Hormonal Induction Research Articles

ßFTZ-F1 and Matrix metalloproteinase 2 are required for fat-body remodeling in Drosophila

During metamorphosis, holometabolous insects eliminate obsolete larval tissues via programmed cell death. In contrast, tissues required for further development are retained and often remodeled to meet the needs of the adult fly. The larval fat body is involved in fueling metamorphosis, and thus it escapes cell death and is instead remodeled during prepupal development. The molecular mechanisms by which the fat body escapes programmed cell death have not yet been described, but it has been established that fat-body remodeling requires 20-hydroxyecdysone (20E) signaling. We have determined that 20E signaling is required within the fat body for the cell-shape changes and cell detachment that are characteristic of fat-body remodeling. We demonstrate that the nuclear hormone receptor ßFTZ-F1 is a key modulator of 20E hormonal induction of fat body remodeling and Matrix metalloproteinase 2 (MMP2) expression in the fat body. We show that induction of MMP2 expression in the fat body requires 20E signaling, and that MMP2 is necessary and sufficient to induce fat-body remodeling.

Epac, not PKA, mediate the antiproliferative effects of cyclic AMP on human arteriolar smooth muscle cells

Peroxisome proliferator-activated receptor-γ (PPAR-γ) is required for the conversion of pre-adipocytes. However, the mechanism underlying activation of PPAR-γ is unclear. Here we showed that cAMP-induced activation of protein kinase A (PKA) and Akt is essential for the transcriptional activation of PPAR-γ. Hormonal induction of adipogenesis was blocked by a phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002), by a protein kinase A (PKA) inhibitor (H89), and by a Rap1 inhibitor (GGTI-298). Transcriptional activity of PPAR-γ was markedly enhanced by 3-isobutyl-1-methylxanthine (IBMX), but not insulin and dexamethasone. In addition, IBMX-induced PPAR-γ transcriptional activity was blocked by PI3K/Akt, PKA, or Rap1 inhibitors. 8-(4-Chlorophenylthio)-2′-O-methyl-cAMP (8-pCPT-2′-O-Me-cAMP) which is a specific agonist for exchanger protein directly activated by cAMP (Epac) significantly induced the activation of Akt. Furthermore, knock-down of Akt1 markedly attenuated PPAR-γ transcriptional activity. These results indicate that both PKA and Akt signaling pathways are required for transcriptional activation of PPAR-γ, suggesting post-translational activation of PPAR-γ might be critical step for adipogenic gene expression.

It’s time to pay attention to the endometrium

The physiologic importance of the endometrium in assisted reproductive technology is now receiving much research attention. The historical importance of ultrasound for determination of endometrial pattern, thickness and contractility is reviewed and speculation provided for a possible mechanism of the adverse effect of thin endometrium on implantation.

Effects of porcine follicle stimulating hormone, luteinizing hormone and estradiol supplementation in culture medium on ultrastructures of porcine cumulus oocyte complexes (pCOCs)

Inhibition of primary oocyte developing to secondary oocyte results from oocyte maturation inhibitor (OMI) which is secreted from oocyte-surrounding cumulus cells (CCs) during forming complexes with an oocyte. Development of primary oocyte occurs when the CCs are dissociated from the oocyte. This research studied the effects of pFSH, LH, and estradiol supplementation in culture medium on ultrastructures of porcine cumulus oocyte complexes (pCOCs) using transmission electron microcopy (TEM) and inverted microscopy. A total of 880 oocytes were isolated from 110 ovaries: an average of 8 oocytes per ovary. The oocytes were round in shape and surrounded by zona pellucida with layers of cumulus cells (CCs), at a diameter ranging between 90 and 150 μm and more than 400 μm. Based on the CCs surrounding an oocyte, pCOCs were classified into 5 types, which were intact-, multi-, partial cumulus cell layers, completely denuded oocyte, and expanded cumulus cell layer, which were found at the percentage of 53.86%, 14.32%, 4.32%, 19.20%, and 8.30%, respectively. The Types I and II pCOCs (intact- and multi-CCs layers) were further cultured at 37 °C with 5% CO 2, 95% air atmosphere, and high humidity for 24–48 h to investigate their morphological changes after hormonal induction. For the pCOCs cultured without hormonal induction, the CCs were still round in shape and remained in contact with an oocyte via a process of granular end point sticking into the zona pellucida. In contrast, for the hormone supplemented groups, morphological alteration of pCOCs were seen after culture of 24–48 h. The CCs shape was changed from round into elongated or columnar in an opposite direction from an oocyte as well as no communication between microvilli of CCs observed. This led into ceasing of OMI secretion. Therefore, changes of CCs morphology were a sign of the beginning of oocyte maturation. Further study is to characterize the granular substance at the end point of CCs that stick into the zona pellucida.

Effect of lactation number, year, and season of initiation of lactation on milk yield of cows hormonally induced into lactation and treated with recombinant bovine somatotropin

Records representing data from 1,500 barren Holstein cows over an 8-yr period from a large commercial dairy farm in northern Mexico were analyzed to determine the effects of lactation number and season and year of initiation of lactation on milk production of cows induced hormonally into lactation and treated with recombinant bovine somatotropin (rbST) throughout lactation. Peak and 305-d milk yields were also assessed as predictors of total milk yield in cows induced into lactation. A significant quadratic relationship was found between 305-d milk yield and number of lactation [7,607±145 and 9,548±181kg for first- and ≥6-lactation cows, respectively; mean ± standard error of the mean (SEM)] with the highest production occurring in the fifth lactation. Total milk yields of cows with ≤2 lactations were approximately 4,500kg less than milk yields of adult cows (the overall average ± standard milk yield was 13,544±5,491kg per lactation and the average lactation length was 454±154 d). Moreover, 305-d milk production was depressed in cows induced into lactation in spring (8,804±153kg; mean ± SEM) and summer (8,724±163kg) than in fall (9,079±151kg) and winter (9,085±143kg). Partial regression coefficients for 305-d milk yield and peak milk yield indicated an increment of 157kg of milk per lactation per 1-kg increase in peak milk yield (r2=0.69). Neither peak milk yield (r2=0.18) nor 305-d milk yield (r2=0.29) was accurate for predicting total milk yield per lactation. Year, parity, and season effects had significant influence on milk yield of cows induced into lactation and treated with rbST throughout lactation, and peak milk yield can assist in the prediction of 305-d milk yield but not total milk yield. This study also showed that hormonal induction of lactation in barren high-yielding cows is a reliable, practical, and affordable technique in countries where rbST treatment and prolonged steroid administration of dairy cows are legally permitted.

Inhibition of the Experimental Induction of Benign Prostatic Hyperplasia: A Possible Role for Fluted Pumpkin (Telfairia occidentalis Hook f.) Seeds

Introduction: Pumpkins are thought to be useful in the management of benign prostatic hyperplasia (BPH). The ability of a 15% Telfairia occidentalis seeds incorporated diet to inhibit hormonal induction of BPH in rats was studied. Materials and Methods: Twenty male Wistar rats were divided into 4 equal groups – one test group and three control groups. The test group was placed on the test diet and was given subcutaneous injections of dihydrotestosterone (DHT) and estradiol valerate (ratio 10:1) every other day for 28 days. One control group, ‘no test diet’ (ND) group, received the hormones, but was placed on a normal diet. The other two control groups, ‘no hormone’ (NH) and ‘no hormone/test diet’ (NHD), received subcutaneous olive oil (vehicle) for the same duration and were placed on the test and normal diets, respectively. Markers of BPH and hormone profile were determined using standard methods. Results: The mean relative prostate weight (×10³) was reduced in the test group (3.6 ± 0.2) relative to the ND group (4.0 ± 0.4). The protein content (mg/tissue) of the rats’ prostates decreased significantly (p < 0.05) from 68.3 ± 2.7 in the ND group to 43.4 ± 3.9 in the test group. Serum prostatic acid phosphatase levels (U/l) decreased significantly (p < 0.05) from 4.8 ± 0.4 in the ND group to 4.0 ± 0.9 in the test group. Histological findings corroborate these data. The testosterone:estradiol ratio (×10³) was significantly (p < 0.05) increased from 7.1 ± 0.1 in the ND group to 8.4 ± 0.4 in the test group. Conclusion: The test diet inhibited the induction of BPH in rats and may act by increasing the testosterone:estradiol ratio.

Estimativa da concentração espermática do sêmen de peixe pelo método de espermatócrito

Um experimento foi realizado com o objetivo de estimar a concentração espermática das espécies dourado (Salminus brasiliensis), curimba (Prochilodus lineatus), jundiá (Rhamdia quelen), cascudo-preto (Rhinelepis aspera) e tilápia-do-nilo (Oreochromis niloticus) pelo método de espermatócrito. Utilizaram-se 19, 58, 51, 43 e 85 reprodutores de dourado, curimba, jundiá, cascudo-preto e tilápia-do-nilo, respectivamente. Com exceção da tilápia-do-nilo, os reprodutores foram submetidos ao processo de indução hormonal e posteriormente submetidos a coleta de sêmen. Foram comparadas as técnicas de mensuração da concentração espermática do sêmen por contagem em câmara hematimétrica de Neubauer e por espermatócrito. Os resultados obtidos foram submetidos à análise de regressão a 5% de probabilidade. As concentrações espermáticas mensuradas por ambas as técnicas apresentaram relação linear, para curimbas, jundiás e tilápias-do-nilo, com equações y = 6,6624 × 10(9) + 3,68553 × 10(8)x; y = 2,153 × 10(9) + 4,426 × 10(8)x e y = -9,0897 × 10(8) + 6,0167 × 10(8), respectivamente. O método de espermatócrito pode ser utilizado para estimar a concentração espermática do sêmen de curimbas, jundiás e tilápias-do-nilo.

CCAAT/enhancer-binding protein-β participates in oxidized LDL-enhanced proliferation in 3T3-L1 cells

Increased circulating oxidized LDL (oxLDL) have been found in obese subjects. Obesity is characterized by an excess of fat mass resulting from an increase in adipocyte number and size. The generation of new adipocytes is a tightly controlled process where multiple factors acting in a signaling cascade follow a precise temporal expression pattern; oxLDL appear to have a role in the impairment of this process. The purpose of this study was to examine the effects of oxLDL on the mechanisms involved in the proliferative stage of the differentiation process in 3T3-L1 cells. After hormonal induction, 3T3-L1 cells undergo approximately two rounds of mitotic clonal expansion (MCE), a process required for adipogenesis. CCAAT/enhancer-binding protein β (C/EBPβ) is immediately expressed after induction, and plays a crucial role in MCE, but its expression must decrease to allow preadipocytes to mature into adipocytes. We found that, in the presence of stimuli to differentiate, oxLDL induced a higher proliferation rate in this cell line, associated with a sustained up-regulation of C/EBPβ, which remained activated inside the nucleus for several days. RNAi-mediated knockdown of C/EBPβ 24 h after oxLDL treatment counteracted the increase in proliferation rate. Both C/EBPβ expression and proliferation processes appear to be influenced by cAMP/protein kinase A (PKA) and extracellular signal-regulated kinases1/2 (ERK1/2) pathways. OxLDL treatment led to increased levels of cAMP, and to a strong, prolonged phosphorylation of ERK1/2 and C/EBPβ. The addition of cAMP and PKA inhibitors, SQ22536 and H-89, respectively, reduced proliferation only in oxLDL-treated cells, whereas the addition of ERK1/2 inhibitor U0126 blocked proliferation in both control and oxLDL-treated cells. C/EBPβ nuclear expression and DNA-binding activity were reduced by U0126, under all tested conditions. These findings show that the altered expression pattern of C/EBPβ is involved in the increase in the number of proliferating cells induced by oxLDL, in hormone-stimulated 3T3-L1 cells.

Hormonal induction of males as a method for studying tropical cladocerans: description of males of four chydorid species (Cladocera: Anomopoda: Chydoridae)

Methyl farnesoate, a crustacean juvenile hormone, successfully induced male development in several littoral cladocerans from Thailand in short-term multispecies cultures. Male morphology is fully described in four species of Chydoridae— Oxyurella singalensis Daday, 1898, Leberis diaphanus (King, 1853), Leydigia cf. ciliata Gauthier, 1939, Disparalona cf. hamata (Birge, 1910). Males of the latter two taxa from Thailand differ from these from the other localities, suggesting the presence of sibling-species in the Indochina region.

Four enzymes cooperate to displace histone H1 during the first minute of hormonal gene activation

Gene regulation by external signals requires access of transcription factors to DNA sequences of target genes, which is limited by the compaction of DNA in chromatin. Although we have gained insight into how core histones and their modifications influence this process, the role of linker histones remains unclear. Here we show that, within the first minute of progesterone action, a complex cooperation between different enzymes acting on chromatin mediates histone H1 displacement as a requisite for gene induction and cell proliferation. First, activated progesterone receptor (PR) recruits the chromatin remodeling complexes NURF and ASCOM (ASC-2 [activating signal cointegrator-2] complex) to hormone target genes. The trimethylation of histone H3 at Lys 4 by the MLL2/MLL3 subunits of ASCOM, enhanced by the hormone-induced displacement of the H3K4 demethylase KDM5B, stabilizes NURF binding. NURF facilitates the PR-mediated recruitment of Cdk2/CyclinA, which is required for histone H1 displacement. Cooperation of ATP-dependent remodeling, histone methylation, and kinase activation, followed by H1 displacement, is a prerequisite for the subsequent displacement of histone H2A/H2B catalyzed by PCAF and BAF. Chromatin immunoprecipitation (ChIP) and sequencing (ChIP-seq) and expression arrays show that H1 displacement is required for hormone induction of most hormone target genes, some of which are involved in cell proliferation.

Obtaining gynogenetic progeny of Siberian sturgeon (Acipenser baerii) using eggs matured and ovulated in vitro

Summary For the first time viable gynogenetic progenies of the sturgeon fishes were obtained from the oocytes matured and ovulated in vitro under the influence of hormonal treatment. In this study, the Siberian sturgeon was used as a model species. The gynogenetic nature of the obtained progenies was confirmed by the results of microsatellite DNA analysis. Our results indicate the possibility of using meiotic gynogenesis combined with hormonal induction of in vitro oocyte maturation and ovulation as an approach to recovery of disappearing sturgeon species with female heterogamety.

Hormonal induction and semen characteristics of tambaqui Colossoma macropomum

In the hatchery-bred tambaqui Colossoma macropomum, spontaneous semen release does not occur, and hand-stripping produces reduced semen volume. The goal of this work is to evaluate the effects of hormonal induction with carp pituitary extract (CPE) on both qualitative (visual aspect, pH, motility, viability and morphological abnormalities) and quantitative (volume, concentration and number of spermatozoa per ejaculate) traits of tambaqui semen. Eleven males were treated with CPE (induced), and 11 were left untreated as a control (non-induced). All analysed parameters except motility and percentage of viable spermatozoa presented significant differences (p < 0.05) between the induced and non-induced treatments. CPE induction resulted in a 25-fold increase in semen volume and a 10-fold increase in the number of spermatozoa collected. However, both sperm concentration and the frequency of sperm with morphological abnormalities (commonly detached heads or bent tails) were significantly lower in CPE-induced fish. The hormonal induction of tambaqui males with CPE is efficient and positively influences some qualitative and quantitative properties of semen. Additionally, semen collection via gentle abdominal massage occurs more readily in CPE-induced fish.

Characterization of ADP-Glucose Pyrophosphorylase Encoding Genes in Source and Sink Organs of Maize

ADP-glucose pyrophosphorylase catalyzes the first and limiting step in starch biosynthesis. Six cDNA sequences encoding three large subunits and three small subunits of maize AGPase from database were mined and subsequently named: agpl1, agpl2, agpl3, agps1, agps2, and agps3. To elucidate the roles of these isogenes, a comprehensive expression analysis of the gene family was conducted by quantitative real-time RT-PCR. Based on the expression patterns, the six genes can be divided into three groups: (1) steady expressers (agpl1, agps1, and agpl2), which were expressed relatively constantly both in leaf and grain; (2) tissue and development-specific expressers (agpl3 and agps2), which were expressed only in grain at middle and late development phases; (3) tissue-specific expressers (agps3), whose transcripts kept constant during grain filling and were observed only in grain. In order to clarify the effects of sugar and plant hormone on maize AGPase genes expression, a serial of treatments were used. The results showed that AGPase genes significantly differed in response to sugar and hormone inductions. Enormous transcript changes of these genes could be observed in glucose and sucrose treatments. Interestingly, synergistic effect of ABA and sucrose on these genes was observed.

Hormonal induction of spermiation, courting behavior and spawning in the southern bell frog, Litoria raniformis

We trialled the efficacy of various exogenous hormones to induce spermiation, courtship behavior, and spawning in the "endangered" southern bell frog, Litoria raniformis. Intralymphatic administration of Lucrin(®), a synthetic nonapeptide luteinizing hormone releasing hormone (LHRH), was used successfully to induce courting behaviors and ejaculation of spermatozoa in males. Various hormones, including Lucrin(®), another synthetic LHRH analog ([des-Gly(10), D-Ala(6)]-LHRH), human chorionic gonadotropin, progesterone, and a dopamine receptor antagonist failed to promote oviposition and spawning in females. This and earlier studies indicate that in the efficacy of hormonal induction in amphibians varies between taxa, hormones, and genders. The lack of response in females may limit the use of reproduction technology in the southern bell frog and closely related species of Australian bell frogs.

Role of ethylene and jasmonic acid on rhizome induction and growth in rhubarb (Rheum rhabarbarum L.)

Experiments were conducted to elucidate the hormonal induction and regulation of rhizome growth in rhubarb (Rheum rhabarbarum L.). It was found that ethylene is the key regulator of rhizome induction and development. The role of jasmonic acid (JA) and its interaction with ethylene in rhizome induction and growth were also examined. Both ethylene and JA have a significant effect on promoting rhizome formation in vitro. Conversely, the ethylene biosynthesis inhibitor aminoethoxyvinylglycine (AVG) (1.5 μM) inhibited rhizome induction in multiple-shoot clumps in vitro, and suppressed the stimulatory effects of exogenously applied ethephon (1 mg l−1) and JA (10 ng l−1) in promoting mini-rhizome formation, further confirming the role of endogenous ethylene in the process. In addition, rhizome growth was significantly enhanced in the presence of both ethylene and JA (ethephon 1 mg l−1 and JA 10 ng l−1) compared to JA alone. These results suggest that endogenous ethylene is the key regulator of rhizome growth in rhubarb and JA promotes rhizome formation, possibly through inducing endogenous ethylene synthesis.

FSH is superior to eCG for promoting ovarian response in Chinese Bamei gilts

The Bamei gilt is a Chinese native breed located in northwest China, which adapts to the extremely dry and cold environment and is distinguished for its excellent reproductive and maternal characters. To ensure sufficient numbers of embryos for transgenic and nuclear transfer research, hormonal induction of gilt estrus and superovulation may be necessary. The objective of this study was to compare the superovulation effects of equine chorionic gonadotropin (eCG, Group A) and FSH (Groups B–D) in Chinese Bamei gilts. The results show that though eCG could produce more corpora lutea (CL, 14.3) than the control (CL, 9.2), and the FSH treatments had significantly increased the number of CL compared with the eCG treatment. Within the different FSH protocols, the numbers of CL were significantly greater in Groups B (CL, 77.8) and C (CL, 66.8) than in Group D (CL, 42.7), however, ovarian cysts were observed in Groups B and C, but not in Group D. These data suggest that Group D (280 IU FSH) is a suitable protocol to facilitate the development of ovarian follicles and increase the number of useful embryos per gilt for embryos recovery. The optimal FSH protocol of superovulation in Bamei gilts appears to be: D13/100 IU, D14/80 IU, D15/60 IU, D16/40 IU plus prostaglandin (PG) 0.2 mg, D17/hCG 1000 IU.

Anosmia Predicts Hypogonadotropic Hypogonadism in CHARGE Syndrome

To test the hypothesis that a smell test could predict the occurrence of hypogonadotropic hypogonadism (HH) in patients with CHARGE syndrome, which is a variable combination of ocular coloboma, heart defects, choanal atresia, retardation of growth/development, genital hypoplasia, and ear anomalies or hearing loss caused by mutations in the CHD7 (chromodomain helicase DNA binding protein 7) gene. We performed endocrine studies and smell testing (University of Pennsylvania Smell Identification Test) in 35 adolescent patients with molecularly confirmed CHARGE syndrome. Complete data on smell and puberty were available for 15 patients; 11 patients had both anosmia and HH, whereas 4 patients had normosmia/hyposmia and spontaneous puberty. In addition, 7 boys were highly suspected of having HH (they were too young for definite HH diagnosis, but all had cryptorchidism, micropenis, or both) and had anosmia. The type of CHD7 mutation could not predict HH because a father and daughter with the same CHD7 mutation were discordant for HH and anosmia. Anosmia and HH were highly correlated in our cohort, and therefore smell testing seems to be an attractive method for predicting the occurrence of HH in patients with CHARGE syndrome. The use of this test could prevent delay of hormonal pubertal induction, resulting in an age-appropriate puberty.

Semen characterization and sperm structure of the Amazon tambaqui Colossoma macropomum

Seminal features of tambaqui were evaluated after hormonal induction of spermiation with common carp pituitary extract. Seventeen adult (6.1 ± 0.9 kg, 62 ± 6 cm) males were collected from earthen ponds and transported to indoor concrete tanks. Semen was evaluated according to volume, pH, osmolality, motility, concentration, viability, sperm morphometry and morphological abnormalities. The semen of tambaqui was white and milky. The volume was 10.2 ± 5.1 mL at pH 8.0 ± 0.1, and yielded a concentration of 9.1 × 109 spermatozoa mL−1 while seminal plasma osmolality was 260 ± 7.3 mOsm kg−1. The percentage of viable sperm cells was determined with 97.0 ± 2.0% and 87.0 ± 8.0% using an eosin-nigrosin staining and fluorescent live-dead kit (propidium iodide and SYBR-14), respectively. A negative correlation (−0.65) between semen pH and sperm motility was observed suggesting this feature might influence the tambaqui sperm kinetics. Morphometrically the spermatozoa were on average 35.48 ± 1.55 μm long with a roundish head (mean length: 2.73 ± 0.21 μm; mean width: 2.58 ± 0.18 μm; n = 250 spermatozoa), without an acrosome, and presented a long midpiece (2.90 ± 0.52 μm) and flagellum (29.84 ± 1.63 μm). About 15.8% of the spermatozoa carried morphological abnormalities, with bent tail being the most frequent defect (7.81 ± 3.12%). The characterization of tambaqui semen favors the development of more precise and efficient procedures for its analysis and utilization in controlled breeding.

Epigenetic control of expression of the human L‐ and M‐ pigment genes

Epigenetics alters gene expression by chromatin modification without changing the sequence of DNA. DNA methylation is an essential signal for epigenetic gene regulation. Methylation of cytosine bases at CpG dinucleotides in DNA results in chromatin condensation resulting in suppression of gene expression. DNA methylation has been shown to play important roles in cell differentiation, genomic imprinting and X-chromosome inactivation. We compared the CpG methylation patterns of the promoters of the L-opsin gene (OPN1LW) and the M-opsin gene (OPN1MW), plus a DNase I hypersensitive (DHS) site located about 8 kb (kilobases) upstream of the OPN1LW gene. Comparisons were made using the human retinoblastoma cell line WERI, which expresses the L and M opsin genes when treated with thyroid hormone (T3), and a lymphoblastoid cell line GM06990 that does not express these genes. The results showed that the great majority of the 14 CpGs located within the proximal 200 bp (base pairs) of each promoter, plus 20 bp of the 5'-untranslated region, were hypo-methylated in WERI-Rb-1 cells, whether or not treated with T3, but almost totally methylated in the lymphoblastoid cell line. Three of the CpGs that are located beyond 200 bp from the transcription start site of OPN1LW were hyper-methylated in both WERI and lymphoblastoid cells. Significant differential methylation was also observed within the DHS region (24 CpGs). This DHS region contains a highly conserved motif that binds CCCTC-binding factor (CTCF), referred to as a 'chromatin insulator or boundary element', that has been shown to regulate gene expression at several genome locations. The results suggest that DNA methylation is likely to contribute to regulation of expression of the L- and M-opsin genes during differentiation, as well as to the retinal L:M cone ratio. In addition, thyroid hormone induction of the opsin genes does not appear to alter DNA methylation.

Morphology of Mouse External Genitalia: Implications for a Role of Estrogen in Sexual Dimorphism of the Mouse Genital Tubercle

We examined the role of androgens and estrogens in mammalian sexual differentiation by morphological characterization of adult wt and mutant mouse external genitalia. We tested the hypothesis that external genitalia development depends on androgen and estrogen action. We studied serial sections of the external genitalia of the CD-1 and C57BL6 wt strains of adult mice (Charles River Laboratories, Wilmington, Massachusetts). We recorded linear measurements of key structures in each specimen, including the urethra, erectile tissue, bone and cartilage. We used similar methodology to analyze mice mutant for estrogen receptor α (αERKO) and androgen receptor (X(Tfm)/Y) (Jackson Laboratory, Bar Harbor, Maine). Morphology in X(Tfm)/Y adult murine external genitalia was remarkably similar to that in wt females. Bone and clitoral length was similar in wt females and X(Tfm)/Y mice. Conversely the αERKO clitoris was 59% longer and bone length in αERKO females was many-fold longer than that in female wt mice or X(Tfm)/Y mutants. The αERKO clitoris contained cartilage, which is typical of the wt penis but never observed in the wt clitoris. Serum testosterone was not increased in female αERKO mice 10 days postnatally when sex differentiation occurs, suggesting that masculinization of the αERKO clitoris is not a function of androgen. Masculinization of the αERKO clitoris suggests a role for estrogen in the development of female external genitalia. We propose that normal external genital development requires androgen and estrogen action.