Effects of heterologous sera on fertilized rabbit ova

Undiluted blood serum of various species was used to culture two-celled rabbit ova for 24 hours. It was found that there is an ovocidal factor present in the serum of man, sheep, cattle, goat, and fowl. The factor is lethal rather than inhibitory; exposure to it for 10 minutes will cause the death of the ova. This factor is unstable, thermolabile (destroyed at 55 degrees C. in 30 minutes), and of large molecular size. The strength or concentration of this factor varies according to the origin of the serum, increasing in the order man, sheep, cattle, goat, fowl. The blood serum of rabbit, horse, dog, guinea pig, rat, and pig contains no ovocidal factor against rabbit ova. The ovocidal factor differs from the spermicidal factor, which is present in all the sera of the different species studied with rabbit spermatozoa. Immunization of the guinea pig against rabbit ova is possible. Normal development of young rabbits was obtained by transplantation of ova cultured in the heated or normal serum of other species after 24 hours.     

Identification of membrane-associated lymphotoxin (LT) on mitogen-activated human lymphocytes using heterologous anti-LT antisera in vitro.

Surface-associated lymphotoxin (LT) molecules have been identified on mitogen-activated human lymphocytes employing heterologous anti-α-LT serum in vitro. These membrane-associated LT molecules are present on PHA- or Con A-activated lymphocytes but do not appear to be expressed on unstimulated cells. Furthermore, these molecules were detected primarily on activated T lymphocytes, with little detectable on activated B- or null-cell populations. The removal of surface LT-bearing lymphocytes, using anti-α-LT serum + C′, does not dramatically affect the capacity of the remaining cells to release LT after mitogen restimulation. In addition, the presence of toxic molecules on the surface of activated lymphocytes suggests that these materials may be expressed in an inactive, noncytotoxic form.     

Serum of rabbits infected with Treponema pallidum (Nichols) inhibits in vitro transformation of normal rabbit lymphocytes.

Serum collected from outbred male New Zealand white rabbits infected intratesticularly with Treponema pallidum (Nichols) was assayed for ability to alter transformation of normal rabbit peripheral blood lymphocytes (PBL) in vitro. Sera collected from 25 infected rabbits inhibited [³H]thymidine incorporation by normal rabbit PBL stimulated with concanavalin A (Con A, 16μg/ml), relative to PBL cultured in normal rabbit serum (NRS). Maximal inhibitory activity was detected in serum collected at the time of peak orchitis. The degree of inhibition was related to the concentration of syphilitic serum in PBL cultures. Inhibition of Con A stimulation was reversed by increased mitogen concentration. Sera which depressed Con A stimulation also depressed lymphocyte transformation induced by oxidation with sodium m-periodate (NaIO₄). Cytotoxic activity was detected in occasional sera. All sera were heat inactivated at 56 °C for 30 min prior to testing. Both freshly collected sera and sera stored at ?70 °C significantly inhibited PBL transformation. These results suggested that serum of syphilitic rabbits contains one or more inhibitors of in vitro lymphocyte transformation.     

Use of salt-stored zonae pellucidae for assessing rabbit sperm capacitation for in vitro fertilization

Zonae pellucidae of tubal and follicular oocytes were collected and prepared for salt storage. Cumulus and corona radiata cells were removed from oocytes with hyaluronidase and a small bore pipette. The oocytes (referred to as zonae since vitelli were rendered nonfunctional) were stored in a salt solution at 4°C. Using in utero capacitated sperm, the penetrability of zonae from tubal and follicular oocytes stored immediately after collection was compared to controls, i.e., in vitro development of tubal ova to the 4-cell stage within 24 hr. The penetration rates were 100% (8 penetrated/ 8 inseminated), 77.8% (7 penetrated/ 9 inseminated), and 100% (10 fertilized/10 inseminated), respectively, and these were not statistically different. The mean (x ) numbers of sperm able to penetrate the zonae, into the perivitelline space (PVS) for tubal (34.0) and follicular (1.1) oocytes were significantly different (P < 0.01). However, following maturational incubation before salt storage, zonae of tubal and follicular origin showed no significant differences in penetrability of in utero capacitated sperm when assessed by percent penetration, or mean numbers of sperm cells reaching the PVS: tubal zonae, 100% (15/15), and follicular zonae, 100% (18/18), and mean number of sperm in the PVS (x [tubal zonae] = 12.4, and x [follicular zonae] = 11.8). The penetrability of tubal zonae with and without maturational incubation was compared, and no significant differences in penetrability by in utero capacitated sperm were present when assessed by percent penetration nonmatured 92.6% (25/27) and matured 93.3% (28/30) and mean number of sperm in the PVS (x [nonmatured] = 3.33 and x [matured] = 2.41). In vitro capacitation of ejaculated rabbit sperm by serum treatment was assessed by the penetration of salt-stored zonae, zonae-free hamster oocytes (ZFHO), and in vitro fertilization of freshly collected tubal oocytes. None of 60 salt-stored zonae and none of 31 tubal oocytes were penetrated, and these values were significantly (P < 0.005) smaller than the 9 of 78 (12%) zona-free hamster ova that were penetrated by sperm cells from the same sample. In vitro capacitation of ejaculated rabbit sperm by washing and preincubation was assessed by the penetration of salt-stored zonae, zona-free hamster oocytes (ZFHO), and fertilization of freshly collected tubal oocytes. Seventy-six of 80 salt-stored zonae were penetrated, and this was significantly (P < 0.005) greater than the 67 of 87 tubal oocytes fertilized and 30 of 35 ZFHO penetrated, which were not significantly different. The salt-stored zonae were more readily penetrated by capacitated sperm when compared to tubal oocytes. However, the ZFHO are more penetrable than salt-stored zonae and tubal oocytes when incompletely capacitated sperm is used. A useful role for this approach in studies dealing with sperm fertilizing ability is anticipated.