Clodronate-Loaded Liposome Treatment Has Site-Specific Skeletal Effects.

Ineffective oral wound healing is detrimental to patients' oral health-related quality of life. Delineating the cellular mechanisms involved in optimal healing will elicit better approaches to treating patients with compromised healing. Osteal macrophages have recently emerged as important positive regulators of bone turnover. The contributions of macrophages to long bone healing have been studied, but their role in oral osseous wound healing following tooth extraction is less clear. Clodronate-loaded liposomes were used as a tool to deplete macrophages in C57BL/6J mice and assess oral osseous bone fill after extraction. In addition to macrophage ablation, osteoclast ablation occurred. Interestingly, depletion of macrophages and osteoclasts via clodronate treatment had differential effects based on skeletal location. In the nonwounded tibiae, clodronate treatment significantly increased CD68+ cells and decreased F4/80+ cells in the marrow, which correlated with increased trabecular bone volume fraction after 7 and 14 d. Serum formation and resorptive markers P1NP and TRAcP 5b were decreased as were tibial TRAP+ osteoclasts. In healing extraction sockets, clodronate treatment increased extraction socket trabecular bone thickness at 14 d, which correlated with decreased TRAP+ osteoclasts and F4/80+ macrophages. Conversely, nonwounded maxillary interseptal bone was unaffected by clodronate treatment. Furthermore, the increase in extraction socket bone fill with clodronate was less than the large increase in trabecular bone observed in a nonwounded long bone. These data suggest a temporal and spatial specificity in the roles of macrophages and osteoclasts in normal turnover and healing.

Effect of local fluency gradient of objects creates search asymmetry.

Search asymmetry is a phenomenon in which search efficiency in a visual-search task differs for searching for an X target among Y distractors from search for a Y target among X distractors. Previous research shows that search asymmetry is mainly produced by a difference in the whole signal strength of items or a difference in item familiarity. This study reports that a difference in the local fluency within items also affects search efficiency and generates search asymmetry. Fluency is a value that correlates with the processing efficiency of an item. In particular, five experiments reveal that search efficiency for two part items depends on whether a fluent part is the top or bottom portion of a target (vs. distractor). We argue that this type of search asymmetry implicates the operation of an unknown mechanism that detects local fluency gradient in visual processing.

A multi-emitter fitting algorithm for potential live cell super-resolution imaging over a wide range of molecular densities.

Multi-emitter fitting algorithms have been developed to improve the temporal resolution of single-molecule switching nanoscopy, but the molecular density range they can analyse is narrow and the computation required is intensive, significantly limiting their practical application. Here, we propose a computationally fast method, wedged template matching (WTM), an algorithm that uses a template matching technique to localise molecules at any overlapping molecular density from sparse to ultrahigh density with subdiffraction resolution. WTM achieves the localization of overlapping molecules at densities up to 600 molecules µm-2 with a high detection sensitivity and fast computational speed. WTM also shows localization precision comparable with that of DAOSTORM (an algorithm for high-density super-resolution microscopy), at densities up to 20 molecules µm-2 , and better than DAOSTORM at higher molecular densities. The application of WTM to a high-density biological sample image demonstrated that it resolved protein dynamics from live cell images with subdiffraction resolution and a temporal resolution of several hundred milliseconds or less through a significant reduction in the number of camera images required for a high-density reconstruction. WTM algorithm is a computationally fast, multi-emitter fitting algorithm that can analyse over a wide range of molecular densities. The algorithm is available through the website. https://doi.org/10.17632/bf3z6xpn5j.1.

Expression of isotocin is male-specifically up-regulated by gonadal androgen in the medaka brain.

Oxytocin, a mammalian neuropeptide primarily synthesised in the supraoptic and paraventricular nuclei of the hypothalamus, mediates a variety of physiological and behavioural processes, ranging from parturition and lactation to affiliation and prosociality. Multiple studies in rodents have shown that the expression of the oxytocin gene (Oxt) is stimulated by oestrogen, whereas androgen has no apparent effect. However, this finding is not consistent across all studies, and no study has examined sex steroid regulation of Oxt or its orthologues in other animals. In the present study, we show that, in the teleost fish, medaka (Oryzias latipes), the expression of the isotocin gene (it), the teleost orthologue of Oxt, in the parvocellular preoptic nuclei (homologous to the mammalian supraoptic nucleus) is male-specifically up-regulated by gonadal androgen, whereas it expression in the magnocellular/gigantocellular preoptic nuclei (homologous to the mammalian paraventricular nucleus) is independent of sex steroids in both sexes. None of the it-expressing neurones appear to co-express androgen receptors, suggesting that the effect of androgen on it expression is indirect. We found that the expression of a kisspeptin gene, kiss2, in the male brain is dependent on gonadal androgen, raising the possibility that the androgen-dependent expression of it may be mediated by kiss2 neurones. Our data also show that the isotocin peptide synthesised in response to androgen is axonally transported to the posterior pituitary to act peripherally. Given that levels of it expression are higher in females than in males, androgen may serve to compensate for the female-biased it expression to ensure a role for isotocin that is equally important for both sexes. These results are unexpectedly quite different from those reported in rodents, indicating that the regulatory role of sex steroids in Oxt/it expression has diverged during evolution, possibly with accompanying changes in the role of oxytocin/isotocin.

Iodoarene-catalyzed oxidative transformations using molecular oxygen.

Molecular oxygen serves as a useful oxidant for the glycol scission of 1,2-diols and the Hofmann rearrangement of primary amides using pentamethyliodobenzene as a catalyst. The use of isobutyraldehyde and Lewis basic nitriles under O2 enabled the iodine(i)/(iii) catalytic cycle, where in situ-generated peracid acts as a terminal oxidant.

Three urocortins in medaka: identification and spatial expression in the central nervous system.

The urocortin (UCN) group of neuropeptides includes urocortin 1/sauvagine/urotensin 1 (UTS1), urocortin 2 (UCN2) and urocortin 3 (UCN3). In recent years, evidence has accumulated showing that UCNs play pivotal roles in mediating stress response and anxiety in mammals. Evidence has also emerged regarding the evolutionary conservation of UCNs in vertebrates, but very little information is available about UCNs in non-mammalian vertebrates. Indeed, at present, there are no reports of the empirical identification of ucn2 in non-mammalian vertebrates or of the distribution of ucn2 and ucn3 expression in the adult central nervous system (CNS) of these animals. To gain insight into the evolutionary nature of UCNs in vertebrates, we cloned uts1, ucn2 and ucn3 in a teleost fish, medaka and examined the spatial expression of these genes in the adult brain and spinal cord. Although all known UCN2 genes except those in rodents have been reported to likely lack the necessary structural features to produce a functional pre-pro-protein, all three UCN genes in medaka, including ucn2, displayed all of these features, suggesting their functionality. The three UCN genes exhibited distinct spatial expression patterns in the medaka brain: uts1 was primarily expressed in broad regions of the dorsal telencephalon, ucn2 was expressed in restricted regions of the thalamus and brainstem and ucn3 was expressed in discrete nuclei throughout many regions of the brain. We also found that these genes were all expressed throughout the medaka spinal cord, each with a distinct spatial pattern. Given that many of these regions have been implicated in stress responses and anxiety, the three UCNs may serve distinct physiological roles in the medaka CNS, including those involved in stress and anxiety, as shown in the mammalian CNS.

Hairworm Infection and Seasonal Changes in Paratenic Hosts in a Mountain Stream in Japan.

For parasites with complex life cycles, the ecological traits determining host competence and seasonal changes in infection in natural habitats are often unclear, making it difficult to predict infection dynamics, including disease outbreaks. Hairworms (phylum Nematomorpha) require both aquatic and terrestrial hosts to complete their life cycle. Although hairworm host competencies have been tested in laboratory experiments, knowledge of the paratenic hosts (aquatic insect larvae) in their natural habitats is limited. This study clarified the species of aquatic insect larvae that are primarily infected by hairworms as paratenic hosts over a year in a mountain stream in central Honshu, Japan. The monthly prevalence and mean abundance of hairworm cysts were high in Ephemera japonica larvae (Ephemeridae: Ephemeroptera) throughout the study period (20.0-88.9 and 0.2-36.8%, respectively). These high prevalence and abundance values may be attributable to their filter-feeding behavior as well as their depositional habitat use. The hairworms also infected leptophlebiids (Ephemeroptera; scrapers), the perlid Calineulia sp., the chloroperlid Haploperla japonica (Plecoptera; predators), and chironomids (Diptera; filter-feeders or predators). The abundance of the cysts tended to be high in aquatic insects inhabiting pools rather than riffles, and the seasonality reflects the reproductive season of the hairworms as well as the phenology of their paratenic hosts. Filter-feeding ephemeropterans inhabiting pools were the major paratenic host of the hairworms in our study site, although their universality and effectiveness as the transporter to definitive hosts remain unclear.

Sandwich ELISA Using a Mouse/Human Chimeric CSLEX-1 Antibody.

BACKGROUND: An assay using a mouse antisialyl Lewis X (sLeX) antibody (CSLEX-1) is used clinically for screening and monitoring patients with breast cancer in Japan. However, the IgM isoform of CSLEX-1 is not preferred for the assay because the bulkiness of IgM generally causes poor accessibility to the antigen. To solve this problem, we developed an antisLeX mouse/human chimeric IgG antibody, CH-CSLEX-1, using transgenic silkworms. The performance of a homologous sandwich ELISA of CH-CSLEX1 was then evaluated. METHODS: To generate CH-CSLEX-1, we used a GAL4/UAS binary gene expression system in transgenic silkworms. The reactivities of CSLEX-1 and CH-CSLEX-1 were determined in a Biacore analysis. To confirm antigen specificity, 3 antigens [sLeX, sLeA, and Lewis Y (LeY)] were used. RESULTS: CH-CSLEX-1 formed correctly as an IgG class of immunoglobulin molecule with an isoelectric point close to the predicted value. The best combination for capturing and probing in a sandwich ELISA was determined as a homologous combination of CH-CSLEX-1. The CH-CSLEX-1 assay specifically detected sLeX, but not sLeA and LeY. A correlation analysis with 107 human samples showed good concordance between the conventional CSLEX-1 assay (homologous sandwich ELISA using CSLEX-1) and the CH-CSLEX-1 assay (r = 0.98). Moreover, the CH-CSLEX-1 assay was not affected by either human antimouse IgG antibodies (HAMA IgG) or HAMA IgM. CONCLUSIONS: The mouse/human chimeric antibody CH-CSLEX-1 allowed the establishment of a highly specific sandwich ELISA for sLeX that was not affected by HAMA.

Effect of Alendronate on Bone Formation during Tooth Extraction Wound Healing.

Alendronate (ALN) is an antiresorptive agent widely used for the treatment of osteoporosis. Its suppressive effect on osteoclasts has been extensively studied. However, the effect of ALN on bone formation is not as clear as its effect on resorption. The objective was to determine the effect of short-term ALN on bone formation and tooth extraction wound healing. Molar tooth extractions were performed in mice. ALN, parathyroid hormone (PTH), or saline (vehicle control) was administered. PTH was used as the bone anabolic control. Mice were euthanized at 3, 5, 7, 10, and 21 d after extractions. Hard tissue healing was determined histomorphometrically. Neutrophils and lymphatic and blood vessels were quantified to evaluate soft tissue healing. Gene expression in the wounds was assessed at the RNA level. Furthermore, the vossicle bone transplant system was used to verify findings from extraction wound analysis. Alkaline phosphatase (ALP) was visualized in the vossicles to assess osteoblast activity. ALN exhibited no negative effect on bone formation. In intact tibiae, ALN increased bone mass significantly more than PTH did. Consistently, significantly elevated osteoblast numbers were noted. In the extraction sockets, bone fill in the ALN-treated mice was equivalent to the control. Genes associated with bone morphogenetic protein signaling, such as bmp2, nog, and dlx5, were activated in the extraction wounds of the ALN-treated animals. Bone formation in vossicles was significantly enhanced in the ALN versus PTH group. In agreement with this, ALN upregulated ALP activity considerably in vossicles. Neutrophil aggregation and suppressed lymphangiogenesis were evident in the soft tissue at 21 d after extraction, although gross healing of extraction wounds was uneventful. Bone formation was not impeded by short-term ALN treatment. Rather, short-term ALN treatment enhanced bone formation. ALN did not alter bone fill in extraction sockets.

Early effects of parathyroid hormone on bisphosphonate/steroid-associated compromised osseous wound healing.

SUMMARY: Administration of intermittent parathyroid hormone (PTH) promoted healing of tibial osseous defects and tooth extraction wounds and prevented the development of necrotic lesions in rats on a combined bisphosphonate and steroid regimen. INTRODUCTION: Osteonecrosis of the jaw (ONJ) has emerged in association with antiresorptive therapies. The pathophysiology of ONJ is unknown and no established cure currently exists. Our objective was to determine the effect of intermittent PTH administration on early osseous healing in the jaw and long bones of rats receiving bisphosphonate and steroid treatment. METHODS: Ovariectomized rats received the combination therapy of alendronate and dexamethasone (ALN/DEX) for 12 weeks. Osseous wounds were created in the jaw and tibia. PTH was administered intermittently and healing at 2 weeks post-op was compared between the jaw and tibia by microcomputed tomography and histomorphometric analyses. RESULTS: ALN/DEX treatment was associated with necrotic open wounds in the jaw but had no negative effects on healing and promoted bone fill in tibial defects. PTH therapy prevented the development of necrotic lesions in the jaw and promoted healing of the tibial defects. PTH therapy was associated with the promotion of osteocyte survival in osseous wounds both in the jaw and tibia. CONCLUSIONS: Wound healing was impaired in the jaw in rats on a combined bisphosphonate and steroid regimen, and PTH therapy rescued necrotic lesions. These findings suggest that PTH therapy could be utilized to prevent ONJ from occurring in patients on combination antiresorptive and steroid therapy.

Role of arginine and fluoride in the prevention of eroded enamel: an in vitro model.

BACKGROUND: The aim of this study was to investigate the effect of arginine and fluoride on the reduction of erosive wear. METHODS: Bovine enamel blocks were randomly allocated into four groups (n = 20) and exposed to: ESPR group (8% arginine, 1450 ppm sodium monofluorophosphate, calcium carbonate and titanium dioxide); ESen group (1450 ppm sodium monofluorophosphate, 5% potassium citrate); positive control PC group (1500 ppm sodium monofluorophosphate) and negative control NC group (water). The samples were submitted to six alternating cycles of demineralization-remineralization (cola, 10 minutes; artificial saliva, 1 hour, respectively). Before and between cyclic demineralization and remineralization, blocks were treated with slurries of the respective toothpastes or water (1 minute). Erosive tissue loss was analysed by microhardness and profilometry. Data were analysed by ANOVA and Tukey tests for individual comparisons among the groups (p < 0.05). RESULTS: In microhardness, the ESPR (217.46 ± 55.45) group was significantly better than the other treatment groups (PC = 302.76 ± 96.10; ESen = 315.56 ± 74.56; p < 0.001). The ESPR group showed a similar loss to NC group (NC = 210.8 ± 49.98; p = 0.991). The mean erosion depth (+/- SE, µm) was detected between NC (14.37 ± 1.72) and dentifrices tested (ESPR (4.11 ± 1.34), ESen group (7.64 ± 1.61) and PC (8.20 ± 2.19) (p = 0.000). CONCLUSIONS: From the results of the present study, the effectiveness of Sensitive Pro Relief in the prevention of erosive surface loss seems to be attributed to the possible effect of the arginine associated with fluoride.

Ultrasound-guided single shot caudal block anesthesia reduces postoperative urinary catheter-induced discomfort.

BACKGROUND: Urinary catheter-induced discomfort during the postoperative period can be distressing, and sometimes results in severe restlessness and agitation, especially in middle-aged and elderly male patients. Recent advances in ultrasound technology have increased the consistency, safety, and ease of a caudal block even in older patients. We speculated that an ultrasound-guided caudal block would be reliable and safe as treatment for such postoperative discomfort. METHODS: Adult male patients (ASA I-II) undergoing cervical laminoplasty were allocated to either the caudal block (CB, N.=24) or non-block (NB, N.=24) group. Following anesthesia induction, urinary catheterization was performed using a 16 French Foley catheter. Thereafter, an ultrasound-guided caudal block was performed with 8 ml of 0.3% ropivacaine and 100 µg of fentanyl for patients in group CB, while group NB did not receive a caudal block. We assessed urinary catheter-induced discomfort as mild, moderate, or severe at 0, 2, 6, 10, and 18 hours after surgery, and compared the incidence and severity of discomfort between the groups using a randomized double-blind design. RESULTS: All caudal blocks were successfully performed with 1 or 2 needle insertions. The incidence of urinary catheter-induced discomfort was significantly reduced in group CB as compared to NB at 0, 2, and 6 hours, while severity was also reduced at 0 and 2 hours. No patient required re-catheterization due to urinary retention after catheter removal. There were no other complications related to the caudal block. CONCLUSION: Preoperative ultrasound-guided single shot caudal block anesthesia safely reduced postoperative urinary catheter-induced discomfort in our male patients.

Intra-oral PTH administration promotes tooth extraction socket healing.

Intermittent parathyroid hormone (PTH) administration increases systemic and craniofacial bone mass. However, the effect of PTH therapy on healing of tooth extraction sites is unknown. The aims of this study were to determine the effect of PTH therapy on tooth extraction socket healing and to examine whether PTH intra-oral injection promotes healing. The mandibular first molars were extracted in rats, and subcutaneous PTH was administered intermittently for 7, 14, and 28 days. In a second study, maxillary second molars were extracted, and PTH was administered by either subcutaneous or intra-oral injection to determine the efficacy of intra-oral PTH administration. Healing was assessed by micro-computed tomography and histomorphometric analyses. PTH therapy accelerated the entire healing process and promoted both hard- and soft-tissue healing by increasing bone fill and connective tissue maturation. PTH therapy by intra-oral injection was as effective as subcutaneous injection in promoting tooth extraction socket healing. The findings suggest that PTH therapy promotes tooth extraction socket healing and that intra-oral injections can be used to administer PTH.

Serum epidermal growth factor receptor levels in patients with malignant melanoma.

BACKGROUND: Epidermal growth factor receptor (EGFR) is known to be abnormally expressed in many human carcinomas, suggesting that there may be an increase in serum EGFR levels in patients with malignant melanoma (MM) and that this might be a possible new tumour marker. AIM: To assess whether serum EGFR levels might be a marker of MM. METHODS: Serum samples were obtained from 66 patients with MM and 12 healthy controls, and EGFR levels were measured by double-determinant ELISA. RESULTS: Patients with in situ or stage I MM had significantly higher serum EGFR levels compared with healthy controls. Interestingly, serum EGFR levels decreased gradually with the stage of the tumour, being highest at stage I and lowest at stage IV. There was also a trend towards a reverse correlation between tumour thickness and serum EGFR levels. Moreover, a longitudinal study identified a trend for serum EGFR levels in patients with preoperative MM to decrease compared with patients with recurrent MM. CONCLUSIONS: To our knowledge, this is the first report investigating the serum EGFR levels of patients with MM, and gives new insight into the relationship between EGFR and MM. We found that serum EGFR levels were significantly increased in patients with early-stage MM such as in situ and stage I tumours. Measurements of serum EGFR levels might be of clinical value in the detection of early-stage MM.

Effect of κ-opioid receptor agonist on the growth of non-small cell lung cancer (NSCLC) cells.

BACKGROUND: It is becoming increasingly recognised that opioids are responsible for tumour growth. However, the effects of opioids on tumour growth have been controversial. METHODS: The effects of κ-opioid receptor (KOR) agonist on the growth of non-small cell lung cancer (NSCLC) cells were assessed by a cell proliferation assay. Western blotting was performed to ascertain the mechanism by which treatment with KOR agonist suppresses tumour growth. RESULTS: Addition of the selective KOR agonist U50,488H to gefitinib-sensitive (HCC827) and gefitinib-resistant (H1975) NSCLC cells produced a concentration-dependent decrease in their growth. These effects were abolished by co-treatment with the selective KOR antagonist nor-BNI. Furthermore, the growth-inhibitory effect of gefitinib in HCC827 cells was further enhanced by co-treatment with U50,488H. With regard to the inhibition of tumour growth, the addition of U50, 488H to H1975 cells produced a concentration-dependent decrease in phosphorylated-glycogen synthase kinase 3ß (p-GSK3ß). CONCLUSION: The present results showed that stimulation of KOR reduces the growth of gefitinib-resistant NSCLC cells through the activation of GSK3ß.

The role of microRNA-150 as a tumor suppressor in malignant lymphoma.

MicroRNA (miRNA; miR) is a class of small regulatory RNA molecules, the aberrant expression of which can lead to the development of cancer. We recently reported that overexpression of miR-21 and/or miR-155 leads to activation of the phosphoinositide 3-kinase (PI3K)-AKT pathway in malignant lymphomas expressing CD3(-)CD56(+) natural killer (NK) cell antigen. Through expression analysis, we show in this study that in both NK/T-cell lymphoma lines and samples of primary lymphoma, levels of miR-150 expression are significantly lower than in normal NK cells. To examine its role in lymphomagenesis, we transduced miR-150 into NK/T-cell lymphoma cells, which increased the incidence of apoptosis and reduced cell proliferation. Moreover, the miR-150 transductants appeared senescent and showed lower telomerase activity, resulting in shortened telomeric DNA. We also found that miR-150 directly downregulated expression of DKC1 and AKT2, reduced levels of phosphorylated AKT(ser473/4) and increased levels of tumor suppressors such as Bim and p53. Collectively, these results suggest that miR-150 functions as a tumor suppressor, and that its aberrant downregulation induces continuous activation of the PI3K-AKT pathway, leading to telomerase activation and immortalization of cancer cells. These findings provide new insight into the pathogenesis of malignant lymphoma.

Skewed T cell receptor repertoire of Vdelta1(+) gammadelta T lymphocytes after human allogeneic haematopoietic stem cell transplantation and the potential role for Epstein-Barr virus-infected B cells in clonal restriction.

The proliferation of Vdelta1(+) gammadelta T lymphocytes has been described in various infections including human immunodeficiency virus (HIV), cytomegalovirus (CMV) and malaria. However, the antigen specificity and functions of the human Vdelta1(+) T cells remain obscure. We sought to explore the biological role for this T cell subset by investigating the reconstitution of T cell receptor (TCR) repertoires of Vdelta1(+) gammadelta T lymphocytes after human allogeneic haematopoietic stem cell transplantation (HSCT). We observed skewed TCR repertoires of the Vdelta1(+) T cells in 27 of 44 post-transplant patients. Only one patient developed EBV-associated post-transplant lymphoproliferative disorder in the present patient cohort. The -WGI- amino acid motif was observed in CDR3 of clonally expanded Vdelta1(+) T cells in half the patients. A skew was also detected in certain healthy donors, and the Vdelta1(+) T cell clone derived from the donor mature T cell pool persisted in the recipient's blood even 10 years after transplant. This T cell clone expanded in vitro against stimulation with autologous EBV-lymphoblastoid cell lines (LCL), and the Vdelta1(+) T cell line expanded in vitro from the same patient showed cytotoxicity against autologous EBV-LCL. EBV-infected cells could also induce in vitro oligoclonal expansions of autologous Vdelta1(+) T cells from healthy EBV-seropositive individuals. These results suggest that human Vdelta1(+) T cells have a TCR repertoire against EBV-infected B cells and may play a role in protecting recipients of allogeneic HSCT from EBV-associated disease.

Effects of in ovo exposure to imazalil and atrazine on sexual differentiation in chick gonads.

We examined the effects of atrazine and imazalil, 2 commonly used pesticides, on sexual differentiation in chickens. Atrazine and imazalil were injected into fertile eggs on d 0. At hatching, sex genotype and phenotype were determined. Gonads were stereomicroscopically and histologically observed. In ovo exposure of atrazine (0.01 to 3 mg/egg) did not influence hatchability, whereas imazalil exposure (2 mg/egg) inhibited hatchability. The sex genotype matched the sex phenotype in controls, atrazine, and imazalil-exposed groups. In control females, the right gonad was regressed at hatching. Regression of the right gonad, however, was inhibited following atrazine and imazalil exposure. In atrazine-exposed female chicks, the left gonads had normal ovary structures, and the remaining right gonads had ovary medulla-like structures. In imazalil-exposed females, some left gonads had an ovary medulla-like structure without the cortex as well as tubules, and the right gonad had testis-like structures. There was no change in male gonads at hatching following atrazine and imazalil exposure. Aromatase activity of the left gonad from female chicks was not changed by any concentration of atrazine exposure. These results suggest that atrazine and imazalil inhibit regression of the right gonad in female chicks, although it is not clear whether the remaining right gonad has aromatase activity. In ovo exposure to atrazine influences sexual differentiation of the ovary by different mechanisms from imazalil, possibly by the induction of aromatase in the right gonad, whereas it is confirmed that imazalil inhibits in vitro aromatase activity in the chick ovary. The results indicated that in ovo exposure to imazalil inhibits sexual differentiation of the ovary by inhibiting aromatase activity.