REPORTS [L Hewitson et dl., Hum. Reprod. 13, 2786 (1998)l. zonae removed with pronase, and the zona-free embry- os recovered individually for 20 min before splitting. After transferring into calcium- and magnesium-free TALP-Hepes medium, blastomeres were dissociated by aspiration through a 30-(~m micropipet. Blastomeres were transferred into empty zonae produced by me- chanical removal of oocyte cytoplasm after zona scor- ing. Each multiple was placed in its own zona to ensure blastomere aggregation. Consequently,zonae were lim- iting, and additional zonae recovered from bovine oo- cytes were used successfully. Surrogate females were selected on serum estradiol and progesterone. 11. Parentage assignments were performed by DNA typ- ing for 13 microsatellite loci amplified by PCR with heterologous human primers for loci D3S1768, D6S276, D6S291. D6S1691. D7S513. D7S794. D8S1106, DlOS1412, D l 1S925, D13S765, D16S403, D17S804, and Dl8572 (Veterinary Genetics Labora- tory. University of California, Davis). 12. L. C. Hewitson et dl., Nature Med. 5, 431 (1999); A. F. Tarantal and A. G. Hendrickx.Am. I. Primatol. 15, 309 (1988). 13. J. Cohen, M. Alikani, J. Trowbridge, Z. Rosenwaks, Hum. Reprod. 7. 685 (1992). 14. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end Labeling (TUNEL; Boehringer Mann- heim) was used to assay apoptosis. Blastocysts were fixed (2% formaldehyde; pH 7.4; 30 rnin), rinsed, and permeabilized with 0.1% Triton X-100-0.1% Na ci- trate (4°C; 2 min) Hoechst 33258 (Sigma) was used to visualize total DNA. To retain their three-dimen- sionality, coverglass spacers (170 pm; >I30 to 150) were placed alongside the blastocysts. Serial confocal imaging (3 p n ) used Leica confocal TCS SP with ultraviolet argon and 488-argon lasers. These gener- ated 3D reconstructions analyzed with Photoshop (Adobe Systems, Mountain View, CA). Reconstruc- tions permitted total cell number by counting nuclei serially. TE cells were distinguished at the periphery, whereas ICM were interior. 15. R. L. Gardner, Development 124, 289 (1997); R. J. Weber et dl., Development 126, 5591 (1999). 16. Supplemental Web material is available at Science On- line at www.sciencemag.org/feature/datal1042714.shl. 17. S. Lehrman, Nature 401, 517 (1998). Severely Reduced Female Fertility in CD9-Deficient Mice Franqois Le Naour, Eric Rubinstein, Claude Jasmin, Michel Prenant, Claude Boucheix* CD9 is a widely expressed cell surface molecule that belongs to the tetraspanin superfamily of proteins. The tetraspanins CD9, KAI-1lCD82, and CD63 are involved in metastasis suppression, an effect that may be related to their association with P1 integrins. Knockout mice lacking CD9 were created to evaluate the physiological importance of CD9. C D 9 Y females displayed a severe reduction of fertility. Oocytes were ovulated but were not successfully fertilized because sperm did not fuse with the oocytes from C D 9 - ' females. Thus, CD9 appears to be essential for sperm-egg fusion, a process involving the 18. H. Leese, I. Donnay,]. G. Thompson. Hum. Reprod. 13, 184 (1998). 19. J. E. Haddow et dl., N. Engl. 1. Med. 341. 549 (1999). 20. M. J. Shamblott etal., Proc. Natl. Acad. Sci. U.S.A. 95, 13726 (1998); J. A. Thompson et dl., Science 282, 1145 (1999). 21. We thank D. Abbott and F. Wegner (Wisconsin Re- gional Primate Research Center); M. C. T. Penedo (Veterinary Genetics Lab, UC Davis); 1. Fanton, M. Garrett, G. Haluska. M. Novy, and Y. Terada; and M. Cook, K. Grund, D. Jacob, B. McVay, T. Swanson, and all at the Oregon Regional Primate Research Center (ORPRC) for thoughtful comments and assistance. Procedures were approved by the Oregon Health Sciences University-ORPRC, Institutional Animal Care and Use Committee. Supported by NIH research awards [National Center for Research Resources (NCRR). National Institute of Child Health and Hu- man Development] to G.P.S. ORPRC infrastructure is sponsored by the NIH-NCRR Regional Primate Re- search Program. 15 June 1999; accepted 10 December 1999 ity (93 to 100% of pregnancies) was observed when CD9- - males were mated with wild- type or heterozygous females. In contrast. only 50 to 60% of C D9 - females produced litters after being maintained in the presence of fertile wild-type, heterozygous, or CD9- - males for up to 2 months. The delay before the beginning of a successful pregnancy was 19 to 30 days on average for C D 9 - ' fe- males, as compared with 4.5 days for wild- type animals. In addition, the litter size was reduced (2 i 0.6 pups in CD9-' females versus 8 i 2.3 pups in CD9+'+ females) and the initial mortality rate was increased to 32 to 55% as compared with less than 2% for wild-type females (Web table 1) (12). No difference in the frequency of vaginal plugs was observed between wild-type and CD9-deficient mice, indicating normal mat- ing behavior (16.9%; n = 46 for CD9-'- versus 17.4%; n = 42 in CD9+'+). The in- fertility was also not due to the absence of sperm at the site of fertilization, as shown by the presence of numerous sperm in the ovi- duct. Histological examination of ovaries of 6-week-old C D 9 - ' mice showed that there was no difference when compared to wild- type females (10) and that the number of ovulated oocvtes was normal (Table 1). How- ever, the naturally ovulated oocytes from CD9- mated females collected at day 0.5 did not divide and became fragmented if maintained in cell culture (Fig. lA), whereas oocytes from CD9+'+ mated females divided and 2 days later reached stage 4 (Fig. 1B). Similar fragmented oocytes (Fig. 1C); instead of blastocysts (Fig. ID), were present in the uterus of C D 9 - ' females 3.5 days after mating. The oocytes recovered from nonmated su- CD9-associated integrin cu6P1. Sequence analysis of CD9 predicts a structure with four transmembrane domains, two extra- cellular loops, and short intracytoplasmic amino and carboxyl tails (I). This structure is shared by all members of the tetraspanin superfamily of proteins (2). CD9 is expressed in multiple tissues but is not ubiquitous (3). The tetraspanins CD9, KAI-1lCD82, and CD63 act as metastasis suppressor molecules (4). Low expression of these molecules is correlated with an increased invasive and metastatic potential (2, 5). In vitro studies show that tetraspanins function in cell adhe- sion, motility, proliferation, differentiation, and signal transduction (2, 6). The tetraspan- ins are physically associated with each other and with several cell surface molecules, in- cluding a subset of p l integrins, which are receptors for extracellular matrix proteins (7). Thus, tetraspanins may function as sur- face organizers or facilitators that group and lnstitut National de la Sante et de La Recherche Medicale (INSERM),unite 268, ~ ~ ~ i t ~ l paul-~rousse, 94800 Villejuif, France. +T~ correspondence should be addressed, E. whom mail: boucheix@infobiogen.fr interconnect specific cell surface proteins in macromolecular complexes and thus increase the formation or the stability of functional signaling complexes, or both (2, 7). To investigate the role of CD9 in vivo, we generated inice in which the CD9 gene was disrupted by gene targeting in embryonic stem (ES) cells (8, 9). The murine CD9 gene consists of eight exons spanning more than 20 kb (10, 11). The promoter and exon 1 were replaced by the neomycin resistance gene (Web fig. 1) (12). The recombination was verified by Southern (DNA) blot (13). Splenocytes from homozygous mice dis- played no detectable CD9 surface expression as determined by flow cytometry analysis or by immunohistochemistry of kidney sections (10). Heterozygous C D 9 + ' mice appeared normal and, when interbred, yielded litters of normal size with a Mendelian genetic distri- bution 25:50:25(%) and with an equal distri- bution between males and females.-~omoz~- perovulated CD9-' mice (Fig. 2A) or gous adult CD9-'- mice showed no obvious CD9+'+ mice (Fig. 2C) 12 hours after human abnormalities and appeared healthy. Howev- chorionic gonadotropin (post-hCG) injection er, when the CD9- mice were intercrossed, had a similar response: 70 and 7496, respec- fertility was severely reduced. Normal fertil- tively, showed a polar body (Web table 1) www.sciencemag.org SCIENCE VOL 287 14 JANUARY 2000 319