John W. Everett

Irritative Deposits from Stainless Steel Electrodes in the Preoptic Rat Brain Causing Release of Pituitary Gonadotropin.

Summary When ovulation was induced by means of stainless steel electrodes in the preoptic area, delivering pulses of varied characteristics, a prominent zone of mild inflammation appeared at the end of the electrode track. When ovulation failed, the zone was characteristically small or absent. Electrolysis with direct current, 10 μA or more for 20 sec or longer, proved uniformly effective in causing ovulation. Substitution of platinum electrodes for stainless steel usually gave negative results. Microinjection of FeCl3 solution into the preoptic tissue induced ovulation, while injection of similar amounts of acidified NaCl did not. Finally, electrolysis stimulated ovulation when electrodes of reagent grade iron wire were used, but not when similar electrodes of platinum were employed. It is concluded that iron and perhaps other metals electrolytically deposited from electrodes of stainless steel (or nichrome) produce irritative foci whose stimulative action on certain neurons continues long after the flow of electric current has ceased.

Ovulation in persistent-estrous rats after electrical stimulation of the brain.

Summary Persistent estrus was induced in female rats by continuous illumination. Through “permanent”bipolar electrodes, stimulation was administered after recovery from anesthesia to either the amygdala (11 rats) or the septum pellucidum (1 rat). The latter and 5 of the former ovulated within 24 hours.

Spontaneous ovulation in the rabbit following combined estrogen-progesterone treatment.

Summary Following treatment with estrogen and progesterone 4/10 rabbits ovulated spontaneously, i.e., without the coital stimulus normally required to induce LH release from the hypophysis in this species.

The Blocking Effect of Nembutal on the Ovulatory Discharge of Gonadotropin in the Cyclic Rat

Summary Brief action of Nembutal during certain critical hours on the day of proestrus delays the ovulatory discharge of hypophyseal gonadotrophin for 24 hours. This is evidence of a 24-hour periodicity in the neural mechanism which incites such discharge.

Strain Differences for Preoptic Stimulation of Ovulation in Cyclic, Spontaneously Persistent-Estrous, and Androgen-Sterilized Rats

The CD (Charles River) strain of rats differs in several important respects from our inbred Osborne Mendel (O-M) strain. The critical period for the ovulatory surge of gonadotropin in proestrus is less sharply limited in CD rats. They are much more refractory to preoptic (POA) electrochemical stimulation for ovulation, not only in normally cycling females, but strikingly so in androgen-sterilized (TP) females, as well as in rats having spontaneous persistent estrus (PE). In proestrous 4-day cyclic CD females ‘blocked’ with pentobarbital, a much larger stimulus (100 µA × 30 sec = 3000 µcoulombs) was required to fully ovulate all subjects than was needed in similar O-M rats (10 µA × 20 sec = 200 µcoulombs). While 5/6 O-M PE rats ovulated in response to massive unilateral POA stimulation (3000 µcoulombs), all of 6 CD PE rats failed to ovulate. Even bilateral POA stimulation with these parameters induced ovulation in only 3/10 rats. O-M TP rats had low POA thresholds close to those of cyclic O-M females, while only 1/6 CD TP rats gave even a partial response (5 tubal ova) to massive bilateral stimulation. CD TP rats also differed from O-M TP rats by secreting abundant prolactin in spite of the constant estrus. This was shown by prominent milk cysts in the mammary glands at 8–9 months of age and by spontaneous pseudopregnancy after corpora lutea were induced by LH injection. O-M TP rats had no mammary development and experienced only a short diestrus after LH injection. Whether there is a common basis for these strain differences remains uncertain.

Nocturnal and diurnal levels of prolactin, LH, FSH, estrogens, and progesterone in middle-aged, spontaneously persistent estrous rats

Reproductive research in aging animals is often performed with very old rats manifesting spontaneous persistent estrus (SPE). However, Charles River (CD) Sprague Dawley rats present SPE as early as 6 months of age and thus are potentially very useful in such studies since the concomitant pathologies associated with old age can be avoided. Serum levels of LH, FSH, prolactin, estrogens, and progesterone were measured by RIA in 8 to 12 month SPE rats after rapid decapitation at 0300, 1000 or 1600 hr (8–11 rats/group). As reported by others in very old SPE rats, proiactin was high at 1600 hr (169±40 ng/ml) compared with levels at 0300 hr (43 ± 7 ng/ml) or 1000 hr (95 ± 25 ng/ml). LH and FSH values did not change with time of day, but those of FSH were consistently above baseline values of cyclic rats. Total estrogens were low (75 ± 9 pg/ml) at 1000 hr but significantly higher at both 0300 hr (127 ± 11.7 pg/ml) and 1600 hr (141 ± 23 pg/ml), resembling levels at about 1000 hr on proestrus in cyclic rats (125 ± 16 pg/ml). Average progesterone concentrations resembled baseline values of cyclic rats but varied widely (0.02 to 25 ng/ml). These findings suggest that the middle-aged SPE rat may be used in studies of the aging reproductive system, and that future studies with SPE rats should consider variations in prolactin, estrogen and progesterone with the time of day.

Vaginal Cytology and Ovarian Status

Beginning with studies reported by Everett (1939), more than one million vaginal smears from over 16,000 rats have been prepared and evaluated in the author’s laboratory during approximately 5 decades. It is appropriate to consider criteria for evaluating the smears and their relationship to ovarian function. Our criteria are based on the five stages of the estrous cycle of rats described by Long and Evans (1922) and presented here in Table 1 essentially as originally summarized. It is important to recognize that each stage represents a stop-action in a steadily moving process. The stages are to be regarded as indirect and somewhat delayed measures of ovarian hormone output. Thus, in general terms, the rise of estrogen in late diestrus results in proliferation of the vaginal epithelium to give the dry, velvety appearance characterizing proestrus (stages I and II).

Strains of Rats

Most studies in this laboratory have been carried out in three strains of rats: two locally inbred strains, designated DA and O-M, respectively, and the commercial CD strain from the Charles River Breeding Laboratories.

Steroid Regulation of the Ovarian Cycle

The inhibitory chronic effects of gonadal secretions on pituitary gonadotropic function were recognized several years before the synthetic steroids became available (cf. Burrows 1949). These negative effects were the basis for the “push-pull” hypothesis of pituitary-gonadal interaction proposed by Moore and Price (1930, 1932). A positive, stimulative effect of gonadal hormones in promoting puberty was noted by Engle (1931), however, and several workers soon reported that the immediate effect of estrogen administration on luteinizing potency of the AP was stimulative (Fevold, Hisaw and Greep 1936; Lane and Hisaw 1934; Lipschutz 1935). More directly, Hohlweg and Chamorro (1937) demonstrated that luteinization could be induced in immature rats by administration of estradiol benzoate. In ewes the induction of ovulation by estrogen therapy was accomplished by Hammond et al. (1942).

Central Neural Control of Spontaneous Ovulation: The Rat Model

The facts that both steroid-induced and spontaneous ovulation in rats can be prevented by treatment with centrally acting drugs and that the controls exhibit circadian rhythmicity implied participation of the central nervous system. Neural control was also implied indirectly by the report by Dempsey and Searles (1943) that LLPE rats formed corpora lutea after copulation. In the estrous rabbit and cat, several workers induced ovulation by electrical stimulation of the hypothalamus or the amygdala (see Harris 1972). Yet not until 1957 was there comparable direct evidence of a role of the brain in species that ovulate spontaneously. In that year Bunn and Everett reported ovulation induced in alert LLPE rats by electrical stimulation through electrodes chronically implanted in the amygdala or septum pellucidum. Critchlow (1957, 1958), employing cyclic rats anesthetized with pentobarbital during the proestrus critical period, succeeded in inducing ovulation by stimulation through electrodes stereotaxically placed deep in the medial hypothalamus. Since then the pentobarbital-blocked rat has become a favorite experimental subject in this and other laboratories.