Artificial Intelligence-Based Quantification of Epithelial Proliferation in Mammary Glands of Rats and Oviducts of Göttingen Minipigs.

Quantitative assessment of proliferation can be an important endpoint in toxicologic pathology. Traditionally, cell proliferation is quantified by labor-intensive manual counting of positive and negative cells after immunohistochemical staining for proliferation markers (eg, Ki67, bromo-2'-deoxyuridine, or proliferating cell nuclear antigen). Currently, there is a lot of interest in replacing manual evaluation of histology end points with image analysis tools based on artificial intelligence. The aim of the present study was to explore if a commercially available image analysis software can be used to quantify epithelial proliferative activity in rat mammary gland and minipig oviduct. First, algorithms based on artificial intelligence were trained to detect epithelium in each tissue. Areas of BrdU- or Ki67-positive nuclei and negative nuclei were subsequently quantified with threshold analysis. Artificial intelligence-based and manually counted labelling indices were strongly correlated and equally well detected the estrous cycle influence on proliferation in mammary gland and oviduct epithelium, as well as the dramatically increased proliferation in rat mammary glands after treatment with estradiol and progesterone. In conclusion, quantification of epithelial proliferation in two reproductive tissues can be achieved in a reliable fashion using image analysis software based on artificial intelligence, thus avoiding time- and labor-intensive manual counting, requiring trained operators.

A comparative study of the turnover of multiciliated cells in the mouse trachea, oviduct, and brain.

BACKGROUND: In mammals, multiciliated cells (MCCs) line the lumen of the trachea, oviduct, and brain ventricles, where they drive fluid flow across the epithelium. Each MCC population experiences vastly different local environments that may dictate differences in their lifetime and turnover rates. However, with the exception of MCCs in the trachea, the turnover rates of these multiciliated epithelial populations at extended time scales are not well described. RESULTS: Here, using genetic lineage-labeling techniques we provide a direct comparison of turnover rates of MCCs in these three different tissues. CONCLUSION: We find that oviduct turnover is similar to that in the airway (~6 months), while multiciliated ependymal cells turnover more slowly.

Biomechanics of epithelial fold pattern formation in the mouse female reproductive tract.

Tubular organs and tissues often show various morphological fold patterns in their luminal epithelia. Computational studies have revealed that these patterns could be explained by mechanical deformation of the epithelia. However, experimental validations of this are sparse, and the mechanisms linking genetic and cellular functions to fold mechanics are poorly understood. In the oviduct of the female reproductive tract, the epithelium forms multiple well-aligned straight folds. Disruption of Celsr1, a planar cell polarity-related gene, causes ectopically-branched folds in mice. Here we discuss the pattern formation of the folds with respect to the growth and mechanics of the epithelium, and the cellular and genetic functions, and compare these with other tubular organs such as the gut.

RAS-related protein 1: an estrogen-responsive gene involved in development and molting-mediated regeneration of the female reproductive tract in chickens.

It is important to identify molecular candidates involved in morphological and functional changes in the female reproductive system. We have discovered several candidate genes that were significantly altered in chick oviducts by exogenous estrogen and those candidates included dexamethasone (DEX)-induced RAS-related protein 1 (RASD1). RAS-related protein 1, a member of the Ras family of monomeric G proteins, is involved in various cellular processes including cell growth, proliferation and differentiation, as well as a cell-signaling protein regulating hormonal actions. Although the RASD1 gene was first identified as a DEX (a corticosteroid) inducible gene, there is evidence that it is also an estrogen-responsive gene. However, hormone-mediated expression and biological functions of RASD1 in the avian female reproductive system are poorly understood. Therefore, we tested the hypothesis that RASD1 may be involved in the development and remodeling of the chicken reproductive system as an estrogen-responsive gene. Here we demonstrate differential expression of RASD1 gene and candidate microRNAs (miRNAs) targeting chicken RASD1 transcripts in chicken oviducts in response to diesthylstilbestrol (DES, a synthetic non-steroidal estrogen) and the estrogen-mediated molting process. Result of the present study indicated that expression of RASD1 messenger RNA (mRNA) increased in the developing oviducts of chicks treated with DES, particularly in the glandular (GE) and luminal (LE) epithelia of the magnum and the shell gland. Also, during induced molting by zinc feeding, RASD1 expression changed in concert with changes in concentrations of estrogen in blood of laying hens. Our results revealed that expression of RASD1 mRNA decreased as the oviduct regressed and then increased as the oviduct underwent re-growth and recrudescence in hens. Furthermore, RASD1 mRNA was expressed predominantly in GE and LE of the oviduct of laying hens during regeneration of the oviduct after induced molting, but not during the period of regression of the oviduct during molting. In addition, the relative expression of candidate miRNAs (miR-30a-5p, miR-30b-5p, miR-30c-5p and miR-30d) regulating RASD1 transcripts changed in response to estrogen stimulation of chick oviducts. These results indicate that transcription of the RASD1 gene and miRNAs regulating post-transcriptional aspects of expression of RASD1 are modulated by estrogen which is critical for growth, development, remodeling and maintenance of function of the chicken oviduct.

Dicer1, AGO3, and AGO4 microRNA machinery genes are differentially expressed in developing female reproductive organs and overexpressed in cancerous ovaries of chickens.

MicroRNA (miRNA)-mediated gene silencing is a key mechanism regulating numerous biological processes such as development of organs and tumorigenesis. The expression of miRNA machinery genes linked to miRNA biogenesis and processing is finely regulated. Despite accumulating evidence for chicken miRNA in the female reproduction system, precise regulatory mechanisms are largely unknown. Therefore, the objective of this study was to determine changes in expression levels of miRNA machinery genes in developmental stages of the oviduct and ovarian carcinogenesis of laying hens. In the present study, differential expression of miRNA machinery genes during ovarian carcinogenesis was determined using cancerous and normal ovaries collected from normal laying hens and hens with cancer. Our results showed that 3 miRNA machinery genes (, , and ) were differentially expressed as laying hens' reproductive organs developed. These genes were simultaneously upregulated in cancerous ovaries compared with those in normal ovaries. Their transcripts were abundantly localized in glandular epithelial cells of cancerous ovaries. Our results indicate that , , and play critical roles in the development of reproductive organs and ovarian carcinogenesis in laying hens, suggesting that simultaneous overexpression of these genes might serve as a prognostic factor for ovarian cancer.

Postnatal development and histofunctional differentiation of the oviduct in the broad-snouted caiman (Caiman latirostris).

Caiman latirostris is a South American crocodilian species characterized as a sentinel of the presence of endocrine-disrupting compounds (EDCs). Evaluating developmental events in hormone-dependent organs, such as the oviduct, is crucial to understand physiological postnatal development, to identify putative periods of exposure sensitive to EDCs, and/or to identify biomarkers useful to evaluate the effects of EDC exposure. In this study, we describe the histomorphological features of C. latirostris oviducts by establishing the ontogeny of changes at cellular, tissue and molecular levels from the neonatal to the pre-pubertal juvenile stages. Since the histological diagnosis of the adenogenic oviduct lies on a group of features, here we defined a histofunctional score system and a cut-off value to distinguish between preadenogenic and adenogenic oviducts. Our results showed that the maturation of the C. latirostris oviduct is completed postnatally and characterized by changes that mimic the pattern of histological modifications described for the mammalian uterus. Ontogenic changes in the oviductal epithelium parallel changes at subepithelial level, and include collagen remodeling and characteristic spatial-temporal patterns of α-actin and desmin. The expression pattern of estrogen receptor alpha and progesterone receptor evidenced that, even at early postnatal developmental stages, the oviduct of C. latirostris is a target organ of endogenous and environmental hormones. Besides, oviductal adenogenesis seems to be an estrogen-dependent process. Results presented here provide not only insights into the histophysiological aspect of caiman female reproductive ducts but also new tools to better characterize caimans as sentinels of endocrine disruption.

Constitutive Notch Signaling Causes Abnormal Development of the Oviducts, Abnormal Angiogenesis, and Cyst Formation in Mouse Female Reproductive Tract.

The Notch signaling pathway is critical for the differentiation of many tissues and organs in the embryo. To study the consequences of Notch1 gain-of-function signaling on female reproductive tract development, we used a cre-loxP strategy and Amhr2-cre transgene to generate mice with conditionally activated Notch1 (Rosa(Notch1)). The Amhr2-cre transgene is expressed in the mesenchyme of developing female reproductive tract and in granulosa cells in the ovary. Double transgenic Amhr2-cre, Rosa(Notch1) females were infertile, whereas control Rosa(Notch1) mice had normal fertility. All female reproductive organs in mutants showed hemorrhaging of blood vessels progressing with age. The mutant oviducts did not develop coiling, and were instead looped around the ovary. There were multiple blockages in the lumen along the oviduct length, creating a barrier for sperm or oocyte passage. Mutant females demonstrated inflamed uteri with increased vascularization and an influx of inflammatory cells. Additionally, older females developed ovarian, oviductal, and uterine cysts. The significant change in gene expression was detected in the mutant oviduct expression of Wnt4, essential for female reproductive tract development. Similar oviductal phenotypes have been detected previously in mice with activated Smo and in beta-catenin, Wnt4, Wnt7a, and Dicer conditional knockouts, indicating a common regulatory pathway disrupted by these genetic abnormalities.

Immunohistochemical localization of progesterone receptor isoforms and estrogen receptor alpha in the chicken oviduct magnum during development.

In this work, the immunohistochemical expression of progesterone receptor (PR) isoforms and estrogen receptor alpha (ER-α), as well as the histomorphometric changes of the magnum region of the left oviduct from 8-day-old chicken embryos to one-month-old chickens were evaluated. Results indicate evident histological changes in the oviduct magnum during development mainly in the magnum's mucosa. Immunohistochemical analysis showed that the oviduct magnum from 8-day-old chicken embryos to one-day-old chickens did not present any PR isoform, but the oviduct magnum of one-week and one-month-old chickens expressed PR in the nuclei of all cell types. In epithelial cells, PR-B was the only isoform expressed; in muscle and serosa cells, PR-A isoform was the only isoform expressed; and stromal cells expressed both isoforms. The results also demonstrate positive ER-α immunostaining in the nuclei of different cells from embryonic life to later developmental stages of the oviduct magnum. Data indicate that the variations of ER-α or PR expression or dominance of either PR expression is differentially regulated depending on the cell type, the development of the oviduct, and in an age-specific manner. These variations in sex steroids hormone receptors are related with histological changes of the oviduct magnum through development.

Atrazine triggers developmental abnormality of ovary and oviduct in quails (Coturnix Coturnix coturnix) via disruption of hypothalamo-pituitary-ovarian axis.

There has been a gradual increase in production and consumption of atrazine (ATR) in agriculture to meet the population rising demands. Female reproduction is necessary for growth and maintenance of population. However, ATR impact on females and particularly ovarian developmental toxicity is less clear. The aim of this study was to define the pathways by which ATR exerted toxic effects on ovarian development of ovary and hypothalamo-pituitary-ovarian (HPO) axis. Female quails were dosed by oral gavage from sexual immaturity to maturity with 0, 50, 250 and 500 mg ATR/kg/d for 45 days. ATR had no effect on mortality but depressed feed intake and growth and influenced the biochemical parameters. Notably, the arrested development of ovaries and oviducts were observed in ATR-exposed quails. The circulating concentrations of E2, P, LH and PRL were unregulated and FSH and T was downregulated in ATR-treated quails. The mRNA expression of GnRH in hypothalamo and LH in pituitary and FSH in ovary was downregulated significantly by ATR exposure and FSH and PRL in pituitary were upregulated. ATR exposure upregulated the level of P450scc, P450arom, 3ß-HSD and 17ß-HSD in ovary and downregulated ERß expression in female quails. However, ATR did not change ERα expression in ovary. This study provides new insights regarding female productive toxicology of ATR exposure. Ovary and oviduct in sexually maturing females were target organs of ATR-induced developmental toxicity. We propose that ATR-induced developmental abnormality of ovary and oviduct is associated with disruption of gonadal hormone balance and HPO axis in female quails.

Diethylstilbestrol regulates expression of avian apolipoprotein D during regression and recrudescence of the oviduct and epithelial-derived ovarian carcinogenesis.

Apolipoprotein D (APOD) is a glycoprotein which is widely expressed in mammalian tissues. It is structurally and functionally similar to the lipocalins which are multiple lipid-binding proteins that transport hydrophobic ligands and other small hydrophobic molecules, including cholesterol and several steroid hormones. Although multiple functions for APOD in various tissues have been reported, its expression, biological function, and hormonal regulation in the female reproductive system are not known. Thus, in this study, we focused on correlations between APOD and estrogen during development, differentiation, regression, and regeneration of the oviduct in chickens and in the development of ovarian carcinogenesis in laying hens. Results of the present study indicated that APOD messenger RNA (mRNA) expression increased (P < 0.001) in the luminal and glandular (GE) epithelia of the chicken oviduct in response to diethylstilbestrol (a nonsteroidal synthetic estrogen). In addition, the expression of APOD mRNA and protein decreased (P < 0.001) as the oviduct regressed during induced molting, and gradually increased (P < 0.001) with abundant expression in GE of the oviduct during recrudescence after molting. Furthermore, APOD mRNA and protein were predominantly localized in GE of cancerous, but not normal ovaries from laying hens. Collectively, results of the present study suggest that APOD is a novel estrogen-stimulated gene in the chicken oviduct which likely regulates growth, differentiation, and remodeling of the oviduct during oviposition cycles. Moreover, up-regulated expression of APOD in epithelial cell-derived ovarian cancerous tissue suggests that it could be a candidate biomarker for early detection and treatment of ovarian cancer in laying hens and in women.

Characterization and miRNA-mediated posttranscriptional regulation of vitelline membrane outer layer protein I in the adult chicken oviduct.

The laying hen is the best model for oviduct growth and development. The chicken oviduct produces the egg components, including the egg white and eggshell. However, the mechanism of egg component production during oviduct development requires further investigation. Vitelline membrane outer layer protein 1 (VMO-1) is found in the outer layer of the vitelline membrane of avian eggs. Comparison of the chicken VMO-1 protein-coding sequence and the human, mouse, rat, and bovine VMO-1 proteins via multiple sequence alignment analysis revealed high degrees of homology of 55%, 53%, 48%, and 54%, respectively. Although the avian homologue of VMO-1 is highly expressed in the magnum of the oviduct, little is known about the transcriptional and posttranscriptional regulation of VMO-1 during oviduct development. The results of this study revealed that estrogen induces VMO-1 messenger RNA (mRNA) expression in oviduct cells in vitro. The expression of genes interacting with VMO-1 by RNA interference (RNAi) functional analysis revealed that ovomucin expression was decreased by VMO-1 silencing. In addition, gga-miR-1623, 1552-3p, and 1651-3p influenced VMO-1 expression via its 3'-UTR, suggesting the posttranscriptional regulation of VMO-1 expression in chickens. Collectively, these results suggest that VMO-1 is an estrogen-induced gene that is posttranscriptionally regulated by microRNAs (miRNAs). The present study may contribute to an understanding of egg component production during chicken oviduct development.

Follistatin is essential for normal postnatal development and function of mouse oviduct and uterus.

Female mice lacking the follistatin gene but expressing a human follistatin-315 transgene (tghFST315) have reproductive abnormalities (reduced follicles, no corpora lutea and ovarian-uterine inflammation). We hypothesised that the absence of follistatin-288 causes the abnormal reproductive tract via both developmental abnormalities and abnormal ovarian activity. We characterised the morphology of oviducts and uteri in wild type (WT), tghFST315 and follistatin-knockout mice expressing human follistatin-288 (tghFST288). The oviducts and uteri were examined in postnatal Day-0 and adult mice (WT and tghFST315 only) using histology and immunohistochemistry. Adult WT and tghFST315 mice were ovariectomised and treated with vehicle, oestradiol-17ß (100ng injection, dissection 24h later) or progesterone (1mg×three daily injections, dissection 24h later). No differences were observed in the oviducts or uteri at birth, but abnormalities developed by adulthood. Oviducts of tghFST315 mice failed to coil, the myometrium was disorganised, endometrial gland number was reduced and oviducts and uteri contained abundant leukocytes. After ovariectomy, tghFST315 mice had altered uterine cell proliferation, and inflammation was maintained and exacerbated by oestrogen. These studies show that follistatin is crucial to postnatal oviductal-uterine development and function. Further studies differentiating the role of ovarian versus oviductal-uterine follistatin in reproductive tract function at different developmental stages are warranted.

Pivotal roles for hormonally regulated expression of the HEP21 gene in the reproductive tract of chickens for oviduct development and in ovarian carcinogenesis.

Hen egg protein (HEP21) is a 21-kDa secreted protein and has a single copy of the Ly6/uPAR domain. Although HEP21 is expressed primarily in the chicken oviduct, its biological function(s) in the reproductive system of chickens is not known. Thus, in the present study, we investigated expression patterns of HEP21 with respect to hormonal regulation, oviduct development, changes in expression in laying hens undergoing induced molting, and in the development of ovarian carcinogenesis in laying hens. Results of present study indicated that HEP21 messenger RNA (mRNA) expression increased (P < 0.001) in the chicken oviduct in response to estrogen. In situ hybridization analyses revealed expression of HEP21 mRNA predominantly in glandular (GE) and luminal epithelia of the magnum of the chicken oviduct in response to estrogen. The expression of HEP21 mRNA decreased (P < 0.001) as the oviduct regressed during induced molting and increased (P < 0.001) with recrudescence of the oviduct following molting. HEP21 mRNA was most abundant in GE of the oviduct during recrudescence, but not during oviduct regression following induced molting. Moreover, we found abundant expression of HEP21 in GE of cancerous ovaries, but not in normal ovaries of hens. Collectively, results of present study suggest that HEP21 is an estrogen-responsive gene in the oviduct of hens that likely regulates development of the chicken oviduct, and egg production and formation. Furthermore, there is increased expression of HEP21 in epithelial-derived ovarian cancer suggesting that HEP21 could be used for diagnosis and monitoring carcinogenesis in laying hens and in women.

Hormonal regulation of beta-catenin during development of the avian oviduct and its expression in epithelial cell-derived ovarian carcinogenesis.

Beta-catenin (CTNNB1) is a dual function molecule that acts as a key component of the cadherin complex and WNT signaling pathway. It has a crucial role in embryogenesis, tumorigenesis, angiogenesis and progression of metastasis. Recently, it has been suggested that the CTNNB1 complex is a major regulator of development of the mouse oviduct and uterus. However, little is known about the CTNNB1 gene in chickens. Therefore, in this study, we focused on the CTNNB1 gene in the chicken reproductive tract and hormonal control of its expression in the chicken oviduct. CTNNB1 was localized specifically to the luminal and glandular epithelium of the four segments of chicken oviduct and DES (diethylstilbestrol, a synthetic non-steroidal estrogen) increased its expression primarily in LE of the magnum. In addition, CTNNB1 mRNA and protein were expressed abundantly in glandular epithelium of endometrioid-type ovarian carcinoma, but not in normal ovaries. Moreover, CTNNB1 expression was post-transcriptionally regulated via its 3'-UTR by binding with target miRNAs including miR-217, miR-1467, miR-1623 and miR-1697. Collectively, these results indicate that CTNNB1 is a novel gene regulated by estrogen in epithelial cells of the chicken oviduct and that it is also abundantly expressed in epithelial cells of endometrioid-type ovarian carcinoma suggesting that it could be used as a marker for diagnosis of ovarian cancer in laying hens and women.

Sex-specific expression of CTNNB1 in the gonadal morphogenesis of the chicken.

BACKGROUND: Beta-catenin (CTNNB1), as a key transcriptional regulator in the WNT signal transduction cascade, plays a pivotal role in multiple biological functions such as embryonic development and homeostasis in adults. Although it has been suggested that CTNNB1 is required for gonad development and maintenance of ovarian function in mice, little is known about the expression and functional role of CTNNB1 in gonadal development and differentiation in the chicken reproductive system. METHODS: To examine sex-specific, cell-specific and temporal expression of CTNNB1 mRNA and protein during gonadal development to maturation of reproductive organs, we collected left and right gonads apart from mesonephric kidney of chicken embryos on embryonic day (E) 6, E9, E14, E18, as well as testes, oviduct and ovaries from 12-week-old and adult chickens and performed quantitative PCR, in situ hybridization, and immunohistochemical analyses. In addition, localization of Sertoli cell markers such as anti-Müllerian hormone (AMH), estrogen receptor alpha (ESR1), cyclin D1 (CCND1) and N-cadherin (CDH2) during testicular development was evaluated. RESULTS: Results of the present study showed that CTNNB1 mRNA and protein are expressed predominantly in the seminiferous cords on E6 to E14 in the male embryonic gonad, and are mainly localized to the medullary region of female embryonic gonads from E6 to E9. In addition, CTNNB1 mRNA and protein are abundant in the Sertoli cells in the testes and expressed predominantly in luminal epithelial cells of the oviduct, but not in the ovaries from 12-week-old and adult chickens. Concomitant with CTNNB1, AMH, ESR1, CCND1 and CDH2 were detected predominantly in the seminiferous cord of the medullary region of male gonads at E9 (after sex determination) and then maintained or decreased until hatching. Interestingly, AMH, ESR1, CCND1 and CDH2 were located in seminiferous tubules of the testes from 12-weeks-old chickens and ESR1, CCND1 and CDH2 were expressed predominantly in the Sertoli cells within seminiferous tubules of adult testes. CONCLUSIONS: Collectively, these results revealed that CTNNB1 is present in gonads of both sexes during embryonic development and it may play essential roles in differentiation of Sertoli cells during formation of seminiferous tubules during development of the testes.

Early life exposure to genistein and daidzein disrupts structural development of reproductive organs in female mice.

In mice, exposure to isoflavones (ISO), abundant in soy infant formula, during the first 5 d of life alters structural and functional development of reproductive organs. Effects of longer exposures are unknown. The study objective was to evaluate whether exposure to a combination of daidzein and genistein in the first 10 compared to 5 d of life results in greater adverse effects on ovarian and uterine structure in adult mice. Thirteen litters of 8-12 pups were cross-fostered and randomized to corn oil or ISO (2 mg daidzein + 5 mg genistein/kg body weight/d) for the first 5 or 10 d of life. The 10-d protocol mimicked the period when infants are fed soy protein formula (SPF) but avoids the time when suckling pups can consume mother's diet. Body and organ weights, and histology of ovaries and uteri were analyzed. There were no differences in the ovary or uterus weight, number of ovarian follicles, number of multiple oocyte follicles, or percent of ovarian cysts with 5 or 10 d ISO intervention compared to respective controls. The 10-d ISO group had higher body weights from 6 d to 4 mo of age and a higher percent of hyperplasia in the oviduct than the respective control. Lower number of ovarian corpus lutea and a higher incidence of abnormal changes were reported in the uteri of both ISO groups compared to their respective controls. Five and 10-d exposure to ISO had similar long-lasting adverse effects on the structure of ovaries and uterus in adult mice. Only the 10-d ISO exposure resulted in greater body weight gain at adulthood.

The morphological and functional observation of the gap junction proteins in the oviduct epithelia in young and adult hamsters.

The histological morphology of oviduct epithelia have been described well, however, the expression pattern of the gap junction proteins in the cells, and the function related with the proteins, such as [Ca2+]i dynamics pattern of living oviduct epithelia at different ages have not been clarified. We used immunohistochemistry to compare the expression pattern of gap junction proteins in the cells of the young and adult groups. Moreover, we used real-time confocal microscopy to observe the spontaneous Ca2+ oscillation (spontaneous fluctuation) in freshly isolated epithelia (ciliated cells) in ampulla potion of oviduct from the two groups. The results show as demonstrated by immunohistochemistry the gap junction proteins (Cx26, Cx32 and Cx43) formed a well-regulated expression in the young animals, but not in the adult animals. In addition, the [Ca2+]i dynamics of ciliated cells in freshly oviduct epithelia have a spontaneous fluctuation pattern that occurs without any stimulation in the young animals, but this pattern was not observed in the adult animals. In conclusions, our findings suggest that gap junctions regulate the spontaneous fluctuation of [Ca2+]i dynamics in ciliated cells of oviduct epithelia in young animals.

Differential expression of alpha 2 macroglobulin in response to dietylstilbestrol and in ovarian carcinomas in chickens.

BACKGROUND: Alpha 2 macroglobulin (A2M; also known as ovostatin), a homotetrameric protein with four disulfide-linked subunits, has the unique feature of inactivating/inhibiting most known proteases including serine-, threonine-, cysteine-, aspartic- and metalloproteases. In chickens, A2M has been identified and characterized biochemically, but little is known of its functional role(s) in the oviduct, hormonal regulation of expression or its expression in ovarian carcinomas in chickens. Therefore, we investigated estrogen regulation of A2M gene expression during development of the chicken oviduct, and its expression in normal and cancerous ovaries from chickens. METHODS: To determine tissue-specific expression of A2M in chickens, we collected various organs from male and female chickens and performed RT-PCR analyses. To examine A2M gene expression in the oviduct of 1-week-old female chicks that received a subcutaneous implant of 15 mg DES in the abdominal region for 20 days, we performed RT-PCR, qPCR and in situ hybridization analyses using cDNAs from control- (n=5) and DES-treated oviducts (n=5), and then each segment of the oviduct from DES-treated chicks. To determine if A2M is a biomarker of ovarian cancer in hens, we collected cancerous (n=10) ovaries from a total of 136 chickens which had completely stopped egg-laying and performed RT-PCR and in situ hybridization analyses. RESULTS: We found that A2M is most abundant in the chicken oviduct, specifically luminal (LE) and glandular epithelia (GE), but it was not detected in any other tissues of either sex. We then determined that DES (dietylstilbestrol, a synthetic nonsteroidal estrogen) increased A2M mRNA only in LE and GE of the oviduct of chicks. Further, expression of A2M was most abundant in GE of endometrioid adenocarcinoma of cancerous, but not normal ovaries of hens. CONCLUSIONS: Collectively, results of the present study indicate that A2M is novel estrogen-stimulated gene expressed in LE and GE of the chicken oviduct and may be used for monitoring effects of therapies for ovarian cancer in laying hens.

Avian SERPINB11 gene: characteristics, tissue-specific expression, and regulation of expression by estrogen.

Serpins, a group of proteins with similar structural and functional properties, were first identified based on their unique mechanism of action: their inhibition of proteases. While most serpins have inhibitory roles, certain serpins are not involved in canonical proteolytic cascades but perform diverse functions including storage of ovalbumin in egg white, transport of hormones (thyroxine- and cortisol-binding globulin), and suppression of tumors. Of these, serpin peptidase inhibitor, clade B, member 11 (SERPINB11) is not an inhibitor of known proteases in humans and mice, and its function is unknown. In the present study, the SERPINB11 gene was cloned, and its expression profile was analyzed in various tissues from chickens. The chicken SERPINB11 gene has an open reading frame of 1346 nucleotides that encode a protein of 388 amino acids that has moderate homology (38.8%-42.3%) to mammalian SERPINB11 proteins. Importantly, SERPINB11 mRNA is most abundant in the chicken oviduct, specifically luminal and glandular epithelia, but it was not detected in any other chicken tissues of either sex. We then determined effects of diethylstilbestrol (DES; a synthetic nonsteroidal estrogen) on SERPINB11 expression in the chicken oviduct. Treatment of young chicks with DES induced SERPINB11 mRNA and protein only in luminal and glandular epithelial cells of the oviduct. Collectively, these results indicate that the novel estrogen-induced SERPINB11 gene is expressed only in epithelial cells of the chicken oviduct and implicate SERPINB11 in regulation of oviduct development and differentiated functions.

Permanent oviduct posteriorization after neonatal exposure to the phytoestrogen genistein.

BACKGROUND: Preimplantation embryo loss during oviduct transit has been observed in adult mice after a 5-day neonatal exposure to the phytoestrogen genistein (Gen; 50 mg/kg/day). OBJECTIVE: We investigated the mechanisms underlying the contribution of the oviduct to infertility. METHODS: Female mice were treated on postnatal days 1-5 with corn oil or Gen (50 mg/kg/day). We compared morphology, gene expression, and protein expression in different regions of the reproductive tracts of Gen-treated mice with those of control littermates at several time points. RESULTS: Neonatal Gen treatment resulted in substantial changes in expression of genes that modulate neonatal oviduct morphogenesis, including Hoxa (homeobox A cluster), Wnt (wingless-related MMTV integration site), and hedgehog signaling genes. An estrogen receptor antagonist blocked these effects, indicating that they were induced by the estrogenic activity of Gen. Oviducts of adults treated neonatally with Gen had abnormal morphology and were stably "posteriorized," as indicated by altered Hoxa gene patterning during the time of treatment and dramatic, permanent up-regulation of homeobox genes (e.g., Pitx1, Six1) normally expressed only in the cervix and vagina. CONCLUSIONS: Neonatal exposure to estrogenic environmental chemicals permanently disrupts oviduct morphogenesis and adult gene expression patterns, and these changes likely contribute to the infertility phenotype.