Despite their severe defects, nectin-2LacZ/LacZ spermatozoa were viable and motile. However, on longer incubation (1224 h), in vitro nectin-2LacZ/LacZ spermatozoan motility and viability declined more quickly than those of the controls (data not shown). It is conceivable that the disorganized mitochondrial sheath impairs the spermatozoan energy metabolism. Alternatively, fewer mitochondria might have been incorporated into nectin-2LacZ/LacZ spermatozoa during spermatogenesis. Reading here
Spermatozoa of Nectin-2-Null Males Fail to Fertilize Oocytes In Vivo
Because nectin-2-null spermatozoa were viable and motile despite their gross morphological defects, the question arose at which step during fertilization these spermatozoa fail to function. In a first set of experiments, we recovered potential zygotes from the oviducts of females after overnight mating for subsequent in vitro culture. Five nectin-2LacZ/LacZ males and three nectin-2wt/LacZ males were each housed with two CD1 females (see Material and Methods). Except for one nectin-2wt/LacZ male, all males mated with at least one female, as assessed by the presence of a copulation plug. Only females with clearly formed copulation plugs were used to recover sperm-exposed oocytes (potential zygotes) to assure that mating had, indeed, occurred. All plugged females had ovulated, and between 3 and 40 potential zygotes were recovered per animal (Table 1).
TABLE 1. Failure of in vivo fertilization by nectin-2LacZ/LacZ males as assessed by in vitro culture of sperm-exposed oviductal oocytes following overnight mating.
|MaleNo.||Nectin-2genotype||Copulationplug||No. of potential zygotes recovered||Pronucleiformation||Blastomereformation|
a N/A, Not applicable.
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).