Effect of in utero Incubation on the Metabolism of Rabbit Spermatozoa

Abstract

IT has been reported that the uptake of oxygen by rabbit spermatozoa increased four-fold after they had been incubated for 6 h in the uterus of the œstrous rabbit¹. It has also been postulated that the capacitation of spermatozoa may be associated with changes in their metabolic pattern to enable them to produce more energy². This communication deals primarily with the metabolism of freshly ejaculated sperm and those incubated in the uterus of the œstrous rabbit. The host rabbit was anæsthetized with ‘Nembutal’ and the uteri were ligated anterior to the cervix and posterior to the uterotubal junction. The freshly ejaculated somen from 3 to 7 bucks, to which streptomycin (2 mg/ml. semen) was added, was divided into 3 portions. One portion was heated at 65° C for 10 min to kill sperm and then injected into the left uterine horn. Another portion with live sperm was injected into the right uterine horn of the same rabbit. The third portion was washed³ and resuspended in Ca⁺⁺ free Krebs–Ringer phosphate⁴, for immediate metabolic studies. Semen deposited in the uterus was collected 6 h later by introducing 5–10 ml. Krebs–Ringer phosphate into each uterine horn and then withdrawing with a syringe. The uterine washing from each horn was diluted with Krebs–Ringer phosphate to make a final volume of about 25 ml. and then centrifuged (1,000g) for 15 min. The supernatant was discarded and the sediment was resuspended in Krebs–Ringer phosphate. The suspension recovered from the left uterine horn contained leucocytes and was free of any live sperm. Counts of sperm and leucocytes were carried out and correction was made for the contamination of leucocytes¹. Aliquots (0.8 ml.) of each of the three suspensions were placed in Warburg vessels (6 ml. capacity) and 0.2 ml. Krebs–Ringer phosphate containing 10–15 µmoles of carbon-14 labelled glucose (specific activity : 5 µc./ml.) was added. In the first three experiments uniformly labelled glucose was used, and in the fourth experiment glucose-1-¹⁴C was used. In experiments 5–7 glucose-1-¹⁴C and glucose-6-¹⁴C were used as substrates. Incubation of fresh sperm in the Warburg apparatus (37° C) was carried out directly after they had been washed, and of the in utero incubated sperm and leucocytes soon after they had been collected. The ¹⁴CO₂ released was trapped with hyamine hydroxide and a strip of filter paper that had been placed in the central well of the Warburg vessels. After incubation for 2 h, 0.1 ml. 3 N perchloric acid from the side-arm was tipped to stop the reaction and to liberate any ¹⁴CO₂ that might have been present in the medium. The radioactivity of both the substrate and the trapped ¹⁴CO₂ with hyamine and filter paper was determined by means of a scintillation counter. Glucose and lactate were estimated as previously described⁵. Lactate production was corrected for any lactate that had been present before incubation.