Lapatinib inhibits meiotic maturation of porcine oocyte-cumulus complexes cultured in vitro in gonadotropin-supplemented medium

ORIGINAL ARTICLES: REPRODUCTIVE BIOLOGY

Lapatinib inhibits meiotic maturation of porcine oocyte-cumulus complexes cultured in vitro in gonadotropin-supplemented medium Eva Nagyova, D.V.M., Ph.D.,a Lucie Nemcova, Ph.D.,a Alzbeta Mlynarcikova, Ph.D.,b Sona Scsukova, Ph.D.,b and Jaroslav Kalous, Ph.D.a a b

Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, Libechov, Czech Republic; and Institute of Experimental Endocrinology, SAS, Bratislava, Slovakia

Objective: To determine whether inhibition of epidermal growth factor (EGF) receptor tyrosine kinase with lapatinib affects oocyte maturation, expression of the cumulus expansion-associated genes such as tumor necrosis factor alpha-induced protein 6 (TNFAIP6) and prostaglandin-endoperoxide synthase 2 (PTGS2), and synthesis of hyaluronan (HA) and progesterone (P) by porcine oocyte cumulus complexes (OCC). Design: Our work focuses on lapatinib, an orally active small molecule that selectively inhibits the tyrosine kinase domain of both EGF receptor and human EGF receptor 2, and downstream signaling. Setting: A reproductive biology laboratory. Patient(s): Not applicable. Intervention(s): Porcine OCC were cultured in vitro in a medium with FSH/LH in the presence/absence of lapatinib. Main Outcome Measure(s): Methods performed: real-time reverse transcriptase-polymerase chain reaction (PCR), immunofluorescence, RIA. Result(s): In FSH/LH-stimulated and expanded cumulus oophorus extracellular matrix, HA was detected with biotinylated HA-binding proteins. However, weaker HA- and weaker cytoplasmic TNFAIP6 were detected were detected in lapatinib-pretreated OCC. The expression of the two cumulus expansion-associated gene transcripts was significantly decreased and synthesis of HA by cumulus cells was reduced. Lapatinib (10 mM) inhibited FSH/LH-induced oocyte meiotic maturation. Progesterone production increased after OCC stimulation with FSH/LH and was significantly decreased by lapatinib (10 mM). Conclusion(s): Lapatinib inhibits oocyte maturation and reduces expression of cumulus expansion-associated transcripts, and synthesis of HA and P in OCC cultured in vitro in FSH/ Use your smartphone LH-supplemented medium. (Fertil SterilÒ 2013;99:1739–48. Ó2013 by American Society for to scan this QR code Reproductive Medicine.) and connect to the Key Words: Lapatinib, oocyte-cumulus complex, oocyte maturation, epidermal growth factor Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/nagyovae-lapatinib-meiotic-maturation-oocyte-cumulus-complex/

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t has been shown that epidermal growth factor (EGF) receptor mediates the ovulatory response to LH in the ovarian follicle and the sustained activity of EGF receptor is an absolute requisite for LH-induced oocyte maturation and

cumulus expansion (1). However, abnormally elevated EGF receptor kinase activity can lead to various pathological states, including proliferative diseases such as cancer. The human EGF receptor (HER) family consists of four closely related

Received September 3, 2012; revised November 20, 2012; accepted December 26, 2012; published online January 30, 2013. E.N. has nothing to disclose. L.N. has nothing to disclose. A.M. has nothing to disclose. S.S. has nothing to disclose. J.K. has nothing to disclose. Supported by GlaxoSmithKline, s.r.o. Czech Republic and grant P502/11/0593 from Grant Agency of the Czech Republic. Reprint requests: Eva Nagyova, D.V.M., Ph.D., Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, 27721 Libechov, Czech Republic (E-mail: [email protected]). Fertility and Sterility® Vol. 99, No. 6, May 2013 0015-0282/$36.00 Copyright ©2013 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2012.12.040 VOL. 99 NO. 6 / MAY 2013

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transmembrane receptors: HER1 (human EGF receptor 1), HER2/c-Erb-B2, HER3/ Erb-B3 and HER4/Erb-B4. These members of the type I receptor tyrosine kinase family are frequently implicated in cancer (2, 3). The HER family-related downstream signaling plays a crucial role in cell proliferation, survival, migration, and differentiation (4, 5). The HER2, although having no endogenous ligand, is the preferred partner for heterodimerization with EGF receptor, HER3, and HER4 as it amplifies the mitogenic signal with potent growth and survival effects. Several reversible 1739

ORIGINAL ARTICLE: REPRODUCTIVE BIOLOGY and irreversible small molecule tyrosine kinase inhibitors with varying degrees of selectivity have been developed (erlotinib, gefitinib, lapatinib, afatinib). These small molecule agents compete with adenosine triphosphate (ATP) binding in the kinase domain of the receptor and inhibit downstream signaling. Our work focuses on lapatinib (GW572016, Tykerb/ Tyverb; GlaxoSmithKline), an orally active small molecule that reversibly and selectively inhibits the tyrosine kinase domain of both EGF receptor and HER2 (6) by binding to the ATP-binding site of the kinase, preventing autophosphorylation or rapid development of resistance to monotherapies (7). The inhibition in EGF receptor-expressing and HER2-overexpressing tumors blocks the activating signaling cascades in the mitogen activated protein kinase (MAPK) and phosphatidyl inositol 3 kinase (PI3K) pathways resulting in growth arrest and/or apoptosis. Female fertility is comprised of highly orchestrated endocrine events. Ovarian follicle development is regulated by the hypothalamic-pituitary-ovarian axis, in which GnRH controls the release of the gonadotropic hormones, FSH and LH, and ovarian steroids exert negative and positive regulatory effects on GnRH secretion (8). Follicular growth and differentiation are controlled by pituitary gonadotropins, as well as paracrine factors produced by granulosa cells (GCs) and theca cells (9). Park et al. (10) presented evidence that LH exerts at least part of its action on ovulation by stimulating the production of EGF family members; amphiregulin (AREG), epiregulin (EREG), and betacellulin (BTC), in mouse preovulatory follicles. In pig, FSH promptly stimulated expression of AREG and EREG, but not BTC in cultured oocyte-cumulus complex (OCC) (11). Thus, EGF-like factors induced by LH are sufficient to switch on cumulus expansion and oocyte maturation and their signaling, although EGF receptor is necessary for LH action. The EGF receptor, a 170-kDa glycoprotein, was the first member of the EGF receptor family to be identified as the receptor for EGF. Furthermore, EGF receptor was found to have kinase activity when stimulated with ligands and to be capable of phosphorylating tyrosine residues on both, itself, and downstream targets (12). In porcine OCC, FSH promotes maturation of the EGF response pathway, as evidenced by a strong increase in EGF-induced EGF receptor tyrosine phosphorylation, synthesis of hyaluronan (HA), and subsequently cumulus expansion (13). In contrast, a specific EGF receptor tyrosine kinase inhibitor, AG1478, is able to block the FSH-induced oocyte maturation. In addition, it reduces synthesis of HA, cumulus expansion, as well as P production by porcine OCC (14). In rat follicles, AG1478 also blocked the LH-induced oocyte maturation and suppressed LH-stimulated expression of cumulus expansion-related genes such as prostaglandin-endoperoxide synthase 2 (Ptgs2), HA synthase 2 (Has2), and tumor necrosis factor (TNF) alpha-induced protein 6 (Tnfaip6) (15). These genes are essential for formation of the viscoelastic HA-rich expanded cumulus oophorus extracellular matrix during maturation of porcine OCC (14, 16–20). The synthesis of HA by porcine cumulus cells in vitro is stimulated with FSH and subsequently HA becomes the major structural element of the expanded viscoelastic extracellular matrix in porcine OCC (16). Of importance, heavy chains of serum-derived 1740

inter-alpha trypsin inhibitor are covalently linked to HA during porcine cumulus expansion and significantly contribute to cumulus matrix organization (17). Furthermore, the presence of TNFAIP6 protein in porcine expanding OCC provided important evidence that TNFAIP6 catalyzes the transfer of heavy chains to HA in pig cumulus matrix (18, 19). At present no studies reported the effect of lapatinib on processes essential for ovulation and pregnancy, such as oocyte meiotic maturation and cumulus expansion. Nevertheless, it has been suggested that the use of biological agents in female cancers increases the probability that some women will conceive while taking a growth factor signaling pathway inhibitor. Finally, there is a report describing the delivery of a baby by a woman who conceived while being treated with lapatinib, suggesting that this drug may not be effective at inhibition of EGF receptor signaling pathway in reproductive tissues (21). Therefore, we wondered whether lapatinib through EGF receptor tyrosine kinase inhibition is able to affect the oocyte meiotic maturation, the expression of cumulus expansion associated genes (PTGS2, TNFAIP6), HA synthesis, and the P secretion by in vitro cultured porcine OCC.

MATERIALS AND METHODS Isolation and Culture of OCC Ovaries were collected from slaughtered gilts at a local abattoir. The follicular fluid (FF) was aspirated from growing follicles 3–5 mm in diameter. The OCC were retrieved from the sediment and washed in the phosphate-buffered saline (PBS)/polyvinyl-pyrrolidone (3 mg/mL; Sigma). For extraction of total RNA, 30 OCC were cultured in 300 mL of M199 (Sevapharma) supplemented with 6.25 mM N-2hydroxyethylpiperazine-N0 -2-ethanesulfonic acid (HEPES), 20 mM sodium bicarbonate, 0.91 mM sodium pyruvate, 1.62 mM calcium lactate, penicillin G (50 mg/mL), and streptomycin (50 mg/mL) (Sigma) with 5% fetal bovine serum (FBS) (Sigma) in four-well dishes (Nunclon) at 38.5 C in an atmosphere of 5% CO2 in air in the presence/absence of lapatinib (a selective inhibitor of both EGF receptor and HER2 tyrosine kinase; GW572016, Tykerb/Tyverb; GlaxoSmithKline). Dimethyl sulfoxide (DMSO) was used as a vehicle for preparation of lapatinib (10 mM stock in DMSO). Maximal final concentration of DMSO in lapatinib containing culture medium was 0.1% (vol/vol). The control groups were treated with the same concentration of DMSO. The effect of different concentrations of lapatinib (1–100 mM) was tested in our experiments. This dose range covers the human dose of 1,500 mg/d (5.6 mM). Expansion of cumulus cells was stimulated by the addition of 100 ng/mL of FSH (Gonal Serono Austria GmbH) and 100 ng/mL LH (Luveris; Serono Austria GmbH) into the culture medium. Lapatinib was added to the culture medium at the time of OCC addition. After 1 hour of culture, both gonadotropins were added. The groups of 15 OCC, obtained at 0 and 4 hours after in vitro culture, were washed twice in PBS/polyvinyl-pyrrolidone solution and used for the extraction of total RNA. To assess the effect of lapatinib on P production, OCC were cultured in the above-mentioned medium VOL. 99 NO. 6 / MAY 2013

Fertility and Sterility® supplemented with 5% FBS and both gonadotropins in the presence of different concentrations of lapatinib (1–100 mM) for 44 hours. At the end of the incubation period, the culture media were collected and stored at 20 C until assayed for P. The Animal Research Committee of Academy of Sciences of the Czech Republic approved all procedures.

RNA Isolation The OCC were cultured in FSH/LH-supplemented medium in the absence/presence of lapatinib (1 and 10 mM) for 4 hours. For the RNA isolation, 15 OCC (representing 1.5  104 cells) were lysed in 350 mL of lysis buffer (Qiagen) and stored frozen at 80 C. Total RNA was extracted with RNeasy Mini kit (Qiagen) following the manufacturer's instructions. RNA was eluted in 30 mL of RNase-free water and stored at 80 C. The concentration of the RNA in the samples was measured by a spectrophotometer Nanodrop ND-1000 (NanoDrop Technologies).

Real-time Reverse Transcription-Polymerase Chain Reaction The effect of lapatinib on the expression of messenger RNA (mRNA) was assessed by a real-time reverse transcriptionpolymerase chain reaction (RT-PCR). Amplification was performed on the RotorGene 3000 cycler (Corbett Research) using One-Step RT-PCR Kit (Qiagen) with gene-specific oligonucleotide primers directed sequences of pig TNFAIP6 (forward: 50 -TCA TAA CTC CAT ATG GCT TGA AC-30 , 396 bp; reverse: 50 -TCT TCG TAC TCA TTT GGG AAG CC-30 , 396 bp (22), PTGS2 (forward: 50 -TCG ACC AGA GCA GAG AGA TGA GAT-30 and reverse: 50 -ACC ATA GAG CGC TTC TAA CTC TGC-30 , 260 bp, NM_214321, and ACTB (forward: 50 -GAG AAG CTC TGC TAC GTC G-30 and reverse: 50 -CCA GAC AGC ACC GTG TTG G-30 , 255 bp, U07786). The concentration of RNA in samples was adjusted to 20 ng/mL with RNase-free water. The 25-mL total reaction volume contained QIAGEN OneStep RT-PCR Buffer (1), dNTP Mix (400 mM of each), reverse and forward primers (both 400 mM), SybrGreenI (0.5 mL of 1:1,000 stock solution; Molecular Probes), RNasine ribonuclease inhibitor (5 IU; Promega), QIAGEN OneStep RT-PCR Enzyme Mix (1 mL), and template RNA (3 mL). The reaction conditions were as follows: reverse transcription at 50 C for 30 minutes; initial activation at 95 C for 15 minutes; cycling comprised of denaturation at 94 C for 20 seconds, annealing at 54 C, 55 C, and 54 C for 20 seconds for TNFAIP6, PTGS2, and ACTB, respectively, and extension at 72 C for 30 seconds; final extension at 72 C for 5 minutes. Fluorescence data were acquired at 3 C below the melting temperature of products to distinguish the possible primer dimers. Products of the RT-PCR were separated by electrophoresis on 1.5% agarose gel and visualized with ethidium bromide staining. The relative abundance of templates in different samples was determined using comparative analysis software (Corbett Research). The results for individual target messages were normalized according to the relative concentration of the internal standard, ACTB. VOL. 99 NO. 6 / MAY 2013

Progesterone RIA After 44 hours of cultivation of 10 OCC in 100 mL media supplemented with FSH/LH and lapatinib, the media were collected and used for determination of P secretion by a RIA (Immunotech SAS). According to data provided by the supplier, the cross-reactivities of the P antibody were less than 20% with other progestins, and less than 0.06% with the androgens and estrogens tested. The intra-assay and interassay coefficients of variation (CV) were below 5.8% or equal to 9.0%, respectively.

Hyaluronan Synthesis Synthesis of HA was measured as described previously (16). Briefly, groups of 10 porcine OCC were cultured for 24 hours in the FSH/LH-supplemented medium with or without lapatinib (1 and 10 mM) in the presence of 2.5 mCi of D-[6-3H] glucosamine hydrochloride (MGP). The cultures were terminated by adding 10 mL of a solution containing 50 mg/mL pronase (Sigma) and 10% Triton X-100 in 0.2 M Tris buffer at pH 7.8. The samples were incubated for 2 hours at 38.5 C and then transferred to Whatman 3MM filter paper circles. The circles were air dried and then washed through three changes of solution containing 0.5% cetylpyridinium chloride and 10 mM nonradioactive glucosamine hydrochloride (MGP) for 45 minutes each. The circles were dried once again and radioactivity was measured using a liquid scintillation counter. Synthesis of HA was measured either in medium plus OCC (total HA) or within the complexes alone (retained HA).

Assessment of Oocyte Maturation and Cumulus Expansion Complexes cultured in FSH/LH-supplemented medium in the absence/presence of lapatinib (1–100 mM) for 44 hours were scored for germinal vesicle (GV), germinal vesicle breakdown (GVBD), and metaphase II. Denuded oocytes were mounted on slides and fixed for 48 hours in acetic acid/ethanol (1:3, vol/ vol). After this procedure, oocytes were stained with 1% orcein and examined under a phase-contrast microscope. Cumulus expansion was evaluated according to a subjective scoring system described previously in our article (16).

Immunofluorescence The OCC cultured in vitro in the presence/absence of lapatinib for 24 hours were fixed at room temperature with 2% (vol/vol) paraformaldehyde-PBS (pH 7.4). After 40 minutes, the OCC were rinsed three times in 0.1 M PBS and blocked for 40 minutes in 0.1 M PBS containing 5% normal goat serum and 0.1% Triton X-100. The fixed OCC were incubated overnight with either biotinylated HA-binding protein (HABP; 1:20 dilution; Merck) or with rabbit anti-human TNFAIP6 (1:100 dilution; Santa Cruz Biotechnology). After washing three times with PBS, the OCC were incubated for 2 hours at room temperature with the fluorescent Alexa Fluor 488 streptavidin-conjugate (1:200) for HABP detection or with Alexa 488 goat anti-rabbit IgG (1:500) for TNFAIP6 detection. As a control for the specificity of the immunostaining, the primary antibodies were omitted during the 1741

ORIGINAL ARTICLE: REPRODUCTIVE BIOLOGY processing of some samples. DNA was stained with 2.5 mg/ mL of DAPI (4,6-diamidino-2-phenylindole; Molecular Probes). Finally, the OCC were washed three times in 0.1 M PBS, mounted in fluorescent mounting medium (DAKO), and examined with an Olympus AX70 microscope equipped with a DP30BW CCD camera. Image files were edited with Adobe Photoshop computer software.

Statistical Analysis Three independent sets of total RNA samples were analyzed in each experiment, and each RT-PCR was run in duplicate. Oneway analysis of variance (ANOVA) followed by the Tukey multiple comparison test was used to evaluate the results of real-time RT-PCR. To compare the influence of the inhibitor on the expression of mRNA, an unpaired t test was used. The GraphPad Prism version 4 (GraphPad Software) was used for the statistical calculations. Differences were considered significant at P< .05. Progesterone, meiotic maturation, and HA data were analyzed by ANOVA followed by the Tukey multiple comparison test. Error bars in each diagram indicate the SEM.

RESULTS Effect of Lapatinib on Oocyte Maturation To investigate the effects of lapatinib on resumption of meiosis, intact OCC were cultured in medium supplemented with FBS, in the presence or absence of FSH/LH and inhibitor lapatinib (1–100 mM) for 44 hours (Table 1). This dose range covers the human dose of 1,500 mg/d (5.6 mM). Stimulation of OCC with FSH/LH caused that 89% of oocytes resumed meiosis and underwent GVBD. Addition of lapatinib (1, 10, 50, and 100 mM) to the culture medium significantly inhibited FSH/LH-induced meiotic maturation (P< .001) as almost all treated oocytes remained at the GV stage (92%, 84%, 82%, and 77 %, respectively). Together 729 denuded oocytes were used for evaluation of meiotic maturation (Table 1).

Cumulus Expansion of OCC Cultured In Vitro in the Presence of Lapatinib Unstimulated complexes were not able to expand in vitro when cultured only in the presence of FBS (Fig. 1A). How-

TABLE 1 Effect of lapatinib on oocyte meiotic maturation. Group F/L F/LþLap 1 mM F/LþLap 10 mM F/LþLap 50 mM F/LþLap 100 mM

GV (% ± SEM)

GVBD (% ± SEM)

No. of oocytes

11.0  5.1a 91.7  4.5b 84.0  2.8b 82.4  3.6b 76.7  7.1b

89.0  5.1a 8.3  4.5b 16.0  2.8b 17.6  3.6b 23.3  7.1b

126 216 272 46 69

Note: Porcine oocyte-cumulus complexes were cultured in FSH/LH (F/L) supplemented medium with/without inhibitor lapatinib (Lap; 1–100 mM) for 44 hours. At the end of the culture period denuded oocytes were used for assessment of meiotic maturation. Different superscripts indicate significant differences within one column (P< .001). Nagyova. Lapatinib inhibits oocyte maturation. Fertil Steril 2013.

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ever, after 24 and 44 hours of FSH/LH stimulation, fully expanded cumulus oophorus extracellular matrix was observed. In contrast, cumulus expansion of OCC was reduced in the presence of lapatinib. The reduction was dependent on the concentration of lapatinib added into the culture medium. Superficial layers of OCC were partially expanded in the presence of 1 mM of lapatinib. Lapatinib at higher concentrations (10 mM; Fig. 1A) markedly reduced cumulus expansion. Given that morphological assessment of the cumulus expansion is a rather subjective assessment, for characterization of differences between expanded cumuli after in vitro culture with/without lapatinib, FSH/ LH-stimulated HA synthesis and its incorporation were measured further.

Effect of Lapatinib on Expression of Cumulus Expansion-related Transcripts (TNFAIP6, PTGS2) To determine whether or not lapatinib affects the expression of cumulus expansion-related genes, we analyzed the mRNA levels of TNFAIP6 and PTGS2 in OCC stimulated in the presence of FSH/LH for 4 hours. The expression of TNFAIP6 and PTGS2 transcripts was substantially upregulated after 4 hours in the presence of FSH/LH in comparison to control without gonadotropins (FBS; P< .001; Fig. 1B). The mRNA level of TNFAIP6 was significantly lower in the presence of lapatinib (10 mM) in FSH/LH-stimulated complexes (P< .01; Fig. 1B), in agreement with a weaker immunofluorescence staining of TNFAIP6 protein within the OCC (Fig. 2). In addition, the expression of PTGS2 transcript was significantly decreased in lapatinib-pretreated (1 and 10 mM) OCC in the presence of FSH/LH (P< .01 and P< .001, respectively; Fig. 1B).

Effect of Lapatinib on HA Synthesis by OCC To determine whether inhibition of EGF receptor signaling pathway (with lapatinib) affects gonadotropin-stimulated HA synthesis and its incorporation within the expanded cumulus oophorus extracellular matrix, compact OCC were cultured with FSH/LH (100 ng/mL for both hormones) for 24 hours in medium supplemented with 5% FBS without/ with lapatinib (1 and 10 mM). Stimulation of intact OCC with FSH/LH resulted in a significant increase in the synthesis of HA compared with that in OCC cultured in the absence of FSH/LH (P< .001; Fig. 1C). Consistent with our morphological observations of cumulus expansion (Fig. 1A), lapatinib (10 mM) significantly reduced total HA synthesis as well as the amount of HA incorporated within the OCC (P< .01; Fig. 1C). After FSH/LH stimulation, the amount of HA incorporated within the expanded cumulus oophorus extracellular matrix represented about 65% of the total HA accumulated in the well (OCC plus medium). After addition of lapatinib (1 or 10 mM) to the culture medium, total HA synthesis was reduced by 43% and 50% (P< .01, P< .001, respectively; Fig. 1C) and the retention of HA within the OCC was decreased to less than 50% of the amount accumulated within the expanded cumulus oophorus extracellular matrix stimulated with FSH/LH alone (43% and 35%, respectively; P< .001; Fig. 1C). VOL. 99 NO. 6 / MAY 2013

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FIGURE 1

(A) Effect of lapatinib (10 mM; Lap10) on FSH/LH-stimulated cumulus expansion assessed after 24 or 44 hours. Oocyte-cumulus complexes (OCC) cultured in fetal bovine serum (FBS) represent control groups. (B) Effect of lapatinib (1, 10 mM; Lap1, Lap10) on expression of the cumulus expansion-related transcripts: TNFAIP6 and PTGS2 messenger RNA after 4 hours of FSH/LH stimulation. Relative abundance of the respective messenger RNA is expressed in arbitrary units as fold-strength increase of specific gene/ACTB ratio more than the level found in unstimulated OCC (FBS). Different superscripts above columns indicate significant differences (P<.01). (C) Effect of lapatinib (1, 10 mM; Lap1, Lap10) on FSH/ LH-stimulated synthesis (total) and retention of hyaluronan (HA) by OCC after 24 hours. Data represent the mean  SEM from at least five replicates for each treatment, prepared in duplicate (at least 50 OCC per treatment). Columns with different letters are significantly different (P<.01). (D) Effect of lapatinib (1–100 mM; Lap1, Lap10) on FSH/LH-stimulated secretion of P by OCC after 44 hours. Values represent the mean  SEM obtained from triplicates of two independent experiments. The absolute value corresponding to control (FBS) was 10.1  1.1 ng/ mL. Bars with different superscripts indicate significant differences (P<.05). Nagyova. Lapatinib inhibits oocyte maturation. Fertil Steril 2013.

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FIGURE 2

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FIGURE 2 Continued Tumor necrosis factor (TNF) alpha-induced protein 6 (TNFAIP6; green) detection in oocyte-cumulus complexes after 24 hours of stimulation with FSH/LH in the absence/presence of lapatinib (Lap; 10 mM). The oocyte-cumulus complexes were incubated with rabbit–anti-human TNFAIP6 and with fluorescent secondary probe AF488 goat–anti-rabbit IgG. Nuclear DNA was counterstained with DAPI (blue). Transmitted light images were obtained with the use of a differential interference contrast (DIC). Nagyova. Lapatinib inhibits oocyte maturation. Fertil Steril 2013.

Effect of Lapatinib on P Secretion by OCC To analyze the involvement of EGF receptor signaling pathway in steroidogenesis of cumulus cells, the effect of lapatinib (1–100 mM) on P production by OCC was investigated. Basal P synthesis by OCC cultured in medium supplemented with FBS was measured after 44 hours (Fig. 1D). Addition of FSH/LH resulted in a significant increase in P levels (35-fold; P< .01; Fig. 1D). Treatment of OCC with 1 mM of lapatinib in the presence of FSH/LH did not affect P production by OCC compared with FSH/LH-stimulated complexes (P>.05; Fig. 1D). However, treatment of the OCC with lapatinib at the three higher concentrations (10, 50, and 100 mM) significantly diminished the P secretion into the culture media compared with gonadotropin-stimulated OCC (3-, 7-, and 19-fold, respectively; P< .05; Fig. 1D).

Immunolocalization of HABP and TNFAIP6 in the Presence/Absence of Lapatinib in OCC Labeling with biotinylated HABP revealed the presence of HA in OCC cultured for 24 hours (Fig. 3). Only a faint HA labeling was detected in nonexpanded OCC cultured in control medium (5% FBS). Well-organized cumulus oophorus extracellular matrix formed after culture in FSH/LH-supplemented medium exhibited strong diffuse patterns of HA staining in the cytoplasm of cumulus cells and in the extracellular matrix of expanded cumulus. In FSH/LH-stimulated OCC cultured in the presence of lapatinib (10 mM), the cumulus expansion was reduced and weaker HA labeling was localized mostly on cytoplasmic membranes of cumulus cells. Tumor necrosis factor alpha-induced protein 6 was detected in OCC subjected to 24 hours of culture (Fig. 2). A faint cytoplasmic TNFAIP6 staining was detected in cumulus cells of nonexpanded OCC after culture in control medium (5% FBS). In addition to diffuse cytoplasmic labeling, a punctate cytoplasmic pattern of TNFAIP6 was found in expanded cumulus oophorus extracellular matrix after culture of OCC in FSH/LH-supplemented medium. When FSH/LH-stimulated OCC were cultured in the presence of lapatinib, weaker cytoplasmic TNFAIP6 labeling pattern was detected in OCC. Surprisingly, an intense fluorescent signal of TNFAIP6 was observed in the ooplasm of FSH/LH-stimulated OCC treated without or with lapatinib.

DISCUSSION Targeting the HER2 in breast cancer patients whose tumors overexpress HER2 and the development of selective protein kinase inhibitors has become an important area of drug discovery for the potential treatment of a variety of solid tumors, VOL. 99 NO. 6 / MAY 2013

such as breast, ovarian, colorectal, and pancreatic cancers, with poor clinical outcome. Konecny et al. (23) demonstrated very well a therapeutic potential of lapatinib, a selective inhibitor of both the EGF receptor and HER2 tyrosine kinases, for the treatment of endometrial cancer cells. They used lapatinib at the dose of 0.052 mM in HER2-overexpressing USPC2 endometrial cancer cells and 10.9 mM in MFE296 cells that express low levels of HER2 and EGF receptor. We have examined the effect of lapatinib (1–100 mM) on resumption of meiosis, expression of cumulus expansion-associated transcripts, synthesis of HA and P production after LH/FSH stimulation in porcine OCC in vitro. Data from the present study show that addition of lapatinib to the culture medium, irrespective of the concentration, significantly blocks FSH/LH-induced oocyte meiotic maturation. We observed that about 84% OCC rested at the GV stage and did not resume meiosis. By contrast, 89% of OCC stimulated with FSH/LH underwent GVBD and resumed meiosis during in vitro culture. Signal transduction through the EGF family of ligands is an integral requirement for oocyte maturation in response to LH (10). The EGF-like factors induced by LH or FSH stimulate oocyte maturation and cumulus expansion and their signaling through EGF receptor has been demonstrated in rat, mouse, and pig models (11, 15, 24). Also in human, EGF-like growth factors play a role in critical periovulatory events as bioactive AREG induced by hCG and accumulated in FF correlated with oocyte maturation (25). To our knowledge, this is the first study that investigates the effect of lapatinib on meiotic maturation of OCC and demonstrates a total inhibition independent of concentration. It is not surprising that the inhibitory effect of lapatinib on resumption of meiosis was similar to the effect of AG1478 (both are inhibitors of EGF receptor tyrosine kinase), recently used in experiments with porcine OCC (14). Furthermore, AG1478 blocked LH stimulation of EGF receptor phosphorylation, meiotic maturation, and expression of cumulus expansionassociated genes (Ptgs2, Tnfaip6, and Has2) in rat follicles (15). In the present article, the inhibition of EGF receptor signaling pathway with lapatinib significantly affected the expression of cumulus expansion-related transcripts (TNFAIP6, PTGS2). Although the expression of both transcripts was significantly increased after FSH/LH stimulation (present results, 11, 14), in the presence of lapatinib (10 mM) the expression was significantly decreased. The expression of TNFAIP6 transcript and protein molecules has been detected in porcine OCC, both in vivo and in vitro (18, 19). In addition, it has been shown that TNFAIP6 is essential for ovulation because Tnfaip6 null female mice have markedly impaired cumulus mucification and a lower ovulation rate than normal (26). Similarly, in 1745

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FIGURE 3

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FIGURE 3 Continued Hyaluronan (HA; green) detection in oocyte-cumulus complexes after 24 hours of stimulation with FSH/LH in the absence/presence of lapatinib (Lap; 10 mM). The oocyte-cumulus complexes were labeled with biotinylated HA-binding protein (HABP) and with fluorescent secondary probe AF488 streptavidin conjugate (green). Nuclear DNA was counterstained with DAPI (blue). Transmitted light images were obtained with the use of a differential interference contrast (DIC). Nagyova. Lapatinib inhibits oocyte maturation. Fertil Steril 2013.

Ptgs2 mutant mice, the defect in ovulation was attributed to an abnormal cumulus oophorus expansion (27). It is well known that gonadotropin-stimulated HA synthesis is required for formation of expanded cumulus oophorus extracellular matrix during the process of cumulus expansion in mouse and pig OCC (16, 17, 28), and for the release of fertilizable ova at ovulation in mouse (29). In agreement, stimulation of intact OCC with FSH/LH resulted in a significant increase in the synthesis of HA compared with that of complexes cultured in the presence of serum alone. In contrast, lapatinib significantly reduced total HA synthesis, as well as the amount of HA incorporated within the complexes. Although after FSH/LH stimulation the amount of HA accumulated within the expanded cumulus oophorus extracellular matrix represented about 65% of the total HA accumulated in the well (OCC medium), lapatinib added to the culture medium reduced the incorporation of HA within the OCC to less than 50% of the amount retained within the expanded OCC stimulated with FSH/LH. Similar results were obtained when gonadotropin-stimulated porcine OCC were cultured with the EGF receptor inhibitor AG1478 (14). Our immunofluorescence data obtained with HABP and TNFAIP6 antibodies clearly show the presence of HA and TNFAIP6 in FSH/LH-stimulated expanded cumulus oophorus extracellular matrix. Recently, both components of expanded cumulus oophorus extracellular matrix were demonstrated to be present in porcine FSH/LH-stimulated OCC (30). On the contrary, in the presence of lapatinib (10 mM), cumulus expansion was reduced. In this case only weaker immunodetection of both cumulus matrix components was observed. Progesterone is produced by the cumulus cells during meiotic maturation of LH-, FSH- or forskolin-stimulated OCC in human (31) and in pig (14, 32, 33). In the present study, addition of FSH/LH resulted in a significant increase in P levels. Treatment of OCC with the lowest concentration of lapatinib (1 mM) in the presence of FSH/LH did not affect P synthesis by OCC. Nevertheless, treatment of the OCC with lapatinib at the three higher concentrations (10, 50, and 100 mM) significantly diminished the P secretion into the culture media compared with gonadotropin-stimulated OCC (3-, 7-, and 19-fold, respectively). Similarly, a significant decrease in P production was observed when porcine OCC cultured with FSH were treated with the specific EGF receptor tyrosine kinase inhibitor AG1478 (14). Shimada and Terada (33) reported almost complete inhibition of P production and meiotic maturation when porcine OCC stimulated with LH/FSH were treated with an inhibitor of P production, aminogluthethimide. Interestingly, the inhibitory effect on meiotic maturation (GVBD) was overcome by additional P. Progesterone is VOL. 99 NO. 6 / MAY 2013

a critical steroid hormone that controls cell proliferation and differentiation. Because progestins are widely used in oral contraception (OC), it is important to clarify mechanisms of P action and cross-reaction with EGF receptor signaling pathway. The relationship between P function and EGF-like factors (AREG, EREG) in porcine OCC was recently described (34). In FSH/LH-stimulated OCC, AREG, EREG, and TACE/ ADAM17 mRNA were expressed. Treatment with a P receptor antagonist, RU486, did not affect AREG, EREG expression. However, the TACE/ADAM17 protein and the TACE/ ADAM17 protease activity (important for shedding of EGFlike factors) were significantly suppressed by RU486, indicating that the level of TACE/ADAM17 protein was regulated by the P-receptor pathway. It was concluded that P-induced TACE/ADAM17 leads to production of soluble EGF domain from cumulus cells, which enhances functional changes of cumulus cells and promotes the progression of meiotic maturation (22). In conclusion, we have shown that lapatinib, through an EGF receptor signaling pathway, is able to inhibit oocyte maturation. The effect of lapatinib was similar to AG1478, as this inhibitor after addition into culture medium, irrespective of the stimulation, completely blocked nuclear maturation of porcine oocytes (14, 35). In addition, lapatinib is able to reduce the expression of cumulus expansion-related transcripts, synthesis of HA, cumulus expansion, and P secretion by OCC cultured in vitro in FSH/LH-supplemented medium. It is in agreement with the previous study where FSH-induced synthesis of both HA and P was reduced, but not abolished, by AG1478, indicating that other signaling pathways elicited by FSH are operating in parallel (14). Taken together, the present data indicate that porcine OCC are sensitive to lapatinibmediated inhibition of the EGFR pathway. Acknowledgments: The authors thank Professor Barbara C. Vanderhyden for reading the manuscript and helpful comments.

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