Emilio O. Fernandez

Precoital Single Doses of Δ9-Tetrahydrocannabinol Block Ovulation in the Rabbit

delta9-Tetrahydrocannabinol (delta9-THC) inhibits pituitary gonadotropin secretion in castrated rhesus monkeys. Also, delta9-THC blocks the ovulatory reflux in rabbits. We report now the dose-response relationship of precoital single doses of delta9-THC on luteinizing hormone (LH) release and ovulation in the rabbit. Forty-five female rabbits in estrous were divided into nine groups of five animals. Groups 1 to 5 received a single intramuscular dose of delta9-THC (5,2.5, 1.25, 0.612, and 0.306 mg/kg, respectively) 2 hours before mating; animals of group 6 received vehicle only. In animals of groups 7 and 8 ovulation was induced with 100 IU of human chorionic gonadotropic (hCG), given intravenously 2 hours after the administration of delta9-THC (5 mg/kg) or vehicle. Rabbit luteinizing hormone (rLH) was measured in plasma 90 to 120 minutes after coitus or hCG administration. After the injection of 5 mg of delta9-THC, luteinizing hormone-releasing factor (LH-RF) (20 microgram intravenously) was administered to the animals of group 9. All animals of groups 6, 7, and 8 ovulated. A dose-response curve was observed in the animals treated with delta9-THC and natural mating. Whereas none of the animals treated with 5 or 2.5 mg/kg ovulated, one of the group treated with 1.25 mg/kg, two of the group treated with 0.612 mg/kg, and all treated with 0.312 mg/kg ovulated. Ovulations correlated with postcoital levels of rLH. All animals of group 9 ovulated, indicating that the site of action of delta9-THC is suprapituitary, probably hypothalamic.

Mechanism of induction of luteal phase defects by danazol.

The effects of danazol on the luteal phase of cycling rhesus monkeys and on the ability of the intact rhesus monkey to respond to hCG stimulation were studied in two experiments. In a third experiment the impact of danazol upon the response of the decapsulated mouse testis to hCG stimulation was evaluated. These experiments demonstrated that danazol shortens the luteal phase and decreases progesterone production in intact monkeys via a direct effect on the gonad.

Presence of a human chorionic gonadotropin-like substance in human sperm

An hCG-like material has been extracted from human sperm. These experiments were designed to characterize this material. Sperms of 10 volunteers were separated from seminal fluid, washed in PBS three times, and resuspended in 0.5 ml of the same buffer. Samples were pooled; cells were disrupted by sonication and extracted in alkaline buffer by constant agitation at 4 degrees C. The extract was ultracentrifuged at 4 degrees C. Supernate was lyophilized and reconstituted in 2 cc of distilled water. This material presented a dose-response curve parallel to those of IS2-hCG and CR119 in beta hCG RIA. When chromatographed in a Sephadex G-150 column the extract eluted within the hCG range and immunoreacted in the specific beta hCG RIA. When absorbed onto a concanavalin A--Sepharose column, all recovered immunoreactive material eluted after exposure to alpha-D-methylglucoside, indicating that it is a glycoprotein. The extract stimulated progesterone and testosterone secretion in porcine granulosa cells and decapsulated rat testis, respectively, indicating its biologic potency.

Demonstration of a chorionic gonadotropin-like substance in rabbit morulae.

Rabbit morulae were treated with specific indirect immunofluorescence and immunoperoxidase histochemical techniques. The first antibody in both systems was rabbit anti-human chorionic gonadotropin (hCG) beta-subunit. Negative controls revealed a complete absence of reaction. All morulae incubated with the double-antibody system showed a positive reaction. Rabbit morulae thus present a substance with antigenic determinants similar to the beta-subunit of hCG. The physiologic role of this substance is unknown.

Immunodetection of a Human Chorionic Gonadotropin-Like Substance in Human Sperm

The presence of a human chorionic gonadotropin (hCG)-like substance in human spermatozoa is reported. A highly sensitive immunocytochemical procedure was utilized (double-antibody immunofluorescence technique). Rabbit anti-hCG or rabbit anti-hCG beta-subunit was used as the first antibody. A positive fluorescence reaction was found in all human specimens analyzed and in positive controls (choriocarcinoma cells). No fluorescence was detected in other species studied (sheep, pig, goat, horse, bull, and guinea pig), nor in the negative controls. These findings open a new research area on the physiologic role of this hCG-like substance in human reproduction.

Evidence for a Human Chorionic Gonadotropin-Like Material in the Rabbit Blastocyst * †

Extracts from (1) 300 day 6 rabbit blastocysts, (2) 300 day 2 unfertilized ova, and (3) uteri from the nonpregnant does were analyzed in the following assays in order to determine the presence of a chorionic gonadotropin: (1) in vitro bioassay for testosterone production by decapsulated rat testes, (2) in vitro bioassay for progesterone production by porcine granulosa cells, (3) in vitro determination of adenylyl cyclase-stimulating activity in rabbit Graafian follicles, (4) gel filtration in a Sephadex G-150 column and assay of the elutions in a radioimmunoassay (RIA) specific for the beta-subunit of human chorionic gonadotropin (hCG), (5) parallelism with hCG standard preparations in beta-hCG RIA, and (6) concanavalin A-column chromatography. The rabbit blastocyst extracts showed an hCG-like material in all of the systems tested. None of the other two extracts presented hCG-like activity in any of the assay. The immunoreactive material in the beta-hCG RIA of the blastocyst extracts after gel filtration presented a profile different from that of pure hCG; this may represent heterogeneity due to a species difference or a slightly different molecular weight. These results confirmed previous findings of several investigators and those from our laboratory in that the preimplanted rabbit embryo contains a gonadotropin with characteristics similar to hCG.

Pharmacologic Modification of the Time Course of Ovum Transport in Guinea Pigs

Various agents were examined for their effects on ovum transport in the guinea pig. Estrogen significantly accelerated ovum transport in this species. The experiments further demonstrated that estrogen did not act by inducing prostaglandin synthesis, nor by altering plasma progesterone levels. The estrogen-induced acceleration was significantly antagonized by tamoxifen, an antiestrogen that acts by interfering with estrogen receptor synthesis. Cycloheximide also antagonized the effects of estrogen on ovum transport. These data suggest that the modification of ovum transport by estrogen is due to the entrance of estrogen into the nuclei of target cells, and subsequent protein synthesis. Although we assume that this action occurs at the level of the oviduct, our experiments do not prove this assumption.