Abstract. Introduction. RBMOnline - Vol 9. No Reproductive BioMedicine Online; on web 26 May 2004

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1 RBMOnline - Vol 9. No Reproductive BioMedicine Online; on web 26 May 2004 Article Cytoplasmic dysmorphisms in metaphase II chimpanzee oocytes Dr Kimie Suzuki graduated from the Nippon Dental School, Tokyo, Japan in 1994, and received a PhD degree from the University of Tokyo in After post-doctoral study in the molecular biology field in Japan and the USA, she joined the chimpanzee stem cell project at Sanwa Kagaku Kenkyusho Co. Ltd, Kumamoto, Japan. Dr Kimie Suzuki Kimie Suzuki 1,3, Nobuhiko Yoshimoto 1, Kohji Shimoda 1, Wataru Sakamoto 2, Yukie Ide 2, Takehito Kaneko 2, Tatsuyuki Nakashima 2, Ikuo Hayasaka 1, Naomi Nakagata 2 1 Kumamoto Primates Research Park, Sanwa Kagaku Kenkyusho Co., Ltd, 990 Nishikuroiwa, Ohtao, Misumi-Cho, Uto-Gun, Kumamoto , Japan 2 Division of Reproductive Engineering, Centre for Animal Resources and Development, Kumamoto University, Honjo, Kumamoto , Japan 3 Correspondence: Fax: ; ki_suzuki@skk-net.com Abstract Prior to fertilization by intracytoplasmic sperm injection (ICSI), cytoplasmic organization was evaluated in metaphase II chimpanzee oocytes obtained from stimulated ovaries. The findings demonstrate a high frequency of anomalies that are remarkably similar to the types of cytoplasmic dysmorphisms reported for human oocytes used in IVF. Similar to the human, the occurrence of these anomalies was oocyte- and animal-specific and associated with reduced competence as indicated by embryo development in vitro to the blastocyst stage. Keywords: chimpanzee oocytes, cytoplasmic dysmorphisms, in-vitro development 54 Introduction It is has long been known that the developmental potential of each mature human oocyte aspirated from a stimulated follicle for IVF is unique (Edwards, 1986). At present, oocyte morphology is one of the primary means by which competence is assessed using specific cellular or cytoplasmic characteristics suggested to be associated with developmental viability. Although controversial, an intact first polar body, the absence of cellular debris in the perivitelline space and a cytoplasm of uniform texture have been suggested to be positive indicators of competence (De Sutter et al., 1996; Xia, 1997; Balaban et al., 1998; Kahraman et al., 2000). However, a variable proportion of oocytes from the same cohort often have distinct cytoplasmic anomalies associated that have been related to reduced embryo competence either during the pre- or post-implantation stages (Alikani et al., 1995; Serhal et al., 1997; Kahraman et al., 2000; Meriano et al., 2001; Mikkelsen and Lindenberg, 2001). Van Blerkom and Henry (1992) identified several common oocyte defects in mature human metaphase II (MII) oocytes that they termed cytoplasmic dysmorphisms and noted the frequency of each dysmorphism was animal-specific, and the severity of each defect was oocyte-specific, and effects on embryo development in vitro were dysmorphism-specific. More recently, Meriano et al. (2001) suggested that for certain infertile patients, some of these dysmorphisms occurred repeatedly in successive IVF cycles and were associated with repeated poor outcomes after embryo transfer. As a species, the chimpanzee is very close to the human in many respects, including a well-established genetic homology (Gibbono, 1998). The authors work with the chimpanzee has centred on their preservation as an endangered species and the use of their embryos for experimental purposes, including the production of stem cell lines. For the generation of chimpanzee embryos by IVF, the same endocrine protocols and embryo culture and transfer methods developed for clinical IVF are used. This study reports that oocytes obtained for IVF from animals of proven fertility have many of the same types of cytoplasmic dysmorphisms as those observed in their human counterparts. These findings are a further indication of species similarity and suggest that the adverse effects of cytoplasmic dysmorphism on oocyte and embryo competence may have common aetiologies in both species.

2 Materials and methods Ovarian stimulation, oocyte retrieval, fertilization, and embryo culture Five chimpanzees (Pan troglodytes, ages 12, 14 (n = 2), 20 and 32 years) with regular menstrual cycles were stimulated only once each in this study. Ovarian stimulation used the short protocol of gonadotrophin releasing hormone agonist (GnRHa) and human menopausal gonadotrophin (HMG) human chorionic gonadotrophin (HCG), with all surgical procedures performed under anaesthesia (ketamine, 5 10 mg/kg body weight, Ketaral, Sankyo Co., Tokyo, Japan). Animals were given a single subcutaneous injection of GnRHa (3.75 mg of leuprolide acetate, Leupron, Takeda Ltd, Osaka, Japan) within 3 days after the start of menstruation. Intramuscular injections of HMG IU (Humegon; NV Organon, Oss, Netherlands) were administered daily beginning on the day following GnRHa administration. Follicle growth was monitored by transvaginal ultrasonography (Sonovista MSC, Mochida Pharm. Co., Tokyo, Japan) every two days in the week preceding ovulation induction with HCG (10,000 IU, Pregnyl; Organon), which occurred when follicles measured over 17 mm (17 18 days after GnRHa administration. Oocytes were aspirated at 31 h following HCG administration, under transvaginal ultrasound guidance, and with a standard human follicle aspiration needle (Cook, Ob/Gyn, Queensland, Australia). Prior to intracytoplasmic sperm injection (ICSI), intact, cumulus-enclosed oocytes were incubated for 2 h at 37 C in fertilization medium (Quinn s Advantage Fertilization Medium, Cooper Surgical, Trumbull, CT, USA), and after removal of cumulus and coronal cells with hyaluronidase and repeated passage through narrow bore glass micropipettes in preparation for ICSI, MII oocytes were identified and inspected by light microscopy. ICSI followed standard protocols used in human IVF, with alignment of the oocyte on the holding pipette such that the polar body was in the 12 or 6 o clock position. The first determination of fertilization occurred at h with pronuclear embryos transferred to new medium (Quinn s Advantage Cleavage Medium) supplemented with 10% serum protein substitute (SPS, Cooper Surgical). Oocyte and embryo cultures used 50 μl droplets under oil in an atmosphere of 5% CO 2, 5% O 2, 90% N 2. Cleavage stage embryos were transferred, between days 2.5 and 3.0 of culture, to blastocyst growth medium (Quinn s Advantage Blastocyst Medium, Cooper Surgical) containing 10% SPS. Morphological evaluations of MII oocytes Morphological evaluations of chimpanzee oocytes were performed at 200 magnification on an inverted light microscope equipped with Hoffman modulation optics. Each oocyte was imaged prior to insemination and tracked separately after ICSI with digital images taken from fertilization to day 5 in those instances where development had not arrested. The identification of cytoplasmic anomalies followed the same classification scheme(s) described for the human oocyte (Van Blerkom and Henry, 1992; Serhal et al., 1997; Meriano et al., 2001) and include the following: (i) extensive vesiculation; (ii) organelle/vesicle clustering; (iii) a well-defined clearing of portions of cortical cytoplasm; (iv) small dense or dark inclusion; (v) a large disc-like structure(s) usually located in central potion of cytoplasm and known to be an abnormal accumulation of smooth endoplasmic reticulum and (vi) relatively large clear vacuoles. The experimental procedures were approved by the Animal Ethical Committee of Kumamoto Primates Research Park, Sanwa Kagaku Kenkyusho Co. Ltd., Kumamoto, Japan. Results Forty-eight oocytes were retrieved from five animals of which the following 46 were classified as MII owing to the presence of a distinct first polar body (Table 1): (i) two, chimpanzees aged 14 years (n = 18); and further individual chimpazees aged (ii) 20 years (n = 4); (iii) 32 years (n = 16) and (iv) 12 years (n = 8). Of these MII oocytes, 74% (34/46) fertilized after ICSI, as demonstrated by the presence of two pronuclei at h. Prior to ICSI, 46 oocytes were examined in detail by light microscopy. Five oocytes with a clear cytoplasm of uniform texture were obtained from four chimpanzees and were classified as normal (Figure 1A). All normal-appearing oocytes fertilized, and three of the five developed to the expanded blastocyst stage after 5 days of culture (Figure 1B). For 41 oocytes subjected to ICSI, cytoplasmic anomalies were observed that were very similar to those seen in the human in relation to phenotype, frequency, oocyte-specificity and ability of the fertilized egg to develop to the expanding blastocyst stage (Table 1). Unless noted, anomalous oocytes were observed in different animals. Twenty-seven per cent (11/41) of the anomalous MII oocytes showed a clustered phenotype considered in the human to be the result of organelle aggregation (asterisk, Figure 1C). The severity of this phenotype ranged from slight to severe with the oocyte shown in this figure classified as moderate. Thirty-six per cent (4/11) of oocytes with slight-to-moderate clustering developed to the blastocyst stage at day 5 (Figure 1D). A single large disc-like inclusion (asterisk, Figure 1E), often delineated by a distinct membrane-like enclosure, occurred in 12% (5/41) of the MII oocytes observed in this study. Forty per cent (2/5) of these oocytes developed to the blastocyst stage, including the one shown in Figure 1F, which was derived from the oocyte, shown in Figure 1E. Ten per cent (4/41) of the MII oocytes appeared highly vesiculated (Figure 1G) and all were obtained from a single female (age 14 years). A similar situation existed for all seven oocytes derived from another female (age 32 years, 17%, 7/41) which showed regions of small dense lipid body accumulations (arrows, Figure 1H) that in some oocytes (asterisk, Figure 1I), seemed to be associated with what has been described in the human as necrotic foci (arrow, Figure 1I). None of the fertilized vesiculated oocytes developed to the blastocyst stage. Only one of the seven oocytes with focal lipid accumulations or presumed necrotic inclusions had developed to the blastocyst stage on day 5 (the blastocyst shown in Figure 1J was derived from the oocyte shown in Figure 1I). Fluid-filled vacuoles of different shapes and size (V1,V2, Figure 1K) were detected in two oocytes (5%, 2/41) and both occurred in the same cohort aspirated from the oldest female (age 32 years) used in this study. One oocyte with an inclusion slightly smaller than the one designated V2 in Figure 1K developed to the blastocyst 55

3 56 Figure 1. Light microscopic images of normal (A) and anomalous (C, G I, K, L) MII chimpanzee oocytes and for some, the corresponding blastocyst (B, D, F and J) that developed on day 5 of culture. Anomalous cytoplasmic phenotypes included clustering (C), a clear disc-like inclusion (E), extensive vesiculation (G), aggregates of dense lipid-like bodies and necrotic foci (H, I), vacuolation (K) and cortical cytoplasmic transparency (L). As discussed in the text, these cytoplasmic anomalies are remarkably similar to cytoplasmic defects and dysmorphisms described for human oocytes obtained from stimulated ovaries.

4 Table 1. Classification of cytoplasmic anomalies in MII oocytes from five chimpanzees according to morphological evaluation. Dysmorphism type Normal Number Blastocysts Dysmorphism type 1: vesiculated; 2: clustering; 3: well-defined cortical clearing; 4: small, dense lipid body accumulations; 5: large disc-like structure; 6: fluid-filled vacuoles. stage (Table 1). Twenty-nine per cent (12/41) of the anomalous oocytes showed areas of translucent cortical cytoplasm that varied in size and appeared devoid of organelles (asterisks, Figure 1L). Nine of these oocytes were retrieved from one female (age 14 years). Of these 12 oocytes, 10 fertilized, two were unfertilized and one developed to the blastocyst stage. This oocyte showed only slight cortical clearing similar in area to the region indicated by the left asterisk in Figure 1L. Discussion While ovarian stimulation and IVF have been used for the nonhuman primate for over 20 years (Gould, 1983; Ozasa and Gould, 1987; Hewitson et al., 1998; Hewitson and Schatten, 2002; Marshall et al., 2003;), the authors are unaware of any notation or detailed description of anomalies in the cytoplasmic organization of MII oocytes. To the best of their knowledge, the only report of IVF in the chimpanzee was the study of Gould (1983), but no mention of oocyte cytoplasmic appearance was included as cumulus cells were not removed before insemination. The present study reports that a very high frequency of MII chimpanzee oocytes aspirated from ovaries stimulated with one of the same hormonal regimens used in clinical IVF exhibit cytoplasmic anomalies that appear similar, if not identical to, cytoplasmic dysmorphisms reported for the human (Van Blerkom and Henry, 1992; Meriano et al., 2001). The finding that the frequency and severity of these dysmorphisms was animal- and oocyte-specific also coincides with clinical findings (Van Blerkom and Henry, 1992; Van Blerkom, 1994; Mikkelsen and Lindenberg, 2001; Meriano et al., 2001). Fertilization rates with dysmorphic chimpanzee oocytes were very similar to those reported with ICSI for human oocytes exhibiting the same apparent cytoplasmic phenotypes (De Sutter et al., 1996; Xia, 1997; Balaban et al., 1998; Kahraman et al., 2000). In clinical IVF, embryos derived from oocytes with severe cytoplasmic clustering, aberrant smooth endoplasmic reticulum (SER) accumulations, and extensive vesiculation or vacuolation show reduced capacity to develop to the blastocyst stage, and high frequencies of posttransfer/implantation demise (Alikani et al., 1995; Serhal et al., 1997; Kahraman et al., 2000; Meriano et al., 2001). Although the number of available chimpanzee oocytes was relatively small, the results indicate comparable rates of fertilization in normal and dysmorphic oocytes but a marked reduction in the ability of embryos to develop to the blastocyst stage when derived from oocytes with certain dysmorphisms. The blastocysts described in this paper were used to establish stem cell lines, however none developed into viable colonies (data not shown). Designations of particular cytoplasmic anomalies or dysmorphisms in human oocytes are subjective and can lead to different conclusions as to their frequency or effect on competence. However, there appears to be a consensus in the clinical IVF literature (see Meriano et al., 2001) that certain dysmorphisms do adversely affect competence after the blastocyst stage, and their occurrence and apparent negative influence on early chimpanzee embryogenesis may be a further demonstration of the similarity between the two species. The present report is thought to be the first to describe dysmorphic cytoplasmic phenotypes in the MII chimpanzee oocytes that closely resemble those found in their nearest cousin, the human. While it is tempting to speculate that dysmorphisms in human and chimpanzee oocytes have common aetiologies, this notion must take into account the fact that their origins in the human are unknown. Morphological similarity of a specific defect viewed by light microscopy does not necessarily demonstrate actual structural or subcellular equivalence between species. Electron microscopy has been used with human oocytes to examine the cellular and subcellular characteristics of the types of defects described here (Van Blerkom, 1990, 1994) and equivalence for the chimpanzee may require similar studies. Although findings indicate that extremely low rates of normal-appearing oocytes occur in both young and old chimpanzees (14, 20 and 32 years old), the number of animals and oocytes obtained is too few to conclude that age and oocyte quality are related in the chimpanzee as they appear to be in the human. The apparent commonality of response to hyperstimulation at the oocyte level may reflect the similar reproductive physiologies of the two species, including the fact that both species have menstrual cycles and maintain reproductive ability for decades. However, the findings also suggest that the human-based ovarian stimulation protocol used here may need to be optimized for the chimpanzee, or that other protocols need to be evaluated in the chimpanzee in order to obtain sufficient numbers of normal blastocysts for stem cell generation. Acknowledgments The authors thank Miwako Takeichi for her assistance in this study. 57

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