Unexpectedly, nectin-2LacZ/LacZ spermatozoa were able to bind to the oolemma at levels comparable those of nectin-2wt/LacZ controls (Table 2). After 90 min of incubation, an average of 42.1 nectin-2LacZ/LacZ versus 47.9 nect^in-2wt^acZ spermatozoa had bound per oocyte (counted as the number of protruding sperm tails at a median focal plane) (Fig. 5). In contrast, penetration of oocytes by nectin-2LacZ/LacZ spermatozoa, as assessed by the presence of decondensing sperm nuclei in the ooplasm, was 75-fold and 32-fold lower after 45 and 90 min of incubation, respectively (Table 2 and Fig. 5). Albuterol Inhaler
After 90 min of incubation in the control group, every oocyte was penetrated by an average of 13.1 spermatozoa, but only 2 of 12 oocytes were penetrated when nectin-2LacZ/ LacZ sperm was used, with an average of 0.42 penetrations per oocyte (counted as the total number of decondensing Sperm nuclei per oocyte). We did observe one oocyte that was penetrated by two nectin-2LacZ/LacZ spermatozoa (Fig. 5b2), which indicates that fusion with nectin-2LacZ/LacZ spermatozoa can occur, albeit at a greatly reduced rate.
TABLE 2. Zona-free hamster egg penetration assay.
|Spermgenotype||Time(min)||Sperm bound per oocyteb||No. of oocytes penetrated||Penetrations per oocyteb|
|nect’m-2LacZ/wt||45||38.2 ± 7.4 (n =||15)||14/15||6.0±3.7|
|nectin-2LacZ/wt||90||47.9 ± 4.8 (n =||15)||15/15||13.1 ± 4.2|
|nectin-2LacZ/LacZ||45||25.8 ± 8.5 (n =||12)||1/12||0.08 ± 0.29|
|nectin-2LacZ/LacZ||90||42.1 ± 6.7 (n =||12)||2/12||0.42 ± 0.79|
a Sperm concentration X 105/ml. b Results are expressed as mean ± SD.
FIG. 5. Nectin-2LacZ/LacZ spermatozoa bind, but fail to fuse, with zona-free hamster oocytes. A and B) Phase-contrast images of an oocyte after 90 min of incubation with nectin-2wt/LacZ (A) or nectin-2LacZ/LacZ spermatozoa (B). a1-b2) Two different focal planes for each oocyte observed under epifluorescence show the small, condensed sperm nuclei of spermatozoa adhered to the egg surface as well as larger, decondensing nuclei of spermatozoa that have penetrated the oolemma (black arrows). Whereas A) depicts a typical result of numerous penetration events obtained with nec-tin-2wt/LacZ spermatozoa, the oocyte in B) represents a rare exception, in which two nectin-2LacZ/LacZ spermatozoa penetrated an oocyte (b2, black arrows). Magnification X200.