John J. Alleva

Inhibition of ovulation in hamsters by the protein synthesis inhibitors diphtheria toxin and cycloheximide.

Summary Diphtheria toxin and cyclohex-imide, structurally unrelated translational inhibitors of protein synthesis, inhibited ovu-lation in hamsters. Inhibition resulted from i.p. injection given well after the 2:30 pm release of pituitary luteinizing hormone (LH) that initiates the 11-hr ovulatory process. Thus it may be concluded that these agents inhibit ovulation by direct action on the ovaries. Both luteinization and follicular rupture were inhibited. Follicles inhibited by cyclo-heximide were similar histologically to control follicles prevented from ovulating by central blockade of LH release with phenobar-bital, whereas follicles inhibited by toxin contained shrunken oocytes and necrotic granu-losa cells. Low doses of both agents inhibited ovulation only when injected within 5 hr after LH release. However, high doses were effective at later times. Similar ovulation inhibition responses to both the microbial toxin and cycloheximide at high doses suggest that protein synthesis essential for ovulation occurs throughout the 11-hr ovulatory process initiated by LH and is not restricted to the early part of this period. We thank Dr. Louis Kasza (Food and Drug Administration) for histological analysis of the sectioned ovaries.

The Zeitgeber for ovulation in rats: Non-participation of the pineal gland

Abstract Ovulation occured between 2: 00 AM and 4: 00 AM whether rats were exposed to light daily from 6: 00 AM to 6: 00 PM (LD 12: 12) or from 4: 00 AM to 8: 00 PM (LD 16: 8). Since the time of ovulation was constantly related to the midpoint of the dark period under both lighting conditions and since the midpoint is determined by the times the light comes on and off, both of these signals together must constitute the environmental cue (Zeitgeber) for ovulation in these rats. Pinealectomized rats ovulated at the same time (2: 00 AM-4: 00 AM) as intact rats, which indicates that the pineal gland is not involved in the mediation of stimuli from the Zeitgeber.

Effect of large daily doses of ascorbic acid on pregnancy in guinea pigs, rats, and hamsters

Abstract l -Ascorbic acid was administered orally during pregnancy in doses of 400 mg/kg/day to guinea pigs and 50, 150, and 450 mg/kg/day to rats and hamsters. No increase in abortion or mortality of offspring was observed.

How Hamsters Keep Time: The 6 PM to 6 AM Light‐Sensitive Period

A recent review of the pineal literature revealed that when hamsters are exposed to 24‐hour light:dark (LD) cycles with < 12 hours of darkness (summerlike, SLD), the nightly period of pineal melatonin synthesis (PPMS) begins close to the midpoint of the dark period (“midnight”) and ends at lights‐on irrespective of the length of the dark period or time of day presented. New evidence based on the onset of behavioral estrus every 4 days indicated that the 24‐hour hamster clock controlling timing of estrus (4:30 PM) and the PPMS has a 12‐hour light‐sensitive period (LSP) set to 6 PM‐6 AM in LD 16:8 (dark 8 PM‐4 AM, SLD) by a balance in opposing actions of evening and morning light [Alleva: Pineal Research Reviews, Volume 5, Alan R. Liss, Inc., New York, 1987]. Present experiments focus on how this balance is maintained. When lights‐off was advanced to 6 PM in SLDs ranging from LD 12.5:11.5 (dark 6:15 PM‐5:45 AM) to 18:6 (dark 9 PM‐3 AM), the onset of estrus later that day was advanced in every SLD. However, when lights‐on was delayed to or beyond 6 AM, the onset of estrus was unaffected. Thus, the balance is maintained by a resistive force (blocking without a delaying action) of evening light and an advancing action of morning light. In this balancing process all evening light from 6 PM to lights‐off but only the first 5 minutes or less of morning light were involved. The advancing action of morning light was characterized in LD 13:11 and 18:6 by imposing on the night before estrus a 5:30 PM‐6:30 AM dark period scanned with a 15‐minute light pulse. Shifts in onset of estrus later that day were plotted vs. time of the light pulse. The resulting phase response curves (PRCs) were similar and comprised only an advancing curve, which rose about 10 PM, peaked at 2 AM, and returned gradually to normal at 6 AM. In contrast, a PRC obtained from LD 12:12 (dark 6 PM‐6 AM) was sinelike, comprising a 6 PM‐9 PM delaying curve followed by an advancing curve similar to those from SLD. An hypothesis based on these findings is presented to explain how hamsters would keep constant AM—PM time throughout summer.

Effect of daily injection of phenobarbital on ovulation in hamsters.

Abstract Ovulation in hamsters normally occurs every fourth day (Day 1) and results from the release of pituitary gonadotropins (PG) into the circulation on the preceding day (Day 4) between 2:00 and 3:00 pm . A central mechanism, sensitive to certain drugs including phenobarbital, controls PG release. This release initiates estrus 2 hr later, and ovulation 11 hr later. The response of the central mechanism to daily exposure to phenobarbital was tested by injecting this drug before 2:00 pm daily for up to 50 days. Ovulatory cycles were followed by daily examination of vaginal washings. Daily injections between 1:00 and 2:00 pm initially blocked ovulation for 1–8 days but eventually were ineffective. Resistance to the daily injections was not overcome by raising the dose or delaying the injection on Day 4. After 4 days rest, injection at 1:30 pm on Day 4 was again effective. As expected, ovulation was never blocked when injections were given daily between 3:30 and 3:45 pm . However, when the last (50th) daily injection was given on Day 4 at 1:30 pm instead of between 3:30 and 3:45 pm , ovulation in all hamsters was blocked. Estrus began at about the normal time in resistant hamsters not given the injection due on Day 4. Resistance apparently results from an altered state of the central mechanism controlling PG release.

The hamster clock phase-response curve from summerlike light: dark cycles and its role in daily and seasonal timekeeping

We address the subject of entrainment of the hamster clock by the day:night cycle in summer when the sun sets after 6 PM and rises before 6 AM (nights<12 h). Summer day:night cycles were simulated by 6 light:dark (LD) cycles with D<12 h (summerlike, SLD) ranging from SLD 12.5h:11.5h (D, 6:15 PM–5:45 AM) to 18h:6h (D, 9 PM–3 AM). These are the near limiting SLDs for constant PM timing (entrainment) of behavioral estrus and wheel running in hamsters. The onset of estrus was observed every 4 d in the same hamsters as a phase marker of their 24h clock. On the day before an experimental estrus, preceded and followed by control onsets, a dark period was imposed to cover a putative 6 PM–6 AM light-sensitive period (LSP). This was scanned with a light pulse (and periodic 5sec bell alarms) lasting 5–240 min. Shifts in onset of estrus on the next day were plotted vs. the end of the light pulse for PM times (“dusk”) and its onset for AM times (“dawn”). The resulting phase shifts from the six SLDs were similar, permitting their combination into a single phase-response curve (PRC) of 1605 shifts. This SLD composite PRC rose at 10:15 PM, peaked at 2 AM (81min advanced shift), fell linearly to 5:55 AM, and then abruptly to normal at 6 AM (no shift). Peak shift was unaffected by light pulse duration or intensity, or hamster age. The SLD composite PRC lacked the 6 PM–9 PM curve of delayed shifts present in reported PRCs from LD 12h:12h and DD. However, a two-pulse experiment showed that all light from 6 PM to L-off was needed to block (balance) the advancing action of a 5min morning light pulse, thereby maintaining entrainment. A working hypothesis to explain daily entrainment and seasonal fertility in the golden hamster is illustrated. A nomenclature for labeling the phases of the hamster clock (circadian time) is proposed.