The acrosome is a unique organelle that plays an important role at the site of sperm-zona pellucida binding during the fertilization process, and is lost in globozoospermia, an inherited infertility syndrome in humans. Although the acrosome is known to be derived from the Golgi apparatus, molecular mechanisms underlying acrosome formation are largely unknown. Here we show that Golgi-associated PDZ-and coiled-coil motif-containing protein (GOPC), a recently identified Golgi-associated protein, is predominantly localized at the trans-Golgi region in round spermatids, and male mice in which GOPC has been disrupted are infertile with globozoospermia. The primary defect was the fragmentation of acrosomes in early round spermatids, and abnormal vesicles that failed to fuse to developing acrosomes were apparent. In later stages, nuclear malformation and an abnormal arrangement of mitochondria, which are also characteristic features of human globozoospermia, were observed. Interestingly, intracytoplasmic sperm injection (ICSI) of such malformed sperm into oocytes resulted in cleavage into blastocysts only when injected oocytes were activated. Thus, GOPC provides important clues to understanding the mechanisms underlying spermatogenesis, and the GOPC-deficient mouse may be a unique and valuable model for human globozoospermia.
To determine whether spermatozoa must be structurally intact before microsurgical injection into oocytes for normal fertilization, intact spermatozoa, as well as sperm heads separated from tails by sonication, were individually injected into oocytes. When whole spermatozoa were injected immediately after their immobilization, the majority of the oocytes were fertilized and developed normally. Sonication in the presence or absence of Triton X-100 decapitated more than 95% of spermatozoa. Although all decapitated spermatozoa were diagnosed as "dead" by live/dead sperm staining, separated sperm heads (nuclei) could participate in normal embryo development when injected into the oocytes. The ability of isolated sperm heads (nuclei) to participate in normal embryo development was maintained under cryopreservation conditions that were not suitable for the survival of plasma membrane-intact spermatozoa. These results indicate that 1) spermatozoa do not need to be structurally intact for intracytoplasmic injection, 2) the plasma and acrosomal membranes and all tail components are not essential for normal embryo development, at least in the mouse, and 3) the cryopreservation conditions required for maintenance of the genetic integrity of sperm nuclei are less stringent than those necessary for keeping plasma membrane-intact spermatozoa alive.
The mouse oocyte can be activated by injection of a single, intact mouse spermatozoon or its isolated head. Isolated tails are unable to activate the oocyte. Active sperm-borne oocyte-activating factor(s) (SOAF) appears during transformation of the round spermatid into the spermatozoon. The action of SOAF is not highly species-specific: mouse oocytes are activated by injection of spermatozoa from foreign species, such as the hamster, rabbit, pig, human, and even fish. Some SOAF can be extracted by simple freeze-thawing of (hamster) spermatozoa; additional SOAF is obtained by sequential treatment of spermatozoa with Triton X-100 and SDS. Electron microscopic examination of sperm heads during SOAF extraction suggests that the relatively insoluble SOAF is associated with perinuclear material. When microsurgically injected into oocytes, Triton X-100-treated sperm heads (with perinuclear material, but without any membranes) can activate the oocytes, leading to normal embryonic development. Whereas perinuclear components have been believed to play a purely structural role, these data suggest an additional function for them in oocyte activation.
This study was undertaken to determine whether primary spermatocyte nuclei can complete meiosis after transfer into maturing or mature oocytes and can participate in normal embryogenesis. When injected into maturing mouse oocytes at prometaphase of the first meiotic division, spermatocyte chromosomes became arranged on a first meiotic metaphase (Met-I) spindle. Thus, oocytes contained two sets of Met-I chromosomes. When these oocytes were matured in vitro and artificially activated, pronuclei and polar bodies of both maternal and paternal origin were formed, and zygotes began development. When single spermatocyte nuclei were injected into fully mature oocytes at metaphase of the second meiosis (Met-II), the spermatocyte nuclei transformed into a Met-I configuration, resulting in the formation of oocytes with both maternal (Met-II) and paternal (Met-I) chromosome complements. After activation of these oocytes, half of each chromosome set was separated into polar bodies. Transfer of nuclei from polar bodies of "paternal" origin into other Met-II oocytes resulted in the formation of oocytes with two sets of Met-II chromosomes, one maternal and one paternal in origin. When activated, two pronuclei and two polar bodies were formed and zygotes began development. It is concluded that nuclei of primary spermatocytes are able to undergo two successive meiotic divisions within oocyte cytoplasm. Thus, factors that drive chromosome condensation, organization of metaphase, and chromosome separation at anaphase in oocytes can drive these same maturation processes in primary spermatocyte nuclei. When a total of 258 two-cell embryos were transferred to foster mothers, only two live pups were born to two mothers. One died shortly after birth and the other 3 wk after birth. The reasons for poor embryonic and neonatal development remain to be determined.
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