24 may have two distinct molecular functions in the anterior pituitary: transcriptional activation of the LHß gene in gonadotropes and control of cell proliferation and/or survival in somatotropes by unknown mechanisms.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The pituitary gland plays an essential role in vertebrates in controlling important aspects of development, reproduction, adaptation, and endocrine homeostasis. Its anterior lobe is composed of distinct glandular cell types defined by the hormones that they synthesize and release; somatotropes produce GH, gonadotropes produce LH and FSH, lactotropes produce PRL, corticotropes produce ACTH, and thyrotropes produce TSH. LH, FSH, and TSH are heterodimeric proteins, consisting of a common {alpha}-glycoprotein hormone subunit ({alpha}GSU) in association with specific ß-subunits. Proliferation of the different cell types and their secretory activities are subject to multiple controls, including neurotransmitters and neuropeptides from the hypothalamus and feedback regulations from target organs. Major advances have recently been made in elucidating the cascades of genes involved in the regulation of pituitary ontogeny (1, 2). In particular, the transcription factor gene Pit-1, which is disrupted in the mouse Snell and Jackson dwarf mutations, has been shown to be required for the specification of three lineages: somatotropes, lactotropes, and thyrotropes (3, 4). Interestingly, this transcription factor is necessary both for the initial activation of the expression of the corresponding hormones and for the proliferation and survival of the three cell types (5, 6, 7). More recently, analysis of another murine pituitary-dependent dwarfism (Ames dwarf) has led to the identification of a novel transcription factor gene, Prophet of Pit-1. Its mutation prevents Pit-1 activation and the initial determination of Pit-1 cell lineage and, therefore, also results in failure to produce GH, PRL, and TSH (8). The Lim homeobox gene Lhx3, which is expressed in the pituitary throughout development, is likely to act upstream of this transcription factor cascade, since its mutation affects the determination of all pituitary cell lineages except the corticotropes (9). Finally, the nuclear receptor steroidogenic factor-1 has been shown to play an essential role in the gonadotrope cell lineage, as the disruption of its gene prevents the expression of gonadotrope-specific markers in the pituitary (10). Although these data reflect important progress, our understanding of the pituitary regulatory cascade is still very fragmentary, and several important regulators remain to be discovered.

Krox-24, also known as Egr-1, NGFI-A, and Zif268, is a zinc finger phosphoprotein that was originally identified as an immediate-early serum response or nerve growth factor response gene product (11, 12, 13, 14). This factor was subsequently shown to be rapidly induced by various stimuli (reviewed in Ref.15). Krox-24 belongs to a multigene family encoding closely related transcription factors (16, 17, 18) that bind to very similar or identical cognate GC-rich sequences and can activate transcription of a nearby gene (19, 20, 21, 22, 23). The expression pattern of Krox-24 during development is rather widespread; the sites of expression include the endothelial system, thymus, muscle, cartilage, bone, and part of the central and peripheral nervous system (24, 25). On the basis of antisense RNA experiments, the gene has been implicated in differentiation of the macrophage lineage and in control of proliferation of T lymphocytes (26, 27). Genomic footprinting experiments have also suggested its involvement in control of transcription of the platelet-derived growth factor-B gene in endothelial cells (28). However, these observations have not yet been corroborated by the analysis of mice carrying a targeted mutation of Krox-24 (29, 30, 31). Furthermore, these latter studies did not confirm the expected involvement of Krox-24 in the control of cell proliferation.

To further investigate the function of Krox-24, we have created a novel targeted allele in which the lacZ gene was introduced into the Krox-24 locus and expected to recapitulate the normal expression of this gene. Observation of these Krox-24-/- mice revealed major differences compared with the previously described mutant animals. Both males and females are sterile, whereas only females were affected in the previous study. Furthermore, in contrast to the other allele, the fertility of the mutant females cannot be restore by injection of LH. Finally, the present mutant allele leads to a reduction in body size and weight, whereas no such defect was reported in the previous study. We show that these defects result from abnormal development of both the anterior pituitary and the ovary, leading to the absence of LH and a significant reduction in GH secretion as well as the absence of LH receptor expression in the ovary. In the pituitary, our data are consistent with a direct involvement of Krox-24 in the control of LHß gene transcription and of the proliferation and/or survival of the somatotrope lineage. Therefore, Krox-24 appears to constitute a novel key regulator of anterior pituitary physiology and to play important, but different, roles in at least two distinct cell lineages.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Targeted Inactivation of the Krox-24 Gene
The Krox-24 transcription unit was disrupted in the plasmid pK24lacZneo by insertion of a cassette consisting of the lacZ gene without promoter and of the G418 resistance gene (neo) under the control of the phosphoglycerate kinase promoter. This cassette was introduced within the Krox-24 5'-untranslated region, 50 bp upstream of the initiation codon (Fig. 1AGo). This modification was expected to prevent efficient transcription and translation of the Krox-24 gene and to place the lacZ gene under the control of the Krox-24 cis-acting elements, making it a useful reporter. In addition, to exclude any low level synthesis of a functional Krox-24 protein from a putative bicistronic or spliced messenger RNA (mRNA), a frameshift mutation was introduced upstream of the DNA-binding domain. The mutant allele was, therefore, expected to be null.



View larger version (23K):
[in this window]
[in a new window]
 
Figure 1. Targeted Inactivation of the Krox-24 Gene

A, Schematic representation of the targeting vector pK24lacZneo, wild-type allele, and mutated allele, showing the lengths of the predicted restriction fragments revealed with the 3'-external probe A. The Krox-24 transcription unit is shown as an open box, and the positions of the translation initiation (ATG) and termination (TGA) codons and of the three zinc fingers (vertical arrowheads) are indicated. The targeting vector contains an insertion of a cassette containing the lacZ gene and the G418 resistance gene (neo) under the control of the phosphoglycerate kinase promoter 50 bp upstream of the Krox-24 initiation codon. In addition, a frameshift mutation has been introduced upstream of the DNA-binding domain (asterisk). Horizontal arrows show the locations of the three oligonucleotide primers used for PCR analysis. B, Southern blot analysis of SpeI-digested DNA corresponding to two positive ES clones (lanes 2 and 7) and several nonhomologous recombinants (lanes 1, 3–6, and 8). C, Example of PCR analysis of the genotype of offspring from heterozygous F1 intercrosses using the mixture of primers shown in A. The pr1-pr2 primer pair allows amplification of a 362-bp fragment from the wild-type allele and of a 5.1-kb fragment, usually not detected, from the disrupted allele. The pr1-pr3 primer pair allows amplification of a 520-bp fragment only from the disrupted allele. Lanes 1–5, DNA from five littermates showing wild-type (4), heterozygous (1, 3, 5) and homozygous mutant (2) patterns.

 
Inactivation of one allele of the Krox-24 gene in ES cells was performed by homologous recombination after electroporation with pK24lacZneo. Recombination events at the Krox-24 locus were identified among G418-resistant colonies by Southern blot analysis of ES cell genomic DNA with a 3'-external probe (Fig. 1Go, A and B). Among 350 G418-resistant colonies analyzed, 24 (7%) showed a pattern suggesting the recombination of one Krox-24 allele. These clones were further studied by Southern blot analysis using internal and 5'-external probes and were analyzed for the presence of the frameshift mutation (data not shown). Eleven clones were found to correspond to the expected replacement of the wild-type allele by a single copy of the disrupted allele with conservation of the frameshift mutation. Their karyotypes were established and were found to be normal. Four of these clones were used for injection into C57Bl6 blastocysts, and all of them led to germ-line transmission. Two chimeras, deriving from different clones, were selected to establish families of heterozygous carriers by crossing with C57Bl6 females. The presence of wild-type or Krox-24- alleles was determined by PCR amplification using three primers located in the Krox-24 and lacZ sequences (Fig. 1CGo). The heterozygous embryos or animals did not show any overt abnormality, except that 16% of the males (18 of 115 animals) were sterile, although they were able to produce vaginal plugs. The absence of Krox-24 mRNA and protein in homozygous mutant mice was verified by in situ hybridization and immunocytochemical analyses performed in several organs (Fig. 2Go, E and F, and data not shown). All of the phenotypes associated with the homozygous mutation that are described below were observed with mice derived from either clone. Therefore, they are likely to result as a direct consequence of the inactivation of Krox-24.



View larger version (116K):
[in this window]
[in a new window]
 
Figure 2. Analysis of the Expression of Krox-24 during Pituitary Development

A, Comparison of the morphology of wild-type (left) and Krox-24-/- (right) pituitaries. Note the reduction in the mutant affecting specifically the anterior pituitary. The arrow indicates the medial region of the anterior lobe, particularly reduced in the mutant animals. B–D, Revelation of ß-galactosidase activity by X-gal staining of pituitary sections from Krox-24+/- 14.5 dpc (B) and 18.5 dpc (C) embryos and 12-week-old animal (D). Note the strong expression in the neural lobe at 14.5 dpc, whereas lacZ is only activated in the anterior pituitary. In contrast, in the adult, the gene is expressed at high and moderate levels in the anterior and intermediate lobes, respectively, but not in the neural lobe, except in the vascular system. E and F, Immunohistochemical detection of the Krox-24 protein on pituitary sections from 12-week-old wild-type and Krox-24-/- mice, respectively. Inset in E, High magnification of the anterior lobe showing the presence of Krox-24 in the cytoplasm of the cells. a, Anterior pituitary; i, intermediate lobe; n, neural lobe. Scale bars: A, 500 µm; B–F, 50 µm.

 
Krox-24 Homozygous Mutant Mice Are Sterile and Have a Reduced Body Size
Homozygous Krox-24 mutant mice were born with the expected frequency from matings of heterozygotes [62 Krox-24-/- (28%), 98 Krox-24+/- (45%), and 58 wild type (27%) of 218 newborn animals]. Although they were of normal weight at birth, analysis of growth curves demonstrated that postnatal weight gain was slower than in the wild-type litter mates (Fig. 3Go). At around 2 months the difference was on the order of 20% and was maintained in older animals. This differential growth was observed in both males and females (Fig. 3Go).



View larger version (16K):
[in this window]
[in a new window]
 
Figure 3. Comparison of the Growth Curves of Wild-Type and Homozygous Mutant Krox-24 Mice

The weights of Krox-24-/- mice and their wild type littermates were measured over a period of 5 months. Each point represents the average of at least six mice. SDs are indicated. At all ages after 2 weeks, the weights of mutant male and female mice were significantly (P < 0.01) lower than those of normal littermates of the same sex.

 
After 8 weeks of age, male and female homozygous Krox-24 mutants were challenged for mating with wild-type, heterozygous, or homozygous mutant partners. Repeated attempts involving more than 40 homozygous mutant individuals consistently failed to lead to pregnancy, with one exception (a homozygous mutant male mated with a wild-type female). Furthermore, homozygous mutant females did not show behavioral signs of estrus, and homozygous mutant males did not exhibit aggressive behavior in the presence of other males, lacked sex drive, and were unable to plug females in estrus. Therefore, we conclude that both male and female Krox-24-/- mice are infertile.

Krox-24 Is Involved in the Control of LH and GH Production in the Anterior Pituitary
We tested whether a slower growth rate and sterility in mutant mice might result from the loss of pituitary functions. Morphological and histological analyses of the pituitary in adult Krox-24-/- animals revealed that the gland was grossly affected by the mutation. Its size was significantly diminished, the reduction affected specifically the anterior lobe, while the posterior or neural lobe appeared normal (Fig. 2AGo). Hormone expression in the anterior pituitary was subsequently studied by immunocytochemistry. Pituitary sections from 12-week-old wild-type and Krox-24-/- mice were analyzed with antibodies directed against each of the six anterior pituitary hormones. Whereas the proportions of ACTH-, TSHß-, FSHß-, and PRL-positive cells were not significantly different in wild-type and homozygous mutant animals, the mutation led to a clear reduction in the number of GH-positive cells and to the complete inhibition of LHß expression (Fig. 4Go). These data were confirmed by analyzing the hormone expression pattern of pituitary cells that has been dispersed and cultured for 24 h. In these experiments, the proportions of cells immunoreactive for ACTH or PRL were similar in wild-type and homozygous Krox-24 mutants, whereas in the mutants, the proportion of GH-positive cells was reduced 2-fold compared with that in wild-type animals, and no cells expressing LHß were detected (data not shown).



View larger version (105K):
[in this window]
[in a new window]
 
Figure 4. Immunohistochemical Analysis of Pituitary Hormone Expression in Mutant and Control Mice

Anterior pituitary sections from 12-week-old wild-type (A–F), Krox-24-/- (G–L), and Krox24+/- (M–R) mice were analyzed by immunohistochemistry for expression of ACTH (A, G, and M), TSHß (B, H, and N), LHß (C, I, and O), FSHß (D, J, and P), GH (E, K, and Q), and PRL (F, L, and R). In addition, in the case of heterozygous mutant pituitaries, lacZ-expressing cells were identified by X-gal staining. Note the absence of LHß-positive cells and a significant reduction in the number of GH-positive cells in the Krox-24-/- pituitary. No significant differences from the controls were observed for ACTH, FSHß, TSHß, or PRL. ß-Galactosidase activity was observed in LHß-, FSHß-, TSHß-, and GH-positive cells (arrows), but not in PRL- or ACTH-positive cells in the Krox-24+/- pituitary. Scale bars: A–L, 25 µm; M–R, 10 µm.

 
To obtain quantitative estimations of the levels of GH and LH in Krox-24-/- anterior pituitaries, we performed both Western blot and immunoassays. Western blot analysis of the levels of PRL, GH, and the two subunits of LH was carried out on pituitary extracts from 2-week-old male and female animals (Fig. 5Go and data not shown). Densitometric analysis of the autoradiograms indicated that the levels of PRL and {alpha}GSU in the mutant were not affected. In contrast, the level of GH was decreased approximately 4-fold in males and 3-fold in females compared with that in the wild-type animals. LHß could not be detected in males or females. Hormone immunoassays were performed, in addition, on older animals. In 2- to 4-month-old males, a 2.5-fold reduction of the GH content of the pituitary was observed in Krox-24-/- animals compared with that in controls [wild-type, 14.8 ± 0.8 µg (n = 4); homozygous mutant, 5.9 ± 0.8 µg (n = 4); P < 0.0003, by Student’s t test]. In females of the same age, the reduction observed in the homozygous mutants was less dramatic [wild-type, 7.7 ± 1 µg (n = 4); homozygous mutant, 5.2 ± 0.4 µg (n = 4); P < 0.06]. Taken together, these data indicate a clear decrease in GH content in homozygous pituitaries and suggest that this phenotype becomes less severe as the animals age. LH levels were below the detection limit of our RIA in the serum of both male and female 1- to 4-month-old Krox-24-/- mice, indicating an at least 6-fold reduction compared with levels in wild-type littermates (data not shown).



View larger version (27K):
[in this window]
[in a new window]
 
Figure 5. Western Blot Analysis of the Presence of Hormones in the Pituitary Gland

Samples were prepared from pituitary glands dissected from wild-type (+/+) or Krox-24-/- (-/-) 2-week-old male littermates. The blots were treated with polyclonal antibodies directed against mouse PRL, GH, denatured rat LHß, or {alpha}GSU as indicated. Positions of mol wt markers are indicated.

 
Krox-24 shows a widespread expression pattern during development and in the adult (24, 25) (data not shown), including, in particular, the hypothalamus, which regulates pituitary hormone secretion. It was thus important to investigate whether the changes in GH and LH observed in the mutant reflected a direct role of Krox-24 in the pituitary. To address this question, we analyzed the presence of the protein in the pituitaries of adult mice by immunocytochemical analysis. Although in the intermediate and neural lobes no signal or very low levels were detected, high levels of Krox-24 were observed in the anterior lobe (Fig. 2EGo). Surprisingly, the majority of the protein appeared to be localized in the cytoplasm (Fig. 2EGo). As expected, no expression was observed in homozygous mutant animals (Fig. 2FGo). In accordance with the immunocytochemical data, analysis of ß-galactosidase activity in heterozygous animals indicated a very high level of expression in the anterior lobe (Fig. 2DGo), confirming that the expression of this reporter faithfully reflects normal Krox-24 expression. ß-Galactosidase activity was also detected in the intermediate lobe, although at a lower level, whereas the neural lobe was completely negative except in the vascular system. To precisely correlate hormone and Krox-24 expression in the anterior lobe, double labeling experiments were performed on heterozygous mutant pituitaries. Specific hormone synthesis was revealed by immunocytochemistry, and Krox-24 expression was detected by X-gal staining, and the sections were examined at high magnification. X-gal staining was highly localized within the cytoplasm (Fig. 4Go), as previously observed in the case of another knock-in experiment (32). The observations suggested that most, and possibly all, GH-, LHß-, FSHß-, or TSHß-positive cells expressed lacZ (Fig. 4Go, N–Q). In contrast, ACTH-positive cells were not labeled by X-gal (Fig. 4MGo). Similarly, most PRL-positive cells did not express lacZ (Fig. 4RGo). Nonetheless, a few cells expressing PRL at a low level were also found to be X-gal positive. It is possible that these latter cells produce both PRL and GH (33, 34).

In conclusion, our data suggest that Krox-24 expression in the anterior pituitary is restricted to specific cell types, including those expressing GH and LH. This supports the idea that the defect in GH and LH production in the homozygous mutant animal is a consequence of the absence of the Krox-24 protein in the corresponding cells.

Krox-24 Is Required for Normal Development of the Anterior Pituitary
If Krox-24 is continuously required for efficient expression of LH and GH in adult mice, its transcriptional activation should precede that of these hormones during pituitary development. Transcription of GH and LHß has been shown to be initiated around 15.5 and 16.5 days postcoitum (dpc), respectively (35). We have determined the pattern of expression of Krox-24 in the pituitary during development by X-gal staining in heterozygous mutant mice. We find that the gene is activated around 14.5 dpc in the anterior pituitary, before GH and LHß activation as anticipated (Fig. 2BGo). Interestingly, at this stage Krox-24 is expressed at a much higher level in the neural lobe. At 18.5 dpc, expression has increased dramatically in the anterior lobe, is maintained in the neural lobe, and begins to appear in the intermediate lobe (Fig. 2CGo). Later, expression disappears in the neural lobe, while it continues to increase in the anterior and intermediate lobes (Fig. 2DGo). Therefore, the pattern of expression of Krox-24, as deduced by X-gal staining, is consistent with its involvement in the control of GH and LH expression during development.

Morphological analysis of the Krox-24-/- pituitary indicated a dramatic and specific reduction of the anterior lobe (Fig. 2AGo). To determine the precise cytological consequences of the mutation, an ultrastructural analysis was performed. In 4- to 6-week-old mutant mice, no obvious alteration was observed for the small secretory granule-bearing cells likely to correspond to corticotropes, gonadotropes, and thyrotropes. In contrast, an abnormal number of large granule-bearing cells displayed dilated endoplasmic reticulum (ER) saculae (Fig. 6Go, A and B). These cells were identified by immunogold labeling as somatotropes (Fig. 6BGo, inset). The ER alteration in these cells varied from slight dilatations of individual ER cisternae to extremely large vacuole-like dilatations occupying most of the cytoplasm of the cell; the few remaining secretory granules were restricted to the cell periphery. Such cells were found to coexist with somatotropes displaying a normal appearance. The distribution of the cells presenting a dilated ER complex varied within the anterior lobe of Krox-24-/- mice; some areas contained many, and others fewer. We also noted that among the pituitaries originating from homozygous Krox-24-/- mice, smaller pituitaries contained more of those abnormal cells compared with larger glands. Such a variability prevented a precise determination of the ratio of somatotropes presenting the abnormal phenotype. Nevertheless, although some somatotropes displaying a slightly dilated ER were also observed in wild-type pituitaries, it was unambiguous that the number of these cells was greatly enhanced in Krox-24-/- pituitaries. Moreover, cells displaying the most pronounced dilatations were only observed in homozygous mutant animals. In conclusion, these data are consistent with a lesion specifically affecting a subset of the somatotropes and possibly explaining the reduction of the number of immunoreactive GH-producing cells in mutant animals.



View larger version (173K):
[in this window]
[in a new window]
 
Figure 6. Electron Microscopic Observation of Mutant and Wild-Type Pituitaries

Low magnification images allow the comparison of the different cell types between wild-type (A) and Krox-24-/- (B) mice. In the wild-type anterior pituitary, the majority of somatotrope-like cells are characterized by large secretory granules (350–400 nm) and possess a lamellar rough ER (arrow). In the Krox-24 homozygous mutant pituitary, typical somatotrope-like cells are present (arrow), but many of the large secretory granules bearing cells appear abnormal, with a dilated ER (large arrowheads). ER dilatations appear extremely pronounced in some of these cells (star). Using immunogold labeling, these abnormal cells were identified as somatotropes (inset). Small arrowheads show labeled secretory granules. Scale bars: A and B, 5 µm; inset, 0.5 µm. n, Nucleus; c, capillary; d, dilated ER.

 
Krox-24 Inactivation Affects the Development of Male and Female Reproductive Systems
As indicated above, both male and female Krox-24-/- animals are sterile. Therefore, we have analyzed their reproductive systems to investigate the origin of this phenotype and its possible link to the pituitary deficiency. In males, the reproductive system was clearly impaired by the Krox-24 mutation. The testes, the seminal vesicles and the prostate were severely reduced in size, whereas the epididymis appeared unaffected (Fig. 7Go, A and D–G). At 5 months, the testes weights of the homozygous mutants were reduced by approximately 38% compared with those of the wild-type animals [wild-type, 0.125 ± 0.005 g (n = 12); homozygous mutant, 0.077 ± 0.007 (n = 12); P < 0.0001], whereas general body weight was only reduced by 20%. In the testes, the sizes of the seminiferous tubules and of the clusters of Leydig cells as well as the number of spermatozoa were reduced (Fig. 7Go, D–G). The amount of seminal fluid in the seminal vesicles was also dramatically diminished (Fig. 7Go, H and I). The sites of Krox-24 expression in the male reproductive apparatus revealed by X-gal staining of Krox-24+/- animals (epithelium of the epididymis and some Sertoli cells in the seminiferous tubules; Fig. 7Go, B and C) are not likely to provide a basis for all of these different phenotypes. In contrast, these phenotypes may all derive from the deficit in LH that is involved in controlling the differentiation of Leydig cells, in particular the synthesis of the androgens that, in turn, are required for normal development of the male reproductive system (Ref. 36 and references therein). Indeed, RIA analysis of the level of testosterone in the serum of 4- to 6-week-old males revealed a significant reduction in Krox-24-/- animals compared with the control value [wild-type, 0.72 ± 0.12 ng/ml (n = 5); homozygous mutant, 0.30 ± 0.03 ng/ml (n = 5); P < 0.04]. Furthermore, a single injection of LH in Krox-24-/- males restored fertility in about half of the animals (data not shown), establishing that LH deficiency is the main cause of male sterility.



View larger version (103K):
[in this window]
[in a new window]
 
Figure 7. Impairment of Male Reproductive System Development by the Krox-24 Mutation

A, Comparison of dissected male reproductive tracts from 16-week-old wild-type (left) and homozygous mutant (right) animals. Note severe reductions in size of the prostate (p), testes (t), and seminal vesicles (sv) in the mutant, whereas the caput (ct) and cauda (ca) of the epididymis appear close to normal. B and C, Analysis of ß-galactosidase activity in paraffin sections through the reproductive system of a Krox-24+/- male. X-gal staining is observed in epithelial cells located at the periphery of the epididymis (B) and in some Sertoli cells of the seminiferous tubules (C). D–I, Paraffin sections through the testes and seminal vesicles from 12-week-old males. Note the decrease in seminiferous tubule (T) size and in the number of both spermatozoa and interstitial Leydig cells (arrows) in the testis from the Krox-24-/- mice (E and G) compared with those in the controls (D and F). In the homozygous mutant, the seminal vesicles (I) are also severely reduced compared with those in the controls (H) and contain much smaller amounts of seminal vesicle fluid (svf). Scale bars: B–G, 40 µm; H–I, 400 µm.

 
Dissection of the female genital apparatus revealed a dramatic reduction in size of the uterus and a smaller effect on the ovary (Fig. 8Go, A, C, and D). In the uterus, the reduction affected both the endometrium and the longitudinal and circular layers of smooth muscle (Fig. 8Go, C and D). Histological analysis of the ovary indicated that the antral follicles were consistently smaller in the mutant, never exceeding 330 µm in diameter, with no evidence of corpora lutea, whereas the largest follicles in the wild-type littermates had diameters between 400–470 µm (Fig. 8Go, E and F, and data not shown). This suggests that the follicles remain immature in Krox-24-/- females and that ovulation does not occur, explaining the infertility. Although such a block is consistent with the absence of LH, the possibility of an additional direct role of Krox-24 in the gonad was not excluded, as analysis of lacZ and Krox-24 expression in Krox-24+/- and wild-type ovaries, respectively, revealed expression in the oocytes, in the thecal and granulosa cells of mature antral follicles, and in the corpora lutea (Fig. 8BGo).



View larger version (64K):
[in this window]
[in a new window]
 
Figure 8. Limited Gonadal Development in Krox-24-/- Females Is Not Solely Due to LH Deficiency

A, Comparison of dissected genital apparatus from wild-type (left) and Krox-24-/- (right) 12-week-old females, showing severe hypogonadism in the mutant. The ovary (o) and the uterus (u) are indicated. B, Analysis of ß-galactosidase activity in a paraffin section from a 12-week-old Krox-24+/- ovary. The lacZ gene is expressed in the corpora lutea (c), in the mature antral follicles (mf), and in the oocyte (arrow). A similar pattern is observed after immunostaining of a wild-type ovary with polyclonal antibody directed against Krox-24 (inset). C–H, Paraffin sections through the uterus and ovary from 12-week-old females, stained with hematoxylin and eosin. Uterine sections of wild-type (C) and homozygous mutant (D) animals demonstrate severe reductions of the mutant endometrium (em) and the longitudinal (lm) and circular (cm) layers of smooth muscle. Sections of wild-type (E) and Krox-24-/- (F) ovaries show the presence of smaller antral follicles (f) and the absence of corpora lutea (c) in the mutant. Histological analysis of uterus (G) and ovary (H) from a Krox-24-/- female after administration of PMSG and hCG. The organs were dissected 24 h after the injection of hCG. Note the significant increase in the size of the uterus (G, compare with D) and the absence of corpora lutea (H), suggesting that ovulation did not occur. Instead, the follicles appear highly hemorrhagic (hf). I and J, Paraffin sections through ovary from wild-type (I) and Krox-24-/- (J) mice immunostained with an antibody directed against the LH receptor. Note the strong reduction of receptor immunostaining in the mutant. The arrows indicate highly expressing areas in the wild type. Scale bars: A, 5 mm; B, 50 µm; C–H, 100 µm; I and J, 40 µm.

 
The Female Genital Phenotype Cannot Be Rescued by Injection of LH
To determine whether the absence of ovulation in Krox-24-/- females was only due to a deficit in LH, we attempted to rescue this phenotype by administration of PMSG/FSH and hCG/LH according to a standard protocol for induction of superovulation. Injected females (n = 24; age, 8–9 weeks) were either presented to wild-type males (n = 12) or killed 24 h after the injection of hCG (n = 12). Pregnancy was never observed, and inspection of the oviduct 24 h after injection revealed no eggs. Furthermore, histological examination of the uterus and the ovary 24 h after hCG injection did not show evidence of ovulation, although an increase in size of both the uterus and the antral follicle was observed (Fig. 8Go, G and H, and data not shown). These enlarged follicles, however, lacked signs of luteinization (i.e. enlarged cells in the thecal layer and dissociation of the cumulus cells surrounding the oocyte). In addition, no corpora lutea were observed; instead, the largest follicles appeared highly hemorrhagic (Fig. 8HGo). This could be due to a defect in follicular steroidogenesis and/or in the receptivity of follicular cells to LH. To investigate these possibilities, an immunohistochemical analysis was performed on ovarian sections from homozygous mutant and wild-type litter mates using specific antibodies directed against 17{alpha}-hydroxylase, a LH-induced steroidogenic enzyme, and LH receptor. Both mutant and wild-type follicles presented an obvious labeling in the theca of the largest follicles after staining with the 17{alpha}-hydroxylase antibody (data not shown). In contrast, the amount of LH receptor on somatic cells of the ovarian follicles was severely reduced in Krox-24-/- mice (Fig. 8Go, I and J). In conclusion, the incapacity to rescue the sterility phenotype of Krox-24-/- females by injection of LH is due to an additional defect in the ovary, involving a reduction in the level of LH receptor.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Krox-24 Constitutes a Novel Marker of Anterior Pituitary Cell Types
Analysis of lacZ expression in Krox-24+/- pituitaries indicated that Krox-24 is expressed in LH/FSH-, TSH-, and GH-positive, but not in ACTH-positive cells or in cells expressing PRL at a high level. This pattern of expression in the different cell lineages is novel, and an interesting question is whether Krox-24 constitutes a cell lineage marker. Although this possibility is consistent with observations suggesting an independent ontogenic pathway for the initial specification of the corticotrope cell lineage (9), it cannot be reconciled with those suggesting that somatotropes and lactotropes are derived from the same progenitor cells (see Refs. 1 and 37 for reviews). It will, therefore, be of interest to compare the expression of Krox-24 during embryogenesis with that of cell lineage markers such as Pit-1 to determine whether Krox-24 is indeed a lineage marker whose expression is lost at late stages in lactotrope development or whether its expression is set up independently in different cell lineages.

As thyrotropes, in contrast to somatotropes and gonadotropes, are apparently unaffected by the mutation, Krox-24 may have no role in this cell type. Alternatively, its absence could be compensated by another factor(s), possibly a member of the Krox-24 multigene family. The presence of apparently normal GH-positive cells in Krox-24-/- mice may be related to a heterogeneity of the somatotrope population and also reflect such a compensation. In line with this hypothesis, we observed that Krox-20 is expressed in a subpopulation of GH-positive cells (P. Topilko, S. Garel, and P. Charnay, unpublished observation), raising the possibility that this closely related gene may compensate for Krox-24 inactivation in this cell subpopulation.

Krox-24 Plays Essential, but Different, Roles in the Gonadotrope and Somatotrope Lineages
The data presented in this report indicate that Krox-24 is required for synthesis of LHß by gonadotropes in the anterior pituitary. This is consistent with the analysis of another mutation of the gene (30), although in the latter case, a low level of LHß was maintained in males, whereas with the present Krox-24 allele, LHß expression was completely eliminated in both males and females. In contrast to LHß, {alpha}GSU, FSHß, and TSHß are normally synthesized in Krox-24 mutants. As LHß and FSHß are synthesized in the same cells (38, 39), these data indicate that the survival and proliferation of the gonadotrope cells are not affected by the Krox-24 mutation, and that LHß synthesis is specifically prevented. In addition, these data reveal that the phenotype does not originate in the hypothalamus, which produces GnRH, as such a defect would affect both LH and FSH synthesis. Indeed, Lee and collaborators (30) have also shown that the LHß promoter contains a Krox-24 binding site and that cotransfection of a reporter construct driven by the LHß promoter with a NGFI-A (Krox-24) expression vector leads to an elevation of LHß promoter activity and that this elevation is dependent on the integrity of the Krox-24 binding site. Together with the gene inactivation studies, these data suggest that the LHß gene constitutes a direct transcriptional target of Krox-24.

We have observed that Krox-24 inactivation leads to a severe reduction in the level of GH in the pituitary of young animals, which may account for the smaller size of the homozygous mutant animals. This reduction in GH level is accompanied by a significant decrease (~2-fold) in the number of GH-positive cells, whereas the numbers of PRL-, FSHß-, TSHß-, and ACTH-positive cells are not affected. Furthermore, the size of the anterior pituitary is reduced in Krox-24-/- mutant mice, and ultrastructural analysis has revealed dramatic cytological abnormalities in many somatotropes. Such abnormalities were not observed in the little, Ames, and Snell dwarf mice (40). They may be a consequence of cell death and/or impairment of normal cell proliferation. In such a case, the Krox-24 mutation would lead to a partial, but selective, elimination of the somatotrope compartment in the anterior pituitary. The absence of effect on the lactotropes suggests that the mutation might not impair the common precursors of the somatotrope and lactotrope lineages.

Although our data do not exclude a direct involvement of Krox-24 in the transcriptional activation of the GH gene, they suggest that a large part of the decrease in GH is due to a reduction of the number of cells responsible for its expression. An argument suggesting that Krox-24 is not required for the direct transcriptional activation of the GH gene is provided by the analysis of the mutation introduced by Lee et al. (30). This mutation is likely to eliminate Krox-24 transcription factor activity (see below); nevertheless, it does not affect GH expression.

In conclusion, the Krox-24 mutation results in a deficit in somatotropes and, in turn, a low level of GH secretion. This situation is clearly different from the gonadotrope lineage, where the Krox-24 mutation does not significantly affect the number of cells, as revealed by analysis of FSH-positive cells. Therefore, Krox-24 performs different roles in gonadotrope and somatotrope lineages. In the former, it appears to act as a transcription factor required for the synthesis of one of the hormones produced by these cells, whereas in the latter, it is required for cell survival and/or proliferation, and this function is not likely to be related to its transcription factor activity.

The Two Krox-24 Mutations Are Associated with Different Phenotypes
In several respects the phenotypes associated with the Krox-24 mutation analyzed in this study contrast those reported by Lee and collaborators (30), who created a distinct mutant allele. These differences include the following. 1) There was a decrease in the level of GH in the homozygous mutants, which probably accounts for the significant reduction in the size of these animals. No such phenotypes were described in the report by Lee and collaborators. 2) Although both mutations affect LHß synthesis, the present mutation appears more stringent, because LH is eliminated in both females and males whereas in the case of Lee and collaborators a low level of LH is maintained in males. This residual level of LH in males might explain why the male reproductive apparatus is only marginally affected in their study, whereas the consequences are much more dramatic with our allele, resulting in sterility. Consistent with this interpretation, we were able to restore the fertility of Krox-24-/- males by injection of LH. 3) In our study, the female sterility phenotype was not rescued by injection of LH and FSH. We have shown that this is due at least in part to an additional defect affecting the ovaries: a severe reduction in the level of LH receptor expression in somatic cells of the ovarian follicles. This is probably a direct consequence of Krox-24 inactivation in the ovaries because the gene is expressed in these cells. Therefore, Krox-24 appears to control the development and physiology of the female reproductive system at two levels along the pituitary-gonadal axis: the synthesis of LH in the anterior pituitary and its receptor in the ovarian follicles. This observation is intriguing, and it will be interesting to assess its significance in terms of physiology and evolution. The fact that the male sterility phenotype can be rescued by LH injection suggests that LH receptor gene expression may not be affected by the Krox-24 mutation in male gonads; therefore, its regulation may be different in males and females.

The Dual Roles of Krox-24 in the Pituitary May Correspond to Different Molecular Functions of the Protein
It is unlikely that the differences in phenotypes associated with the two Krox-24 mutant alleles could be explained by differences in the genetic background, as the two mutations have been maintained in the same hybrid background (129/C57Bl6) (31). Although this is not a pure background, all of the phenotypes observed in homozygous mutant animals presented a full penetrance, except male sterility, with one case of fertility of 40 homozygous males. The differences in phenotypes are therefore likely to originate from the mutations themselves. Their comparison may provide us with some clues on the dual function of the protein. The present mutation involved the insertion of a lacZ-neo cassette containing a polyadenylation site in between the Krox-24-coding sequence and its promoter. This was expected to prevent transcription of the gene. In addition, a frameshift mutation was introduced into the coding sequence at the level of a NdeI restriction site corresponding to the beginning of the DNA-binding domain (Fig. 1AGo). Therefore, the resulting allele was expected to be null, and indeed, we were unable to detect either Krox-24 protein (Fig. 2FGo and data not shown) or its mRNA (data not shown) in pituitaries and other organs from Krox-24-/- mice. In contrast, the targeting construction of Lee and collaborators involved the insertion of a neo cassette at the same NdeI site. Although this is likely to eliminate the DNA-binding activity of the protein, the presence of a truncated protein corresponding to the N-terminal half of Krox-24 cannot be excluded. Therefore, we propose the following interpretation of the phenotypic differences associated with the two mutations in somatotropes. The Krox-24 protein carries at least two molecular activities. The first one, requiring the DNA-binding domain, results in transcriptional activation of target genes, for example LHß in gonadotropes. The second activity requires exclusively the N-terminal half of the protein and controls cell proliferation or survival in somatotropes by unknown mechanisms. We propose that the latter activity is not completely eliminated in the case of the mutation generated by Lee and collaborators, and that this is sufficient to allow normal survival and function of somatotropes. This model, however, does not account for the other phenotypic differences between the two alleles.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Construction of the Targeting Vector
A 12-kilobase (kb) XbaI genomic fragment containing the Krox-24-coding sequence was cloned into the XbaI site of Bluescript KS-. A 3.8-kb HindIII-XhoI fragment containing the lacZ gene and the phosphoglycerate kinase (PGK) promoter driving the neor gene from the pßGalPGKneopA plasmid (a gift from P. Soriano) was blunt ended using the Klenow fragment of DNA polymerase I and cloned into an SmaI site located 50 bp upstream of the Krox-24 initiation codon in the previous construct. In addition, an in-frame mutation was introduced at the level of a unique NdeI site localized at the beginning of the region encoding the first zinc finger by filling in the 5'-ends with the Klenow enzyme. The resulting 18.8-kb targeting vector was linearized with SalI before electroporation.

ES Cell Culture, Transfection, and Screening
ES cells (CK35 line derived from the 129/SV Pasteur strain) (41) were maintained on a feeder layer of mouse embryonic fibroblasts that were mitotically inactivated by mitomycin treatment in DMEM containing 0.05 mM ß-mercaptoethanol, 1 mM pyruvate, 2 mM glutamine, 100 U/ml penicillin/streptomycin, and 1000 U/ml leukemia inhibitory factor (all products from Life Technologies, Gaithersburg, MD) and supplemented with 15% FCS (Techgene, Les Ulis, France). Electroporation and G418 selection were performed as described previously (42). From a total of 6 x 107 transfected ES cells, 350 G418-resistant colonies were picked and transferred into separate 1.5-cm wells. These clones were subsequently expanded into two 35-mm diameter petri dishes for freezing and DNA analysis. Genomic DNA extraction and Southern blot analysis were performed as described previously (42).

Generation and Screening of Krox-24 Mutant Mice
Krox-24+/- ES cells were injected into C57Bl/6J blastocysts to obtain chimeras (42). Male chimeras were bred with C57Bl/6J females to test for germ-line transmission of the agouti coat color marker. The presence of the Krox-24-disrupted allele was detected by PCR analysis of tail genomic DNA. A mixture of three oligonucleotides was used for PCR amplification: a common 5'-primer mapping in the Krox-24 coding sequence (5'-GAGTGTGCCCTCAGTAGCTT-3') and two different 3'-primers, mapping in the Krox-24-coding sequence (5'-GGTGCTCATAGGGTTGTTCGCT-3') and in the lacZ-coding sequence (5'-AACGACTGTCCTGGCCGTAACC-3'), respectively. PCR amplification was performed with Taq polymerase (Eurobio) under the conditions recommended by the supplier and involved 30 cycles consisting of 1 min at 92 C, 2 min at 60 C, and 2 min at 72 C. Animal studies were conducted in accord with The Endocrine Society Guidelines for the Care and Use of Experimental Animals.

ß-Galactosidase Staining and Histological and Immunohistochemical Analyses
For analysis of the lacZ expression pattern in whole embryos (14.5–18.5 dpc), the animals were fixed for 30 min in Kryofix (Merck, Rahway, NJ), cryoprotected in 30% sucrose, embedded in Tissue-Tek, (Sakuraus Finetek, Torrance, CA), and frozen at -60 C. Ten-micron cryostat sections were prepared and stained with X-gal as described previously (22). Testes, ovaries, and pituitaries from postnatal animals were dissected; fixed overnight in 35% methanol, 35% acetone, and 5% acetic acid; embedded in paraffin; and sectioned at 6 µm. Some sections were stained with hematoxylin and eosin. Immunohistochemistry was performed at the indicated dilutions with polyclonal antisera against mouse PRL (AFP 131078; 1:300), rat GH (AFP 411S; 1:500), rat LHß (AFP 22238790; 1:300), rat FSHß (85GP9691bFSHb; 1:300), rat TSHß (AFP 1274789; 1:1000), rat ACTH (AFP 39013082; 1:1000; all antibodies from the National Hormone and Pituitary Program, NIDDK, Bethesda, MD), the 19 amino acids mapping at the C-terminal terminus of mouse Krox-24 (1:50; Santa Cruz Biotechnology, Santa Cruz, CA), and rat LH receptor (amino acids 15–38; 1:500). Alkaline phosphatase-conjugated secondary antibodies (guinea pig, rabbit, and human kits, Vector Laboratories, Burlingame, CA) were used with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate as chromogen. Normal serum was substituted for the primary antibody in negative controls. In the case of the LH receptor, immunostaining was performed with an avidin-biotin-immunoperoxidase procedure.

Western Blotting
Single pituitary glands from wild-type and Krox-24-/- mice were homogenized directly in 50 µl Laemmli sample buffer and boiled for 3 min. Aliquots of the cleared samples were analyzed by SDS-PAGE (43), and the total protein concentration was estimated by Coomassie blue staining of the gels and densitometric analysis. The protein concentration was then normalized by addition of Laemmli sample buffer. The samples were boiled again, and the proteins were resolved by SDS-PAGE on 15% polyacrylamide gel and then electrophoretically transferred to a polyvinylidene difluoride membrane (Amersham, Arlington Heights, IL). The presence of the hormones was revealed by incubating the membrane overnight with antihormone antibodies at the appropriate dilution followed by 1-h treatment with a peroxidase-conjugated secondary antibody and the ECL (Amersham) detection system. Densitometric analysis was performed with an Eagle Eye image analysis system (Stratagene, La Jolla, CA).

Hormone Immunoassays
For the different hormones, samples from the same litter were analyzed within the same assay. GH enzyme immunoassay was performed on pituitary extracts. The pituitaries were snap-frozen and stored at -80 C until analysis. They were homogenized in 10 mM phosphate buffer (pH 7.4), 0.9% NaCl supplemented with sodium deoxycholate (0.5%), Nonidet P-40 (0.5%), 200 µM phenylmethylsulfonylfluoride, and 1 µM pepstatin. The lysates were centrifuged (15,000 x g, 10 min, 4 C) after a brief ultrasonic disruption. Aliquots of the supernatants were analyzed using a rat GH double antibody enzyme immunoassay for rat GH, validated for mouse GH (Ezan E. et al., submitted for publication). Results were expressed as micrograms of rGH equivalent of the NIDDK standard rat GH RP-2 per anterior pituitary. Free LHß in the serum was quantitated using a Reprokit (Sanofi, Milan, Italy). Testosterone was quantified in the blood obtained after decapitation using the RIA described and validated by Hochereau de Reviers et al. (44). The intraassay coefficient of variation was 8.5%, and the sensitivity of the assay was 0.1 ng/ml.

Electron Microscopy
Pituitaries were dissected from 4- to 6-week-old mice, cut in small pieces, and immersed for 3 h at 4 C in the fixative solution [2% paraformaldehyde and 1% glutaraldehyde diluted in 0.1 M phosphate buffer, pH 7.5 (PB)]. In some experiments, mice were fixed by intraaortic perfusion with the same fixative before being postfixed by immersion as described above. After fixation, the tissues were rinsed in PBS, postfixed in 1% osmium tetroxide in PB for 1 h, rinsed in 80 mM maleate buffer (pH 4.45), and treated en bloc with uranyl acetate (2% in 85 mM maleate buffer, pH 4.75) before being dehydrated and embedded in Araldite. For immunogold detection of GH, ultrathin sections were obtained and pretreated with 5% hydrogen peroxide diluted in PBS (5 min) and with 50 mM glycine in PBS (10 min), then blocked in PBS containing 5% BSA and 10% FCS (30 min). Sections were incubated with an anti-GH rabbit antiserum (Chemical Credential (Aurora, OH) 1/100 in PBS containing 1% BSA, 2% FCS, and 0.1% Tween-20) for 3 h at room temperature, followed by a 15-nm colloidal gold-conjugated goat antirabbit antiserum (Biocell, Cardiff, U.K.; 1:100 dilution in PBS containing 1% BSA, 2% FCS, and 0.01% gelatin, 1 h at room temperature). Each incubation step was followed by three rinses in PBS. Finally, the sections were postfixed with 1% glutaraldehyde in PBS, rinsed in water, and contrasted using uranyl acetate and lead citrate.


    ACKNOWLEDGMENTS
 
We thank Dr. C. Kress for the gift of the CK35 ES cells, Dr. I. J. Mason for the gift of the antibody against 17{alpha}-hydroxylase, Dr. R. Counis for the gift of the antibodies against LHß and {alpha}GSU, Dr. P. Roche for providing the LH receptor antibodies, Drs. S. Bao and Z. M. Lei for LH receptor immunohistochemical analysis, Dr. A. Tixier-Vidal for useful discussions, and Drs. C. Kordon, J. Ghislain and M. Hausser for critical reading of the manuscript. We acknowledge the National Hormone and Pituitary Program, the NIDDK, the NICHHD, and the USDA for the gifts of antibodies directed against pituitary hormones. We acknowledge the expert technical help of the staff of the animal house.


    FOOTNOTES
 
Address requests for reprints to: Dr. Patrick Charnay, U-368, INSERM, Biologie Moléculaire du Développement, Ecole Normale Supérieure, 46 rue d’Ulm, 75230 Paris Cedex 05, France.

This work was supported by grants from Institut National de la Santé et de la Recherche (INSERM), Ministère de l’Enseignement Superieure et de la Recherche (MESR), European Economic Community (EEC), Associaton de Recherche sur le Cancer (ARC), Ligue Nationale Française Contre le Cancer (LNFCC), Associazione Italiana per la Ricerca sul Cancer (AIRC), Associazione Italiana Sclerosi Multiple (AISM), and Telethon (Italy; project D33).

Revision received October 10, 1997. Accepted for publication October 16, 1997.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Treier M, Rosenfeld MG 1996 The hypothalamic-pituitary axis: co-development of two organs. Curr Opin Cell Biol 8:833–843[CrossRef][Medline]
  2. Gage PJ, Brinkmeier ML, Scarlett LM, Knapp LT, Camper SA, Mahon KA 1996 The Ames dwarf gene, df, is required early in pituirary ontogeny for the extinction of Rpx transcription and initiation of lineage-specific cell proliferation. Mol Endocrinol 10:1570–1581[Abstract]
  3. Camper SA, Saunders TL, Katz RW, Reeves RH 1990 The Pit-1 transcription factor gene is a candidate for the murine Snell dwarf mutation. Genomics 8:586–590[CrossRef][Medline]
  4. Li S, Crenshaw ED, Rawson EJ, Simmons DM, Swanson LW, Rosenfeld MG 1990 Dwarf locus mutants lacking three pituitary cell types result from mutations in the POU-domain gene pit-1. Nature 347:528–533[CrossRef][Medline]
  5. Godfrey P, Rahal JO, Beamer WG, Copeland NG, Jenkins NG, Mayo KE 1993 GHRH receptor of little mice contains a missence mutation in the extracellular domain that disrupts receptor function. Nat Genet 4:227–232[CrossRef][Medline]
  6. Lin S-C, Lin CR, Gukovsky I, Lusis AJ, Sawchenko PE, Rosenfeld MG 1993 Molecular basis of the little mouse phenotype and implications for cell type-specific growth. Nature 364:208–213[CrossRef][Medline]
  7. Rhodes SJ, Chen R, DiMattia GE, Scully KM, Kalla KA, Lin S-C, Yu VC, Rosenfeld MG 1993 A tissue-specific enhancer confers Pit-1-dependent morphogen inducibility and autoregulation on the pit-1 gene. Genes Dev 7:913–932[Abstract/Free Full Text]
  8. Sornson MW, Wu W, Dasen JS, Flynn SE, Norman DJ, O’Connell SM, Gukovsky I, Carriere C, Ryan AK, Miller AP, Zuo L, Gleiberman AS, Andersen B, Beamer WG, Rosenfeld MG 1996 Pituitary lineage determination by the prophet of Pit-1 homeodomain factor defective in Ames dwarfism. Nature 384:327–333[CrossRef][Medline]
  9. Sheng HZ, Zhadanov AB, Mosinger B, Fujii T, Bertuzzi S, Grinberg A, Lee EJ, Huang S-P, Mahon KA, Westphal H 1996 Specification of pituitary cell lineages by the LIM homeobox gene Lhx3. Science 272:1004–1007[Abstract]
  10. Ingraham HA, Lala DS, Ikeda Y, Luo X, Shen WH, Nachtigal MW, Abbud R, Nilson JH, Parker KL 1994 The nuclear receptor steroidogenic factor 1 acts at multiple levels of the reproductive axis. Genes Dev 8:2302–2312[Abstract/Free Full Text]
  11. Milbrandt J 1987 A nerve growth factor-induced gene encodes a possible transcriptional regulatory factor. Science 238:797–799[Abstract/Free Full Text]
  12. Christy BA, Lau LF, Nathans D 1988 A gene activated in mouse 3T3 cells by serum growth factors encodes a protein with "zinc finger" sequences. Proc Natl Acad Sci USA 85:7857–7861[Abstract/Free Full Text]
  13. Lemaire P, Revelant O, Bravo R, Charnay P 1988 Two genes encoding potential transcription factors with identical DNA-binding domains are activated by growth factors in cultured cells. Proc Natl Acad Sci USA 85:4691–4695[Abstract/Free Full Text]
  14. Sukhatme VP, Cao X, Chang LC, Tsai-Morris C-H, Stamenkovich D, Ferreira PCP, Cohen DR, Edwards SA, Shows TB, Curran T, Le Beau MM, Adamson ED 1988 A zinc finger-encoding gene coregulated with c-fos during growth and differentiation, and after cellular depolarization. Cell 53:37–43[CrossRef][Medline]
  15. Gashler A, Sukhatme VP 1995 Early growth response protein 1 (Egr-1): prototype of a zinc-finger family of transcription factors. Prog Nucleic Acids Res Mol Biol 50:191–224[Medline]
  16. Chavrier P, Zerial M, Lemaire P, Almendral J, Bravo R, Charnay P 1988 A gene encoding a protein with zinc fingers is activated during G0/G1 transition in cultured cells. EMBO J 7:29–35[Medline]
  17. Crosby SD, Puetz JJ, Simburger KS, Fahrner TJ, Milbrandt J 1991 The early response gene NGFI-C encodes a zinc-finger transcriptionnal activator and is a member of the GCGGGGGCG (GSG) element-binding protein family. Mol Cell Biol 11:3835–3841[Abstract/Free Full Text]
  18. Patwardhan S, Gashler A, Siegel MG, Chang LC, Joseph LJ, Shows TB, Le Beau MM, Sukhatme VP 1991 EGR3, a novel member of the Egr family of genes encod-ing immediate-early transcription factors. Oncogene 6:917–928[Medline]
  19. Christy BA, Nathans D 1989 DNA-binding site of the growth factor-inducible protein Zif268. Proc Natl Acad Sci USA 86:8737–8741[Abstract/Free Full Text]
  20. Chavrier P, Vesque C, Galliot B, Vigneron M, Dollé P, Duboule D, Charnay P 1990 The segment-specific gene Krox-20 encodes a transcription factor with binding sites in the promoter region of the Hox-1.4 gene. EMBO J 9:1209–1218[Medline]
  21. Lemaire P, Vesque C, Schmitt J, Stunnenberg H, Frank R, Charnay P 1990 The serum-inducible mouse gene Krox-24 encodes a sequence-specific transcriptional activator. Mol Cell Biol 10:3456–3467[Abstract/Free Full Text]
  22. Sham MH, Vesque C, Nonchev S, Marshall H, Frain M, Das Gupta R, Whiting J, Wilkinson D, Charnay P, Krumlauf R 1993 The zinc finger gene Krox20 regulates HoxB2 (Hox2.8) during hindbrain segmentation. Cell 72:183–196[CrossRef][Medline]
  23. Swirnoff AH, Milbrandt J 1995 DNA-binding specificity of NGFI-A and related zinc finger transcription factors. Mol Cell Biol 15:2275–2287[Abstract]
  24. McMahon AP, Champion JE, McMahon JA, Sukhatme VP 1990 Developmental expression of the putative transcription factor Egr-1 suggest that Egr-1 and c-fos are coregulated in some tissues. Development 108:281–287[Abstract]
  25. Watson MA, Milbrandt J 1990 Expression of the nerve growth factor-regulated NGFI-A and NGFI-B genes in the developing rat. Development 110:173–183[Abstract]
  26. Perez-Castillo A, Pipaon C, Garcia I, Alemany S 1993 NGFI-A gene expression is necessary for T lymphocyte proliferation. J Biol Chem 268:19445–19450[Abstract/Free Full Text]
  27. Nguyen HQ, Hoffman-Liebermann B, Liebermann DA 1993 The zinc finger transcription factor Egr-1 is essential for and restricts differentiation along the macrophage lineage. Cell 72:197–209[CrossRef][Medline]
  28. Khachigian LM, Lindner V, Williams AJ, Collins T 1996 Egr-1-induced endothelial gene expression: a common theme in vascular injury. Science 271:1427–1431[Abstract]
  29. Lee SL, Tourtellotte LC, Wesselschmidt RL, Milbrandt J 1995 Growth and differentiation proceeds normally in cells deficient in the immediate early gene NGFI-A. J Biol Chem 270:9971–9977[Abstract/Free Full Text]
  30. Lee SL, Sadovsky Y, Swirnoff AH, Polish JA, Goda P, Gavrilina G, Milbrandt J 1996 Luteinizing hormone deficiency and female infertility in mice lacking the transcription factor NGFI-A (Egr-1). Science 273:1219–1221[Abstract]
  31. Lee SL, Wang Y, Milbrandt J 1996 Unimpaired macrophage differentiation and activation in mice lacking the zinc finger transcription factor NGFI-A (EGR1). Mol Cell Biol 16:4566–4572[Abstract]
  32. Murphy P, Topilko P, Schneider-Maunoury S, Seitanidou T, Baron-Van Evercooren A, Charnay P 1996 The regulation ofKrox-20 expression reveals important steps in the control of peripheral glial cell development. Development 122:2847–2857[Abstract]
  33. Nikitovitch-Winer MB, Atkin J, Maley BE 1987 Colocalization of prolactin and growth hormone within specific adenohypophyseal cells in male, female, and lactating female rats. Endocrinology 121:625–635[Abstract]
  34. Frawley LS, Boockfor FR 1991 Mammosomatotropes: presence and functions in normal and neoplastic pituitary tissue. Endocr Rev 12:337–355[CrossRef][Medline]
  35. Japon MA, Rubinstein M, Low MJ 1994 In situ hybridisation analysis of anterior pituitary hormone gene expression during fetal mouse development. J Histochem Cytochem 42:1117–1125[Abstract]
  36. Saez JM 1994 Leydig cells: endocrine, paracrine, and autocrine regulation. Endocr Rev 15:574–626[CrossRef][Medline]
  37. Rhodes SJ, DiMattia GE, Rosenfeld MG 1994 Transcriptional mechanisms in anterior pituitary cell differentiation. Curr Opin Genet Dev 4:709–717[CrossRef][Medline]
  38. Pelletier G, Leclerc R, Labrie F 1976 Identification of gonadotropic cells in the human pituitary by immunoperoxidase technique. Mol Cell Endocrinol 6:123–128[CrossRef][Medline]
  39. Childs GV, Ellison DG 1980 An immunocytochemists’s view of gonadotropin storage in the adult male rat: cytochemical and morphological heterogeneity in serially sectioned gonadotropes. Am J Anat 158:397–409[CrossRef][Medline]
  40. Cheng TC, Beamer WG, Phillips JA, Bartke A, Mallonee RL, Dowling C 1983 Etiology of growth hormone deficiency in little, Ames and Snell dwarf mice. Endocrinology 113:1669–1678[Abstract]
  41. Camus A, Kress C, Babinet C, Barra J 1996 Unexpected behavior of a gene trap vector comprising a fusion between the Sh ble and the lacZ genes. Mol Reprod Dev 45:255–263[CrossRef][Medline]
  42. Schneider-Maunoury S, Topilko P, Seitanidou T, Levi G, Cohen-Tannoudji M, Pournin S, Babinet C, Charnay P 1993 Disruption of Krox-20 results in alteration of rhombomeres 3 and 5 in the developing hindbrain. Cell 75:1199–1214[CrossRef][Medline]
  43. Laemmli UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685[CrossRef][Medline]
  44. Hochereau de Reviers MT, Copin M, Seck M, Monet-Kuntz C, Cornu C, Fontaine I, Perreau C, Elsen JM, Boomarov O 1990 Stimulation of testosterone production by PMSG injection in the ovine male: effect of breed and age and application to males carrying the F Booroola gene. Anim Reprod Sci 23:21–28[CrossRef]



This article has been cited by other articles:


Home page
Mol. Endocrinol.Home page
Y. Shima, M. Zubair, T. Komatsu, S. Oka, C. Yokoyama, T. Tachibana, T. A. Hjalt, J. Drouin, and K.-i. Morohashi
Pituitary Homeobox 2 Regulates Adrenal4 Binding Protein/Steroidogenic Factor-1 Gene Transcription in the Pituitary Gonadotrope through Interaction with the Intronic Enhancer
Mol. Endocrinol., July 1, 2008; 22(7): 1633 - 1646.
[Abstract] [Full Text] [PDF]


Home page
Mol. Endocrinol.Home page
T. B. Salisbury, A. K. Binder, and J. H. Nilson
Welcoming {beta}-Catenin to the Gonadotropin-Releasing Hormone Transcriptional Network in Gonadotropes
Mol. Endocrinol., June 1, 2008; 22(6): 1295 - 1303.
[Abstract] [Full Text] [PDF]


Home page
Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
P. R. Reynolds, S. D. Kasteler, M. G. Cosio, A. Sturrock, T. Huecksteadt, and J. R. Hoidal
RAGE: developmental expression and positive feedback regulation by Egr-1 during cigarette smoke exposure in pulmonary epithelial cells
Am J Physiol Lung Cell Mol Physiol, June 1, 2008; 294(6): L1094 - L1101.
[Abstract] [Full Text] [PDF]


Home page
J EndocrinolHome page
H. L Henderson, J. Townsend, and D. J Tortonese
Direct effects of prolactin and dopamine on the gonadotroph response to GnRH
J. Endocrinol., May 1, 2008; 197(2): 343 - 350.
[Abstract] [Full Text] [PDF]