Estradiol, or testosterone, and a variety of C-19 and C-18 synthetic steroids with androgen- or estrogen-like biological activity given to the neonate induce sterility in the adult. Pregnanes are not active in this respect. Those competing with estrogens and androgens (progesterone) protect the neonate against the deleterious effect. These compounds are active in the neonate whether injected repeatedly or given only in one injection (Table 4.1). In most studies rodents (rats or mice) were the test animal of choice, but other mammals and even birds are sensitive.
The account of "neonatal sterilization" is the story of the advocates of direct effect of steroids on the gonads and those who believed in the indirect influence, mediated through the hypothalamus and
Pharmacological doses of many hormones during uterine life or shortly after birth cause severe disturbances in reproductive functions, affect the development of sex organs and sex behavior, and predispose some tissue to the development of neoplastic lesions. The neonate is very sensitive to steroid hormone insult. A single injection of as little as 1µg of estradiol benzoate and 5–10 µg of testosterone propionate is sufficient to produce sterility in the adult. Females exposed to such small amounts may sexually mature and ovulate for several weeks, or even a few months, before becoming sterile. The rodent neonate was held to be sensitive to the insult only during a “critical” period defined to last about 10 days after birth.
It has been my privilege and pleasure during the past half century to participate in the unfolding of present-day concepts of the mammalian female reproductive cycles. When the studies recorded here b
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.
With respect to the ovarian status and the estrous cycle as expressed by the vaginal cytology, it was emphasized that there is only a rough correspondence between the vaginal stages and the days of the cycle. The 4-day cycle tends to be the most frequent, with the 5-day cycle a normal variant. Spontaneous persistent vaginal estrus (SPE), an acyclic state occurring in older adult rats, reflects the presence in the ovaries of large vesicular follicles failing to luteinize and, hence, the absence of corpora lutea once the condition becomes well established. Pseudocyclic fluctuations in the vaginal smear during SPE resemble those in ovariectomized rats treated chronically with estrogen. The age of onset of SPE varies among different rat strains, appearing as early as 5 or 6 months in the DA and CD strains, but rarely before 12 months in the O-M strain. O-M/DA hybrids were intermediate. In the DA strain there was also a marked influence of length of daily illumination on the occurrence of SPE: exposure to 10 hours or less of light per day restored cycling to rats that had already shown SPE while exposed to longer days. An hereditary influence of age was also apparent in the rapidity with which continuous illumination induced persistent estrus (LLPE). Young DA females rapidly entered LLPE within 10 days, while young O-M females remained cyclic for 5 weeks. Hybrids again were intermediate. At middle age. O-M rats became as responsive as young rats of the DA strain. Like SPE in DA rats. LLPE was reversible, for estrous cycles returned after daily exposure to light was reduced. The special sensitivity of DA rats to lighting may have been a trait acquired from outcrossing with wild gray rats in years past. Normal cycling could be restored in SPE rats by daily injection of progesterone at low dosage. The same effect followed isolated treatments with progesterone upon return of proestrus/estrus after interruption of SPE. This was the first demonstration of positive feed-back of progesterone, the first sign of its biphasic action, and an indication that progesterone facilitates the action of estrogen in promoting ovulation. When progesterone treatment was delayed after proestrus/estrus there was progressively lower effectiveness during the next 10 days. Indirect support of regular ovulatory cycles resulted from treating DA SPE rats with prolactin (PRL) daily at low doses, provided that an initial set of corpora lutea was first induced by other means.(ABSTRACT TRUNCATED AT 400 WORDS)
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; Lipschütz 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).
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.
In retrospect, the Long and Evans (1922) monograph contained the first evidence that different AP secretions are responsible in rats for ovulation and for corpus luteum function in early pregnancy or pseudopregnancy. It was almost 20 years later that Astwood (1941) purified material from rat pituitaries that was distinct from FSH and LH and had the selective power of maintaining luteal secretion. Evans et al. (1941) identified the luteotropic material as prolactin.
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).
Boling et al. (1941) correlated ovulation time in rats with the interval from the beginning of heat behavior. Inasmuch as the beginning of heat in these rodents is itself related to the time of day (Hemmingsen and Krarup 1937), it follows that ovulation is governed in some manner by events related to circadian periodicity. It is now common knowledge that a major determinant is a pronounced surge of LH secretion on the afternoon of proestrus, beginning during a so-called “critical period” of a few hours, cued in turn by the photoperiod to which the animal is exposed. Awareness of the close relationship of ovulation time to clock hours in rats came from the studies with progesterone and estrogen outlined in the previous section.
An outstanding characteristic of the DA strain and its fertility problem was the suspension of cyclic estrus early in adult life among segregated females and replacement of cycles by more or less continuous vaginal cornification. This persistent vaginal “estrus” when well established was associated with the presence in the ovaries of prominent vesicular follicles and the absence of corpora lutea. Cycling DA females also rapidly developed the condition when exposed to continuous illumination. In addition, there were certain seasonal influences relating to the length of daily illumination as detailed below.
The generally accepted sequence of biosynthetic steps involved in the production of aldosterone from cholesterol is shown in Fig. 1. Other possible pathways have been suggested by Wettstein (1961), who incubated tritiated progesterone with beef adrenal homogenate and found that it was incorporated into 18-oxoprogesterone and 21-deoxyaldosterone, two possible intermediates of aldosterone biosynthesis. Tritium-labelled 18-hydroxy-11-deoxycorticosterone (18-OH-DOC) and 18-hydroxyprogesterone were converted to aldosterone by bullfrog adrenal slices (Nicolis and Ulick,1965).11-Dehydrocorticosterone-4-14C was converted to aldosterone by rabbit adrenal tissue (Fazekas and Kokai, 1967) as well as by homogenates of monkey and frog adrenals (Sharma, 1970). Addition of substrate amounts of 11β-hydroxyprogesterone to beef adrenal tissue slices (Stachenko and Giroud, 1964) and of 21-deoxyaldosterone, 11-dehydroaldosterone, or 21-hydroxypregnenolone to beef adrenal homogenate (Kahnt and Neher, 1965) stimulated aldosterone production in vitro. Thus, the reactions listed in Fig. 1 can theoretically occur in a number of different sequences. However, no direct evidence has yet been presented which would indicate that activation of different pathways of aldosterone production could be of physiological importance, although this possibility has been suggested by Baniukiewicz et al. (1968) and Blair-West et al. (1970b).
There is little direct evidence that angiotensin1 has a stimulating effect on aldosterone biosynthesis by adrenocortical tissue. However, it is a very active stimulator of aldosterone secretion in several animal species and, according to indirect evidence, enhances aldosterone secretion by stimulating biosynthesis rather than by stimulating the release of preformed steroid. Thus, the increments in aldosterone output of isolated dog or sheep adrenals induced by small doses of angiotensin (Ganong et al., 1962; Davis, 1962; Blair-West et al., 1962) were much larger than the total aldosterone content of normal adrenal glands2. In the rat, systemically infused angiotensin not only led to a significant increase in aldosterone secretion but also to a sixfold increase in the adrenal content of aldosterone (Dufau and Kliman, 1968a).
Aldosterone is synthesized by homogenized adrenocortical tissue from endogenous as well as from exogenous precursors. According to Kahnt and Neher (1965a), the endogenous precursor of aldosterone production by beef adrenocortical tissue homogenate is most likely free cholesterol. After centrifugal fractionation of sheep (Raman et al., 1966), bullfrog (Psychoyos et al., 1966) and rat (Marusic and Mulrow, 1967 b) adrenal homogenates, only the mitochondrial fractions were found to actively convert corticosterone to aldosterone. The enzyme systems necessary for the conversion of corticosterone to 18-hydroxycorticosterone or aldosterone could not be obtained in soluble form following ultrasonic treatment of mitochondria (Raman er al., 1966).
