E2 Benzoate Research Articles

Reproductive outcomes of prepubertal Bos indicus beef heifers raised in a pasture-based feeding system submitted to ovulation induction strategies prior to a timed-artificial insemination protocol

Reproductive outcomes were evaluated in Nelore (Bos indicus) heifers submitted to one, two or no ovulation induction protocols based on progesterone (P4) and estradiol (E2) prior to a timed-artificial insemination (TAI) protocol. A total of 1,437 heifers (13.0 ± 0.8 mo old; 3.1 ± 0.1 of body condition score [BCS] and 279.9 ± 25.8 kg of body weight [BW]) were randomly assigned to 1 of 3 treatments: 0IND (n = 486): no ovulation induction protocol; 1IND (n = 481): one ovulation induction protocol; or 2IND (n = 470): two ovulation induction protocols. On Day −47, heifers from 2IND received a disinfected intravaginal P4 device (2 g, previously used for 21 d), kept until Day −40, when 0.5 mg of E2 cypionate (EC) was given. On Day −19, heifers from 2IND and 1IND underwent the same protocol. On Day 0, all heifers were submitted to the same TAI protocol, starting with a P4 device (0.5 g), 0.5 mg of cloprostenol sodium (PGF), and 1.5 mg of E2 benzoate. On Day 7, P4 device was removed, 0.5 mg of PGF, 0.5 mg of EC, and 200 IU of equine chorionic gonadotropin (eCG) were administered. The TAI was performed 2 d later (Day 9). Blood samples were collected on Days −47 and 0, to determine the presence of CL (circulating P4 concentrations ≥ 1.0 ng/mL). Ultrasound was performed on Days 40, 75 and between Day 150 and parturition to assess pregnancy per AI (P/AI) and pregnancy loss (PL). Statistical analyses were performed using SAS 9.4 (a-cP ≤ 0.05; A,B0.05 < P ≤ 0.10). The proportion of heifers with CL on Day −47 was similar among groups (3.4%). A greater proportion of heifers from 1IND had CL on Day 0, followed by 2IND, then 0IND (87.9a; 80.4b; 28.8c%). There was an effect of treatment on expression of estrus (2IND: 66.6a; 1IND: 67.2a; 0IND: 57.4b%), P/AI on Day 40 (2IND: 53.4a; 1IND: 43.9b; 0IND: 46.5b%), P/AI on Day 75 (2IND: 49.8a; 1IND: 40.5b; 0IND: 44.4ab%) and final P/AI (2IND: 45.5a; 1IND: 35.8b; 0IND: 40.5ab%). No differences were observed in PL (40–75 = 6.3%; 75-final = 9.6%; Total = 15.3%). Particularly within lighter heifers, there was an effect of treatment on P/AI on Day 40 (0IND: 39.2b; 1IND: 43.3ab; 2IND: 53.9a%) and on Day 75 (0IND: 36.6B; 1IND: 39.0AB; 2IND: 48.5A%). At the first pregnancy diagnosis, more nonpregnant heifers from 2IND had CL on Day 40 than 0IND, but 1IND did not differ from the other groups (85.4a; 74.8b; 80.8ab%). In conclusion, ovulation induction protocols performed prior to the TAI protocol increased the proportion of heifers with CL on Day 0. The use of two induction protocols resulted in greater fertility, particularly in lighter heifers, and increased cyclicity among nonpregnant heifers. These results indicate that this strategy may be an optimized method for inducing cyclicity and enhancing fertility of prepubertal Nelore heifers raised in pasture-based feeding systems.

Treatment alternatives to induce follicular wave emergence for timed-AI in lactating dairy Cows

Two experiments evaluated the effect of different hormonal treatments to synchronize follicle wave emergence on follicle dynamics and pregnancies per AI (P/AI) in estradiol (E2)/progesterone (P4) timed-AI (TAI) protocols in lactating dairy cows. In Experiment 1, lactating, primiparous Holstein cows (n = 36) received a P4 releasing device (Day 0) and were allocated at random to one of the following three treatment groups: Group EB received 2 mg E2 benzoate (EB) intramuscularly (i.m.), Group EB + GnRH received 2 mg EB+20 μg buserelin (GnRH) i.m., or Group EB + P4 received 2 mg EB + 100 mg of injectable P4 (iP4) in oil i.m. All cows received 0.150 mg D-Cloprostenol on Days 7 and 8 followed by P4 device removal, 400 IU eCG and 1 mg ECP on Day 8. Daily ultrasound examinations revealed that although the interval from P4 device removal to ovulation was not affected by treatment, cows that received EB + GnRH had an earlier (P < 0.05) emergence of the new follicular wave (Day 2.6 ± 0.2) than the other two treatment groups (Days 3.5 ± 0.3 and 6.1 ± 0.3, for EB and EB + P4, respectively). In Experiment 2, 808 lactating cows were assigned randomly to the three treatments evaluated in Experiment 1, and all the cows were TAI to determine P/AI. Cows in the EB + GnRH group had greater P/AI (57.4 %, P < 0.01) than those in the EB (44.6 %) or EB + P4 (45.7 %) groups. In conclusion, the administration of GnRH, but not iP4, on the day of insertion of a P4 device improves P/AI in lactating dairy cows synchronized for TAI with an estradiol/P4-based protocol.

Abstract 077: 17b-estradiol Implants With An Antiandrogen Or Castration Attenuate Body Weight And Total Lean Mass But Differentially Modulate Total Cholesterol And The Pressor Response To Chronic Angiotensin II In The Male Rat

Background: Transfemales are biological males at birth, but identify as female by gender. Gender-Affirming Hormone Therapy ( GAHT ) is used to alter hormone levels to match gender identity. GAHT in transfemales involves administration of estradiol ( E2 ) with an androgen antagonist ( AA ) or orchiectomy. Sex is an independent predictor of cardiovascular ( CV ) risk, but the role of sex hormones on CV risk in transfemales is unknown. Thus, this study tested the hypothesis that GAHT in male rats increases CV risk. Methods: Male Sprague Dawley ( SD ) rats were randomly assigned at 14 weeks of age to three groups that underwent either mean arterial pressure ( MAP , N=3-5/group) or metabolic (N=8/group) monitoring. Control ( C ): empty silastic capsule. E2+AA: silastic capsule with 17-beta E2 benzoate (5 mg/21 days) plus AA, spironolactone (10 mg/kg/day, incorporated in diet). E2+CTX: silastic capsule with E2 plus castration ( CTX ). Silastic capsules (+/- E2) were replaced every 21 days starting at 14 weeks. AA, started at 14 weeks of age, was continued in E2+AA but discontinued after CTX at 16 weeks of age in E2+CTX. Results: (Table 1) : The pressor response to angiotensin II ( ANG II ) was similar in C and E2+CTX but was blunted in E2+AA despite similar baseline MAP, increase in E2 and decrease in testosterone. Cholesterol was only increased in E2+AA. Conclusion: These results suggest that feminizing therapy in male rats is associated with similar changes in sex steroids but the pressor response to ANG II and lipid profile differ based on the method of androgen reduction. The use of a transfemale rodent model will allow study of the pathophysiology of increased CV risk associated with GAHT across the lifespan.

Hormonal combinations aiming to improve reproductive outcomes of Bos indicus cows submitted to estradiol/progesterone-based timed AI protocols

The aim was to study reproductive outcomes of Nelore (Bos indicus) cows submitted to a 7-d estradiol (E2)/progesterone (P4)-based timed artificial insemination (TAI) protocol, receiving various combinations of doses and hormones. Primiparous (n = 962) and multiparous (n = 1935) cows were submitted to synchronization (n = 2012) and resynchronization (n = 885 non-pregnant cows at pregnancy diagnosis 30 d after TAI) protocols, following a 2 × 2 × 2 factorial arrangement of eight treatments. At the initiation of the TAI protocol (Day −9), all cows received a 1.0 g intravaginal P4 insert, 2.0 mg E2 benzoate and received (PGF1) or not (PGF0) 0.5 mg cloprostenol sodium (PGF). On Day −2, the P4 insert was removed, all cows received 0.5 mg PGF, 300 IU equine chorionic gonadotropin (eCG) and 0.5 (EC0.5) or 1.0 mg estradiol cypionate (EC1.0). On Day 0, cows were treated (G1) with 8.4 μg buserelin acetate (GnRH) or not (G0), concurrently with TAI. The eight treatments were generated: 1) PGF0-EC0.5-G0 (n = 364), 2) PGF0-EC0.5-G1 (n = 363), 3) PGF1-EC0.5-G0 (n = 363), 4) PGF1-EC0.5-G1 (n = 360), 5) PGF0-EC-1.0-G0 (n = 360), 6) PGF0-EC1.0-G1 (n = 363), 7) PGF1-EC1.0-G0 (n = 361), and 8) PGF1-EC1.0-G1 (n = 363). Pregnancy per AI (P/AI) was greater at first AI compared with resynchronization (58.9 [n = 2012] vs. 54.9% [n = 885]). Presence of CL on Day −9 resulted in more cows expressing estrus (81.3 [n = 680] vs. 67.1% [n = 2033]) and greater P/AI (66.0 [n = 692] vs. 54.9% [n = 2106]). There was no difference in P/AI between cows that received or not PGF on Day -9 (58.7 [n = 1447] vs. 56.6% [n = 1450]). In contrast, PGF tended to increase P/AI of cows with CL on Day −9 (with PGF = 69.1 [n = 375] vs. without PGF = 62.5% [n = 317]). Cows that received 1.0 mg EC expressed more estrus than those treated with 0.5 mg (73.8 [n = 1414] vs. 67.9% [n = 1398]) and had greater P/AI (60.2 [n = 1447] vs. 55.1% [n = 1450]). P/AI was greater in cows treated with GnRH at TAI (59.8 [n = 1449] vs. 55.5% [n = 1448]), particularly in cows that did not show estrus (52.7 [n = 393] vs. 38.1% [n = 420]). Moreover, GnRH on Day 0 increased P/AI in cows with BCS < 3.0 (57.1 [n = 723] vs. 48.6% [n = 698]), in primiparous (50.1 [n = 465] vs. 41.9% [n = 497]) and in cows that received 0.5 mg EC (58.9 [n = 723] vs. 51.3% [n = 727]). In conclusion, 1.0 mg of EC on Day −2 and GnRH at TAI improved P/AI, but the combination of a higher dose of EC and GnRH treatment at AI did not enhance this effect. Furthermore, GnRH improved P/AI especially in Bos indicus cows with lower expression of estrus, such as primiparous, thinner cows, and cows treated with 0.5 mg of EC.

Protein kinase inhibitors infused intraventricularly or into the ventromedial hypothalamus block short latency facilitation of lordosis by oestradiol.

An injection of unesterified oestradiol (E2 ) facilitates receptive behaviour in E2 benzoate (EB)-primed, ovariectomised female rats when it is administered i.c.v. or systemically. The present study tested the hypothesis that inhibitors of protein kinase A (PKA), protein kinase G (PKG) or the Src/mitogen-activated protein kinase (MAPK) complex interfere with E2 facilitation of receptive behaviour. In Experiment 1, lordosis induced by i.c.v. infusion of E2 was significantly reduced by i.c.v. administration of Rp-cAMPS, a PKA inhibitor, KT5823, a PKG inhibitor, and PP2 and PD98059, Src and MAPK inhibitors, respectively, between 30 and 240minutes after infusion. In Experiment 2, we determined whether the ventromedial hypothalamus (VMH) is one of the neural sites at which those intracellular pathways participate in lordosis behaviour induced by E2 . Administration of each of the four protein kinase inhibitors into the VMH blocked facilitation of lordosis induced by infusion of E2 also into the VMH. These data support the hypothesis that activation of several protein kinase pathways is involved in the facilitation of lordosis by E2 in EB-primed rats.

Effect of Acupuncture Combined with Gavage of Rule Granule on Serum Prolactin, Estradiol and Progestone Contents and Their Receptor Expression in Breast in Mammary Gland Hyperplasia Rats

To observe the effect of acupuncture combined with medication (Rule Granule) on serum prolactin (PRL)，estradiol (E2) and progestone (P) contents and expressions of prolactin receptor (PRLR), estrogen receptor (ER) and progestrone receptor (PR) proteins in mammary gland (MG) tissues of MG hyperplasia (MGH) rats, so as to explore its mechanisms underlying improvement of MGH. A total of 55 female SD rats were randomized into blank control group (n＝10), model group (n＝10), acupuncture group (n＝11), medication group (n＝10), and acupuncture plus medication group (n＝9). The MGH model was established by muscular injection of E2 benzoate(0.5 mg/kg, once daily for 20 days), followed by injection of P (5 mg/kg) into the lateral muscle of the hind-limb, once daily for 5 days, and the rats of the control group were treated by muscular injection of normal saline at the same site and in the same procedures. Acupoint group A composed of bilateral Tianzhong (SI 11), Ganshu (BL 18) and Zusanli (ST 36), and group B composed of Tanzhong (CV 17), and bilateral Wuyi (ST 15) and Hegu (LI 4) were alternatively punctured with filiform needles and stimulated swiftly by twirling the needle in each acupoint for 20－30 sec, once daily for 30 days. Rats of the medication group and acupuncture ＋medication group were treated by gavage of Rule Granule fluid containing Baishao (Paeonia Iactiflora Pall), Danggui (Angelica sinensis), etc. (1.5 mL/100 g for each rat) and those of the other groups treated by gavage of distilled water (1.5 mL/100 g). The treatment was given once daily for continuous 30 d. At the end of the experiments, the rats' abdominal aorta blood was collected for assaying the contents of serum PRL, E2 and P with ELISA, and the pathological changes of breast tissue (the left 2nd pairs) were observed under microscope after sectioning and H．E. staining. The expression of PRLR, ER and PR proteins in the breast tissue was detected by immunohistochemistry. After modeling, the hyperplasia of mammary gland in the model group was obvious; the serum PRL and E2 contents and the immunoactivities of PRLR, ER and PR were significantly increased (P<0.01); and the content of serum P was considerably decreased in the model group in comparison with the blank control group (P<0.01). After the treatment, the hyperplasia of mammary gland was improved; the serum PRL and E2 contents and the expression of PRLR, ER and PR were significantly decreased (P<0.05, P<0.01); and the content of P was notably increased in the acupuncture, medication and acupuncture plus medication groups relevant to the model group (P<0.05，P<0.01). The therapeutic effects of acupuncture plus medication were significantly superior to those of both simple medication and simple acupuncture in down-regulating serum PRL and E2 contents and PRLR, ER and PR immunoactivity, as well as in up-regulating serum P content (P<0.05). No significant differences were found between the medication and acupuncture groups in the above mentioned 3 serum and 3 mammary indexes (P>0.05)．. Acupuncture, Rule Granule, and acupuncture combined with Rule Granule can improve hyperplasia of mammary gland in rats, which may be related to their effects in reducing serum PRL and E2 and breast ER, PR and PRLR expression levels, and in increasing serum P level. The therapeutic effects of acupuncture plus Rule Granule are better than those of simple acupuncture and simple Rule Granule.

Lordosis facilitated by GPER-1 receptor activation involves GnRH-1, progestin and estrogen receptors in estrogen-primed rats

The present study assessed the participation of membrane G-protein coupled estrogen receptor 1 (GPER-1) and gonadotropin releasing hormone 1 (GnRH-1) receptor in the display of lordosis induced by intracerebroventricular (icv) administration of G1, a GPER-1 agonist, and by unesterified 17β-estradiol (free E2). In addition, we assessed the participation of both estrogen and progestin receptors in the lordosis behavior induced by G1 in ovariectomized (OVX), E2-benzoate (EB)-primed rats. In Experiment 1, icv injection of G1 induced lordosis behavior at 120 and 240min. In Experiment 2, icv injection of the GPER-1 antagonist G15 significantly reduced lordosis behavior induced by either G1 or free E2. In addition, Antide, a GnRH-1 receptor antagonist, significantly depressed G1 facilitation of lordosis behavior in OVX, EB-primed rats. Similarly, icv injection of Antide blocked the stimulatory effect of E2 on lordosis behavior. In Experiment 3, systemic injection of either tamoxifen or RU486 significantly reduced lordosis behavior induced by icv administration of G1 in OVX, EB-primed rats. The results suggest that GnRH release activates both estrogen and progestin receptors and that this activation is important in the chain of events leading to the display of lordosis behavior in response to activation of GPER-1 in estrogen-primed rats.

Obligatory role of hypothalamic neuroestradiol during the estrogen-induced LH surge in female ovariectomized rhesus monkeys

Negative and positive feedback effects of ovarian 17β-estradiol (E2) regulating release of gonadotropin releasing hormone (GnRH) and luteinizing hormone (LH) are pivotal events in female reproductive function. While ovarian feedback on hypothalamo-pituitary function is a well-established concept, the present study shows that neuroestradiol, locally synthesized in the hypothalamus, is a part of estrogen's positive feedback loop. In experiment 1, E2 benzoate-induced LH surges in ovariectomized female monkeys were severely attenuated by systemic administration of the aromatase inhibitor, letrozole. Aromatase is the enzyme responsible for synthesis of E2 from androgens. In experiment 2, using microdialysis, GnRH and kisspeptin surges induced by E2 benzoate were similarly attenuated by infusion of letrozole into the median eminence of the hypothalamus. Therefore, neuroestradiol is an integral part of the hypothalamic engagement in response to elevated circulating E2 Collectively, we will need to modify the concept of estrogen's positive feedback mechanism.

Effects of Neonatal Exposure to Zearalenone on Puberty Timing, Hypothalamic Nuclei of AVPV and ARC, and Reproductive Functions in Female Mice.

It is now established that mycoestrogen zearalenone (ZEN) disrupts reproductive physiology, but the specific mechanisms by which this occurs remain unknown, especially in brain. Growing evidence suggests that populations of estradiol (E2)-sensitive neurons in anteroventral periventricular (AVPV) and arcuate (ARC) nuclei, especially kisspeptin neurons, play a pivotal role in the timing of puberty onset, ovulation, and normal reproduction. The present study was conducted to find whether the ZEN can cause estrogen-like actions during the critical period of neonatal differentiation. In this study, we compared the effect of neonatal exposure to sesame oil, E2 benzoate (EB, 20 µg/kg body weight [bw]), and 3 various doses: 0.2, 1, and 2 mg/kg bw of ZEN (0.2, 1, and 2 ZEN) on the onset of puberty and estrus cyclicity as well as ovarian follicular profile, kisspeptin expression, and neuronal density in AVPV and ARC hypothalamic nuclei and E2 and luteinizing hormone (LH) levels on postnatal day 70. Control mice received no treatment. Vaginal opening was significantly advanced by EB and 2 ZEN. Disrupted estrus cycles and decreased follicular profiles were observed in EB, 1 ZEN, and 2 ZEN animals. In addition, EB, 1 ZEN, and 2 ZEN reduced the expression of kisspeptin and neuronal density of AVPV and ARC nuclei and caused a decrease in the LH and an increase in E2 plasma levels. Taken together, our observations provide physiological evidence that neonatal exposure to ZEN exerts estrogen-like actions in the estrogen-sensitive hypothalamic AVPV and ARC nuclei, controlling reproductive functions in adult female mice.

Production of antibodies and development of an enzyme-linked immunosorbent assay for 17β-estradiol in milk

ABSTRACTThe residue of 17β-estradiol (E2) in milk could potentially lead to the occurrence of various reproductive diseases; therefore, a rapid and sensitive method for monitoring E2 residues in milk was highly necessary. In this study, we produced new polyclonal and monoclonal antibodies using E2-3-O-carboxymethyl ether as a hapten and developed an indirect competitive enzyme-linked immunosorbent assay (icELISA) for the detection of E2 in milk. The results showed that the sensitivity of polyclonal antibody was higher than that of monoclonal antibody, providing a half maximum inhibition concentration (IC50) against E2 of 0.17 ng/mL, high cross-reactivity (CR) to E2 benzoate (150%) and oestriol (18.02%), and negligible CR with other oestrogen compounds. Under optimized conditions, the developed icELISA based on the polyclonal antibody had a limit of detection values of 0.093 μg/L, which was enough sensitive to detect E2 in milk. In spiked samples (0.5, 1, and 2 μg/L), the recoveries ranged from 83.12% to 94.58% with coefficients of variation <12.8%. These results indicated that the icELISA method we developed was suitable for screening of E2 residue in milk.

Estrogen receptor α and β are involved in the activation of lordosis behavior in estradiol-primed rats

The present study was designed to assess the participation of estrogen receptors alpha (ERα) and beta (ERβ) in the short-term facilitation of lordosis behavior in ovariectomized (ovx), estradiol (E2) primed rats. In experiment 1, dose response curves for PPT and DPN (ERα and ERβ agonists, respectively) facilitation of lordosis behavior (lordosis quotient and lordosis score) were established by infusing these agonists into the right lateral ventricle (icv) in female rats injected 40h previously with 5μg of E2 benzoate. PPT doses of 0.08 and 0.4ng produced high lordosis quotients starting at 30min and continuing at 120 and 240min post-injection. DPN induced high levels of lordosis behavior at all times tested. However, the intensity of lordosis induced by both agonists was weak. In experiment 2, we tested the involvement of each ER in facilitation of lordosis by icv infusion of MPP (ERα-selective antagonist) or PHTPP (ERβ-selective antagonist) prior to infusion of 2ng of free E2. Icv infusion of either MPP or PHTPP 30min before free E2 significantly depressed E2 facilitation of lordosis. The results suggest that both forms of ER are involved in the short-latency facilitation of lordosis behavior in E2-primed rats.

Prolonged infusion of estradiol benzoate into the stalk median eminence stimulates release of GnRH and kisspeptin in ovariectomized female rhesus macaques.

Our recent study indicates that a brief infusion (20 min) of estradiol (E2) benzoate (EB) into the stalk-median eminence (S-ME) stimulates GnRH release with a latency of approximately 10 minutes. In contrast to the effect induced by a brief infusion of EB, it has previously been shown that systemic EB administration suppresses release of GnRH, kisspeptin, and LH with a latency of several hours, which is known as the negative feedback action of E2. We speculated that the differential results by these 2 modes of EB administration are due to the length of E2 exposure. Therefore, in the present study, the effects of EB infusion for periods of 20 minutes, 4 hours, or 7 hours into the S-ME of ovariectomized female monkeys on the release of GnRH and kisspeptin were examined using a microdialysis method. To assess the effects of the EB infusion on LH release, serum samples were also collected. The results show that similar to the results with 20-minute infusion, both 4- and 7-hour infusions of EB consistently stimulated release of GnRH and kisspeptin from the S-ME accompanied by LH release in the general circulation. In contrast, sc injection of EB suppressed all 3 hormones (GnRH, kisspeptin, and LH) measured. It is concluded that regardless of the exposure period, direct E2 action on GnRH and kisspeptin neurons in the S-ME, where their neuroterminals are present, is stimulatory, and the E2-negative feedback effects do not occur at the S-ME level.

Neuroestradiol in the Hypothalamus Contributes to the Regulation of Gonadotropin Releasing Hormone Release

Release of gonadotropin releasing hormone (GnRH) from the medial basal hypothalamus (MBH)/median eminence region (S-ME) is essential for normal reproductive function. GnRH release is profoundly regulated by the negative and positive feedback effects of ovarian estradiol (E2). Here we report that neuroestradiol, released in the S-ME, also directly influences GnRH release in ovariectomized female monkeys, in which the ovarian source of E2 is removed. We found that (1) brief infusion of E2 benzoate (EB) to the S-ME rapidly stimulated release of GnRH and E2 in the S-ME of ovariectomized monkeys, (2) electrical stimulation of the MBH resulted in GnRH release as well as E2 release, and (3) direct infusion of an aromatase inhibitor to the S-ME suppressed spontaneous GnRH release as well as the EB-induced release of GnRH and E2. These findings reveal the importance of neuroestradiol as a neurotransmitter in regulation of GnRH release. How circulating ovarian E2 interacts with hypothalamic neuroestrogens in the control of GnRH release remains to be investigated.

Distribution and Estrogen Regulation of Membrane Progesterone Receptor-β in the Female Rat Brain

Although several studies have reported the localization of membrane progesterone (P(4)) receptors (mPR) in various tissues, few have attempted to describe the distribution and regulation of these receptors in the brain. In the present study, we investigated expression of two mPR subtypes, mPRα and mPRβ, within regions of the brain, known to express estradiol (E(2))-dependent [preoptic area (POA) and hypothalamus] and independent (cortex) classical progestin receptors. Saturation binding and Scatchard analyses on plasma membranes prepared from rat cortex, hypothalamus, and POA demonstrated high-affinity, specific P(4)-binding sites characteristic of mPR. Using quantitative RT-PCR, we found that mPRβ mRNA was expressed at higher levels than mPRα, indicating that mPRβ may be the primary mPR subtype in the rat brain. We also mapped the distribution of mPRβ protein using immunohistochemistry. The mPRβ-immunoreactive neurons were highly expressed in select nuclei of the hypothalamus (paraventricular nucleus, ventromedial hypothalamus, and arcuate nucleus), forebrain (medial septum and horizontal diagonal band), and midbrain (oculomotor and red nuclei) and throughout many areas of the cortex and thalamus. Treatment of ovariectomized female rats with E(2) benzoate increased mPRβ immunoreactivity within the medial septum but not the medial POA, horizontal diagonal band, or oculomotor nucleus. Together, these findings demonstrate a wide distribution of mPRβ in the rodent brain that may contribute to functions affecting behavioral, endocrine, motor, and sensory systems. Furthermore, E(2) regulation of mPRβ indicates a mechanism through which estrogens can regulate P(4) function within discrete brain regions to potentially impact behavior.

Anomalously rapid effect of the estrogen receptor- α agonist PPT on food intake in ovariectomized rats.

Studies in knockout mice indicate that the feeding-inhibitory effect of estradiol (E2) requires the estrogen receptor alpha (ER-α). Consistent with that, Roesch [(2006) Physiological Behavior, 87, 39] reported that daily injections of the ER-α agonist PPT, but not the ER-α agonist DPN, reduces feeding in ovariectomized rats. However, whether PPT mimics the physiological effect of E2 on food intake is unknown. We therefore studied the effects of PPT, DPN and E2 on food intake in ovariectomized rats. Two microgram E2 benzoate was injected sc at mid-light once each 4th day, a near physiological regimen that decreases food intake and produces the cornified pattern of vaginal cytology typical of estrus 30–42 h later (i.e., during the second nocturnal phase after injection). Similarly timed injections of PPT (250 μg/rat) decreased food intake both at 6–18 and 30–42 h, i.e. during the first and second dark phase after injection. However, PPT produced vaginal cornification only at 30–42 h, i.e. at the same time as E2. DPN (100 μg/rat) did not change food intake at any time. The faster onset of inhibition of feeding after PPT in comparison to E2 indicates that PPT does not mimic the physiological effect of E2 on feeding and suggests that PPT's action on feeding may not depend on activation of nuclear ER-α. agonist PPT, but not the ER-α agonist DPN, reduces feeding in ovariectomized rats. However, whether PPT mimics the physiological effect of E2 on food intake is unknown. We therefore studied the effects of PPT, DPN and E2 on food intake in ovariectomized rats. Two microgram E2 benzoate was injected sc at mid-light once each 4th day, a near physiological regimen that decreases food intake and produces the cornified pattern of vaginal cytology typical of estrus 30-42 h later (i.e., during the second nocturnal phase after injection). Similarly timed injections of PPT (250 μg/rat) decreased food intake both at 6–18 and 30–42 h, i.e. during the first and second dark phase after injection. However, PPT produced vaginal cornification only at 30–42 h, i.e. at the same time as E2. DPN (100 μg/rat) did not change food intake at any time. The faster onset of inhibition of feeding after PPT in comparison to E2 indicates that PPT does not mimic the physiological effect of E2 on feeding and suggests that PPT's action on feeding may not depend on activation of nuclear ER-=.

Effects of Long-Term Treatment with Resveratrol and Subcutaneous and Oral Estradiol Administration on the Pituitary-Thyroid-Axis

The lack of estrogen during menopause is associated with various symptoms including osteoporosis, cardiovascular diseases, and menopausal symptoms. For many years, conventional hormone replacement therapy has been successfully used to treat these conditions. However, in light of recent studies that draw attention to potential hazards of conventional HRT, various attempts were undertaken to search for alternatives of classical HRT. Phytoestrogens are supposed to ameliorate various discomforts associated with menopause. Resveratrol (RES) is present in red wine, grapes and peanuts and has been implicated in cardioprotection and prevention of adverse side effects observed after regular HRT. As the pituitary-thyroid axis is a target of estrogen action, we first assessed the effects of E2 administration on thyroid hormone stimulating hormone releasing hormone (TRH)-induced thyroid stimulating hormone (TSH) secretion from pituitary cell cultures in vitro. Our data reveal that E2 treatment augments the TRH-induced TSH secretion. We furthermore designed a long-term study of three months to assess the effects of subcutaneous and oral administration of 17beta-estradiol (E2), as well as the actions of RES on the pituitary-thyroid axis in ovariectomized (OVX) female rats. Our results demonstrate that serum levels of 1.0 and 8.1 microM RES lead to a significant increase in total serum triiodthyronine (T3) levels. OVX induces TSHbeta mRNA in the adenohypohysis and E2 treatment attenuates this effect. Treatment of rats with subcutaneous implants of E2 does not affect the pituitary-thyroid axis, whereas orally applied E2 benzoate (E2B) increases plasma TSH and total thyroxine (T4) in OVX rats. In all animals, we could not detect changes in thyroid morphology as assessed by hematoxylin-eosin (HE) and Perjod-Acid Schiff's (PAS) staining.

Sexually Dimorphic and Estrogen-Dependent Expression of Estrogen Receptor β in the Ventromedial Hypothalamus during Rat Postnatal Development

The ventromedial hypothalamus (VMH) is a sexually dimorphic region of the brain related to female reproductive behavior. The effect of estrogen in the adult rat VMH is thought to be mediated predominantly via estrogen receptor (ER)alpha, because this receptor is expressed at considerably higher levels than ER beta. The present study revealed, using in situ hybridization and immunohistochemistry, that both ER beta mRNA and protein were expressed in the ventrolateral portion of the caudal VMH, at remarkably higher levels during early postnatal development than in adulthood. In addition, the expression was sexually dimorphic, with females having significantly more ER beta-immunoreactive (-ir) cells than males, between postnatal d 5 (P5) and P14, although the sex difference was not significant by P21. Double-label immunofluorescence revealed that 66% of ER beta-ir cells coexpressed ER alpha in the caudal VMH of the P5 female rat. Furthermore, neonatal treatment with E2 benzoate down-regulated ER beta mRNA in the female rat VMH at P5 and decreased VMH ER beta-ir cells during the period between P5 and P14. In contrast to females, no differences in expression of ER beta mRNA or protein were detected between control and E2 benzoate-treated males. These results suggest that estrogen is involved in regulating the sexually dimorphic expression of ER beta in the VMH during early postnatal development of the rat.

Immunocytochemical Detection of Estrogen Receptor-α in the Female Rabbit Forebrain: Topography and Regulation by Estradiol

Two antibodies (H222 and Zymed) directed towards different sites of the estrogen receptor-α (ERα) were used for the following objectives: (1) to map the ERα in the forebrain of ovariectomized (ovx) rabbits by immunocytochemistry and (2) to determine the effect of endogenous (intact non-pregnant animals) and exogenous (ovx, estrogen-treated animals) estradiol (E2) on the population of ERα in the forebrain. Similar results were obtained with both antibodies used: dense aggregations of ERα-immunoreactive (IR) neurons were found in the infundibular nucleus (IN), the medial preoptic area (POA), the bed nucleus of the stria terminalis (BNST), and some nuclei of the amygdala. By contrast, no ERα-IR neurons were present in the ventromedial hypothalamic nucleus (VMN), but a dense aggregation of ERα-IR neurons occurred lateral to it in nucleus X. Numerous ERα-IR neurons were observed in the paraventricular hypothalamic nucleus, but not in the supraoptic or suprachiasmatic nuclei. The hippocampus proper lacked ERα-IR neurons, but the ventral subiculum in the hippocampal formation had a dense group of such cells. Intact non-pregnant rabbits showed less ERα-IR neurons in all regions tested than ovx animals. This difference was particularly clear in the medial POA, amygdala and BNST, while the IN showed only a marginal decrease. The dorsal, but much less the ventral, part of nucleus X also showed a decrease in the number of ERα-IR neurons compared with ovx animals. E2 benzoate (5 µg/day for 5 days) reduced even further the number of ERα-IR neurons in all regions except in a circumscribed area of the IN and the ventral part of nucleus X. These results show the existence of both sensitive and insensitive neurons to the down-regulatory effect of E2 on the presence of ERα. Sensitive neurons are located in the telencephalon, POA and several hypothalamic nuclei (PVN), while insensitive neurons are mainly restricted to the IN and the ventral part of nucleus X in the basal hypothalamus.

Increased Expression of Mullerian-Inhibiting Substance Correlates with Inhibition of Follicular Growth in the Developing Ovary of Rats Treated with E2 Benzoate

Müllerian-inhibiting substance (MIS) is an essential factor for male sexual differentiation. In the present study, we examined whether the expression of MIS and several of its related transcription factors is altered in the ovaries of rats treated with the synthetic estrogen, E2 benzoate (EB; 10 μg/0.02 ml), from postnatal day 1 (P1) to P5. The EB-treated rats had a significantly reduced number of layered follicles at P6 in comparison with the control rats that were treated with vehicle alone. The expression levels of both MIS mRNA and protein in the granulosa cells of small growing follicles in the ovary at P6 were higher in the EB-treated rats than in the controls. These results indicate that the inhibitory effect of EB on the follicular stratification may correlate with the inappropriately increased expression level of MIS. Furthermore, the expression levels of one of its transcriptional activators, steroidogenic factor 1, and ER-β in granulosa cells of small growing follicles were higher in EB-treated ovaries than in the control ovaries. These results suggest the role of MIS in the regulation of follicular growth and the possible involvement of steroidogenic factor 1and/or ER-β in this molecular cascade may contribute to postnatal ovarian development.

Increased Expression of Müllerian-Inhibiting Substance Correlates with Inhibition of Follicular Growth in the Developing Ovary of Rats Treated with E2 Benzoate

Müllerian-inhibiting substance (MIS) is an essential factor for male sexual differentiation. In the present study, we examined whether the expression of MIS and several of its related transcription factors is altered in the ovaries of rats treated with the synthetic estrogen, E2 benzoate (EB; 10 microg/0.02 ml), from postnatal day 1 (P1) to P5. The EB-treated rats had a significantly reduced number of layered follicles at P6 in comparison with the control rats that were treated with vehicle alone. The expression levels of both MIS mRNA and protein in the granulosa cells of small growing follicles in the ovary at P6 were higher in the EB-treated rats than in the controls. These results indicate that the inhibitory effect of EB on the follicular stratification may correlate with the inappropriately increased expression level of MIS. Furthermore, the expression levels of one of its transcriptional activators, steroidogenic factor 1, and ER-beta in granulosa cells of small growing follicles were higher in EB-treated ovaries than in the control ovaries. These results suggest the role of MIS in the regulation of follicular growth and the possible involvement of steroidogenic factor 1 and/or ER-beta in this molecular cascade may contribute to postnatal ovarian development.