Reduced fertilization after ICSI and abnormal phospholipase C zeta presence in spermatozoa from the wobbler mouse

Reproductive BioMedicine Online (2010) 21, 742 749 www.sciencedirect.com www.rbmonline.com ARTICLE Reduced fertilization after ICSI and abnormal phospholipase C zeta presence in spermatozoa from the wobbler mouse Elke Heytens a,1, Thomas Schmitt-John b, Jakob M Moser b, Nanna Mandøe Jensen b, Reza Soleimani a,1, Claire Young c, Kevin Coward c,2, John Parrington c, Petra De Sutter a, * a Department of Reproductive Medicine, Ghent University Hospital, De Pintelaan 185, B-9000 Ghent, Belgium; b Molecular Biology Department, Aarhus University, Forskerparken, Gustav Wieds Vej 10, DK 8000 Aarhus C, Denmark; c Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, UK * Corresponding author. E-mail address: Petra.DeSutter@UGent.be (P.D. Sutter). 1 Present address: Institute for Fertility Preservation, Department of Obstetrics and Gynecology, New York Medical College, Valhalla, NY, USA. 2 Present address: Nuffield Department of Obstetrics and Gynaecology, University of Oxford, Level 3 Women s Centre, John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK. Dr Elke Heytens has obtained an MSc degree in biochemistry and a Master s degree in molecular medical biotechnology at Ghent University, Belgium. She joined the research team of the Centre for Reproductive Medicine at Ghent University Hospital in 2005. During her PhD research she was working on oocyte activation in assisted reproduction, and more particularly in case of male infertility. Abstract Failed fertilization after intracytoplasmic sperm injection (ICSI) can be due to a reduced oocyte-activation capacity caused by reduced concentrations and abnormal localization of the oocyte-activation factor phospholipase C (PLC) zeta. Patients with this condition can be helped to conceive by artificial activation of oocytes after ICSI with calcium ionophore (assisted oocyte activation; AOA). However some concern still exists about this approach. Mouse models could help to identify potential oocyteactivation strategies and evaluate their safety. In this study, the fertilizing capacity of wobbler sperm cells was tested and the efficiency of AOA with two exposures to ionomycin to restore fertilization and embryo development was studied. The quality of the obtained blastocysts was assessed and embryo transfer was performed to evaluate post-implantation development. The presence of PLCzeta in the spermatozoa and testis of the wobbler mouse was evaluated by PLCzeta immunostaining and quantitative reverse-transcription polymerase chain reaction. Sperm cells from wobbler mice had reduced fertilizing capacity and abnormalities in PLCzeta localization, but not in its expression. Artificially activating the oocytes restored fertilization and embryo development. Therefore, the wobbler mouse can be a model for failed fertilization after ICSI to study PLCzeta dynamics and aid in optimization of the AOA method. RBMOnline ª 2010, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: assisted oocyte activation, ICSI, ionomycin, PLCzeta, wobbler 1472-6483/$ - see front matter ª 2010, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.rbmo.2010.07.006

PLCf in spermatozoa and testis from the wobbler mouse 743 Introduction Globozoospermia is a rare disorder characterized by round-headed sperm cells that lack an acrosome (Schirren et al., 1971). Consequently the spermatozoa cannot interact with the oocyte and patients with globozoospermia were thus considered infertile for a long time. But even since the introduction of intracytoplasmic sperm injection (ICSI), which bypasses the interaction of the gametes, little success in treating globozoospermia has been achieved (Dam et al., 2007). Often, completely failed fertilization is observed when a spermatozoon from a globozoospermia patient is used for ICSI (Battaglia et al., 1997). A mouse oocyte-activation test revealed that these sperm cells have a reduced oocyte-activation capacity (Rybouchkin et al., 1996). Recently it has been shown that this is linked to reduced expression, abnormal localization and changes in amino acid sequence of the oocyte-activation factor phospholipase C f (PLCf) (Heytens et al., 2009; Yoon et al., 2008). PLCf induces several rises in intracellular calcium concentration in the oocytes, so-called calcium oscillations, which are necessary for the completion of all the events of oocyte activation and thus successful fertilization (Cuthbertson et al., 1981; Miyazaki et al., 1993; Saunders et al., 2002). Some globozoospermia patients have been helped to conceive by artificially increasing calcium concentrations and thus activating the oocytes after ICSI by using a calcium ionophore (assisted oocyte activation; AOA) (Rybouchkin et al., 1997). This method is still being used today and several healthy babies have been born after applying the AOA procedure (Heindryckx et al., 2008). However, concerns still exist about this approach. The precise pattern of calcium oscillations observed during normal mammalian fertilization are not mimicked by an ionophore (Heytens et al., 2009) and this could be of considerable importance given that studies have indicated that the pattern of calcium oscillations may influence development at later stages (Ozil, 1990; Ozil et al., 2006). Therefore optimization of the method is mandatory and it is sensible to evaluate preimplantation, but more importantly post-implantation development after AOA. An oocyte-activation-deficient infertile mouse model could be used to evaluate embryo development following artificial oocyte-activation procedures. The current study identified a suitable mouse model the wobbler mouse. This mouse strain suffers from motor neuron disease caused by a naturally occurring mutation and in addition male wobbler mice are infertile (Duchen and Strich, 1968). Intriguingly, the morphology of the sperm cells from these mice is similar to the round-headed sperm cells from men with globozoospermia (Heimann et al., 1991; Schirren et al., 1971). Thus the wobbler mouse could be a useful model for globozoospermia and could also be used to study abnormalities in PLCf expression and localization in the spermatozoa associated with such a condition. This study tested the fertilizing capacity of wobbler mouse spermatozoa and evaluated the expression and localization of PLCf within such spermatozoa. In addition, the expression of PLCf in wobbler mouse testis was studied. Subsequently, the model was tested by evaluating embryo development after AOA. Materials and methods Unless otherwise indicated, all chemicals were purchased from Sigma Chemicals (Bornem, Belgium). All procedures involving live animal handling and euthanasia were performed according to standard animal protocols approved by the Ghent University Hospital Ethical Committee for Laboratory Animals (ECP 06/03). Collection of mouse oocytes Seven-to-12 week-old female B6D2/F1 mice (Charles River Laboratories, Brussels, Belgium) were stimulated to superovulate with 5 IU equine chorionic gonadotrophin (Folligon; Intervet, Oss, the Netherlands) and 48 h later with 5 IU human chorionic gonadotrophin (HCG; Chorulon, Intervet). Oocytes were collected at 14 h post HCG, freed from cumulus cells by a short incubation in hyaluronidase (200 IU, 3 5 min) and cultured in potassium simplex optimized medium (KSOM; Lawitts and Biggers, 1993) supplemented with 0.4% bovine serum albumin (BSA; Calbiochem, Bierges, Belgium) at 37 C and 6% CO 2 until the start of ICSI. To obtain fertilized oocytes or zygotes (in-vivo controls), the stimulated mice were paired with B6D2/F1 males and checked for a vaginal plug the following morning. Zygotes were collected 22 h post-hcg and freed from cumulus cells by a short incubation in hyaluronidase and cultured in KSOM. Collection of spermatozoa Spermatozoa from the epididymis of C57BL/6J-wr and C57BL/6J male mice (8 10 weeks) were suspended in KSOM HEPES containing 0.01% polyvinyl alcohol (KSOM/PVA), washed and resuspended in nuclear isolation medium (NIM; 123 mmol/l KCl, 2.6 mmol/l NaCl, 7.8 mmol/l NaH 2 PO 4, 1.4 mmol/l KH 2 PO 4, 3 mmol/l EDTA, ph 7.2) containing 0.01% polyvinyl alcohol (NIM/PVA). Spermatozoa were sonicated mildly to remove tails, and resuspended in NIM supplemented with 50% glycerol for storage at 20 C until required (Kuretake et al., 1996). Before use, the sperm suspensions were thawed and washed with injection buffer. For evaluation of post-implantation development, chemically inactivated sperm cells were used (breeding of the wobbler mouse strain is not permitted at the study facility). Chemically inactivated spermatozoa were obtained by alkaline carbonate extraction of the sperm heads (100 mmol/l Na 2 CO 3, ph 11.5, 10 min at 4 C; Kurokawa et al., 2005). Intracytoplasmic sperm injection For ICSI, sperm heads were transferred into NIM/PVA and washed twice to remove glycerol. ICSI was performed in KSOM/PVA at room temperature. Randomly selected sperm heads were delivered into the oocytes cytosol by application of piezo pulses to penetrate the zona pellucida and plasma membrane using a piezo micropipette-driving unit (Prime Tech, Ibaraki, Japan). Four experimental groups were set up: (i) injection with C57BL/6J-wr or chemically inactivated sperm heads; (ii) injection with C57BL/6J sperm heads (positive control); (iii) sham injection of medium

744 E Heytens et al. (negative control); and (iv) non-manipulated oocytes to examine the occurrence of spontaneous parthenogenetic activation (medium control). Zygotes were the positive control for culture conditions. Oocytes were transferred into KSOM and cultured at 37 C under 6% CO 2. Assisted oocyte activation The protocol of AOA consists of two applications of ionomycin, which is used in the Department of Reproductive Medicine of the Ghent University Hospital (Heindryckx et al., 2008; Rybouchkin et al., 1997). Thirty minutes after injection of the wobbler or inactivated spermatozoa, the oocytes were incubated with 10 lmol/l ionomycin in KSOM with physiological calcium concentrations (1.7 mmol/l) for 10 min, followed by a second exposure of the same duration 30 min later. Two-cell and blastocyst formation were evaluated at 24 h post activation and 120 h post HCG respectively. Cell counting of blastocysts To evaluate preimplantation development, differential staining of inner cell mass (ICM) and trophoblastic ectoderm (TE) cells of blastocysts was performed at 120 h post HCG according to Thouas et al. (2001). Blastocysts were first incubated in propidium iodide/1% Triton X-100 for up to 10 s and were then immediately transferred into fixative solution consisting of 100% ethanol and 25 lg/ml bisbenzimide (Hoechst 33258) and stored at 4 C overnight. Finally, the blastocysts were transferred to a drop of glycerol on a microscopic slide and covered with a cover slip. ICM nuclei labelled with bisbenzimide only appeared blue and TE nuclei labelled with both bisbenzimide and propidium iodide appeared pink-to-red. Numbers of ICM and TE nuclei were counted directly under a fluorescence microscope and expressed as the mean ± SD. Embryo transfer Recipient CD1 female mice were paired with vasectomized CD1 males (Charles River Laboratories) to induce a state of pseudopregnancy. Females were checked for a vaginal plug the following morning, to confirm that mating had occurred and that the female was at day 1 of pseudopregnancy. Ten 2-cell embryos were transferred to the left oviduct of pseudopregnant mice on day 1 of pseudopregnancy. Foster mothers from each group were left to carry to term and deliver the pups. The pups were weighed at day 7 and from then twice a week until they were 8 weeks old. Development was scored by the time of pinna unfolding, generalized hair growth and eye opening. At the age of 12 weeks, the mice were mated to evaluate their fertility. In the AOA group, chemically inactivated spermatozoa were used. PLCf immunostaining Spermatozoa were fixed in 4% paraformaldehyde/ phosphate-buffered saline (PBS), permeabilized with 0.5% (v/v) Triton X-100/PBS and kept at 4 C until use. Sperm smears were incubated in 3% BSA/PBS and labelled with anti-plcf (Young et al., 2009) in 0.05% BSA/PBS overnight at 4 C. After three washes with PBS, samples were labelled with Alexa Fluor 555 goat anti-rabbit IgG (1:400; Invitrogen, Merelbeke, Belgium), counterstained with 5 lg/ml Hoechst 33258 and 5 lg/ml fluorescein-conjugated Pisum sativum agglutinin and mounted in glycerol-dabco. Spermatozoa (n = 100) were examined using a confocal laser-scanning microscope (Radiance 2100 blue laser diode; Bio-Rad, Hercules, CA, USA). Cryosections of mouse testes (10 lm) were fixed in 4% paraformaldehyde/pbs and blocked with 2% BSA/PBS for 1 h at 37 C. Sections were incubated in anti-plcf diluted 1:50 in 2% BSA/PBS overnight at 4 C. After washing, secondary antibody (anti Rabbit-Cy3, 1:400) and peanut lectin agglutinin for acrosome staining (1:400) were added and samples incubated at 37 C for 45 min. After washing, sections were counterstained with Hoechst stain, covered with elvanol and inspected by fluorescence microscopy. Quantitative reverse-transcription polymerase chain reaction Dissected testes were immediately frozen in liquid nitrogen and stored at 80 C until use. Testes from five male wr/wr and five male wild-type mice at the age of 60 days (±2 days) were pooled. Total RNA was isolated from testes according to standard protocols. The concentration of total RNA was determined by its OD 260/280 reading. Aliquots were run on formaldehyde agarose gels to monitor RNA integrity. Total RNA (5 lg) was subjected to reverse transcription using random-hexamer primers with RevertAid H Minus First Strand cdna Synthesis Kit (Fermentas). cdna concentrations were determined by OD 260/280 measurement and diluted to 1 lg/ll. For quantitative reverse-transcription polymerase chain reaction (qpcr), a final concentration of 100 ng cdna/reaction were used. TaqMan probes against hypoxanthine phosphoribosyltransferase 1, murine VASA homologue (Mvh, also termed Ddx4), protamin 1 (Prm1) and PLCf (Plcz1) were obtained from Applied Biosystems (assay ID Mm01545399_m1, Mm00802445_m1, Mm01342731_g1 and Mm01255074_m1, respectively). qpcr conditions were according to manufacturer s protocol. All samples were tested twice as triple triplicates. All experiments were conducted on an MX3005p (Stratagene) machine and data were analysed using the comparative C t method. Statistical analysis Chi-squared tests and Fisher s exact tests were used for statistical analysis of oocyte activation and post and preimplantation embryo development. Parameters of blastocyst quality (cell numbers for ICM, TE, total cell number (TCN) and ICM/TCN) and weight of the pups were analysed with one-way analysis of variance and least significant difference post-hoc test. The qpcr-data were analysed with the two-sided t-test. Levene s test for equality of variances (a = 0.01) and normality tests (Kolmogorov Smirnov, a = 0.01) were performed to choose the appropriate test. A significance level of a = 0.05 was used throughout unless otherwise stated.

PLCf in spermatozoa and testis from the wobbler mouse 745 Results PLCf analysis To determine whether PLCf protein expression or localization in spermatozoa is abnormal in the wobbler mouse strain, immunostaining with a specific anti-plcf antibody was performed on sperm cells and testis cryosections of wobbler and wild-type mice in order to visualize PLCf protein. In sperm cells from wild-type mice, PLCf was located primarily in the post-acrosomal region of the sperm head, as shown in Figure 1 and in line with previous observations using this antibody (Young et al., 2009). By contrast, in spermatozoa from wobbler mice which had abnormal head morphology and lacked the acrosome, no prominent PLCf staining was observed (Figure 1). In addition, non-specific staining in the tail, as seen in a previous study (Young et al., 2009), was also observed. Immunostaining of PLCf in wild-type testis was in the inner areas of seminiferous tubules, suggesting that PLCf protein expression is only present during later stages of spermatogenesis. In addition, some tubules, even adjacent ones, showed no immunoreactivity, again consistent with a late pattern of expression for PLCf protein. In contrast, in wobbler mice seminiferous tubules, PLCf staining could also be found but the number of PLCf-positive tubules on the cross-sections appeared to be lower than in wild-type testes (Figure 2). In order to analyse PLCf gene expression in wild-type and wobbler mice testes, qpcr was performed on RNA prepared from these testes. RNA from four individuals was pooled, reverse transcribed and two identical qpcr analyses were Figure 1 Immunocytochemistry of spermatozoa from (A D) a wild-type mouse and (E H) a wobbler mouse. (A, E) Phospholipase C f staining showing absence of PLCf in the sperm head, (B, F) sperm nucleus stained with Hoechst 33342, (C, G) FITC-PSA-lectin staining identifying acrosome, and (D, H) bright-field image. (I L) show merged fluorescent and merged fluorescent and bright-field data from (I, K) a wild-type spermatozoon and (J, L) a wobbler spermatozoa. Bars = 7.5 lm.

746 E Heytens et al. Figure 2 Immunostaining of two representative testis sections from a wild-type mouse and a wobbler mouse. Cryosections were stained with anti-phospholipase C f (PLCf; red), peanut lectin agglutinin FITC (PNA; green) to visualize the acrosome and Hoechst 33342 (blue). Same magnification throughout; bar = 50 lm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.) performed for each genotype, each comprising three triplicates. The data indicated a slight but significant (P < 0.001) down-regulation of Plcz1 mrna in wobbler mouse testis, but also down-regulation of protamine 1, a marker for post meiotic spermatids, while the expression of the pre-meiotic germ-cell marker gene VASA (Mvh/Ddx4) was not altered (Figure 3). Fertilizing capacity and in-vitro embryo development after ICSI and assisted oocyte activation Injection of spermatozoa from the wobbler mouse resulted in lower fertilization rates in comparison with injection of fertile mouse spermatozoa (11% versus 88% in the control group, P < 0.001, Table 1). The developmental potential of oocytes injected with spermatozoa from control and wobbler mice as well as oocytes injected with wobbler spermatozoa and activated with ionomycin (AOA) is also shown in Table 1. Post-injection survival rates of control or wobbler spermatozoa and medium-injected control oocytes were not significantly different. The cleavage rate of oocytes that were injected with wobbler spermatozoa and activated with ionomycin was not significantly different from the control group (79 versus 88%, respectively). The proportion of cleaved oocytes progressing to the blastocyst stage was significantly lower in the AOA group Figure 3 Quantitative reverse-transcription PCR of murine VASA homologue, protamin 1 (Prm1) and phospholipase C f (PLCz1) mrna in wild-type (B16-WR+/+) and homozygous wr/wr (B16-WR / ) testes. RNA from four individuals was pooled and two identical qpcr analyses were performed for each genotype, each comprising three triplicates. **P < 0.01; ***P < 0.001 in comparison with the ICSI group (22 versus 72%, P < 0.001). Differential staining was performed successfully on 40 blastocysts of the in-vivo control group, 22 blastocysts of the ICSI group and 13 in the AOA group. Only the blastocysts that were completely labelled and that were not damaged during the staining procedure were included. The percentage of blastocysts analysed was not significantly

PLCf in spermatozoa and testis from the wobbler mouse 747 Table 1 Evaluation of in vitro embryo development after ICSI. No. of oocytes (%) Injected Survived Cleaved Developed to blastocysts Control spermatozoa 55 43 (78) 38 (88) a 31 (72) a Wobbler spermatozoa 58 44 (76) 5 (11) b 0 (0) b Wobbler spermatozoa + AOA 108 85 (79) 67 (79) a 19 (22) c Medium-injection control 53 44 (83) 4 (9) b 0 (0) b In-vivo control NA 62 62 (100) c 56 (90) d AOA = assisted oocyte activation. a d Values with a different letter in the same column are significantly different (P 0.018; chi-squared and Fisher s exact test). different between the different groups (71, 71 and 68% for in-vivo control, ICSI and AOA, respectively). The ICM, TE and TCN reached by the blastocysts obtained with in-vivo controls (10.9 ± 0.8, 70.0 ± 3.0 and 77.6 ± 3.9) were significantly different from the ICSI group (6.3 ± 1.1, 33.9 ± 4.2 and 39.3 ± 3.9, P < 0.002) and the AOA group (5.8 ± 1.8, 35.0 ± 7.8 and 39.3 ± 2.8, P < 0.001; Table 2). There was no difference in ICM, TE cell numbers and TCN between the ICSI and AOA group. Blastocysts in the ICSI and the AOA group showed an ICM/TCN ratio of 0.20 ± 0.03 and 0.17 ± 0.02, respectively, and this ratio did not differ significantly from the in-vivo group (0.16 ± 0.01). Evaluation of post-implantation development Two foster mothers in each group (in-vivo, ICSI and AOA) carried to term and delivered pups. In the AOA group, chemically inactivated spermatozoa were used because the study facility was not permitted to breed wobbler mouse. The implantation rate was not significantly different between the three groups (nine, eight and six pups in the in-vivo, ICSI and AOA groups, respectively, out of 20 embryos transferred) and also the number of viable pups per embryo transferred was similar (six, eight and six pups, respectively). The AOA pups did not show any abnormalities and their postnatal growth was normal in comparison to in-vivo control pups (bodyweight at 1 week up to 2 months; Figure 4). The weight of the pups originating from the ICSI embryos was lower at 2 and 3 weeks for both male and female pups, but this difference was not observed at later time points. Development, as assessed by time of pinna unfolding, generalized hair growth and eye opening, was not different between pups delivered after embryo transfer of in-vivo control, ICSI or AOA embryos (data not shown). The AOA mice successfully mated and healthy, normal pups were born. Figure 4 Post-natal growth rate of (A) male and (B) female pups during first 8 weeks after birth of intracytoplasmic sperm injection (ICSI), assisted oocyte activation (AOA) and in-vivo control pups. Data are mean ± SEM. *Significantly different from in-vivo control (one-way analysis of variance, P < 0.05). Discussion Spermatozoa with abnormal acrosomes cannot interact with the oocyte and also have a reduced oocyte-activation capacity. In humans, it has recently been shown that men with such a condition have expression, localization and amino acid sequence abnormalities of PLCf in their sperm cells (Heytens et al., 2009; Yoon et al., 2008). PLCf is the Table 2 Differential staining of day 5 blastocysts. ICM TE TCN ICM/TCN Control spermatozoa 6.3 ± 1.1 a 33.9 ± 4.2 a 39.3 ± 3.9 a 0.20 ± 0.03 Wobbler spermatozoa + AOA 5.8 ± 1.8 a 35.0 ± 7.8 a 39.3 ± 2.8 a 0.17 ± 0.02 In-vivo control 10.9 ± 0.8 b 70.0 ± 3.0 b 77.6 ± 3.9 b 0.16 ± 0.01 ICM = inner cell mass; TCN = total cell number; TE = trophoblastic ectoderm. a,b Values with a different letter in the same column are significantly different (one way ANOVA followed by LSD post hoc test; P < 0.002).

748 E Heytens et al. oocyte-activation factor and induces the increases in intracellular calcium concentration necessary for oocyte activation (Saunders et al., 2002). Spermatozoa from the wobbler mouse are very similar in morphology to the spermatozoa from globozoospermic men, which are deficient in oocyte activation capability. Mouse models have the potential to help understanding of the causes of globozoospermia, identification of new strategies for treating this type of infertility and, in addition, evaluation of the safety of such artificial oocyte-activation approaches. The wobbler mouse could be such a useful animal model. The current findings showed that sperm cells from the wobbler mouse have a reduced fertilizing capacity and abnormalities of PLCf localization in the spermatozoa. Importantly, this mouse model made it possible to evaluate the possibility of abnormalities of PLCf expression during spermatogenesis that might occur in globozoospermic individuals. Furthermore, while the pattern of expression for PLCf mrna during spermatogenesis has been studied in pig testis (Yoneda et al., 2006), as far as is known this is the first time that the pattern of expression of PLCf protein during spermatogenesis has been studied. The current study, immunostaining demonstrated a somehow lowered abundance of PLCf protein in both wobbler sperm cells and testes, but mainly an altered pattern of localization in the spermatozoa. Throughout spermatogenesis a lower immunoreactivity for PLCf was found in wobbler testes as compared with wild type. The qpcr data also indicated a slight down-regulation of PLCf mrna expression in wobbler testes. This probably reflected a lower number of maturating sperm cells in wobbler mice, since protamine 1 gene was also downregulated. The number of germ cells appeared not to be altered since murine VASA homologue expression was found not to be different between wobbler and wild-type testes. Previously, PLCf was found to be mislocalized and reduced in quantity in sperm samples from globozoospermia patients (Heytens et al., 2009; Yoon et al., 2008). Accordingly, mislocalization of PLCf in wobbler sperm cells might to some extent explain, or contribute to, the observed deficiency of wobbler sperm cells to activate oocytes after ICSI. It is not clear how this would work, since a whole spermatozoon is injected in an oocyte but could, for instance, involve defects in the normal pattern of activation of PLCf during gamete fusion. Other sperm factors might be compromised in spermatozoa from both wobbler mouse and globozoospermia patients and the wobbler mouse can also be useful for this future research. Activating the oocytes by artificially increasing the calcium concentration restored fertilization and similar blastocyst percentages as those following parthenogenetic activation of mouse oocytes were attained (Heytens et al., 2008). However, blastocyst development is still significantly lower than after ICSI and the wobbler mouse can be useful to develop new AOA strategies to improve embryo development. In addition, live offspring were obtained using this approach and the resulting pups were healthy and fertile. Since breeding the wobbler mouse in the research facility was not permitted, chemically inactivated spermatozoa were used instead of the wobbler spermatozoa to perform embryo transfer. However, this approach shows the potential of the method and also demonstrates that it could be used to propagate the wobbler mouse or other globozoospermia model strains. In summary, the wobbler mouse shows failed fertilization after ICSI and atypical localization of PLCf, but no abnormal expression of PLCf. This makes the wobbler a good candidate as a mouse model for the evaluation and optimization of new AOA procedures, which could include injection of PLCf protein into the oocyte along with the spermatozoa during ICSI. 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