The deleted in azoospermia like (DAZL) gene has been identified in many vertebrate species. DAZL shows high homology with deleted in azoospermia (DAZ) genes that identified only in humans, great apes and Old World monkeys, and boule homolog (BOLL) that identified in many vertebrate species. These genes encode RNA binding proteins (RBP), which regulate the post-transcriptional functions of several genes. In humans, DAZ copies are linked to Y chromosome, while DAZL and BOLL are linked to chromosomes 3 and 2, respectively. DAZ copies has been reported to express in prenatal and postnatal germ cells, particularly in the premeiotic spermatogonia. BOLL has been reported to express during the meiotic G2/M transition in germ cells. DAZL has been reported to express in all stages of germ cells. Compared to humans and mice, the detailed functionalities of DAZL is not clear in many vertebrate species. In our studies, we use chickens as an animal model to examine the expression profiling of DAZL gene in germ cells right from the early embryonic development to the adult. Also, we are studying the effects of small interfering RNA (siRNA) mediated knockdown of DAZL and Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/CRISPR associated protein 9 (CRISPR/Cas9) mediated knockout of DAZL in the chicken primordial germ cells (PGCs). In the chicken, DAZL is linked to chromosome 2 (2p1.3-p1.2), and encodes a 289 amino acids protein. By in situ hybridization, we detected a strong expression of DAZL in the germ plasm of chicken oocytes. Later, the expression of DAZL was strongly detected in all stages of intrauterine development and post-ovipositional development especially in the PGC specifying cells. Moreover, the expression of DAZL was strong and constant in the male and female germ cells until adult stage. The siRNA mediated knockdown of DAZL significantly reduced the PGCs proliferation and increased the apoptosis in vitro. We examined the knockout efficiency of DAZL using CRISPR/Cas9 technique in chicken DF1 fibroblast cell line, prior to test in the PGCs. The results of T7 endonuclease I (T7E1) assay and subsequent sequencing indicates clear mutations on the DAZL gene in DF1 cells, and the method could be applicable to cause mutations on the DAZL gene in PGCs. In conclusion, chicken DAZL express in all stages of germ cells as a germ line marker, and alteration in the gene expression causes germ cells impairment.

SERPINB3 (also known Squamous cell carcinoma antigen 1, SCCA1) is involved in apoptosis, immune response, cell migration and invasiveness of cells. It has been investigated in various types of squamous cell carcinoma. Therefore we investigated the functional role of SERPINB3 gene in human epithelial ovarian cancer (EOC) using laying hens, the most relevant animal model. In 136 laying hens, EOC was found in 10 (7.4%). We compared the expression and localization of SERPINB3 using RT-PCR, quantitative RT-PCR, in situ hybridization and immunohistochemistry, and SERPINB3 activation was detected in chicken and human ovarian cancer cell lines using immunofluorescence microscopy. Thereafter, we examined the prognostic value of SERPINB3 expression in patients with EOC by multivariate linear logistic regression and Cox’ proportional hazard analyses. In present study, SERPINB3 mRNA was induced in cancerous ovaries (p< 0.01), and it was only expressed in the glandular epithelium(GE) of cancerous ovaries of laying hens. SERPINB3 protein was localized predominantly to the nucleus of glandular epithelium in cancerous ovaries of laying hens, and it was abundant in the nucleus of both chicken and human ovarian cancer cell lines. In 109 human patients with EOC, 15 (13.8%), 66 (60.6%) and 28 (25.7%) of those patients showed weak, moderate and strong expression of SERPINB3 protein, respectively. Strong expression of SERPINB3 protein was a prognostic factor for platinum resistance (adjusted OR, 5.94; 95% Confidence Limits, 1.21-29.15). Therefore SERPINB3 may play an important role in ovarian carcinogenesis and be a novel biomarker for predicting platinum resistance and a poor prognosis for survival in patients with EOC. This research was funded by the World Class University (WCU) program (R31-10056), Basic Science Research Program (2010- 0013078) through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology and by the Next-Generation BioGreen 21 Program (No.PJ008142), Rural Development Administration, Republic of Korea.

Polycystic ovarian syndrome (PCOS) is a heterogeneous syndrome associated with follicle growth arrest, dysregulated sex hormone profile, hyperthecosis and insulin resistance. Chemerin, a novel adipokine, is associated with obesity and metabolic syndrome. Although obese women and in PCOS subjects have elevated plasma chemerin levels, whether and how chemerin is involved in the regulation of follicular growth/steroidogenesis and pathogenesis of PCOS is unknown. Our objective is to better understand the complex regulatory mechanisms involved in the control of these processes and gain insights in their dysregulation in the pathogenesis of PCOS. We hypothesize that: (a) hyperandrogenism induces small and medium antral follicle growth arrest and ovarian structural changes, resulting from granulosa cell and oocyte apoptosis and theca cell survival, and (b) chemerin regulates follicular growth and steroidogenesis and contributes to the pathogenesis of PCOS. Using immature rats (day 13～15 for follicle culture and day 21～24 for granulosa cells culture) and a chronically androgenized rat model [dihydrotestosterone (DHT); 83 μg daily, day 21～105] which recapitulates the reproductive and metabolic phenotypes of human PCOS, we have examined the granulosa cell expression patterns of chemerin and its receptor CMKLR1 and their steroidogenic and follicle growth capability. DHT treatment resulted in decreased follicle numbers in preantral to preovulatory stages and absence of corpus luteum, but increased numbers of condensed atypical follicles. Atypical follicles, constituted predominantly of theca cells, exhibited high expression of calpain and down‐regulation of the cytoskeletal protein substrates vimentin, fodrin and β‐tubulin. Granulosa cell aromatase expression was significantly down‐regulated, a response accompanied by increased activated caspase‐3 content and DNA fragmentation. While PTEN levels were considerably higher in granulosa cells in the PCOS rats than controls, phospho‐Akt (Ser473) content was lower. In addition, DHT also activated granulosa cell caspase‐3, decreased XIAP, PARP and phospho‐Akt contents and induced apoptosis in vitro, responses that could be attenuated by forced expression of XIAP. These findings are consistent with our hypothesis that dysregulated follicular growth in PCOS is associated with changes in follicular growth dynamics and follicle cell fate, a consequence of dysregulated interactions of pro‐survival (p‐Akt, XIAP, PARP) and proapoptotic (calpain, PTEN, caspase‐3) modulators in a cell‐specific manner. Chemerin and CMKLR1 were expressed in granulosa cells and negatively regulated by gonadotropin in vivo and in vitro. Serum and ovarian chemerin levels in DHT‐treated rats were elevated, and associated with arrested early antral follicular growth, remodeling of the follicle wall and decreased expression of p450 side‐chain cleavage enzyme (p450- scc), aromatase and hydroxysteroid dehydrogenases. Recombinant chemerin inhibited FSH ‐ induced estradiol secretion in granulosa cells from DHT‐treated rats in vitro. Chemerin also suppressed basal and FSH‐ and GDF9‐induced follicle growth and estradiol/ progesterone production in preantral follicle cultures. Moreover, chemerin suppressed FSH‐induced p450scc/aromatase expression and progesterone/estradiol secretion in immature rat granulosa cells in vitro. These studies demonstrate that chemerin is a novel negative regulator in FSH‐induced follicular growth and steroidogenesis and support the notion that the dysregulation of chemerin expression and function contributes to pathogenesis of PCOS. Our observations also suggest that this chronically androgenized rat model may be useful not only for studies on the long term effects of androgen on folliculogenesis, but also on the pathophysiology of PCOS. * This work was supported by grants from the Canadian Institutes of Health Research (CIHR; MOP‐119381) and the World Class University (WCU) program through the Ministry of Education, Science and Technology funded by the National Research Foundation of Korea (R31‐10056).

Serpins are a superfamily of related protease inhibitors with common structural features and inhibitory mechanisms. However, SERPINA 14 in mammals does not have inhibitory activity against most known proteases. Rather, it may have an immunoregulatory role in mammals to prevent rejection of the fetal allograft by inhibiting lymphocyte proliferation and natural killer cell function. In the pig, SERPINA14 is involved in iron transport to the fetus by binding to and stabilizing the iron-binding protein uteroferrin (ACP5). In chickens, these very little known about serpins in chickens. Therefore, we investigated the expression patterns of serpin genes in the oviduct of adult hens and in the oviduct of 37-day-old chicks treated with an estrogen analogue, diethylstilbestrol (DES). Results indicated that SERPINB3 and SERPINB11 genes were highly expressed in oviducts of DES-treated chicks, but not in oviducts of control chicks. Both SERPINB3 and SERPINB11 transcripts were localized specifically to the gland-like areas of oviducts of DES-treated chicks. Immunohistochemical analyses confirmed that SERPINB3 and SERPINB11 proteins were present in the gland-like area and luminal epithelium of the oviducts of DES-treated chicks. Collectively, the results suggest that SERPINB3 and SERPINB11 are expressed in response to estrogens and they have distinct functions related to development and differentiation of the mature oviduct in hens.

S-adenosylhomocysteine hydrolase-like protein 1 (AHCYL1), also known as IP3 receptor- binding protein released with IP3 (IRBIT), is a member of the AHCY-like protein family. AHCYL1 protein regulates IP3-induced Ca2+ release in the cytoplasm of cells and, therefore, is likely to be an important gene regulating various biological processes in the oviduct of chickens. Inmammals, expression is greatest during activation of dendritic cells which are antigen presenting cells associated with immunoregulatory processes in blood and skin. However, the identification of the AHCYL1 gene in chickens has not been investigated. In the present study, we first used RT-PCR to demonstrate AHCYL1 gene expression in adult chicken organs and oviducts of immature chickens treated with DES (diethylstilbesterol, a synthetic estrogen agonist). The results indicated that AHCYL1 mRNA is expressed in chicken reproductive organs (testis, ovary and oviduct). Inaddition, expression of AHCYL1 mRNA increased in response to DES-treated immature oviducts compared to the non-treated control immature oviducts of chickens. Interestingly, AHCYL1 was abundant in the cytoplasm of luminal and glandular epithelia, but not in other cell-types such as stroma and connective tissues, of the chicken oviduct. These results suggest that AHCYL1 is a novel estrogen-stimulated gene associated with development of the chicken oviduct, as well as functions of oviductal glandular and luminal epithelia that may include activation of resident immune cells, such as dendritic cells.