The protein of the ejaculatory bulb (PEB) in Drosophila melanogaster. 1. Its stage specificity and sex dimorphism

PEB is the major protein (35-39 kDa) of highly differentiated ejaculatory bulbs in D. melanogaster. A minor ejaculatory bulb protein (hPEB) of about 80 kDa was detected using immunoblotting technique. Both proteins exhibit parallel genetic variation in electrophoretic mobility. This suggests that they are coded by the same gene. The proteins are present in adult males and are not detected in virgin females. During development they are first detected in male pupa at the stage of eye pigmentation (that is shortly before imago eclosion). The quantities of PEB and hPEB increase and reach the constant level at 6-10 day of imago development.     

Genetic control and expression of the major ejaculatory bulb protein (PEB-me) inDrosophila melanogaster

PEB-me is a predominant protein of matureDrosophila melanogaster ejaculatory bulbs. It is resolved into four or five closely spaced subfractions (apparent molecular weight 35–39 kD) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Four electrophoretic variants of PEB-me differing in apparent molecular weight by 200–800 daltons were found. These appear to be controlled by four alleles of a gene (peb) located by recombination and deletion mapping to the 60F1-2 region of chromosome 2. A minor ejaculatory bulb protein of ca. 80 kD (hPEB) was found to be immunochemically related to PEB and possibly encoded bypeb. PEB is not detected by immunoblotting techniques in virgin females, in male tissues other than the ejaculatory bulb, or during developmental stages preceding the formation of this organ. The results of transplantations of genital imaginal discs and of immature ejaculatory bulbs between two strains having different PEB alleles suggest that the ejaculatory bulb is the site of PEB synthesis. In flies mutant fortra, tra-2, dsx, orix, tissue specificity of PEB localization is retained and the protein is found whenever the ejaculatory bulb is formed, regardless of the chromosomal sex of the fly. The protein is transferred into the female genital duct during mating, where it can be detected for up to 12 hr. Possible functions of PEB inDrosophila reproduction are discussed.     

The reversible inhibition of steroid-induced sexual behavior by intracranial cycloheximide

Cycloheximide(Cyclo), an inhibitor of protein synthesis by a direct action on protein synthesis at the ribosomal level, was used to reversibly inhibit estrogen-induced sexual receptivity. Cyclo (100 μg per rat) was infused into the preoptic area(POA) of ovariectomized rats at varying times before, simultaneously with, and after 3 μg of subcutaneous estradiol benzoate (EB). All animals received 0.5 mg progesterone (P) 36 hr after EB, and were tested for sexual receptivity 4–6 hr after P. The females were placed with stud males and a lordosis quotient was computed for each female (lordosis quotient = number of lordosis responses/20 mounts by the male × 100). Females receiving Cyclo 6 hr before, simultaneously with, or 12 hr after EB showed significantly lower levels of sexual receptivity when compared to females receiving Cyclo 36 hr before and 18 and 24 hr after EB. When those animals that showed low levels of sexual behavior after Cyclo infusion were reprimed with EB and P 7 days later and presented with a male they showed high levels of sexual receptivity. Thus, the effect of Cyclo was reversible. Only Cyclo infusions into the POA (bilateral) and third ventricle were effective in suppressing sexual behavior. Caudate nucleus, lateral ventricle, and unilateral POA infusions were without effect.The data presented are in agreement with earlier work that utilized actinomycin D to inhibit steroid-induced sexual behavior. Cyclo was found to be less toxic than actinomycin D. All of the available evidence is consistent with the hypothesis that estrogen stimulates RNA and/or protein synthesis in its facilitation of sexual behavior in the female rat.     

The regulation of precopulatory behavior by ovarian hormones in the female Mongolian gerbil

The hormonal regulation of precopulatory behavior in the female Mongolian gerbil was studied using two groups (N = 6) of sexually experienced females. A novel testing procedure was used which involved females living continuously with test males for several days. The test males showed either full sexual behavior (copulating males, C) or only precopulatory behavior (noncopulating males, NC). Experiment 1 investigated changes during the estrous cycle and following ovariectomy in females. Experiment 2 studied the effects of hormonal treatment of these ovariectomized females with 6 micrograms estradiol benzoate (EB) followed by 0.4 mg progesterone (P) or by 0.04 ml arachis oil. When tested with NC males, females displayed a greater range of precopulatory behavior. The patterns could be classified into three groups according to the manner of response to ovariectomy and hormone treatment. Group I patterns (approach, leave, and olfactory investigation of the male's head) were affected by neither ovariectomy nor EB treatment relative to Day 3 levels (Day 3, day preceding estrus; Day 4, estrus), but they were increased to estrous levels by EB and P. Group II patterns (darting, foot-stomping, and the present and piloerection postures) appeared only during estrus, did not appear after ovariectomy, and reappeared only after sequential EB and P treatment. Group III patterns (investigation of the male's anogenital area, allogrooming, ventral gland marking, and sand-rolling) were reduced relative to both estrus and Day 3 levels by ovariectomy and increased above Day 3 levels by EB alone; EB and P treatment further increased Group III patterns to the level of estrus. It is suggested that female precopulatory behavior patterns differ in their responsiveness to ovarian hormones. Estrogen appears to affect those patterns associated with the earliest stages of estrus (Group III).     

The proteins of the ejaculatory bulbs in different species of Drosophila

A comparative electrophoretic study or ejaculatory bulb proteins in 29 different Drosophila species has been carried out. In all analyzed species, ejaculatory bulb contains a major component (designated as PEB). It has molecular mass of 61-65 kDa in the species of virilis group, 33-36 kDa in species of obscura group, and 34-56 kDa in species of melanogaster group. Using immunoblotting technique, we have demonstrated that PEB is introduced into organs of female sex tract during mating. The nature and significance of revealed interspecific differences in PEB proteins has been discussed.     

Phycobiliprotein-bilin linkage diversity. II. Structural studies on A- and D-ring-linked phycoerythrobilins

The discovery that the phycocyanobilin group attached to Cys-155 of the beta subunit of C-phycocyanin is D-ring linked (Bishop, J. E., Lagarias, J. C., Nagy, J. O., Schoenleber, R. W., Rapoport, H., Klotz, A. V., and Glazer, A. N. (1986) J. Biol. Chem. 261, 6790-6796) prompted examination of the linkage mode for phycoerythrobilin (PEB) groups attached at the corresponding position in other biliproteins. Appropriate small peptides were obtained by exhaustive enzymatic digestion of Porphyridium cruentum R-phycocyanin (peptide R-PC beta-2TP PEB) and B-phycoerythrin (peptide B-PE beta-2TP PEB). These peptides had the following structures R-PC beta-2TP PEB Gly-Asp-Cys(PEB)-Ser-Ser B-PE beta-2TP PEB Cys(PEB)-Thr-Ser. The spectroscopic and chemical properties of these peptides were compared with those of P. cruentum B-phycoerythrin peptide alpha-1 PEB, Cys(PEB)-Tyr-Arg, in which the bilin is A-ring linked (Schoenleber, R. W., Leung, S.-L., Lundell, D. J., Glazer, A. N., and Rapoport, H. (1983) J. Am. Chem. Soc. 105, 4072-4076). The PEB groups in peptides R-PC beta-2TP PEB and B-PE beta-2TP PEB were shown to be D-ring linked on the basis of the following criteria. Secondary ion mass spectrometry showed the bilins in these peptides and in alpha-1 PEB to have the same mass. The 18'-CH3, 18'-H, and 15-H resonances in the 1H NMR spectra of R-PC beta-2TP PEB and B-PE beta-2TP PEB appear significantly upfield from the corresponding thioether-linked ring A resonances seen in the spectrum of peptide alpha-1 PEB. The CD spectra of the two former peptides showed a strong positive Cotton effect at 300 nm. Such a Cotton effect is absent from the CD spectrum of peptide alpha-1 PEB and those of other A-ring-linked PEB peptides. Refluxing in methanol led to a near-quantitative release of PEB from alpha-1 PEB but no release from R-PC beta-2TP PEB and less than 20% release from B-PE beta-2TP PEB. In conjunction with earlier studies, these results show that distinctive amino acid sequences are found about the attachment sites for A-ring-linked, D-ring-linked, and dilinked (A- and D-ring-linked) bilins on the alpha and beta subunits of cyanobacterial and red algal phycobiliproteins and that the mode of linkage can be correctly predicted from inspection of the amino acid sequence.     

评价空肠弯曲菌外膜蛋白PEB1介导的重组BCG与上皮细胞的结合能力

目的：评价PEBl介导的屋尘螨抗原（Derp2）重纽BCG疫苗（PEBI-Derp2．rBCG）与人上皮细胞的结合能力。方法：采用体外细胞培养的方法,分别将普通BCG、胞壁型Derp2-rBCG和胞壁型融合蛋白PEBl-Derp2-rBCG与HeLa细胞及人类肠粘膜上皮细胞（HIEC）进行共孵育,利用HE和抗酸染色法对各组细胞与疫苗的黏附结果进行染色,光学显微镜下计数各组的黏附率,并进行比较;对以上各组分别加入PEBl蛋白,进行黏附阻断,观察对结合能力的影响。结果：孵育24小时后,无论HeLa细胞还是H匝CPEBl-Derp2．rBCG组较普通BCG组和Derp2-rBCG组的黏附率明显提高,差异有显著性（P〈0．05）;PEBl蛋白的加入对PEBl．Derp2-rBCG的黏附功能有明显抑制作用（P〈O．05）;但是,Derp2-rBCG组与普通BCG组比较没有明显差异（P〉o．05）,PEBl蛋白的加入对二者的黏附亦无影响（P〉0．05）。结论：PEBl具有介导增强PEBl-Derp2-rBCG与上皮细胞黏附的能力。     

评价空肠弯曲菌外膜蛋白PEB1 介导的重组BCG 与上皮细胞 的结合能力

目的:评价PEB1介导的屋尘螨抗原(Derp2)重组BCG疫苗(PEB1-Derp2-rBCG)与人上皮细胞的结合能力。方法:采用体外细胞培养的方法,分别将普通BCG、胞壁型Derp2-rBCG和胞壁型融合蛋白PEB1-Derp2-rBCG与HeLa细胞及人类肠粘膜上皮细胞(HIEC)进行共孵育,利用HE和抗酸染色法对各组细胞与疫苗的黏附结果进行染色,光学显微镜下计数各组的黏附率,并进行比较;对以上各组分别加入PEB1蛋白,进行黏附阻断,观察对结合能力的影响。结果:孵育24小时后,无论HeLa细胞还是HIECPEB1-Derp2-rBCG组较普通BCG组和Derp2-rBCG组的黏附率明显提高,差异有显著性(P<0.05);PEB1蛋白的加入对PEB1-Derp2-rBCG的黏附功能有明显抑制作用(P<0.05);但是,Derp2-rBCG组与普通BCG组比较没有明显差异(P>0.05),PEB1蛋白的加入对二者的黏附亦无影响(P>0.05)。结论:PEB1具有介导增强PEB1-Derp2-rBCG与上皮细胞黏附的能力。     

Radical mechanism of cyanophage phycoerythrobilin synthase (PebS)

PEB (phycoerythrobilin) is a pink-coloured open-chain tetrapyrrole molecule found in the cyanobacterial light-harvesting phycobilisome. Within the phycobilisome, PEB is covalently bound via thioether bonds to conserved cysteine residues of the phycobiliprotein subunits. In cyanobacteria, biosynthesis of PEB proceeds via two subsequent two-electron reductions catalysed by the FDBRs (ferredoxin-dependent bilin reductases) PebA and PebB starting from the open-chain tetrapyrrole biliverdin IXα. A new member of the FDBR family has been identified in the genome of a marine cyanophage. In contrast with the cyanobacterial enzymes, PebS (PEB synthase) from cyanophages combines both two-electron reductions for PEB synthesis. In the present study we show that PebS acts via a substrate radical mechanism and that two conserved aspartate residues at position 105 and 206 are critical for stereospecific substrate protonation and conversion. On the basis of the crystal structures of both PebS mutants and presented biochemical and biophysical data, a mechanism for biliverdin IXα conversion to PEB is postulated and discussed with respect to other FDBR family members.     

The virulence factor PEB4 (Cj0596) and the periplasmic protein Cj1289 are two structurally related SurA-like chaperones in the human pathogen Campylobacter jejuni

The PEB4 protein is an antigenic virulence factor implicated in host cell adhesion, invasion, and colonization in the food-borne pathogen Campylobacter jejuni. peb4 mutants have defects in outer membrane protein assembly and PEB4 is thought to act as a periplasmic chaperone. The crystallographic structure of PEB4 at 2.2-? resolution reveals a dimer with distinct SurA-like chaperone and peptidyl-prolyl cis/trans isomerase (PPIase) domains encasing a large central cavity. Unlike SurA, the chaperone domain is formed by interlocking helices from each monomer, creating a domain-swapped architecture. PEB4 stimulated the rate of proline isomerization limited refolding of denatured RNase T(1) in a juglone-sensitive manner, consistent with parvulin-like PPIase domains. Refolding and aggregation of denatured rhodanese was significantly retarded in the presence of PEB4 or of an engineered variant specifically lacking the PPIase domain, suggesting the chaperone domain possesses a holdase activity. Using bioinformatics approaches, we identified two other SurA-like proteins (Cj1289 and Cj0694) in C. jejuni. The 2.3-? structure of Cj1289 does not have the domain-swapped architecture of PEB4 and thus more resembles SurA. Purified Cj1289 also enhanced RNase T(1) refolding, although poorly compared with PEB4, but did not retard the refolding of denatured rhodanese. Structurally, Cj1289 is the most similar protein to SurA in C. jejuni, whereas PEB4 has most structural similarity to the Par27 protein of Bordetella pertussis. Our analysis predicts that Cj0694 is equivalent to the membrane-anchored chaperone PpiD. These results provide the first structural insights into the periplasmic assembly of outer membrane proteins in C. jejuni.     

Characterization of the activities of the CpeY, CpeZ, and CpeS bilin lyases in phycoerythrin biosynthesis in Fremyella diplosiphon strain UTEX 481

When grown in green light, Fremyella diplosiphon strain UTEX 481 produces the red-colored protein phycoerythrin (PE) to maximize photosynthetic light harvesting. PE is composed of two subunits, CpeA and CpeB, which carry two and three phycoerythrobilin (PEB) chromophores, respectively, that are attached to specific Cys residues via thioether linkages. Specific bilin lyases are hypothesized to catalyze each PEB ligation. Using a heterologous, coexpression system in Escherichia coli, the PEB ligation activities of putative lyase subunits CpeY, CpeZ, and CpeS were tested on the CpeA and CpeB subunits from F. diplosiphon. Purified His(6)-tagged CpeA, obtained by coexpressing cpeA, cpeYZ, and the genes for PEB synthesis, had absorbance and fluorescence emission maxima at 566 and 574 nm, respectively. CpeY alone, but not CpeZ, could ligate PEB to CpeA, but the yield of CpeA-PEB was lower than achieved with CpeY and CpeZ together. Studies with site-specific variants of CpeA(C82S and C139S), together with mass spectrometric analysis of trypsin-digested CpeA-PEB, revealed that CpeY/CpeZ attached PEB at Cys(82) of CpeA. The CpeS bilin lyase ligated PEB at both Cys(82) and Cys(139) of CpeA but very inefficiently; the yield of PEB ligated at Cys(82) was much lower than observed with CpeY or CpeY/CpeZ. However, CpeS efficiently attached PEB to Cys(80) of CpeB but neither CpeY, CpeZ, nor CpeY/CpeZ could ligate PEB to CpeB.     

Regulation of urine marking in male and female mice: effects of sex steroids

Effects of sex steroids on urine-marking activity were studied in male, female, and neonatally androgenized female mice. Urine marking was estimated by suspending ceramic tubes that were connected in a horizontal row with a steel rod into the home cage of an isolated mouse. Intact males showed high marking activity, which was diminished after castration. Both testosterone propionate (TP) and estradiol benzoate (EB) were effective in restoring the marking activity of castrated males, while 5-alpha-dihydrotesterone (DHT) did not have any stimulative effects. Intact normal females showed quite low marking activity and ovariectomy further depressed it. TP and DHT enhanced the marking of ovariectomized females, but EB restored the activity only to the preovariectomy level. In intact females which were neonatally androgenized, the marking activity was much higher than that of normal females. The pattern of the change induced by gonadectomy and hormone treatment in these females resembled that in males. Thus, ovariectomy reduced the activity and both TP and EB restored the level. These results indicate that the sexual dimorphism in the urine marking in mice is primarily determined by hormonal environment during early postnatal age. Hormonal control of scent marking is discussed in relation to the studies in other rodents.     

The effects of testosterone propionate and estradiol benzoate on the vitro synthesis of FSH.

The effects of estradiol benzoate (EB) and testosterone propionate (TP) on pituitary and plasma concentrations of follicle stimulating hormone (FSH) and the in vitro synthesis of FSH in pituitary tissue was studied in mature male rats. By the 4th day of treatment with EB, pituitary content and concentration of FSH had declined, and content had fallen to 6% and concentration to 3% of pretreatment values. Similar results occurred during in vitro synthesis. However, serum levels of FSH did not show any decline until the 21st and 28th days of treatment. Administration of TP produced a progressive increase in pituitary content and concentration of FSH, though serum levels remained unchanged for the 1st 7 days, after which they fell slightly. The effect of TP on the in vitro synthesis of FSH showed no consistent pattern, though in no case was a decrease in the uptake of labeled leucine into immunoprecipitable FSH observed. The results suggest that EB and TP have different effects on pituitary FSH in normal adult male rats.     

Mapping of the gene coding for the major protein of the ejaculatory bulb in Drosophila melanogaster

The major protein of ejaculatory bulb of Drosophila melanogaster males (PEB-me) is represented by a group of 4-5 sufbractions. Four PEB variants differing in SDS-PAGE mobility were found. The results of genetic analysis suggest that electrophoretic mobility of the entire complex of protein PEB bands is controlled by a single locus (peb). The locus is therefore supposed to contain the structural gene for the subfractions of PEB. Its genetic and cytological location is 2-107.2-2-108.0 and 60E11-60F5, respectively.     

The inhibition of steroid-induced sexual behavior by intrahypothalamic actinomycin-D

Lordosis behavior can be elicited in the ovariectomized rat after treatment with estradiol benzoate (EB) and progesterone (P) injections, but the EB must act for an extended period before P can facilitate this behavior. The possibility that this action of EB involves the stimulation of RNA or protein synthesis was tested by implanting actinomycin D (Act-D) directly into the preoptic area, one probable site of estrogen action. A total dose of 0.18 μg Act-D in bilateral cocoa butter pellets significantly inhibited lordosis behavior when implanted 12 hr after the injection of 3 μg. but not 15 μg EB. Implantation of this dose of Act-D subcutaneously, or intrahypothalamically 32 hr after EB injection, was without effect. Act-D placed in the ventromedial hypothalamus also suppressed lordosis, but implants in the caudate nucleus were without effect. At the time of the behavioral tests the animals were in excellent condition as determined by calculation of a health score, and no physical lesions were evident at the site of the implants. However, it was impossible to test the reversibility of this suppression of lordosis behavior since the animals became ill and many died within 1–2 weeks of implantation. The present results are consistent with, but not proof of, the concept that RNA synthesis may be essential for steroidinduced sexual behavior.     

Female-typical sexual behavior of rhesus and defeminization by androgens given prenatally

Adult rhesus monkeys were observed in standardized tests for female-typical sexual and related social responses. In the first experiment reported, 7 castrated males and 5 spayed females were paired with each of 4 intact males on two occasions following intramuscular injection with estradiol benzoate (EB) (6 micrograms/kg X 14 days) and on two other occasions without such treatment. In tests without EB, males and females did not behave differently toward the intact male partners, and all responses were displayed at low frequencies. In tests with EB, females showed reliably higher frequencies than males of approaching, sitting close to, grooming, and soliciting, and they presented to a higher proportion of the male partner's sexual contacts. EB reliably increased the frequency of display of all of these same five responses in females but not in castrated males. The intact male partners displayed reliably fewer approaches, sexual contacts, mounts, intromissions, and ejaculations to castrated males than to spayed females regardless of estrogenization. In a second experiment 10 intact adult pseudohermaphroditic females and 6 intact control females were tested following EB injections with each of the same 4 intact males. Pseudohermaphrodites were experimentally produced by injecting pregnant females with either testosterone propionate (TP) or dihydrotestosterone propionate (DHTP). Pseudohermaphrodites, regardless of type of androgen used in their production, showed reliably fewer solicits than controls to male partners. Moreover, they displayed most of the other responses at lower average frequencies than controls. Frequencies of intromission and ejaculation by intact male partners were reliably lower with pseudohermaphrodites than with control females, but frequencies of approach, sexual contact, and mount were not reliably different. We conclude that in this testing and measurement situation male and female rhesus monkeys differ markedly in the degree of expression of female-typical sexual behaviors, and genotypic males are behaviorally less responsive to estrogens than females. Exposing genotypic females to androgens during fetal life decreases the expression of female-typical, estrogen-influenced responses, and the effect is most pronounced on those soliciting responses that subserve proceptivity.     

Tamoxifen's effects on the zebra finch song system are estrogenic, not antiestrogenic.

Antiestrogens fail to block the masculine ontogeny of the zebra finch song system that is hypothesized to occur as a result of early estrogen action. Moreover, they hypermasculinize the male, and masculinize the female song systems. In experiment 1, we assessed whether these antiestrogenic effects might mimic estrogenic actions. Zebra finch chicks received one of two treatments. They were given estradiol benzoate (EB) or vehicle daily for the first 20 days after hatching and sacrificed at 60 days of age, or they received EB or vehicle for the first 25 days after hatching, at which time they were sacrificed. In the day 60 group, certain attributes of the song system were hypermasculinized in males and masculinized in females by EB, when compared with controls. In the day 25 group, males treated with EB were partially demasculinized, while the females were partially masculinized. In experiment 2, we assessed whether simultaneous treatment with tamoxifen was capable of antagonizing the effects of EB obtained in experiment 1 (day 60 group). Sixty-day-old females, previously treated with both EB and tamoxifen for the first 20 days after hatching, had more masculine song regions than females treated with either EB alone or tamoxifen alone. In males, the effects of the combined treatment of EB and tamoxifen over those produced by tamoxifen alone were not as dramatic as in the female. These results are similar to those obtained in systems where tamoxifen is purely estrogenic and suggest that in the song system, tamoxifen acts as an estrogen, not an antiestrogen.     

Effect of forebrain implants of testosterone or estradiol on scent-marking and sexual behavior in male and female rabbits

Chinning consists of rubbing the chin against an object, thereby depositing secretions from the submandibular glands. As mating, chinning is stimulated in male and female rabbits by testosterone and estradiol, respectively. To investigate the brain sites where steroids act to stimulate chinning and mating we implanted into the ventromedial hypothalamus (VMH) or the medial preoptic area (MPOA) of gonadectomized male and female rabbits testosterone propionate (TP; males) or estradiol benzoate (EB; females) and quantified chinning and sexual behavior. EB implants into the VMH or MPOA reliably stimulated chinning in females. Most of those implanted into the VMH and around half of the ones receiving EB into MPOA or diagonal band of Broca (DBB) showed lordosis. Chinning, but not sexual behavior, was stimulated in males by TP implants into the MPOA or DBB. Neither chinning nor mounting were reliably displayed by males following TP implants into the VMH. Results indicate that, in females, the VMH is an estrogen-sensitive brain area that stimulates both chinning and lordosis while the MPOA seems to contain subpopulations of neurons involved in either behavior. In males, androgen-sensitive neurons of the MPOA, but not the VMH, are involved in chinning stimulation but it is unclear if these areas also participate in the regulation of copulatory behavior.     

Effect of forebrain implants of testosterone or estradiol on scent-marking and sexual behavior in male and female rabbits

Chinning consists of rubbing the chin against an object, thereby depositing secretions from the submandibular glands. As mating, chinning is stimulated in male and female rabbits by testosterone and estradiol, respectively. To investigate the brain sites where steroids act to stimulate chinning and mating we implanted into the ventromedial hypothalamus (VMH) or the medial preoptic area (MPOA) of gonadectomized male and female rabbits testosterone propionate (TP; males) or estradiol benzoate (EB; females) and quantified chinning and sexual behavior. EB implants into the VMH or MPOA reliably stimulated chinning in females. Most of those implanted into the VMH and around half of the ones receiving EB into MPOA or diagonal band of Broca (DBB) showed lordosis. Chinning, but not sexual behavior, was stimulated in males by TP implants into the MPOA or DBB. Neither chinning nor mounting were reliably displayed by males following TP implants into the VMH. Results indicate that, in females, the VMH is an estrogen-sensitive brain area that stimulates both chinning and lordosis while the MPOA seems to contain subpopulations of neurons involved in either behavior. In males, androgen-sensitive neurons of the MPOA, but not the VMH, are involved in chinning stimulation but it is unclear if these areas also participate in the regulation of copulatory behavior.     

Behavioral demasculinization of female quail is induced by estrogens: studies with the new aromatase inhibitor, R76713.

The injection before Day 12 of incubation of estradiol benzoate (EB) into Japanese quail eggs produces a complete behavioral demasculinization of adult males that will hatch from these eggs. These males never show copulatory behavior even after administration of high levels of exogenous testosterone (T). It is usually assumed that such a demasculinization normally takes place in female embryos under the influence of endogenous estrogens but few experimental data are available to confirm the validity of this model. A series of four experiments was performed during which R76713, a triazole derivative that specifically inhibits aromatase (estrogen synthetase) activity, was injected into quail eggs at different stages of incubation to prevent the production of endogenous estrogens. The consequences of these embryonic treatments on the T-activated sexual behavior in adults were then quantified. When injected before Day 12 of incubation, R76713 completely blocked the behavioral demasculinization of females without affecting the behavior of the males. After a treatment with T, almost all R76713-treated females showed as adults a masculine copulatory behavior that was undistinguishable from the behavior of intact males. This effect was fully reversed by the injection in egg of EB demonstrating that the effects of R76713 were specifically due to the suppression of endogenous estrogens. Injection of R76713 during the late phase of the incubation (Day 12 or Day 15) only maintained weak copulatory behavior in females which confirmed that the behavioral demasculinization in quail takes place mainly though not exclusively during the early stages of ontogeny. In a last experiment, we combined an early R76713 treatment with an injection of EB either on Day 9 or on Day 14 of incubation. This showed that the sensitivity to differentiating effects of estrogens varies with age in a sexually differentiated manner. The EB injection on Day 9 demasculinized both male and female embryos. If this injection was delayed until Day 14, it was no longer effective in males but still caused a partial demasculinization of females. This demonstrates that even if females are not yet behaviorally demasculinized on Day 9 of incubation (suppression of aromatase activity at that age will maintain the behavior), their sensitivity to estrogens is already different from that of males.