Persistent Estrus Research Articles

Vaginal microflora in a rat model of polycystic ovarian disease

Exposure of female rats to continuous light for prolonged periods induces persistent estrus (PE), an anovulatory disorder. Instead of cyclic fluctuations, the vaginal smears from PE rats show only cornified cells as the hormonal control of the estrous cycle becomes inappropriate, and ovaries become cystic when ovulation ceases. The reproductive tract, a key target organ in PE, shows numerous manifestations of chronic estrogenic influence.In this paper, we report our findings on bacterial colonizations and ultrastructural examinations of the vaginal surface epithelium of PE rats with polycystic ovaries (PCO). Quantitation of bacteria in the vaginal lavage was done with a standard volume of sterile normal saline and studied in five different hormonal states: diestrus day 1 (D-1), diestrus day 2 (D2), proestrus (Pro E), estrus (E), and persistent estrus (PE). Scanning electron microscopy (SEM) was used to examine the vaginal surface epithelium from PE rats with PCO and compared to the controls. In all rats, cardiovascular perfusion was performed for in vivo fixation of the organs using 2.5% glutaraldehyde in 0.1 M Na Cacodylate buffer (pH 7.3) for 30 minutes.

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.

The LHRH neuronal system in female rats: relation to the medial preoptic nucleus.

Previously we have found that small lesions confined to the medial preoptic nucleus (MPN) or the suprachiasmatic nucleus (SCN) blocked the cyclic release of gonadotropins in the female rat, inducing a persistent estrous state. Since the MPN is located just caudal to the organum vasculosum of the lamina terminalis (OVLT) where LHRH cell bodies are most concentrated, we applied an immunocytochemical technique to examine the possibility that the lesions had simply disrupted LHRH neurons or fibers. Using a new anti-LHRH provided by Dr. V. D. Ramirez, we found that the distribution pattern of immunoreactive LHRH cell bodies and fibers was similar to that previously reported, although the staining was more intense and extensive with low background. There was no concentration of LHRH cell bodies and fibers in the MPN or SCN and, in fact, these nuclei generally showed a lower density of stained elements than did surrounding tissue. In persistent estrous animals with lesions confined to the MPN there was no detectable reduction of stained fibers in the median eminence. These results, along with the results of other workers, suggest that persistent estrus following lesions of the MPN or SCN is not due to reduction of LHRH neurons or fibers. Rather, they support the hypothesis that these nuclei are critical for triggering the ovulatory release of LHRH.

Dissociation of estrous cycle and activity rhythm in rats

When rats were kept in continuous light there was a time-lag between the onset of activity arrhythmia and that of persistent estrus. When animals showing both arrhythmias in continuous light were kept under a LD 21:3 photoperiod, the activity rhythm returned to normal but the estrous cycle did not.

Nafoxidine administered to newborn female GR mice arrests the development of their mammary glands.

Mice of the GR strain develop many hormone-dependent mammary tumors in response to estrogen and progesterone stimulation. Since this strain is so sensitive to steroid hormones, we administered a single dose of the antiestrogen Nafoxidine to female GR mice within 24 hours after their birth. This treatment arrested the development of their mammary glands and when the mice were adults, 10 weeks old, they did not cycle normally but were in a state of persistent estrus. Whole mounts of mammary glands from Nafoxidine-treated mice revealed cystic areas within some ducts and bulbous swellings at the ends of others. No hyperplastic alveolar nodules (HAN) were identified in the glands. In contrast, a single dose of 17 beta estradiol administered within 24 h after birth, resulted in a highly branched gland displaying typical end buds, a few alveoli and more HAN than were observed in glands of control adult mice of the same strain. Thus Nafoxidine treatment not only arrested the development of the mammary glands in female GR mice (causing them to appear "masculinized") but it also produced abnormalities within the glands.

Effects of prolonged caging with fertile males on reproductive functions in aging female rats.

Previous studies showed that retired breeder female rats ceased to exhibit regular estrous cycles beginning around 10–12 months of age (Lu et al., 1979). Since the onset of reproductive dysfunction occurs in multiparous females soon after being separated from fertile males, the present study examined whether prolonged caging with males could maintain normal reproductive functions in aging female rats. Eight-month-old multiparous female rats (Long-Evans) were housed in groups of 5, either with (mated; n=47) or without (nonmated: n=49) a young fertile male. At 13 months of age, the mated females were separated from the males. Between 8 and 13 months of age, 45 of 47 mated females became pregnant and delivered pups. With advancing age, the number of females which delivered pups decreased markedly, but the average litter size did not change. At 13 months of age, 49% of the mated females exhibited regular estrous cycles, whereas only 20% of the nonmated females were regularly cyclic. At 15 and 17 months of age, the mated group still showed a significantly higher percentage of regularly cyclic animals than the nonmated group. Both groups of animals showed a progression from regular cyclicity to irregular cyclicity to persistent estrus or repetitive pseudopregnancy with advancing age. The profile of change in reproductive patterns in the mated group during 13–17 months of age was remarkably similar to that which occurred during 9–13 months of age in the nonmated controls. The patterns of periovulatory luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion were similar in 4-month-old regular cyclic rats and 1 5-month-old mated females displaying regular estrous cycles. These results demonstrate that regular estrous cyclicity and normal gonadotropin secretion can be maintained for an extended period of time in aging female rats by caging with fertile males, and suggest that the cessation of regular cyclicity beginning around 10–12 months of age in multiparous females may be due in part to separation from the males.

Effects of constant bright illumination on reproductive processes in the female rat

Physiological and behavioral reproductive changes in the female rat which occur under constant bright illumination (LL) are examined. The development of LL-induced persistent estrus (PE) is discussed first in relation to other conditions in which PE is displayed. Next, mechanisms are reviewed which may account for the LL-induced changes. These include: (1) role of the retina, the retinohypothalamic tract and the suprachiasmatic nucleus; (2) influence of adrenal, pineal and Harderian glands; and (3) disruptions in either the 4-day endocrine rhythms or circadian neural component of the estrous cycle. Additional topics which are examined include the ontogeny of age-induced PE and the effects of LL on hormone receptor binding, puberty, sexual receptivity and mating.

Neonatal exposure to zearalenone causes persistent anovulatory estrus in the rat.

The effect of neonatal administration of zearalenone on the female reproductive system was studied in the rat. A single subcutaneous injection of 1.0 mg zearalenone to 3- or 5-day-old rats caused persistent vaginal estrus in adulthood. Ovaries in these animals contained many large follicles but no newly formed corpora lutea. The same effects were observed in rats which had received 100 micrograms estradiol-17 beta in the neonatal period. Most rats which had received 100 micrograms zearalenone or 10 micrograms estradiol-17 beta showed regular 4-day estrous cycles and had newly formed corpora lutea in their ovaries. These results demonstrate that neonatal exposure to zearalenone produced persistent anovulatory estrus in the rat, the potency being about one tenth that of estradiol-17 beta.

Changes in plasma hormone profiles and in hypothalamic catecholamine turnover rates in neonatally androgenized rats during the transition phase from cyclicity to persistent estrus (delayed anovulatory syndrome).

Changes in plasma hormone profiles and in hypothalamic catecholamine turnover rates in neonatally androgenized rats during the transition phase from cyclicity to persistent estrus (delayed anovulatory syndrome).

Ultrastructure of the Surface Epithelium of the Polycystic Ovary in Constant Light Rats

It is now well established that exposure of cyclic female rats to continuous light induces persistent estrus (PE), a syndrome which in many respects resembles the human polycystic ovary (PCO) syndrome. As in the human PCO, light microscopic studies of cystic ovaries of the constant light rat show numerous atretic follicles, and the surface epithelium grossly appears smooth and white. In the present study, we have utilized scanning (SEM) and transmission electron microscopy (TEM) to study changes on the surface epithelium of PCO in the constant light rat model.

Responses to copulatory stimulation in neonatally androgenized female rats.

A series of experiments evaluated the extent to which copulatory stimulation could ameliorate the anestrus and sterility exhibited by neonatally androgenized female rats. The age at which animals began to exhibit persistent vaginal estrus and the degree of sexual receptivity exhibited under several testing paradigms were found to be inversely related to the dose of testosterone propionate (TP) injected neonatally. With increasing numbers of mounts received, both the number of androgenized animals exhibiting sexual receptivity and the quality of the estrous behavior exhibited tended to increase. The extent to which copulatory stimulation modified receptivity varied with the dose of TP injected neonatally and the condition of testing. Animals injected with high doses of TP (500 microgram) usually showed little or no receptive behavior even in the most extensive behavioral tests. However, under some testing conditions animals receiving 50 micrograms of TP neonatally, while showing little or no receptivity during initial mounts, showed increased receptivity as behavioral tests were extended. Following matings that included one to five ejaculations, only control animals were observed to become pregnant. However, when androgenized females cohabited with males for an extended period, animals that had neonatally received .5 microgram of TP, but not higher doses, did become pregnant. It is concluded that the capacity of systems mediating reproductive physiology and behavior to be facilitated by stimuli associated with males and that mating is a characteristic of the female rat which can be manipulated by injection of hormones during the neonatal period.

Discrete lesions reveal functional heterogeneity of suprachiasmatic structures in regulation of gonadotropin secretion in the female rat.

The present study examines the function of several cytologically distinct suprachiasmatic structures in the regulation of ovulation and positive feedback effects of estrogen and progesterone on gonadotropin release in the rat. Small (0.6-0.8 mm dia.) electrolytic lesions were placed at four loci along the rostrocaudal extent of the suprachiasmatic region in regularly cycling female rats. Anovulatory persistent estrus occurred only when lesions were located either in the suprachiasmatic nucleus (SCN) or the medial preoptic nucleus (MPN), a small periventricular cell group lying immediately caudal to the organum vasculosum lamina terminalis (OVLT). Lesions restricted to the OVLT and adjacent ventral prechiasmatic region (VPC-L), or the anterior suprachiasmatic region (ASR) between the MPN and SCN resulted in irregular estrous cycles frequently marked by periods of prolonged diestrus. Following administration of 50 microgram estradiol benzoate (EB) a daily afternoon surge of gonadotropin was observed in control animals. This circadian release of gonadotropins was completely abolished by SCN, ASR and MPN lesions. EB-induced gonadotropin surges were also greatly attenuated by VPC-L lesions. Subsequent administration of 1.5 mg progesterone (P) induced large surges of luteinizing hormone and follicle-stimulating hormone in VPC-L and ASR lesioned animals as well as controls. P administration also elicited gonadotropin surges in SCN lesioned animals, although surges were markedly attenuated in magnitude compared to controls. Only lesions that destroyed the MPN and immediately adjacent periventricular tissue completely and invariably eliminated P-induced gonadotropin release. Thus, anovulatory-persistent estrus appears to be associated specifically with lesions that interfere with the-positive feedback effect of P (MPN and SCN lesions). Animals with lesions that block or attenuate EB effects without interfering with P sensitive neural substrates can maintain long-term spontaneous ovulation (VPC-L and ASR lesions). An hypothesis is advanced to account for the differential effect of MPN and SCN lesions on P-induced gonadotropin release.

Structural and Functional Regression of Polycystic Ovaries by Danazol

The effects of danazol on ovarian steroidogenesis and follicular size was studied in androgen-sterilized rats bearing polycystic ovaries. Daily treatment with 4 or 20 mg of the drug caused a reduction in ovarian weight and regression of cystic follicles. Serum levels of luteinizing hormone (LH), testosterone, and estrogen were significantly decreased. The persistent estrus smears were changed to diestrus, indicating inhibition of estrogenic activity.

Scanning Electron Microscopy of the Polycystic Ovary in Constant Light Rats

Exposure of female rats to continuous light eventually induces persistent estrus (PE), a syndrome which in many respects resembles the human polycystic ovary syndrome. Light microscopic studies on the polycystic ovaries of constant light PE rats have been previously reported. In the present study, we have utilized scanning electron microscopy (SEM) to provide information regarding three-dimensional changes occurring on the surface of the polycystic ovary.Following exposure to continuous light for 50 days, PE was induced in a group of Sprague-Dawley female rats according to the procedure described elsewhere (2). Polycystic ovaries from constant light PE rats and ovaries from controls were fixed in 2.5% glutaraldehyde in 0.1M Na cacodylate buffer, pH 7.3 at room temperature for 24 hours. They were then cut into two equal halves along their long axis and fixed for an additional 3-4 hours. The specimens were processed for SEM studies by a modified OTOTO technique, dehydrated and critical point dried with CO2.

Plasma prolactin and progesterone responses to mating are altered in ages rats.

The effects of varying amounts of copulatory stimulation on patterns of plasma concentrations of prolactin and progesterone were evaluated in 3- and 12-month-old female rats. The 12-month-old group included rats which still exhibited oestrous cycles and rats in persistent vaginal oestrus (PVO). The extent of copulatory stimulation was defined by the number of intromissions received during mating: less than or equal to 5, 15 or greater than 50. Blood samples were drawn over the 8 days after mating through a cannula inserted into the right external jugular vein. Plasma from the samples was assayed for prolactin and progesterone. In aged but still cyclic rats, pregnancy rates were positively correlated with the number of intromissions received during mating. Only one rat in PVO became pregnant. All animals which became pregnant and rats in PVO which, after mating, exhibited a disruption of the pattern of PVO, showed the nocturnal surge of plasma prolactin characteristic of pregnant and pseudopregnant rats. While these surges persisted until day 8 after mating in pregnant animals, they were absent by this time in the rats in PVO. Prolactin surges were present in some but not all of the aged rats which did not become pregnant. Progesterone concentrations were raised in all pregnant animals except the one pregnant rat in PVO and, while not related to the number of intromissions, concentrations were higher 8 days after mating in young compared with those in aged pregnant rats. Plasma progesterone was low in rats in PVO regardless of disruption of the pattern of PVO. We have concluded that the failure of limited copulatory stimulation to induce pregnancy in older rats results, at least in part, from its failure to initiate nocturnal prolactin surges. Nevertheless, our data suggest that matings which are not experimentally limited should provide ample stimulation to establish such surges. Although reduced plasma concentrations of prolactin and progesterone at pro-oestrus and reduced plasma progesterone through part of gestation may contribute to decreasing fertility in aged rats, other unidentified factors appear to be involved in mediating the capacity of extensive copulatory stimulation to induce pregnancy in these animals.

The role of hypothalamic adrenergic receptors in preventing testosterone-induced androgenization in the female rat brain.

Androgenization of the female neonatal rat brain by testosterone and the subsequent development of persistent estrus in the adult can be blocked by drugs which interfere with normal hypothalamic neuronal function. The mechanism of action of these drugs was studied. A 25-micrograms dose of testosterone propionate at 5 days of age produced a 69% incidence of persistent estrus at 90 days of age. alpha-Methyl-p-tyrosine given with testosterone propionate resulted in an 81% incidence of persistent estrus. Therefore, hypothalamic norepinephrine depletion did not prevent androgenization. Tyramine (100 micrograms) inhibited androgenization to an incidence of 27% by 90 days. Phenoxybenzamine (50 micrograms) and phentolamine (50 micrograms) each reduced the incidence to 0%. Combining the beta-antagonist propranolol with phenoxybenzamine or phentolamine reversed the block in androgenization, resulting in 37% and 50% incidences of persistent estrus, respectively. Tyramine causes the release of neuronal stores of norepinephrine which is turn stimulates alpha- and beta-adrenergic receptors. The action of tyramine implicates adrenergic stimulation as a mechanism for the inhibition of androgenization, but does not define which receptor type is involved. In addition to postsynaptic alpha-blockade, phenoxybenzamine and phentolamine can cause presynaptic alpha-blockade, resulting in increased neuronal norepinephrine release and beta-stimulation. When the beta-antagonist propranolol was added to the alpha-antagonists, the block in androgenization was reversed. Therefore, we conclude that beta-adrenergic receptor stimulation prevents androgenization of the neonatal rat brain.

Oestrogen receptors and metabolic activity in the genital tract after ovariectomy of ewes with permanent infertility caused by exposure to phytooestrogens.

Characteristics of the uterus anc cervix after ovariectomy of ewes with permanent phytooestrogen infertility (PPI) were compared with controls. Ewes with PPI had more oestrogen-binding sites in the cervix, but not in the uterus. There was no difference between the two groups of ewes in the binding affinity constant of receptors from the uterus or cervix. There were more keratinized cells in the vaginal epithelium of ewes with PPI, and the rates of protein and glycoprotein synthesis in the uterus and cervix were higher in ewes with PPI. These results offer further evidence that PPI in adult ewes is similar to the "persistent oestrus' syndrome in rodents oestrogenized neonatally.

The relationship of the oestrogen and progestin receptors in the abnormal uterus of the adult anovulatory rat. Effects of neonatal treatment with testosterone propionate or clomiphene citrate.

The neonatal administration of testosterone propionate to Wistar rats resulted in anovulatory adults in persistent vaginal oestrus. Clomiphene citrate had a similar effect. In both groups of adults, hyperplasia of the uterine epithelium and occasional metaplasia was observed. The uterine nuclear and cytosol oestrogen and progestin receptors of these anovulatory rats were found to have affinities for their respective ligands similar to those of normal females. The nuclear oestrogen receptor comprised occupied and unoccupied components, as in normal females. The content of the nuclear oestrogen receptor was comparable with that of females in the late dioestrous or pro-oestrous phase. This content was higher in the clomiphene-treated group. Despite the relatively high nuclear oestrogen receptor content the content of progestin receptors, a putative index of the oestrogenic response, was lower in the treated rats than in normal adult females throughout the cycle. Administration of oestradiol to both treatment groups resulted in depletion of cytosol oestrogen receptor content 1 h later, which, however, was not reflected by an increase in the content of nuclear oestrogen receptors. There was no measurable increase in progesterone receptor content in treated rats after daily administration of oestrogen (5 microgram/rat) for 3 days. These changes in sex-hormone-receptor interactions involving an impairment of the normal oestrogenic response may be associated with the abnormal differentiation of the uterus in these sterile, anovulatory animals.

Light‐dependent and light‐independent estrous cycles and locomotor activity rhythms in rats regulating their cage illumination∗

Abstract Female rats were individually housed in a cage placed inside a small light‐proof chamber. They were allowed to switch on and off the ceiling lamp of the chamber by pressing a lever. Adult rats, raised under LD 12:12 and exhibiting regular 4‐day estrous cycles, developed persistent estrus during a 60‐day stay in this cage, where self‐selected LD alternations were quite irregular depending on animals. Female rats, born and raised under irregular LD cycles controlled by their mother and isolated in such a cage, also produced irregular LD cycles but their extrous cycles repeated regularly. Mating, pregnancy, parturition and lactation took place normally. Their locomotor activity exhibited an ultradian rhythmicity. These rats demonstrated very regular 4‐day estrous cycles and 24‐h locomotor activity rhythms, when they were transferred to the LD 12:12 environment. Female rats, either born and raised under the illumination control by their mother and by themselves or raised under LD 12:12, developed persistent estrus, if they were housed in a light‐proof chamber in which the ceiling lamp was switched on and off unpredictably by a certain rat in a remote separate cage. From these results, it is suggested that the clock mechanism involved in the regulation of the estrous cycle might function independently from the environmental LD cycles unless it is irreversibly entrained by the 24‐h LD cycles or disturbed by unpredictably irregular LD alternations. The ultradian pattern of the locomotor activity rhythm might also differentiate and become well‐organized into a circadian type after the exposure of the 24‐h environmental time cues.

Induction of anovulatory sterility by neonatal treatment with 5 beta-dihydrotestosterone in female rats.

Wistar female rats were injected with testosterone (T) or 5 beta-dihydrotestosterone (5 beta-DHT) for the first 5 days of life. Neonatal treatment with 1 mg of T resulted in anovulatory persistent oestrous syndrome in 100% of the animals. In the females injected with 1 mg of 5 beta-DHT, 74% of the treated rats became sterile at 120 days of age. In addition, 0.5 mg 5 beta-DHT was also effective in inducing anovulatory persistent oestrus; the incidence of sterility was 10 and 80% at 60 and 120 days of age, respectively. When daily dose of 5 beta-DHT was reduced to 0.1 mg, however, only 33% of the rats resulted in sterility. These results suggest that the free form of T and non-aromatizable androgen, 5 beta-DHT, can permanently suppress the development of female type of neuroendocrine regulation. The possible participation of the process other than the central aromatization in androgenization will be discussed.