Sperm-exposed oocytes were then cultured in vitro and periodically observed microscopically until the blastomere stage. We found that all but one oocyte of females mated to nectin-2wt/LacZ males were fertilized and proceeded to develop normally to the blastomere stage. In contrast, none of 73 sperm-exposed oocytes isolated from five females that had mated to nectin-2LacZ/LacZ males initiated the developmental program. Neither could we observe the formation of male pronuclei in this set of sperm-exposed oocytes, an observation suggesting that fertilization had not occurred (Table 1).
Spermatozoa of Nectin-2-Null Males Are Deficient in Migration to the Oviduct
To address the question of whether the observed absence of fertilized eggs in females mated to nectin-2LacZ/LacZ males results solely from defects in gamete interaction, we assessed the ability of spermatozoa to enter the oviducts. For this purpose, oviducts were dissected from females after natural overnight matings with either nectin-2LacZ/LacZ or nectin-2wt/ LacZ control males. The contents of the oviducts were flushed out, and spermatozoa were processed as described in Materials and Methods. We found that nectin-2LacZ/LacZ spermatozoa were approximately 4-fold less efficient in reaching the oviducts compared to the heterozygous controls. On average, 880 spermatozoa were isolated from the oviducts of females mated to control males, but only 204 spermatozoa were found in the case of nectin-2LacZ/LacZ males (Fig. 3). This difference was determined to be statistically highly significant (P = 0.0002). Thus, it seems likely that the reduced ability of nectin-2LacZ/LacZ spermatozoa to reach the oviducts contributes to the observed infertility phenotype in nectin-2-deficient males. Ventolin Inhaler
FIG. 3. Nectin-2LacZ/LacZspermatozoa are less likely to reach the oviducts. Spermatozoa were recovered from the oviducts of ovulated CD1 females following overnight mating with either nectin-2wt/LacZ (left bar) or nectin-2LacZLacZ males (right bar). Matings of five individual nectin-2wt/LacZ males and eight nectin-2LacZ/LacZ males were scored. Approximately 4-fold fewer nectin-2LacZ/LacZ spermatozoa were found in the oviducts compared to nec-tin-2wt/LacZ spermatozoa (P = 0.0002, two-tailed unpaired t-test).