Different Doses Of Estradiol Benzoate Research Articles

Effects of different doses of estradiol benzoate used in a cervical relaxation protocol on the success of non-surgical embryo recovery and luteal function in superovulated ewes

This study investigated the effectiveness of different doses of estradiol benzoate (EB) to promote cervical relaxation and their effects on luteal function and outcomes of non-surgical embryo recovery (NSER) in sheep. Multiparous (MULT) and nulliparous (NULL) crossbred Lacaune X Santa Inês ewes were superovulated and naturally bred. Seven days after progesterone withdrawal, females were randomly assigned to one of three distinct cervical relaxation protocols, consisting of i.m. treatment with 37.5 µg d-cloprostenol and different doses of EB: 0.0 mg (0.0EB group; n = 3 NULL and 14 MULT); 0.5 mg (0.5EB group; n = 4 NULL and 12 MULT) or 1.0 mg (1.0EB group, n = 6 NULL and 11 MULT) 16 h before NSER. All ewes received 50 IU of oxytocin 20 min before NSER (D17). Blood samples were collected and ultrasound exams (B-mode and color Doppler) were performed at two timepoints: immediately before d-cloprostenol and EB treatments and prior to NSER. Estrous behavior, corpora lutea count and NSER success outcomes were not affected by EB treatments nor parity (P > 0.05). Embryo recovery rate was greater for ewes in the 0.5EB group and in the NULL ewes (P < 0.05). Ovarian biometrics differed between the two evaluation timepoints in all groups (P < 0.05). Plasma estradiol increased over time, reaching a significant greater level in 1.0EB ewes compared to controls on D17 (P < 0.05), whereas progesterone concentrations decreased over time in all groups (P > 0.05). In conclusion, treatments did not affect NSER success but they did affect luteal function by altering P4 and E2 concentrations. Therefore, the NSER technique can be successfully performed in ewes with or without prior treatment with EB.

Detection of estradiol in rat brain tissues: Contribution of local versus systemic production

Estrogens play important roles in regulating brain development, brain function, and behavior. Many studies have evaluated these effects using ovariectomized (OVX) rats or mice with different doses of estrogen replacement, assuming that estradiol levels in all regions of the brain are the same as levels achieved in the serum. It is well known, however, that the brain contains all the enzymes necessary to produce estrogens, and that estrogen levels in the brain are determined by both systemic and local production and are region-specific. The present study conducted a detailed analysis of the relationship between systemic levels of 17-β-estradiol (E2) achieved by estrogen replacement and levels achieved in specific regions of the brain. Levels of E2 were measured in both brain and serum in OVX rats treated with different doses of estradiol benzoate (EB) using a novel and recently validated UPLC–MS/MS method. Results confirmed significantly higher levels of E2 in the brain than in serum in brain regions known to contain aromatase (ARO) activity, both in OVX controls and in rats treated with physiological doses of EB. Additional studies compared the level of E2 and testosterone (T) in the brain and serum between testosterone propionate (TP) treated OVX and male. This demonstrated higher levels of E2 in certain brain regions of males than in TP treated OVX females even though T levels in the brain and serum were similar between the two groups. Studies also demonstrated that the differences between serum and brain levels of E2 can be eliminated by letrozole (ARO inhibitor) treatment, which indicates that the differences are due to local ARO activity. Collectively the results provide a detailed analysis of brain region-specific E2 concentrations in OVX, E2-, and T-treated rats and demonstrate the degree to which these concentrations are ARO-dependent.

Exogenous Exposure to Estradiol Benzoate or Flutamide at the Weaning Age Alters Expression of Connexin Isoforms in the Initial Segment of Male Rat

ABSTRACTConnexin (Cx) is a complex which allows direct communication between neighboring cells via exchange of signaling molecules and eventually leads to functional harmony of cells in a tissue. The initial segment (IS) is an excurrent duct of male reproductive tract and expression of numerous genes in the IS are controlled by androgens and estrogens. The effects of these steroid hormones on gene expression in the IS during postnatal development have not extensively examined. The present research investigated expressional modulation of Cx isoforms in the IS by exogenous exposure to estrogen agonist, estradiol benzoate (EB), or androgen antagonist, flutamide (Flu), at weaning age. Two different doses of EB or Flu were subcutaneously administrated in 21-day old of male rats, and expressional changes of Cx isoforms in the adult IS were analyzed by quantitative real-time PCR. Treatment of a low-dose EB (0.015 μg/kg body weight) resulted in an increased expression of Cx31 gene and a decreased expression of Cx37 gene. A high-dose EB (1.5 μg/kg body weight) treatment caused an increase of Cx31 gene expression. Increased levels of Cx30.3 and Cx40 transcripts were observed with a low-dose Flu (500 μg/kg body weight) treatment. Treatment of high-dose Flu (50 mg/kg body weight) led to expressional increases of Cx30.3, 40, and 43 genes. Our previous and present findings suggest differential responsiveness on gene expression of Cx isoforms in the IS by androgens and estrogens at different postnatal ages.

Exogenous exposure to estradiol benzoate or flutamide at the weaning age alters expression of connexin isoforms in the initial segment of male rat.

Connexin (Cx) is a complex which allows direct communication between neighboring cells via exchange of signaling molecules and eventually leads to functional harmony of cells in a tissue. The initial segment (IS) is an excurrent duct of male reproductive tract and expression of numerous genes in the IS are controlled by andevrepogens and estrogens. The effects of these steroid hormones on gene expression in the IS during postnatal development have not extensively examined. The present research investigated expressional modulation of Cx isoforms in the IS by exogenous exposure to estrogen agonist, estradiol benzoate (EB), or andevrepogen antagonist, flutamide (Flu), at weaning age. Two different doses of EB or Flu were subcutaneously administrated in 21-day old of male rats, and expressional changes of Cx isoforms in the adult IS were analyzed by quantitative real-time PCR. Treatment of a low-dose EB (0.015 μg/kg body weight) resulted in an increased expression of Cx31 gene and a decreased expression of Cx37 gene. A high-dose EB (1.5 μg/kg body weight) treatment caused an increase of Cx31 gene expression. Increased levels of Cx30.3 and Cx40 transcripts were observed with a low-dose Flu (500 μg/kg body weight) treatment. Treatment of high-dose Flu (50 mg/kg body weight) led to expressional increases of Cx30.3, 40, and 43 genes. Our previous and present findings suggest differential responsiveness on gene expression of Cx isoforms in the IS by andevrepogens and estrogens at different postnatal ages.

Different doses of estradiol benzoate induce conditioned place preference after paced mating

The ovarian hormones estrogen and progesterone are required for the complete display of sexual behavior in female rats. Paced mating produces a reward state in intact cycling and ovariectomized (OVX), hormonally primed females as evaluated by the conditioned place preference (CPP) paradigm. Most of the studies that have evaluated CPP induced by paced mating in OVX females have used relatively high doses of estradiol benzoate (EB). In the present study we determined if different doses of EB, combined with progesterone (P), could induce CPP after paced mating. For this purpose OVX female rats were divided in five groups that received one of different doses of estradiol benzoate (5, 2.5, 1.25 or 0.625μg estradiol+0.5mg of progesterone) before being allowed to pace the sexual interaction and conditioned in a CPP paradigm. We found that the lowest dose of EB used (0.625μg) significantly reduced the lordosis quotient and the lordosis coefficient. Even though these females paced the sexual interaction, they didn't change its original preference, suggesting that sexual interaction did not induce a positive affective, reward state. Females allowed to pace the sexual interaction with higher doses of EB developed CPP after paced mating. These results indicate that a threshold of estradiol is required for paced mating to induce CPP.

Dose-dependent effects of chronic estrogen treatment on anxiety-related behavior in female mice

Though estrogens are well known to regulate anxiety or fear-related behavior, controversial effects of estrogen replacement to ovariectomized rodents were reported. This inconsistency may be due to differences in the treating condition of estrogens, such as duration of administration and/or their total amount. The present study was conducted to address these issues by examining the effect of chronic treatment of estradiol benzoate (EB) in ovariectomized ICR/Sea mice with several different doses of EB and intermittent observation of anxiety-related behavior in several tests [open field (OF), elevated plus maze (EPM), light-dark transition (LDT)]. The behavioral parameters of the tests were subjected to factor analysis. As the results of comparing the factorial scores for two extracted factors, it was indicated that the mice implanted with relatively higher-dose EB capsule were more anxious than the animals treated with vehicle and/or lower-dose EB. While, the mice treated with the lower-dose EB did not show apparent change of anxiety. Together, the findings in the present study suggest that the chronic estrogen treatment may have anxiogenic effects in higher doses, while the effects were not clear in lower doses.

Cortisol Inhibits Sexual Receptivity, but Has No Effect on Attractivity or Proceptivity in Ovariectomized Ewes Treated with Different Doses of Estradiol Benzoate.

Cortisol Inhibits Sexual Receptivity, but Has No Effect on Attractivity or Proceptivity in Ovariectomized Ewes Treated with Different Doses of Estradiol Benzoate.

211 EFFECT OF ESTRADIOL BENZOATE ON THE FIRST WAVE DOMINANT FOLLICLE IN HOLSTEIN HEIFERS

The objective of this study was to evaluate the effect of different doses of estradiol benzoate on the first wave dominant follicle in Holstein heifers. Estrous cycle of cyclic Holstein heifers (n = 16; 13.84 ± 0.44 months of age; 335.5 ± 6.65 kg) was synchronized using CIDR (14 days; VetoCIDR®, Aburaihan, Tehran, Iran) in association with GnRH analogue (12.5 μgi.m. Vetaroline®, Aburaihan) on Day 6 and PGF2 analogue (500 μg; Vetaprost®, Aburaihan) on day 13 after CIDR insertion. On Days 5 to 7 of the ensuing cycle (Day 0 of the experiment), heifers were assigned into 4 experimental groups. Heifers in Groups 1, 2, and 3 (n = 4 in each group) received 2, 5, and 10 mg of estradiol benzoate (i.m. Vetastrol®, Aburaihan), respectively, concurrent with 100 mg of progesterone (i.m. Vetagesterone®, Aburaihan). Heifers in the control group (n = 4) received distilled water as a placebo intramuscularly. Ovarian follicles were monitored daily, from Days 0 to 16, using an ultrasound scanner (Sonovet 2000, Korea) equipped with a 7.5-MHz transrectal probe. Initiation of follicle regression was defined when there was a sudden decrease of 1 to 2 mm followed by a progressive decrease in the diameter of the ovarian follicle. Emergence of anew follicle wave was defined as the day that the dominant follicle was, retrospectively, identified at a diameter of 4 to 5 mm. Data were analyzed using Kruskal-Wallis one-way ANOVA in SAS (SAS Institute, Cary, NC). On Day 0, there was a functional CL (19.62 ± 0.57 mm) and a growing dominant follicle (11.25 ± 0.62 mm) on the ovary of all heifers. The dominant follicle regressed on Day 3.5 ± 1.04, 3.2 ± 0.85, 2.7 ± 0.75, and 8.5 ± 0.50 in Groups 1, 2, 3, and control, respectively (P &lt; 0.05). A new follicle wave emerged on Day 7.7 ± 0.25, 8.2 ± 0.48, 9.7 ± 1.44, and 7.2 ± 1.11 in Groups 1, 2, 3, and control, respectively (P &gt; 0.05). Estrus was detected on Day 12.0 ± 1, 11.7 ± 0.34, 13.0 ± 0.00, and 15.2 ± 1.75 in Groups 1, 2, 3, and control, respectively (P &gt; 0.05). The interval between steroid injections (Day 0) and ovulation were 13 ± 0.41, 13 ± 0, 14.3 ± 0.33, and 16.2 ± 1.75 days for Groups 1, 2, 3, and control, respectively (P &gt; 0.05). Interovulatory intervals were 18.7 ± 0.33, 18.7 ± 0.33, 20.7 ± 0.67, and 20.5 ± 1.55 days for Groups 1, 2, 3, and control, respectively (P &gt; 0.05). In conclusion, estradiol benzoate induced early regression of the dominant follicle of the first wave but did not affect the interval between steroid treatment and ovulation in Holstein heifers. This research was funded by Aburaihan Pharmaceutical Company (Tehran, Iran).

Estradiol modulates behavioral arousal and induces changes in gene expression profiles in brain regions involved in the control of vigilance

Estrogens reduce lipocalin-type prostaglandin D synthase (L-PGDS) expression in a region-dependent manner in the mouse preoptic area (POA). This result linked sex hormones with sleep-wake cycle regulation. In turn, the somnogenic effects of prostaglandin D(2) have been shown to be mediated via increases in adenosine, and a select group of sleep-active ventrolateral preoptic nucleus (VLPO) neurons are directly activated by adenosine 2A (A(2A)) agonists. We hypothesized that increased arousal after estrogen administration is mediated by a reduction of L-PGDS and lowered A(2A) receptor expression in the POA. To test this hypothesis, running wheel activity (RWA) of ovariectomized female mice treated with oil or different doses of estradiol benzoate (EB) was studied, followed by quantitative reverse-transcriptase polymerase chain reaction to determine mRNA expression of genes related to sleep and arousal in brain region extracts from oil-treated and EB-treated mice. RWA was increased in estrogen-treated mice, and these effects followed an inverted-U dose-response curve. The most effective dose (1.25 microg EB/capsule) increased RWA more than 2.5-fold, as compared with control animals, and EB doses that were higher or lower were less effective. Increases in RWA were accompanied by decreased L-PGDS mRNA in the POA and decreased A(2A) receptor mRNA in the POA and VLPO. Given that EB-treated animals have higher motor activity and lower levels of L-PGDS and A(2A) receptor mRNAs in sleep-active areas, these correlational findings support the hypothesis that EB may increase behavioral arousal by decreasing the levels of well-known sleep-inducing molecules within the preoptic region.

Estrogen inhibition of MPA‐Induced mouse mammary tumor transplants

Estrogen compounds were used to treat mice bearing syngeneic transplants of medroxyprogesterone acetate(MPA)-induced BALB/c mammary adenocarcinomas. Both MPA-dependent and MPA-independent tumor lines were used. These lines expressed estrogen (ER) and progesterone receptors (PR). We demonstrate that different doses of estradiol benzoate (EB) and 17-beta-estradiol (E2) inhibit tumor growth and induce tumor regression in both MPA-independent and -dependent tumors, even in the presence of MPA or progesterone (P). EB was unable to induce regression of (ER-) hormone-independent tumor lines. A few MPA-dependent tumors became resistant to the estrogenic treatment; in subsequent passages some of these tumors retained their MPA-responsiveness, although estrogen sensitivity was not recovered.

GABAergic drugs and lordosis behavior in the female rat

Agents modifying GABAergic neurotransmission were administered to ovariectomized rats treated with different doses of estradiol benzoate (EB) + progesterone (P) or with EB alone. Hormone treatments were designed to induce an intermediate level of receptivity in order to be able to observe both stimulatory and inhibitory effects on lordosis behavior. Both the GABA A receptor agonist THIP and the GABA B receptor agonist baclofen inhibited lordosis behavior at doses from 20 and 5 mg/kg, respectively. The GABA transaminase inhibitorγ-acetylen GABA (GAG) and the GABA agonist 3-aminopropanesulfonic acid had no effects, even when high doses were administered. The GABA A receptor antagonist bicuculline had no effect by itself nor did it block the effects of THIP. It is therefore suggested that the GABA A receptor is of slight importance in the control of lordosis behavior. No evidence could be found supporting the hypothesis that an interaction between P and GABA is important for hormoneinduced receptivity. It does not appear likely that motor disturbances are responsible for the inhibitory effects of baclofen and THIP. The exact mechanism by which these drugs inhibit lordosis behavior is not clear at present.

Morphometric investigations on endocrine glands: VII. The influence of some estrogens on the formation of castration cells in the anterior pituitary of rats

After ovarectomy there are observed changes in body weight increase, uterus weight and area size of the gonadotropic cells in the adenohypophysis. A replacement by estrogens may diminish such variations, but their extent is different for each of the single parameters; therefore no high correlation coefficients resulted in calculations on the basis of all data of a test. The administration of estradiol benzoate, estriol and estrone twice a week (about once per cyclus period) caused moderate changes in body weight increase and marked differences in uterus weights, but in the tested doses no alteration in the cell size of the gonadotropes, compared to ovarectomized animals without further treatment. Daily application of different doses of estradiol benzoate or estrone signify a higher efficacy of the first steroid. Different treatment conditions - starting shortly after excision of the ovaries or one week later - yielded only slight divergencies. Progesterone injected together with estrone caused no further significant deviations. Mestranol had only a moderate effect on the gonadotropes, but the uterus weight may increase under such a replacement up to values similar to those in untreated animals. On the other hand, there was observed a marked shrinking of the gonadotropes following higher doses of estriol, but in this case the uterus weight was less affected. In relation to equal doses of estrone, the uterus weights are similar following estrone cyanate, but in contrast to the parent compound, the effect on the gonadotropes was only low.

Sites of action of testosterone and estradiol on longitudinal bone growth.

In this study the mechanisms by which sex steroids influence body growth were investigated. The effect of different doses of testosterone propionate on longitudinal bone growth and body weight gain was studied in a) gonadectomized male rats, b) gonadohypophysectomized male rats, and c) gonadohypophysectomized male rats given replacement therapy with bovine growth hormone (bGH). The effect of different doses of estradiol benzoate on the same growth parameters was studied in female rats divided into the same experimental groups as the males. Accumulated longitudinal bone growth was determined using oxytetracycline as an intravital marker. Testosterone caused a dose-dependent increase in longitudinal bone growth in gonadectomized male rats. In contrast, testosterone exerted no significant increase in longitudinal bone growth in gonadohypophysectomized male rats with and without bGH replacement therapy. Treatment with estrogen inhibited longitudinal bone growth and body weight gain. The inhibitory effect of estradiol was approximately the same in gonadohypophysectomized female rats given bGH replacement therapy as in gonadectomized female rats. The results suggest that testosterone exerts its stimulatory effect on body growth mainly by modulating hypothalamopituitary functions, e.g., by altering the secretory pattern of GH. On the other hand, it seems that changes in the hypothalamopituitary functions are less significant for the inhibitory effect of estradiol on body growth. It is concluded from this study that the sites of action for estrogen and testosterone in modulating body growth in the rat are different.

The effect of hypophysectomy on the quail's oviduct response to low and high doses of estradiol benzoate.

The response of the quail's (Coturnix coturnix Japonica) oviduct to different doses of estradiol benzoate (EB) was explored in ovariectomized and ovariectomized-hypophysectomized animals. Doses of EB ranged from 10 microgram to 1 mg/animal daily for 6 consecutive days; animals were sacrificed 24 h after the last injection. Controls were injected with olive oil. Parameters thought to reflect stimulation of the oviduct were 1) wet and dry weight, 2) DNA, 3) RNA, 4) soluble proteins, 5) ovalbumin, and 6) morphological changes at the level of magnum's mucosa. A statistical significant difference in several of the parameters listed was found between the effect of different doses of EB and whether the injection was done in ovariectomized or in ovariectomized-hypophysectomized animals. The dose response with EB for cell proliferation and induced proteins accumulation (ovalbumin) show that it takes considerably more EB to maximally stimulate ovalbumin accumulation when compared with cell proliferation. Hypophysectomy decreases the oviduct responses to high EB doses. Ultrastructural studies of the magnum mucosa confirm these biochemical results. The evidence presented suggesting an EB dose-dependent difference induction of specific cellular responses in the presence and absence of the pituitary in quails are the basis to postulate the contributing role of the pituitary in the parameters measured.

Undernutrition, puberty and the development of estrogen positive feedback in the female rat.

The development of the positive feedback of estrogen in normally fed animals and its possible alterations in animals subjected to restricted food intake was studied. Different doses of estradiol benzoate (EB), administered s.c. as a single injection to normal prepubertal rats of different ages, resulted in an increase in uterine weight which became more prominent as the animals approached puberty. Significant gonadotropin release was induced by EB only from Day 26 on. The LH response to EB measured in the afternoon (1600 h) of the second day following the injection increased with age; the older rats showing an elevation in LH levels after the administration of doses of EB which were ineffective in younger rats. In rats injected with the highest dose of EB (5 µg/100 g bw), gonadotropin release occurred 1 day earlier. Modification of the growth rate of rats by varying the milk supply during the nursing period, followed by normal postweaning feeding, revealed that attainment of a critical body weight is not essential for puberty to occur since animals reached puberty at the same age, but at different body weights. Other animals maintained after weaning under restricted diet did not reach puberty for as long as their access to food was restricted (Day 57). When food was made available ad libitum, vaginal opening and ovulation occurred within a week. At 42 days of age, the underfed rats had small uteri and restoration of food produced a dramatic increase in uterine weight and gonadotropin levels. At this age, the underfed rats responded to EB injection with a remarkable increase in gonadotropins which was even greater than that induced by a similar dose of EB in younger normally fed rats of similar body weight. Restoration of food followed by ES injection resulted in ovulation within 3 days. The results indicate that 1) the gonadotropin response to estrogen positive feedback develops gradually and quantitatively as the animal matures and 2) undernutrition-induced delayed puberty is not caused by inability of the hypothalamic-pituitary unit to respond to positive estrogen feedback, but rather to ovarian failure to release estrogen in amounts sufficient to trigger a gonadotropin surge. However, the fact that in underfed rats, basal gonadotropin levels were not elevated indicates that the steroid negative feedback was operative in these animals.

On the Feedback Actions of Estrogen on Gonadotropin and Prolactin Release in Infantile Female Rats1

A fetoneonatal estradiol-binding plasma protein, often referred to as alpha fetoprotein (AFP), is present in high levels in serum of neonatal rats. The existence of elevated serum FSH levels, in spite of high estrogen titers and the presence of adult type estrogen receptors in the hypothalamus and pituitary, suggests that the lack of a fully expressed estrogen negative feedback in these animals is not due to a decreased function of the receptors, but rather to binding of estrogen to AFP. To evaluate this hypothesis, rats were ovariectomized at day 10 and injected sc daily for 3 days with different doses of estradiol benzoate (Eb), for which AFP has a very high binding affinity, diethylstilbestrol (DES), which binds very weakly to AFP, or ll-β-methoxyethynylestradiol (RU2858), which does not bind to AFP. Only the largest doses of EB (0.05 and 0.10 μg/100 g BW) were effective in preventing the postcastration rise in FSH. The smallest dose (0.0025 μg/100 g BW) of DES significantly reduced the postcastration ...

Further studies on the maturation of the estrogen negative feedback on gonadotropin release in the female rat.

To evaluate estrogen negative feedback in infantile female rats, 9-day-old rats were ovariectomized (OVX) and treated with different doses of estradiol benzoate (Eb, s.c. once daily for 2 days); plasma LH, FSH and estradiol (E2) levels were then determined by RIA. The responses of these rats were compared with those of 25-day-old rats subjected to the same experimental procedures. In intact sham-operated controls, plasma FSH was elevated on days 9--13 and low on days 25--29, whereas LH did not change significantly throughout the period studied. OVX at day 9 or 25 increased the levels of both hormones, with the response faster and larger in the older rats. Treatment with Eb was more effective in suppressing the post-castration rise in gonadotropin levels in 27-day-old than in 11-day-old rats. At day 27 plasma E2 was elevated to pre-castration values by as little as 10 ng of Eb, whereas at day 11 this dose produced plasma E2 titers even higher than pre-castration values. Plasma E2 titers were high at 9--13 days of age and low at days 25--29. OVX on day 9 decreased E2 2--4 days later to about 1/2 the initial concentration, whereas adrenalectomy (ADRX) or ADRX-OVX was followed by an almost complete disappearance of the steroid. When E2 was injected at day 10 in intact rats to elevate plasma E2, E2 remained elevated when measured 3 to 120 min after its injection, but on day 25, 50% of the injected E2 had disappeared from plasma in 90 min. These results provide additional support for the view that estrogen negative feedback, even though present in infantile rats, is less effective than later in life and hence indicate that high gonadotropin titers observed at days 9--13 in the presence of high E2 may be caused by the relative ineffectiveness of the feedback at these early ages. The high E2 titers appear to be caused by an enhanced rate of production of E2 by the adrenals and ovaries and by a reduced metabolic clearance of the steroid.

Development of dopaminergic and estrogenic control of prolactin release in the female rat.

Plasma prolactin as determined by radioimmunoassay was barely detectable in 21-day-old fetuses and on the day of birth (day 0). It had declined by days 3–6 and then rose gradually to attain adult levels at day 35. To further evaluate (he development of the prolactin control system, the ability of estrogen to elicit prolactin release was studied at various ages by injecting different doses of estradiol benzoate (Eb) SC once daily lor 2 days and measuring plasma prolactin 24 hr after the last injection. At 11 days of age, there was no significant increase in prolactin with any of the doses of Eb used (0.05–0.4 µg/100 g body wt). In 27-day-old rats, the 0.4 µg dose induced a dramatic rise in plasma prolactin and the 0.05, 0.10 and 0.2 µg dose induced much smaller but significant increases in prolactin. By 37 days, the 0.2 µg dose became highly effective and the 0.4 µg dose induced an even larger increase. The response of adult females to this dose was significantly less than that observed in 37-day-old anima...