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We tested whether the presence of wild-type sperm in the ejaculate could compensate for the defect in the knockout sperm, enabling them to enter the UTJ.
This could happen if sperm from the knockouts lack a protein that would stimulate the opening of the UTJ.
This medium caused the oocytes to stick to the bottom of the dish and to shrink away from the zona pellucida, enabling a 30-gauge needle to be used to open a slit in the zona.
Following the partial zona dissection, eggs were detached from the dish by introducing BSA-containing medium into the drop.
In mammals, spermatozoa spread from the site of insemination toward the site of fertilization in a controlled way that allows the encounter of male and female gametes in a viable and competent state, and in ratios adequate to assure fertilization with minimum risk of polyspermy.
Numerous studies in several species have shown that this process is far more complex than would be predicted if sperm movement were completely random [1, 2].
This could happen, for example, if sperm from the knockouts are recognized as “foreign” by some mechanism that triggers closing of the UTJ.
Previous studies indicate that sperm from mice with disrupted calmegin genes (−/−) are impaired in their ability to ascend into the oviduct .
After mutant males are mated to wild-type females, very few sperm can be found in or above the uterotubal junction (UTJ).
Sperm were obtained from the epididymides of sperm/ml.
After 15, 60, and 120 min of incubation, 10 μl of sperm suspension were transferred to a Makler counting chamber (Sefi Medical Instrument, Haifa, Israel) for analysis of sperm movement using an HTM-IVOS Ver.10 (Hamilton Thorne Research, Beverly, MA). Ltd., Tokyo, Japan), followed 48 h later by 5 IU of h CG (Teikoku Zoki Co. Oocytes were recovered from the oviducts 12 h after h CG, placed in TYH medium, and inseminated with sperm (1– 2 × 10 on the X chromosome (X*).