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. Author manuscript; available in PMC: 2009 Feb 6.
Published in final edited form as: Genesis. 2008 Sep;46(9):499–505. doi: 10.1002/dvg.20428

Generation of Cyp17iCre transgenic mice and their application to conditionally delete estrogen receptor alpha (Esr1) from the ovary and testis

Phillip J Bridges 1,*, Yongbum Koo 2,*, Dong-Wook Kang 3, Susan Hudgins-Spivey 1, Zi-Jian Lan 4, Xueping Xu 4, Francesco DeMayo 4, Austin Cooney 4, CheMyong Ko 1
PMCID: PMC2637183  NIHMSID: NIHMS88607  PMID: 18781648

Abstract

A transgenic mouse line that expresses iCre under regulation of the Cytochrome P450 17α-hydroxylase/17, 20-lyase (Cyp17) promoter was developed as a novel transgenic mouse model for the conditional deletion of genes specifically in the theca/interstitial cells of the ovary and Leydig cells of the testis. In this report we describe the development of Cyp17iCre mice and the application of these mice for conditional deletion of the estrogen receptor alpha (Esr1) gene in the theca/interstitial and Leydig cells of the female and male gonad, respectively. These mice will prove a powerful tool to inactivate genes in the gonad in a cell-specific manner.

Targeted gene deletion has become a powerful tool in the study of gene function with the utilization of this technology leading to marked progress in our understanding of both physiological and pathophysiological systems. The ovary is one organ where targeted genetic deletion has proven fruitful with the establishment of transgenic mice with Cre expression targeted to granulosa cells (Lécureuil et al., 2002), somatic cells (Bingham et al., 2006) and the oocyte (Lan et al., 2004; Lewandoski et al., 1997). However, our understanding of ovarian function cannot advance at optimal pace without a tool to specifically delete genes of interest from the other major endocrine cell population of the ovary, the theca/interstitial cells. Hence, our primary goal was to develop the mice necessary to allow the specific deletion of genes in the theca/interstitial cells of the ovary and in this report we describe the generation of such a line of transgenic mice, with codon-improved Cre (iCre) driven by the promoter to Cytochrome P450 17α-hydroxylase/17, 20-lyase (Cyp17).

Cytochrome P450 17α-hydroxylase/17, 20-lyase, the product of Cyp17 gene expression, plays a major role in the control of sex steroid hormone synthesis by mediating the17α-hydroxylation of pregnenolone or progesterone to dehydroepiandrostenedione or androstenedione, respectively. In the female mouse, Cyp17 expression is primarily restricted to the ovary and placenta (Su et al., 2002) and within the ovary, Cyp17 is abundant in the gonadotropin-primed theca/interstitial cell population, but not the granulosa cells or oocyte (Zhang et al., 2001), making it ideal for iCre targeting. Furthermore, coincident to the need of a transgenic mouse line with iCre targeted to the theca/interstitial cells, is one also designed to allow the deletion of Leydig cell specific genes from the male gonad. Fortunately, the specificity of Cyp17 expression to the Leydig cells of the testis (Zhang et al., 2001) makes this line of mice also ideal as a tool to inactive genes specifically within that endocrine population of cells. Hence, this report was widened as a functional characterization of these mice inclusive to both the sexes.

Three lines of Cyp17iCre founder mice were generated (A, B and C) by pronuclear injection of a KpnI/SalI DNA fragment derived from a Cyp17iCre expression plasmid (Figure 1). Then, to facilitate expression analysis, founder mice were crossed with the iCre reporter strain (Gt[ROSA]26Sortm1Sor; ROSA26) (Soriano, 1999). These mice have the lacZ reporter gene expressed when iCre excises a loxP-flanked polyadenylation sequence. Therefore, with iCre/loxP recombination, only tissues with functional iCre activity will express β-galactosidase which stains intensely blue to X-gal (Bell et al., 2005). Analysis of X-gal staining in the ovaries and testis collected from each of the three lines of Cyp17iCre/ROSA26 mice revealed no dramatic differences in the pattern of staining, however extra-gonadal X-gal staining was observed in two of the three lines. In line B, mottled X-gal staining was observed in the lungs of adult mice and in line C, disperse X-gal staining was noted in embryos collected 16.5 days after conception. These two lines are described in only a limited fashion hereafter and were not evaluated for the functional analysis (generation of Esr1 flox/floxCyp17iCre transgenic mice), described below.

Figure 1.

Figure 1

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Schematic representation of the Cyp17iCre transgene construct. A) Plasmid map of the completed Cyp17iCre construct. B) Linearlized structure of Kpn 1 /Sal 1 fragment of the construct. This DNA fragment was used for oocyte injection.

In the Cyp17iCre (line A) female, gross analysis of the ovary, oviduct and uterus after staining of these tissues as an intact unit with X-gal revealed strong ovarian, weak oviductal and very sparce uterine expression of β-galactosidase (Figure 2). Further histological evaluation of gonadotropin-primed Cyp17iCre/ROSA26 ovaries revealed X-gal staining throughout the majority of the theca/interstitium with minor staining observed in some granulosa cells in the ovary (Figure 2). In the Cyp17iCre/ROSA26 testis, X-gal staining was observed specifically in the Leydig cells (Figure 2). Evaluation of fetal gonadal X-gal staining on embryonic day 16.5 revealed strong staining in the testis but not the ovary (Figure 2), consistent with the pattern of expression of mRNA for Cyp17 in these tissues (Heikkila et al., 2002).

Figure 2.

Figure 2

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β-galactosidase expression in gonadal tissues of Cyp17iCre/ROSA26 mice. Upper panels: Direct X-gal staining of the adult female and male gonads. Sectioned images of the ovary and testis are shown. The sections were counterstaining with nuclear fast red. Lower panels: Direct X-gal staining of embryonic day 16.5 female and male gonads. Sectioned images of the ovary and testis are shown. The sections were counterstaining with nuclear fast red.

Gross morphological analysis of the placenta and embryos collected from pregnant females on embryonic day 16.5 revealed the expected placental staining (Arensburg et al., 1999; Durkee et al., 1992) (Figure 3). Similar to the sexually dimorphic staining of X-gal to the embryonic gonads, distinct staining was observed in the male but not the female embryonic adrenal (Figure 3). However, some inconsistency is apparent in the reported expression levels of mRNA for Cyp17 in the male versus female embryonic adrenal (Heikkila et al., 2002; Keeney et al., 1995). Within the pituitary gland, X-gal staining was apparent in the intermediate lobe whereas the anterior and posterior lobes remained free of expression (Figure 3), an unexpected finding that was consistent among the three original founder lines. Gross morphological analysis of the kidney, adrenal gland, spleen and whole brain revealed only occasional and very sparse staining. Minor mottled staining was observed in some areas of the lung, whereas the liver stained intensely blue (a consistent finding among the three transgenic lines). Whether the extra-gonadal expression of iCre to the liver translates to aberrant metabolic function is unlikely though as all transgenic mice maintained good health and developed at the expected rate of growth. Overall, X-gal staining indicated relatively specific incorporation of iCre.

Figure 3.

Figure 3

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β-galactosidase expression in non-gonadal tissues of Cyp17iCre/ROSA26 mice. Upper panels: Direct X-gal staining of the pituitary and placenta of a female mouse. Lower panels: Direct X-gal staining of the embryonic day 16.5 adrenal gland. Sectioned images of the male and female embryonic adrenal gland are shown. The sections were counterstaining with nuclear fast red.

To determine the functional ability of iCre to excise a floxed gene in vivo, Cyp17iCre mice was crossed with those having loxP sites flanking estrogen receptor alpha (Esr1) (Gieske et al., 2008), with the goal of specifically deleting Esr1 from the ovarian theca/interstitium and testicular Leydig cells. Because Esr1 is localized to the theca/interstitial cells (Schomberg et al., 1999) and the surface epithelium of the ovary (Urzua et al., 2006), these mice were well suited to test the ability to delete a gene specifically from our targeted population of cells. Furthermore, total genetic deletion of Esr1 leads to the development of severe hemorrhagic ovarian cysts (Dupont et al., 2000), making mice with a conditional deletion of this gene an excellent model for further analysis of the role of Esr1 in ovarian health and reproductive function. Founder lines had also undergone multiple generations of breeding before crossing with Esr1 flox/flox mice, allowing analysis of functional excision via a transgene with a now stable insertion. Briefly, Esr1flox/flox mice were created by a targeting strategy used to generate Esr1−/− mice (Dupont et al., 2000), as described previously (Gieske et al., 2008). To generate Esr1flox/flox Cyp17iCre mice, Esr1flox/flox mice were first crossed with Cyp17iCre mice. Esr1flox/+Cyp17iCre F1 heterozygotes were then bred with Esr1flox/flox mice to generate four potential genotypes: Esr1flox/flox Cyp17iCre (the desired transgenic), as well as Esr1flox/+Cyp17iCre, Esr1flox/flox and Esr1flox/+. Analysis of Esr1 flox/flox Cyp17iCre mice indicated successful application of the Cyp17iCre line as a conditional deleter of Esr1 within the gonad. Both in the ovary and testis of Esr1 flox/flox Cyp17iCre mice, deletion of Esr1 was restricted to the gonads and abundant Esr1 expression was maintained in the uterus, oviduct, and epididymis, respectively (Figure 4).

Figure 4.

Figure 4

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Gonad-specific deletion of Esr1 gene by Cyp17iCre mice. Upper panels: Expression of Esr1 in the reproductive tissues of a female Esr1 flox/flox Cyp17iCre mouse. Note strong Esr1 expression both in oviduct and uterus but not in the ovary except for ovarian surface epithelial cells. Lower panels: Expression of Esr1 in the reproductive tissues of a male Esr1 flox/flox Cyp17iCre mouse. Note strong Esr1 expression in the efferent ductile and epididymis but not the testis.

Within the ovary, normal Cyp17 protein expression was observed by immunohistochemical analysis in the theca/interstitial cells of both Esr1 flox/flox and Esr1 flox/floxCyp17iCre mice (Figure 5). In contrast, Esr1 expression, while apparent in the theca/interstitial cells and ovarian surface epithelium of Esr1 flox/flox mice, was only detected in the ovarian surface epithelium of Esr1 flox/floxCyp17iCre mice (Figure 5), indicating successful deletion of Esr1 from the iCre-expressing theca/interstitium and maintained expression in the non-targeted ovarian surface epithelium. Similar results were observed in the testis. Normal Cyp17 protein expression was observed in the Leydig cells of both Esr1 flox/flox and Esr1 flox/floxCyp17iCre mice (Figure 5). Esr1 expression was detected in the Leydig cells of Esr1 flox/flox mice but not in Esr1 flox/floxCyp17iCre transgenic mice (Figure 5), again indicating successful deletion of Esr1 from iCre-expressing cells after excision of the loxP sequence.

Figure 5.

Figure 5

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Successful deletion of Esr1 by the application of Cyp17iCre mice. Upper panels: Expression of Cyp17 and Esr1 in the ovary of Esr1 flox/flox and Esr1flox/flox Cyp17iCre mice. Note that Esr1 expression is absent in the theca/interstitial cells of the Esr1flox/flox Cyp17iCre mouse ovary while Cyp17 expression persists. Lower panels: Expression of Cyp17 and Esr1 in the testis of Esr1 flox/flox and Esr1flox/flox Cyp17iCre mice. Note that Esr1 expression is absent in the Leydig cells of the Esr1flox/flox Cyp17iCre mouse testis while Cyp17 expression persists.

In summary, we report the development and application of a novel strain of transgenic mice with iCre targeted to the Cyp17 promoter. The use of these mice to conditionally delete genes of interest from the theca/interstitial cells of the ovary and Leydig cells of the testis will allow rapid advancement of our understanding of gene function as it relates to fertility and reproductive health.

METHODS

Generation of Cyp17iCre Transgenic Mice

Three transgenic mouse lines were constructed that expressed iCre recombinase under the control of the mouse Cyp17 promoter (Cyp17iCre). An iCre fragment was amplified from the iCre plasmid (pBlue.iCre, generously provided by Dr. R. Sprengel, Max-Planck Institute for Medical Research, Germany) by polymerase chain reaction (PCR), digested and subcloned into another plasmid (pGL3B-iCre) to generate a promoter-free iCre expression cassette (Shimshek et al., 2002). Genomic DNA fragments corresponding to the promoter region of Cyp17 were amplified from mouse (C57-B6) genomic DNA, and cloned into the promoter-free iCre expression cassette (pGL3B-iCre) to produce a Cyp17iCre expression plasmid (pGL3B-Cyp17iCre). The nucleotide sequences of this transgene vector, pGL3B-Cyp17iCre, were confirmed by sequence analysis. A KpnI/SalI DNA fragment from pGL3B-Cyp17iCre vector which contained the 3.34 kb Cyp17 promoter, a 1.1 kb iCre coding sequence and a SV40 late poly A signal (Schorpp et al., 1996) was cut, purified and microinjected into the pronuclei of fertilized eggs from C57-B6/SJL mice as previously described (Jorgez et al., 2006; Lan et al., 2004; Li et al., 2005; Shimshek et al., 2002). The microinjected eggs were transferred to pseudopregnant mothers, yielding three viable Cyp17iCre founder mice that transferred this transgene to their progeny.

Animal use and Genotyping

Genomic DNA was extracted from ear punches or embryonic tails using the Easy DNA kit (Invitrogen), according to the manufacturer's directions. The presence of iCre or LacZ was determined by PCR using the following primer pairs: Cre-F (5′-tct gat gaa gtc agg aag aac c-3′) and Cre-R (5′-gag atg tcc ttc act ctg att c-3′); LacZ-F (5′-gcg tta ccc aac tta atc g-3′) and LacZ-R (5′-tgt gag cga gta aca acc-3′) (Jorgez et al., 2006). For the functional analysis, the primer sets ERαP2F (5′-gtg tca gaa aga gac aat-3′) and ERαP3 (5′-ggc att acc act tct cct ggg agt ct-3′) were used to determine the presence or absence of loxP sequences and the primer sets ERαP1 (5′-ttg ccc gat aac aat aac at-3′) and ERαP3 were used to determine whether or not exon 3 had been deleted (Gieske et al., 2008). Animal use was approved by the respective University of Kentucky and Baylor College of Medicine Animal Care and Use Committees.

Histochemical Analysis

X-gal staining for β-galactosidase expression was performed on fresh tissues collected at sacrifice with the reagents and protocol recommended for direct staining by the manufacturer (Speciality Media, NJ). Briefly, tissues were fixed with the supplied paraformaldehyde based-fixative for 1 h on ice, rinsed with two PBS-based rinse solutions at room temperature and then incubated overnight at 37°C in the dark with X-gal stock solution diluted 1:40 in the supplied base solution. After the overnight incubation, tissues were washed and embedded in either paraffin or OCT. Paraffin-embedded sections were cut to 7 μm, deparaffinized by treatment with xylenes, rehydrated through a graded series of alcohols then rinsed and counterstained with nuclear fast red. Tissues embedded in OCT were cut to 10 μm, rehydrated and counterstained with nuclear fast red.

Immunohistochemical analysis of Esr1 and Cyp17 was performed on ovaries and testis collected at sacrifice, fixed with 4% neutral buffered paraformaldehyde and then embedded in paraffin blocks. Sections were cut to 4 μm and immunostaining performed on deparaffinized sections using an EnVision detection kit (DAKO, Carpenteria, CA), according to the manufacturer's instructions. Antigen-retrieval was perfomed using 10 mM citrate buffer (pH 6.0) in an autoclave for 15 min at 120°C and the endogenous peroxidase activity was blocked with 5% hydrogen peroxide for 15 min. Sections were then incubated with primary antibodies that recognized Esr1 (mouse monoclonal 6F11, 1:40 dilution, Novocastra, Newcastle, UK), and Cyp17 (rabbit polyclonal, 1:100 dilution, kindly provided by Dr. A.J. Conley, Department of Population Health and Reproduction, University of California-Davis (Pattison et al., 2007)). 3,3′-diamino-benzidine (DAB, DAKO) or 3-amino-9-ethylcarbazole (AEC, DAKO) were then used as a chromogen and counterstaining was accomplished with Mayer's hematoxyline. Staining was examined using a BX51 microscope (Olympus, Japan) with images acquired using the DP-70 imaging system (Olympus).

ACKNOWLEDGMENTS

This work was supported by the University of Kentucky Start-Up Fund (C Ko), NIH grants (IR01HD052694, P20RR15592; C KO) and College of Health Sciences NR research Support Funds (C Ko). Dong Wook Kang is a recipient of a Korea Research Foundation Grant (KRF-2004-214-E00042, MOEHRD). The authors would like to thank Dr. A.J. Conley, Department of Population Health and Reproduction, University of california-Davis for kindly providing the Cyp17 antisera and Dr. Myung Chan Gye, Scott Huffman and Sung Eun Lee (University of Kentucky) for technical assistance.

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