Aryl Phosphate Derivatives of Bromo-Methoxy-Azidothymidine: MATERIALS AND METHODS(13)
Control and test sperm suspensions (2 X 106) in < 100 ^l of BWW-3.5% BSA medium were added, and the gametes were coincubated for 4 h at 37°C in 5% CO2 in air. After incubation, the eggs were washed three times with BWW medium to remove the loosely associated sperm. The eggs were fixed and stained as previously described. Bound and decondensed fluorescent sperm were viewed with a Olympus model BX-60 epifluorescent microscope equipped with an excitation filter BP 360-370 and DM 400 barrier filter BA 420. The number of sperm bound per egg, the number of swollen sperm heads per egg, and the percentage of penetrated eggs were determined for control and test sperm. An egg was considered penetrated when it contained at least one swollen sperm head in the cytoplasm. asthma inhalers
In Vivo Fertility Trials
Sexually mature adult female CD-1 mice were superovulated by a i.p. injection of 5 IU of eCG (Gestyl; Diosynth B.V., Oss, Holland) followed by an i.p. injection of 5 IU of hCG (Steris Laboratories, Phoenix, AZ) 46-48 h later. Mice were randomly assigned to one of the two treatment groups (27 or 24 per group): 1) vehicle controls received cream base (Taro Pharmaceuticals, Hawthorne, NJ) with 1% DMSO; 2) the test group received cream base with 1% WHI-07 in 1% DMSO. These treatments were given intra-vaginally (50 ^l) prior to artificial insemination. Caudae epididymal sperm were obtained from proven breeder adult CD-1 males and suspended in a modified Krebs-Ringer-bicarbonate medium (M2; Gibco-BRL) supplemented with pyruvate, lactate, and glucose. Sperm were suspended in M2 medium-5% BSA for 1 h prior to use. For each experiment, cauda epididymal sperm pooled from 25 male mice and adjusted to 1-2 X 106 motile sperm/50 ^l were used for insemination. A 50-^l volume per mouse was ejected through a 1-ml syringe with a blunted 18-gauge needle. Preparations were analyzed for sperm concentration, motility, and sperm motion parameters by CASA using software designed for oval sperm head morphology (Hamilton Thorne). On Day 8, individual females representing the control and test group were killed, and their uteri were examined for the presence or absence of embryos. A total of three independent fertility experiments were performed.