Insulin Receptor and IGF1R Are Not Required for ...

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Oct 23, 2009 - the insulin receptor (INSR) and the type 1 IGF receptor. (IGF1R), both of which are expressed in developing oocytes. IGF1 plays an important ...
Original Article Sex Dev DOI: 10.1159/000252813

Received: January 29, 2009 Accepted: June 25, 2009 Published online: October 23, 2009

Insulin Receptor and IGF1R Are Not Required for Oocyte Growth, Differentiation, and Maturation in Mice J.L. Pitetti a D. Torre a B. Conne a M.D. Papaioannou a C.R. Cederroth a S. Xuan b R. Kahn c L.F. Parada d J.D. Vassalli a A. Efstratiadis b S. Nef a a

Department of Genetic Medicine and Development, University of Geneva Medical School, University of Geneva, Geneva, Switzerland; b Department of Genetics and Development, Columbia University Medical Center, New York, N.Y., c Joslin Diabetes Center and Harvard Medical School, Boston, Mass., d Department of Developmental Biology, University of Texas Southwestern Medical Center, Dallas, Tex., USA

Key Words Differentiation ⴢ Fertility ⴢ Growth ⴢ Igf1r ⴢ Insr ⴢ Maturation ⴢ Mouse ⴢ Oozyte

Abstract In mammals, insulin and insulin-like growth factors (IGFs: IGF1 and IGF2) act through 2 structurally related receptors, the insulin receptor (INSR) and the type 1 IGF receptor (IGF1R), both of which are expressed in developing oocytes. IGF1 plays an important role in female reproduction, and female Igf1 knockout mice fail to ovulate and are infertile. On the other hand, little is known about the in vivo role of the insulin signaling pathway in oocytes during follicular development, although exposure to insulin or IGF1 in vitro improves oocyte maturation. To further address the significance of insulin/IGF signaling, we used conditional mutant mice and ablated the function of the genes encoding INSR, IGF1R, or both receptors specifically in developing mouse oocytes. Our genetic evidence showed unexpectedly that the female reproductive functions are not affected when Insr, Igf1r or both Insr;Igf1r are ablated in oocytes, as the female mice are fertile and exhibit normal estrous cyclicity, oocyte development and maturation, parturition frequency,

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and litter size. In view of these novel observations indicating that the insulin/IGF signaling is not essential in oocytes, the IGF1-dependent female fertility is re-evaluated and discussed. Copyright © 2009 S. Karger AG, Basel

Mammalian oocytes are highly specialized cells which undergo meiosis and monospermic fertilization. As a consequence, sperm chromatin is reprogrammed into a functional pronucleus, zygotic genome activation is induced, and early embryonic development commences. This developmental potential is acquired during oogenesis and requires the coordinate action of both circulating hormones and locally derived factors that are essential for follicular development including recruitment, selection, and dominance [for review see van den Hurk and Zhao, 2005]. In particular, progression through the successive stages of follicular development requires bi-directional communication between the oocyte and granulosa cells

J.L.P. and D.T. contributed equally to this work.

Serge Nef Department of Genetic Medicine and Development University of Geneva Medical School 1, rue Michel-Servet, CH–1211 Geneva 4 (Switzerland) Tel. +41 22 379 5193, Fax +41 22 379 5260, E-Mail serge.nef @ unige.ch

[Eppig, 2001] and involves factors of the transforming growth factors- family (e.g., GDF-9, BMPs, AMH, Inhibin, and Activin) as well as a variety of growth factors such as growth hormone (GH), epidermal growth factor (EGF), and insulin-like growth factors [van den Hurk and Zhao, 2005]. The latter have long been known to play an important role in female reproduction [Dupont and Holzenberger, 2003]. IGF1 has been reported to be selectively expressed in the granulosa cells of developing follicles whereas IGF1R is expressed both in the granulosa cells and in oocytes of murine and human follicles, suggesting a potential paracrine action of the IGF1 ligand [Zhou et al., 1991; Zhou and Bondy, 1993]. In humans, IGF1R is expressed in follicles throughout all stages of follicular development, from the primordial to the preovulatory stage [Zhou and Bondy, 1993]. The insulin receptor (INSR) has been reported to be expressed in developing oocytes in mice, rats, and humans [Samoto et al., 1993; Acevedo et al., 2007]. In addition to the presence of INSR and IGF1R in mouse oocytes during oocyte growth and maturation [Zhou and Bondy, 1993], key intermediate enzymes of the insulin/IGF signaling cascade such as the 3-phosphoinositide-dependent protein kinase-1 (PDPK1), thymoma viral proto-oncogene 1 (AKT1), and the downstream glycogen synthase kinase 3 (GSK3) are present in fully grown oocytes, the latter being activated during maturation [Acevedo et al., 2007]. Overall, active insulin/ IGF signaling operates during oocyte maturation. The importance of insulin/IGF action on oocyte and follicular development has been revealed by studies using transgenic animals and oocyte culture systems. In ovarian follicles IGF1 stimulates the proliferation and differentiation of granulosa cells [Cara and Rosenfield, 1988], while it stimulates steroidogenesis in theca cells [Levy et al., 1992]. In addition, IGF1 has been reported to promote the in vitro maturation of follicles and denuded oocytes [Gomez et al., 1993]. Female Igf1-null mice that survive to adulthood are infertile due to an arrest of follicular development at the late preantral stage [Liu et al., 1993; Zhou et al., 1997] thus confirming the essential role of IGF1 during folliculogenesis. IGF1 regulates antrum formation and FSH action by increasing the aromatase activity in granulosa cells [Baker et al., 1996; Zhou et al., 1997]. In contrast, female mice lacking both Igf1r and Igf2r are fertile [Ludwig et al., 1996], suggesting that the IGFs are not signaling exclusively through IGF1R but also through INSR. These data are consistent with the notion that INSR mediates part of the reproductive functions of IGF1 or IGF2 [Efstratiadis, 1998]. 2

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Whether insulin or IGFs are acting exclusively on granulosa cells or also on oocytes remains an open question. In vitro studies in several species have demonstrated a role for both insulin and IGF1 in oocyte maturation and early embryo development. Thus, the presence of insulin in the maturation medium leads to accelerated meiotic progression [Stefanello et al., 2006] and also exerts mitogenic and anti-apoptotic actions [Augustin et al., 2003]. Similarly, when IGF1 is added to the maturation medium, meiotic progression is accelerated and the number of blastocysts is increased [Sakaguchi et al., 2002]. Currently, however, there is no in vivo evidence for the role of the insulin/IGF family of growth factors on the development, growth, and meiotic maturation of mouse oocytes. We sought, therefore, to investigate these functions by deleting Insr, Igf1r, or both receptors specifically in oocytes during follicle/oocyte growth and meiotic progression.

Materials and Methods Animals Igf1rflox (Igf1rfx/fx) mice and Gdf9-Cre (Gdf9:Cre) mice were provided by A. Efstratiadis and A.J. Cooney, respectively, and were genotyped at weaning (P21) from tail biopsies by classic PCR as described [Xuan et al., 2002; Lan et al., 2004]. Irflox (Insrfx/fx) mice were provided by R. Kahn [Bruning et al., 2000] and genotyped using a set of 3 primers (21157: 5ⴕ-CACACACACACGCCTACAC-3ⴕ, 21160: 5ⴕ-TCTCCCTACACCCACTCACA-3ⴕ, and 20138: 5ⴕ-CTGAATAGCTGAGACCACAG-3ⴕ) discriminating between Insrwt (⬃320 bp), Insrfx (⬃450 bp) and Insr (⬃250 bp) alleles. To analyze all different allelic inactivations, female mice carrying both Insr and Igf1r floxed alleles (Insrfx/fx;Igf1rfx/fx) were crossed with transgenic males expressing the Cre-recombinase under the control of the Gdf9 promoter to generate 50% Insrfx/wt;Igf1rfx/wt and 50% Insrfx/wt;Igf1rfx/wt;Gdf9:Cre mice. These animals were subsequently intercrossed to generate oocyte-specific deletions of Insr (Insrfx/fx;Gdf9:Cre), Igf1r (Igf1rfx/fx;Gdf9: Cre), or both Insr and Igf1r (Insrfx/fx;Igf1rfx/fx;Gdf9:Cre) as well as control animals (Insrfx/fx;Igf1rfxfx or Insrfx/wt;Igf1rfx/wt;Gdf9:Cre). Protocols for the use of animals were approved by the Commission d’Ethique de l’Expérimentation Animale of the University of Geneva Medical School and the Geneva Veterinarian Office. Estrous Cycles Using vaginal smears, the pattern of estrous cycles was determined in 8 weeks old females for 21–28 consecutive days as previously described [Nelson et al., 1982]. Vaginal smears were performed daily at the same time, and cytology was then used to identify the phase of the estrous cycle. Proestrus was determined by the presence of small nucleated cells whereas estrus was scored by the presence of cornified cells. Large round cells with irregular borders were indicative of metestrus, and high density of leukocytes indicated the diestrus stage.

Pitetti et al.

Fertility Tests Insr (Insrfx/fx;Gdf9:Cre), Igf1r (Igf1rfx/fx;Gdf9:Cre), or both Insr and Igf1r (Insrfx/fx;Igf1rfx/fx;Gdf9:Cre) mutant females as well as control Insrfxfx;Igf1rfxfx females (n = 3–4) were each bred with one 8-week-old wild type C57B/6 male for a period of 6 months. Number of litters and pups per litter were recorded at weaning. Histology and Immunohistochemistry For histology and ovarian morphometry purposes, mice exhibiting 2 consecutive estrus stages were sacrificed at the proestrus stage. Ovaries were fixed overnight either in 4% paraformaldehyde (PFA) or in Bouin’s fixative and embedded in paraffin. Sections of 5 m were stained with hematoxylin and eosin (H&E) or processed for immunohistochemistry (IHC). For IHC analysis, PFA fixed sections were incubated overnight at 4 ° C with an anti galactosidase antibody (ab9361, Abcam, 1:500). For fluorescent staining, Alexa-conjugated secondary antibodies (Invitrogen) were used for signal revelations. All images were obtained with a Zeiss Axioscop microscope and processed using the the AxioVision software. Ovarian Morphometry Thirty transverse sections of ovaries from 3 different animals per genotype were randomly selected to evaluate the number of follicles at each developmental stage. Primordial, primary, secondary, pre-antral, and antral follicles were identified according to established morphological criteria [Pedersen and Peters, 1968]. For antral follicles, we measured and took into account the largest diagonal of the oocyte only if it crossed the pronucleus.

Real-Time Quantitative RT-PCR qRT-PCRs were performed as previously described [Cederroth et al., 2007; Papaioannou et al., 2009], with minor modifications. In brief, total RNA from a pool of 100 wild-type germinal vesicle (GV) oocytes dissected from 4-weeks-old control females was extracted using the RNeasy micro kit (Qiagen) according to the manufacturer’s protocol. RNA integrity and quantity were assessed using RNA 6000 nanochips with an Agilent 2100 bioanalyzer (Agilent Technologies). Total RNAs were reverse transcribed with the Omniscript RT-kit (Qiagen) according to manufacturer’s instructions, and 1/5 of the total cDNA was used as template for each gene. For real-time quantitative RT-PCR (qRT-PCR), cDNA was PCR amplified in triplicates on a Corbett Rotor-Gene 6000 (Qiagen) using SYBR GreenER master mix (Invitrogen). Raw threshold-cycle (Ct) values were obtained from Sequence Detection Systems 2.0 software (Applied Biosystems). Primers used for qRT-PCR, which all have efficiencies close to 2, were: Insr : 5ⴕggaccatgcctgaagctaag-3ⴕ(sense) and 5ⴕ-atggaaacacatcactggca-3ⴕ (antisense); Igfr1: 5ⴕ-aggctgagaagctgggctgca-3ⴕ (sense) and 5ⴕacagaagcatacagcactcca-3ⴕ (antisense). Statistical Analysis Results are expressed as means 8 SE of n experiments. The nonparametric unpaired t test was used for statistical analysis. Differences were considered statistically significant if the p value was !0.05.

Results Zymography For each lane, 3 oocytes (3 animals/genotype) were solubilized in 10 l of 0.25% triton X-100 (Merck), 2 mg/ml bovine serum albumin (BSA; SIGMA) in water, mixed with an equal volume of double strength electrophoresis sample buffer (2! ESB; 0.1 M Tris HCl, pH 6.8, 2% SDS, 20% Glycerol, 0.005% bromophenol blue), and electrophoresed under non-reducing conditions in a polyacrylamide gel, using the Laemmli buffer system [Laemmli, 1970]. To reveal the enzymatic activity of tissue plasminogen activator (tPA) in oocytes, zymographic assays were performed according to Vassalli et al. [1984] and allowed to develop at 37 ° C for 24– 48 h. Photographs were taken using dark field illumination. Western Blots Oocytes collected at germinal vesicle (GV) stages were washed in phosphate-buffered saline (PBS), lysed in 5 l Laemmli buffer [Laemmli, 1970], and heated 5 min at 95 ° C. Protein samples were separated in a 8% polyacrylamide gel, transferred onto a nitrocellulose membrane (Immobilon-P, Millipore: IPVH00010), and visualized using Coomassie blue staining. Unspecific binding sites were blocked using 5% non-fat milk in TBS (Tris 10 mM, NaCl 0.154 M, pH 7.4). Primary antibodies against INSR (SC-711), IGF1R (SC-712), and actin (Chemicon, MAB1501) were used at 1: 1,000 in TBS, 0.1% Triton X-100, 3% bovine serum albumin (Sigma) and incubated overnight at 4 ° C. INSR and IGF1R signals were revealed using a HRP conjugated goat anti-rabbit secondary antibody (Bio-Rad, 1:10,000) and actin signal using a HRP conjugated goat anti-mouse secondary antibody (Bio-Rad, 1:10,000). Both signals were detected using Lumigen TMA-6 (GE Healthcare) according to manufacturer’s instructions.

Dispensable Role of Insulin Receptors in Mouse Oocytes

Level of Insr and Igf1r Expression in Mouse Oocytes The abundance of Insr and Igf1r transcripts was measured by quantitative real-time RT-PCR using total RNA template extracted from a pool of isolated oocytes at the GV stage. Expression levels as assessed by Ct values showed that both receptors were present in oocytes, but Igf1r transcripts were approximately 30 times more abundant than Insr transcripts (fig. 1A). INSR and IGF1R proteins were also detected in wild type GV oocytes by Western blot analysis (fig. 1B). Taking into consideration the robust expression of IGF1 in granulosa cells [Zhou et al., 1991; Zhou and Bondy, 1993] and the presence of INSR and IGF1R in oocytes, it is reasonable to suggest that the IGF1 ligand exerts a paracrine action on oocytes. Generation of Mice with an Oocyte-Specific Deletion of Insr, Igf1r, or Insr;Igf1r Whether the insulin signaling is required for in vivo oocyte development and maturation is unknown. A significant limitation when working with mice constitutively lacking a member of the insulin/IGF receptor is their neonatal lethality; Insr mutant pups die within 4 days after birth of ketoacidosis [Accili et al., 1996; Joshi et al., 1996] while Igf1r and Insr;Igf1r mutant mice die at birth Sex Dev

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1,000

Fig. 1. Expression of insulin receptors in mouse oocytes. A RTPCR shows that Insr and Igf1r are both expressed in germinal vesicle (GV) oocytes. Differences in Ct indicate that Igf1r transcripts are ⬃30 times more abundant than Insr transcripts. B INSR and IGF1R proteins are detected in WT oocytes at 86 kDa and 130 kDa, respectively. INSR: insulin receptor; IGF1R: IGF1 receptor. The amount of protein extracts loaded per lane represents either 20 or 50 WT oocytes.

due to respiratory failure [Liu et al., 1993; Louvi et al., 1997]. To bypass this lethality and to generate female mice carrying a conditional deletion of Insr, Igf1r, or both Insr;Igf1r in oocytes, we used mice carrying floxed alleles of Insr [Bruning et al., 1998] and Igf1r [Dietrich et al., 2000] and derived through appropriate crosses with mice carrying an oocyte-specific Gdf9:Cre transgene [Lan et al., 2004] Insrfx/fx;Gdf9:Cre, Igf1rfx/fx;Gdf9:Cre and Insrfx/fx;Igf1rfx/fx;Gdf9:Cre progeny (see Materials and Methods). For simplicity, these animals are hereafter referred to as Oo-Insr, Oo-Igf1r, and Oo-Insr;Igf1r, respectively. The Gdf9:Cre transgene is exclusively expressed in oocytes of follicles from the primordial to the antral stage, from postnatal day 3 (P3) onwards [Lan et al., 2004]. We confirmed the specificity of Cre recombinase activity by -gal IHC using ROSA26R reporter mice [Soriano, 1999]. The penetrance of the Cre recombinase expression was evaluated by IHC with an antibody against -galactosidase using ovarian sections of control and double OoInsr;Igf1r mice bearing the R26R reporter transgene. As expected, we observed a strong and specific staining in oocytes, indicating that the Cre recombinase was able to induce LacZ gene expression in reporter mice. galactosidase staining was observed in oocytes of primordial follicles as well as in primary, secondary, and antral stages (fig. 2A–F). In order to show that the Gdf9-Cre transgene was able to eliminate completely both Insr and 4

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Igf1r in oocytes, we went further and assessed by genomic DNA PCR whether Insrfx and Igf1rfx maternal alleles were both deleted in pups generated from Oo-Insr;Igf1r females. For this purpose, Oo-Insr;Igf1r females were mated with Insrfx/fx;Igf1rfx/fx males. If the Gdf9-Cre transgene is fully efficient and penetrant, both Insrfx and Igf1rfx alleles will be deleted in oocytes (Insr;Igf1r ) and, as a consequence, the genotype of pups will be Insrfx/;Igf1rfx/. As expected, we found that PCR assays of a large number of embryos originating from Oo-Insr;Igf1r females lead without exception to the expected Insrfx/;Igf1rfx/ genotype (fig. 2G), Overall, these data indicate that a complete and specific ablation of both Insr and Igf1r had been achieved in developing oocytes of Oo-Insr;Igf1r mice. Oo-Insr, Oo-Igf1r, and Oo-Insr;Igf1r Mutant Animals Show No Histological Abnormalities in Reproductive Organs Oo-Insr, Oo-Igf1r, and Oo-Insr;Igf1r mutant animals were all viable, grew to adulthood normally, and did not display any gross abnormalities of the external genitalia or abnormal sexual behavior when compared to control littermates. Histological analysis of Oo-Insr, Oo-Igf1r, and Oo-Insr;Igf1r mutant ovaries at 6 weeks showed no gross anatomical defects and contained a similar number of primordial, primary, and secondary follicles (fig. 3A). These results indicate that ovaries with an oocyte-specific deletion of Insr, Igf1r, or both Insr;Igf1r are all capable of initiating folliculogenesis. Estrus Cycle and Oocyte Maturation Are Not Affected in Oo-Insr, Oo-Igf1r, and Oo-Insr;Igf1r Mutant Mice To further investigate the reproductive physiology of Oo-Insr, Oo-Igf1r, and Oo-Insr;Igf1r mice, estrous cyclicity was evaluated by performing daily vaginal smears. Similar to control females, both Oo-Insr and Oo-Igf1r mutant mice exhibited normal estrous cyclicity, whereas OoInsr;Igf1r mutant females showed a slight, but significant increase in the number of days between 2 periods of ovulation (fig. 3B), which might reflect a subtle alteration in the maturation process of follicles from the primordial to the antral stage. To address this issue, we performed a quantitative evaluation of all types of follicles at different stages of maturation of control and mutant mice. Numbers of primordial, primary, secondary, preantral, and antral follicle populations did not differ significantly between controls and mutants, indicating that oocytes mature normally in the ovaries of mutant littermates (table 1). Translational control is a critical feature of meiotic maturation; fully grown primary oocytes contain a pool Pitetti et al.

Fig. 2. Complete and specific ablation of both Insr and Igf1r in developing oocytes. Anti- Gal immunofluorescence (red) reveals that oocytes from primordial (A), primary (B), secondary (C), pre-antral (D), as well as antral (E) follicles of Gdf9: Cre;R26R mice are positive for -galactosidase, thus showing that Cre activity is oocyte specific and already begins at the primordial stage. As expected, oocytes from R26R (Gdf9:Cre negative) mice were negative for -galactosidase staining (F). Scale bars: 50 m. G Representative PCR genotyping for Insr and Igf1r loci performed on genomic DNA of embryos generated from Oo-Insr;Igf1r females mated with Insrfx/fx;Igf1rfx/fx males. As expected, the genotype is Insrfx/;Igf1rfx/ , confirming the ablation of both Insr and Igf1r in developing oocytes. Ctl: control.

A

B

C

D

E

F

G

Fig. 3. Both INSR and IGF1R are dispensable for oocyte maturation. A Photomicro-

graphs of hematoxylin/eosin stained sections of the ovary of control, Oo-Insr, Oo-Igf1r, and Oo-Insr;Igf1r animals at 3 months of age revealed no histological abnormalities in the mutants. Scale bar: 200 m. B Estrous cycle measured over a period of 3 weeks was not affected in Oo-Insr, Oo-Igf1r mutant mice when compared with controls (n = 3 for each genotype). In contrast, Oo-Insr;Igf1 double mutants (n = 3) exhibited a slight, but statistically significant increase in cycle length. C tPA (tissue plasminogen activator) activity, as revealed by zymograms, was unaffected in Oo-Insr;Igf1 oocytes at 2 different stages of maturation, i.e., it was absent from GV (germinal vesicle) and present in GVBD (germinal vesicle breakdown) oocytes regardless of genotype, * p ! 0.05 vs. control.

Dispensable Role of Insulin Receptors in Mouse Oocytes

A

C

B

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Fig. 4. Insulin signaling does not regulate oocyte size. Oocyte size of control (A) or Oo-Insr;Igf1r (B) animals at 3 months of age was evaluated by measuring the large diameter of oocytes in hematoxylin/eosin stained sections crossing the oocyte nucleus in pre-antral or antral follicles. C Control and Oo-Insr;Igf1r mutant oocytes displayed a diameter of 73.42 8 0.85 m (n = 3 mice, n = 36 follicles) and 71.77 8 0.83 m (n = 3 mice, n = 36 follicles), respectively. Scale bar: 100 m.

A

B C

Table 1. Quantitative assessment of follicular stages in control, Oo-Insr, Oo-Igf1r, and Oo-Insr;Igf1r mutant

ovaries Genotype

Primordial

Primary

Secondary

Pre-antral

Antral

WT Oo-Insr Oo-Igf1r Oo-Insr;Igf1r

38.881.66 36.882.17 37.087.33 37.587.33

18.5782.72 18.3781.58 21.0082.07 20.4782.51

19.2782.57 18.1081.44 22.0086.12 17.2084.81

17.6081.01 18.5082.51 15.0080.26 19.0780.35

5.7780.85 8.2083.60 4.0080.87 3.6780.80

The numbers of follicles per ovary from control, Oo-Insr, Oo-Igf1r, and Oo-Insr;Igf1r animals (n = 3 each) are shown. Values are expressed as means 8 SEM. No significant differences between mutant and control animals could be observed (p ≥ 0.05).

of dormant mRNAs that are translationally activated during meiotic maturation and after fertilization [Stutz et al., 1998]. It has been demonstrated that the tPA mRNA is present in primary oocytes but is only translated during meiotic maturation of oocytes; more precisely, tPA appears only in oocytes that have progressed beyond the GV breakdown (GVBD) stage [Huarte et al., 1985]. In order to assess whether Insr and Igf1r potentially affect oocyte maturation, we took advantage of the zymography technique which is based on the proteolytic capacity of tPA and evaluated whether its translation was affected in GV and GVBD oocytes of control and mutant females [Granelli-Piperno and Reich, 1978]. Consistent with the absence of defective follicular maturation previously described, we found an equivalent lysis area in control and mutant females, indicating that tPA translation and oocyte maturation had not been affected (fig. 3C). 6

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Oocyte Size is Not Affected in Oo-Insr;Igf1r Mutant Mice In Drosophila melanogaster the insulin signaling pathway plays an essential role in controlling body, organ, and cell size [Bohni et al., 1999]. In mammals, however, the involvement of the insulin signaling pathway in mediating cell size has never been described. The rapid and extensive growth of mouse oocytes during folliculogenesis might represent a sensitive assay to investigate whether cell size is also modulated by the insulin/IGF signaling pathway in mammalian cells. For this purpose, we measured the diameter of oocytes from antral follicles in both control and Oo-Insr;Igf1r mice (fig. 4). We did not observe any difference between WT and mutants, suggesting that the insulin signaling mediated by both Insr and Igf1r is not required for oocyte growth and cell size increase.

Pitetti et al.

Fig. 5. Oo-Insr;Igf1r mutant mice are fertile. A The number of lit-

ters obtained after 6 months of mating was similar between control and Oo-Insr;Igf1r mice (5.3 8 1.2 litters and 6.7 8 0.9 litters, respectively; 3 mating cages). B However, the number of pups per litter was slightly reduced in Oo-Insr;Igf1r females compared to control animals (6.5 8 0.04 versus 8.0 8 0.35 pups per litter, respectively). * p ! 0.05 vs. control.

Reproductive Function in Oo-Insr;Igf1r Mutant Mice Is Indistinguishable from Control Littermates Despite the absence of apparent histological differences in mice lacking insulin signaling specifically in oocytes, we further investigated the effects of these mutations on reproductive functions. Oo-Insr;Igf1r mutant mice and 6-week-old female controls were each mated with a C57B/6 wild type male for a period of 6 months, and frequencies of parturition and litter size were recorded. Mutant females were able to give birth to normal progeny; the number of litters appeared to be unaffected, whereas the number of pups per litter was slightly reduced in mutants compared with controls (fig. 5). Despite the slight but significant differences in litter size recorded between control and Oo-Insr;Igf1r mutant mice, these results suggest that loss of function of both Insr and Igf1r in oocytes does not drastically impair reproductive ability.

Discussion

In this study, we have generated mutant mice with targeted disruption of Insr, Igf1r, or both receptors specifically in developing oocytes. Through the characterization of different parameters of female reproductive funcDispensable Role of Insulin Receptors in Mouse Oocytes

tion, such as parturition frequency, litter size, pups per litter, estrous cyclicity, and oocyte maturation, we show that Insr and Igf1r are dispensable for oocyte and follicular development, maturation, as well as early embryonic development. The absence of a reproductive phenotype in mice lacking Insr and Igf1r in oocytes raises a question on the efficiency of Cre-mediated deletion. This aspect of potentially incomplete Cre-mediate excision and leftover function is particularly relevant since our conclusions are based on negative data, considering that floxed loci may respond differently to the same Cre transgene, while different Cre transgenes may have different effects on the same floxed locus [Stratikopoulos et al., 2008]. However, our evidence indicates that the absence of a phenotype is not linked to incomplete deletion of the Insr and Igf1r loci in oocytes. Indeed, we have presented strong genetic evidence documenting that Oo-Insr;Igf1r females systematically transmit Insr and Igf1r deleted alleles to their progeny, which indicates that complete and specific ablation of both Insr and Igf1r had been achieved in developing oocytes (fig. 2G). Finally, we have unpublished data showing that when Oo-Insr;Igf1r females are mated with males bearing a germ cell-specific deletion of Insr and Igf1r (Ngn3:cre;Insrfx/fx;Igf1rfx/f x animals), 100% of the embryos obtained appear constitutive double mutants, i.e., the phenotype displays a male-to-female sex-reversal similar to what we observed in Insr;Igf1r; and insulin receptor related receptor (Irr) triple constitutive mutant embryos [Nef et al., 2003]. Collectively, these data indicate that we have achieved specific and efficient deletion of insulin signaling in oocytes. Therefore, the absence of reproductive phenotypes apparently reflects the non-essential role of these receptors in oocytes for follicular development and maturation. In Drosophila the insulin signaling pathway plays an essential role in controlling body, organ, and cell size. Mutations in insulin signaling protein homologues such as insulin receptor substrates (Chico), PI(3)K (Dp110), PTEN (dPTEN), Akt/PKB and (DAkt1/dPKB) all lead to changes in cell size and cell number [Brogiolo et al., 2001]. In contrast, the involvement of the insulin/IGF signaling pathway in mediating cell size in mammals has never been shown: mice lacking both Insr and Igf1r did not reveal any particular effect on cell size [Baker et al., 1993; Liu et al., 1993; Nef et al., 2003]. However, we believe that oocyte growth during folliculogenesis might represent the most stringent system to assess whether the insulin/ IGF signaling pathway mediates, even partially, mammalian cell size. Indeed, the developing oocyte grows extenSex Dev

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sively during follicular maturation and ultimately reaches a maximal diameter of 70 m corresponding to a cellular volume several orders of magnitude higher than that of a somatic cell. Due to the perinatal lethality of Insr and Igf1r constitutive mutants, it had not previously been possible to assess oocyte growth in these animals. When measuring the oocyte diameter of antral follicles in control and mutant animals with an oocyte-specific deletion of both Insr and Igf1r, we did not find any reduction in size. These data confirm that in mammals, cell size is not mediated by the insulin/IGF growth factors even in extreme cases of cell size increase such as oocyte growth. Previous studies have reported the expression of both INSR and IGF1R in oocytes and have indicated a promoting role of insulin and IGF1 in the in vitro acceleration of meiosis in nude oocytes [Sakaguchi et al., 2002; Stefanello et al., 2006]. A potential explanation for these seemingly conflicting results might be the difference in experimental design. In this report, we performed loss-offunction experiments while the above mentioned in vitro data could be considered as gain-of-function experiments, owing to the large amount of insulin/IGF in the culture medium. In fact, gain of function in the insulin/ IGF1 pathway could be involved in premature ovarian failure (POF). In humans, POF seems to be related to increased insulin signaling. Supporting the effects of a gain of function in the insulin/IGF pathway, oocyte-specific deletion of Pten, a negative regulator of the insulin signaling, leads to premature activation of primordial follicles [Reddy et al., 2008]. These phenotypes are reminiscent of human premature ovarian failure. PTEN negatively regulates PI3-Kinase and AKT which in turn suppress FOXO3a. Consistent with these findings, knock-out of Foxo3a leads to similar phenotypes [Castrillon et al., 2003]. In contrast, selective disruption of Pten in granulosa cells promotes granulose cell survival and results in increased ovulation in the adult mouse ovary [Fan et al.,

Acknowledgments The authors wish to thank Françoise Kühne and Virginie Godino for technical assistance. This work was supported by grants to S.N. from the Swiss National Science Foundation (3100A0119862 and 3100A0-108245). C.R.C is funded by a grant from the Ernst & Lucie Schmidheiny Foundation.

Accili D, Drago J, Lee EJ, Johnson MD, Cool MH, et al: Early neonatal death in mice homozygous for a null allele of the insulin receptor gene. Nat Genet 12:106–109 (1996). Acevedo N, Ding J, Smith GD: Insulin signaling in mouse oocytes. Biol Reprod 77: 872–879 (2007). Augustin R, Pocar P, Wrenzycki C, Niemann H, Fischer B: Mitogenic and anti-apoptotic activity of insulin on bovine embryos produced in vitro. Reproduction 126: 91–99 (2003).

References

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2008]. The fact that both FSH and IGF share common signaling pathways, such as the PI3-Kinase and AKT pathway in granulosa cells, suggests that FSH response is potentiated by autocrine/paracrine action of IGF1 in these supporting cells. The absence of a phenotype in oocytes lacking Insr, Igf1r, or both receptors is in striking contrast with the effects observed in mice lacking constitutively Igf1, which are infertile due to an arrest of follicular development at the preantral or early antral stage [Liu et al., 1993; Zhou et al., 1997]. Whatever the exact function of IGF1 in the ovulatory process may be, this function is retained in OoInsr;Igf1r mice. This difference in phenotype may simply reflect the mode of action of IGF1 which probably acts exclusively on granulosa cells. Indeed, IGF1 is essential for normal basal and estrogen-induced granulosa cell proliferation and follicular growth [Kadakia et al., 2001]. It remained unclear whether IGF1 action is mediated through an autocrine/paracrine or/and an endocrine (liver-derived) fashion. In fact, both female growth hormone receptor mutants lacking endocrine IGF1 or female mice possessing only endocrine IGF1 are (sub)fertile suggesting that both the endocrine and autocrine/paracrine IGF1 functions can sustain female fertility [Chandrashekar et al., 2004; Stratikopoulos et al., 2008]. We propose that both endocrine and autocrine/paracrine IGF1 act primarily on follicular/granulosa cells but not on oocytes to promote follicular development and oocyte maturation.

Sex Dev

Baker J, Liu JP, Robertson EJ, Efstratiadis A: Role of insulin-like growth factors in embryonic and postnatal growth. Cell 75:73–82 (1993). Baker J, Hardy MP, Zhou J, Bondy C, Lupu F, et al: Effects of an Igf1 gene null mutation on mouse reproduction. Mol Endocrinol 10: 903–918 (1996). Bohni R, Riesgo-Escovar J, Oldham S, Brogiolo W, Stocker H, et al: Autonomous control of cell and organ size by CHICO, a Drosophila homolog of vertebrate IRS1–4. Cell 97: 865– 875 (1999).

Pitetti et al.

Brogiolo W, Stocker H, Ikeya T, Rintelen F, Fernandez R, Hafen E: An evolutionarily conserved function of the Drosophila insulin receptor and insulin-like peptides in growth control. Curr Biol 11: 213–221 (2001). Bruning JC, Michael MD, Winnay JN, Hayashi T, Horsch D, et al: A muscle-specific insulin receptor knockout exhibits features of the metabolic syndrome of NIDDM without altering glucose tolerance. Mol Cell 2:559–569 (1998). Bruning JC, Gautam D, Burks DJ, Gillette J, Schubert M, et al: Role of brain insulin receptor in control of body weight and reproduction. Science 289:2122–2125 (2000). Cara JF, Rosenfield RL: Insulin-like growth factor I and insulin potentiate luteinizing hormone-induced androgen synthesis by rat ovarian thecal-interstitial cells. Endocrinology 123:733–739 (1988). Castrillon DH, Miao L, Kollipara R, Horner JW, DePinho RA: Suppression of ovarian follicle activation in mice by the transcription factor Foxo3a. Science 301:215–218 (2003). Cederroth CR, Schaad O, Descombes P, Chambon P, Vassalli JD, Nef S: Estrogen receptor alpha is a major contributor to estrogen-mediated fetal testis dysgenesis and cryptorchidism. Endocrinology 148:5507–5519 (2007). Chandrashekar V, Zaczek D, Bartke A: The consequences of altered somatotropic system on reproduction. Biol Reprod 71: 17–27 (2004). Dietrich P, Dragatsis I, Xuan S, Zeitlin S, Efstratiadis A: Conditional mutagenesis in mice with heat shock promoter-driven Cre transgenes. Mamm Genome 11:196–205 (2000). Dupont J, Holzenberger M: Biology of insulinlike growth factors in development. Birth Defects Res C Embryo Today 69: 257–271 (2003). Efstratiadis A: Genetics of mouse growth. Int J Dev Biol 42: 955–976 (1998). Eppig JJ: Oocyte control of ovarian follicular development and function in mammals. Reproduction 122:829–838 (2001). Fan HY, Liu Z, Cahill N, Richards JS: Targeted disruption of Pten in ovarian granulosa cells enhances ovulation and extends the life span of luteal cells. Mol Endocrinol 22:2128–2140 (2008). Gomez E, Tarin JJ, Pellicer A: Oocyte maturation in humans: the role of gonadotropins and growth factors. Fertil Steril 60: 40–46 (1993). Granelli-Piperno A, Reich E: A study of proteases and protease-inhibitor complexes in biological fluids. J Exp Med 148: 223–234 (1978).

Dispensable Role of Insulin Receptors in Mouse Oocytes

Huarte J, Belin D, Vassalli JD: Plasminogen activator in mouse and rat oocytes: induction during meiotic maturation. Cell 43: 551–558 (1985). Joshi RL, Lamothe B, Cordonnier N, Mesbah K, Monthioux E, et al: Targeted disruption of the insulin receptor gene in the mouse results in neonatal lethality. EMBO J 15:1542– 1547 (1996). Kadakia R, Arraztoa JA, Bondy C, Zhou J: Granulosa cell proliferation is impaired in the Igf1 null ovary. Growth Horm IGF Res 11: 220– 224 (2001). Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685 (1970). Lan ZJ, Xu X, Cooney AJ: Differential oocytespecific expression of Cre recombinase activity in GDF-9-iCre, Zp3cre, and Msx2cre transgenic mice. Biol Reprod 71: 1469–1474 (2004). Levy MJ, Hernandez ER, Adashi EY, Stillman RJ, Roberts CT Jr, LeRoith D: Expression of the insulin-like growth factor (IGF)-I and -II and the IGF-I and -II receptor genes during postnatal development of the rat ovary. Endocrinology 131:1202–1206 (1992). Liu JP, Baker J, Perkins AS, Robertson EJ, Efstratiadis A: Mice carrying null mutations of the genes encoding insulin-like growth factor I (Igf-1) and type 1 Igf receptor (Igf1r). Cell 75: 59–72 (1993). Louvi A, Accili D, Efstratiadis A: Growth-promoting interaction of IGF-II with the insulin receptor during mouse embryonic development. Dev Biol 189:33–48 (1997). Ludwig T, Eggenschwiler J, Fisher P, D’Ercole AJ, Davenport ML, Efstratiadis A: Mouse mutants lacking the type 2 IGF receptor (IGF2R) are rescued from perinatal lethality in Igf2 and Igf1r null backgrounds. Dev Biol 177:517–535 (1996). Nef S, Verma-Kurvari S, Merenmies J, Vassalli JD, Efstratiadis A, et al: Testis determination requires insulin receptor family function in mice. Nature 426:291–295 (2003). Nelson JF, Felicio LS, Randall PK, Sims C, Finch CE: A longitudinal study of estrous cyclicity in aging C57BL/6J mice: I. Cycle frequency, length and vaginal cytology. Biol Reprod 27: 327–339 (1982). Papaioannou MD, Pitetti JL, Ro S, Park C, Aubry F, et al: Sertoli cell dicer is essential for spermatogenesis in mice. Dev Biol 326: 250–259 (2009). Pedersen T, Peters H: Proposal for a classification of oocytes and follicles in the mouse ovary. J Reprod Fertil 17:555–557 (1968). Reddy P, Liu L, Adhikari D, Jagarlamudi K, Rajareddy S, et al: Oocyte-specific deletion of Pten causes premature activation of the primordial follicle pool. Science 319: 611–613 (2008).

Sex Dev

Sakaguchi M, Dominko T, Yamauchi N, Leibfried-Rutledge ML, Nagai T, First NL: Possible mechanism for acceleration of meiotic progression of bovine follicular oocytes by growth factors in vitro. Reproduction 123: 135–142 (2002). Samoto T, Maruo T, Ladines-Llave CA, Matsuo H, Deguchi J, et al: Insulin receptor expression in follicular and stromal compartments of the human ovary over the course of follicular growth, regression and atresia. Endocr J 40:715–726 (1993). Soriano P: Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet 21: 70–71 (1999). Stefanello JR, Barreta MH, Porciuncula PM, Arruda JN, Oliveira JF, et al: Effect of angiotensin II with follicle cells and insulin-like growth factor-I or insulin on bovine oocyte maturation and embryo development. Theriogenology 66: 2068–2076 (2006). Stratikopoulos E, Szabolcs M, Dragatsis I, Klinakis A, Efstratiadis A: The hormonal action of IGF1 in postnatal mouse growth. Proc Natl Acad Sci USA 105:19378–19383 (2008). Stutz A, Conne B, Huarte J, Gubler P, Volkel V, et al: Masking, unmasking, and regulated polyadenylation cooperate in the translational control of a dormant mRNA in mouse oocytes. Genes Dev 12:2535–2548 (1998). van den Hurk R, Zhao J: Formation of mammalian oocytes and their growth, differentiation and maturation within ovarian follicles. Theriogenology 63: 1717–1751 (2005). Vassalli JD, Dayer JM, Wohlwend A, Belin D: Concomitant secretion of prourokinase and of a plasminogen activator-specific inhibitor by cultured human monocytes-macrophages. J Exp Med 159:1653–1668 (1984). Xuan S, Kitamura T, Nakae J, Politi K, Kido Y, et al: Defective insulin secretion in pancreatic beta cells lacking type 1 IGF receptor. J Clin Invest 110:1011–1019 (2002). Zhou J, Bondy C: Anatomy of the human ovarian insulin-like growth factor system. Biol Reprod 48:467–482 (1993). Zhou J, Chin E, Bondy C: Cellular pattern of insulin-like growth factor-I (IGF-I) and IGF-I receptor gene expression in the developing and mature ovarian follicle. Endocrinology 129:3281–3288 (1991). Zhou J, Kumar TR, Matzuk MM, Bondy C: Insulin-like growth factor I regulates gonadotropin responsiveness in the murine ovary. Mol Endocrinol 11:1924–1933 (1997).

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