Testis-specific novel transcripts in chicken: in situ localization and expression pattern profiling during sexual development.

Rengaraj, Deivendran,Kim, Duk Kyung,Zheng, Ying Hui,Lee, Sang In,Kim, Heebal,Han, Jae Yong Society for the Study of Reproduction [etc.] 2008 BIOLOGY OF REPRODUCTION Vol.79 No.3

<P>Tissue-specific novel transcripts expressed during sexual development were examined by RT-PCR, quantitative RT-PCR (qRT-PCR), and in situ hybridization to provide data for chicken genomics. Public databases for transcript data have been constructed with known and unknown sequences of various tissues from different animals. However, the expression patterns and functions of the transcripts are less known. From the The Institute for Genomics Research Gallus gallus library, we examined 291 tentative consensus (TC) sequences that assembled 100% with transcripts by RT-PCR during male and female sexual development from Embryonic Day 6 to 25 wk of age. We found 85 TC sequences that were specific to testicular development; of these, 43 TC sequences were exclusively upregulated in 25-wk-old testis. Another 52 TC sequences were not specific to one tissue, but occurred in the testis and ovary at different developmental ages. Twelve testis-specific TC sequences upregulated in 25-wk-old testis were randomly selected and further examined with qRT-PCR. For precise localization, these 12 testis-specific TC sequences were examined by in situ hybridization with 25-wk-old adult testis. Six TC sequences were strongly expressed in secondary spermatocytes and haploid spermatids until spermatozoa release. Another six TC sequences were differentially expressed in the adluminal compartment of seminiferous tubules. Among the testis-specific TC sequences, TC120901 is a known gene, phospholipase C, zeta (PLCZ1). Our data provide potential insight into gene expression and genomic information on novel transcripts that are important to avian reproduction.</P>

Effect of endoplasmic reticulum stress on porcine oocyte maturation and parthenogenetic embryonic development in vitro.

Zhang, Jin Yu,Diao, Yun Fei,Oqani, Reza K,Han, Rong Xun,Jin, Dong Il Society for the Study of Reproduction [etc.] 2012 BIOLOGY OF REPRODUCTION Vol.86 No.4

<P>X-box-binding protein 1 (XBP1) is an important regulator of a subset of genes active during endoplasmic reticulum (ER) stress. In the present study, we analyzed XBP1 level and location to explore the effect of ER stress on oocyte maturation and developmental competency of porcine embryos in an in vitro culture system. First, we examined the localization of XBP1 at different meiotic stages of porcine oocytes and at early stages of parthenogenetic embryo development. Fluorescence staining showed that expression of functional XBP1 was weak in mature oocytes and at the 1-, 2-, and 8-cell stages of embryos but abundant at the germinal vesicle (GV), 4-cell, morula, and blastocyst stages. In addition, RT-PCR revealed that both spliced XBP1 (XBP1-s) and unspliced XBP1 (XBP1-u) were expressed at the GV, 4-cell, morula, and blastocyst stages. Tunicamycin, an ER stress inducer, induced active XBP1 protein in nuclei of 4-cell embryos. Next, porcine embryos cultured in the presence of tauroursodeoxycholate, an ER stress inhibitor, were studied. Total cell numbers and the extent of the inner cell mass increased (P < 0.05), whereas the rate of nuclear apoptosis decreased (P < 0.05). Moreover, expression of the antiapoptotic gene BCL2 increased, whereas expression of the proapoptotic genes BCL2L1 (Bcl-xl) and TP53 decreased. The results indicated that inhibition of ER stress enhanced porcine oocyte maturation and embryonic development by preventing ER stress-mediated apoptosis in vitro.</P>

Avian prostatic acid phosphatase: estrogen regulation in the oviduct and epithelial cell-derived ovarian carcinomas.

Bae, Hyocheol,Lim, Whasun,Bae, Seung-Min,Bazer, Fuller W,Choi, Youngsok,Song, Gwonhwa Society for the Study of Reproduction [etc.] 2014 BIOLOGY OF REPRODUCTION Vol.91 No.1

<P>Prostatic acid phosphatase (ACPP) is a glycoprotein that is mainly synthesized and secreted by glandular epithelial cells (GE) of the prostate, and it is well known as a biomarker for prostate cancer. Although ACPP was used as prognostic/diagnostic indicator and studied to elucidate regulatory mechanism(s) during several decades in humans, its role is not clearly understood. Gene profiling data using a chicken DNA microarray revealed that ACPP increased significantly during remodeling and recrudescence of the oviduct in response to estrogen. Thus, in this study, we investigated the expression and hormonal regulation of ACPP gene in the reproductive tracts of chickens. ACPP was specifically detected in the luminal cells (LE) and GE of chicken oviduct, and diethylstilbestrol (a synthetic nonsteroidal estrogen) stimulated its expression during development of the oviduct. In addition, ACPP mRNA and protein were localized to LE and GE during the regeneration phase of the oviduct of laying hens during induced molting. Furthermore, ACPP mRNA and protein were abundant in GE of ovarian carcinoma, but not in normal ovaries. Moreover, strong expression of ACPP protein was detected in epithelial cells of cancerous ovaries from women. Collectively, results of the present study are the first to show that ACPP is a novel estrogen-stimulated gene in the oviductal epithelial cells of the chicken and that its expression increases significantly in epithelial cells of ovarian carcinoma, which indicates that it may be a candidate biomarker for diagnosis of epithelia-derived ovarian cancer in women.</P>

Comprehensive analysis of reproductive ADAMs: relationship of ADAM4 and ADAM6 with an ADAM complex required for fertilization in mice.

Han, Cecil,Choi, Eunyoung,Park, Inju,Lee, Boyeon,Jin, Sora,Kim, Do Han,Nishimura, Hitoshi,Cho, Chunghee Society for the Study of Reproduction [etc.] 2009 BIOLOGY OF REPRODUCTION Vol.80 No.5

<P>A Disintegrin And Metalloprotease (ADAM) family members expressed in male reproductive tissues are divided phylogenetically into three major groups. In the present study, we analyzed six ADAMs in one of the groups (ADAMs 4, 6, 24, 26, 29, and 30) of which function is largely unknown. Our results showed that most of the ADAMs undergo unique processing during sperm maturation and are located at the surface of sperm head. We found that the levels of ADAM4 and ADAM6 are dramatically reduced in Adam2 and Adam3 knockout sperm defective in various fertilization processes. We observed premature processing of ADAM4 in the Adam3-null mice. Furthermore, we obtained a result showing complex formation of ADAM6 with ADAM2 and ADAM3 in testis. Taken together, these results disclose involvement of ADAM4 and ADAM6 in a reproductive ADAM system that functions in fertilization.</P>

Analysis of the lysophosphatidic acid-generating enzyme ENPP2 in the uterus during pregnancy in pigs.

Seo, Heewon,Choi, Yohan,Shim, Jangsoo,Kim, Mingoo,Ka, Hakhyun Society for the Study of Reproduction [etc.] 2012 BIOLOGY OF REPRODUCTION Vol.87 No.4

<P>Lysophosphatidic acid (LPA), a simple phospholipid, plays a critical role in the establishment of pregnancy in pigs. LPA production is mediated by the action of ENPP2, a secreted lysophospholipase D (lysoPLD) that converts lysophosphatidylcholine to LPA. However, the mechanism that regulates LPA production by ENPP2 in the porcine uterus is not well understood. In this study, we evaluated ENPP2 expression during the estrous cycle and pregnancy in the uterine endometrium and in early stage conceptuses. We also evaluated lysoPLD activity in the uterine lumen. ENPP2 transcripts and proteins were detected in the uterine endometrium at all stages of the estrous cycle and pregnancy, with higher levels on Day (D) 12 and D15 of the estrous cycle and pregnancy. ENPP2 expression was localized mainly in luminal and glandular epithelial cells in the endometrium and was also detected in conceptuses on D12 of pregnancy. Secreted ENPP2 protein was detected in fluid flushing samples from the uterine lumen on D12 of the estrous cycle and pregnancy, with higher levels on D12 of pregnancy. LysoPLD activity was detected in uterine flushings on D12 of the estrous cycle and pregnancy, with higher levels on D12 of pregnancy. This study showed that uterine endometrium and conceptuses produce ENPP2 and secreted it into the uterine lumen where it has lysoPLD activity. These results suggest that ENPP2 may play an important role in the establishment of pregnancy in pigs by regulating LPA production at the maternal-conceptus interface.</P>

Contributions of an animal scientist to reproductive biology.

Society for the Study of Reproduction [etc.] 2011 BIOLOGY OF REPRODUCTION Vol.85 No.2

<P>I became interested in biology as an undergraduate in a premedical curriculum but developed a passion for the field of reproductive biology because of a course in physiology of reproduction taken to meet requirements for admission to veterinary school. My career path changed, and I entered graduate school, obtained the Ph.D., and have enjoyed an academic career as a reproductive biologist conducting research in uterine biology and pregnancy in animal science departments at the University of Florida and at Texas A&M University. However, I have never allowed academic boundaries to interfere with research and graduate education as that is contrary to collegiality, the cornerstone of great universities. I consider that my major contributions to science include 1) identification of proteins secreted by cells of the uterine endometrium that are critical to successful establishment and maintenance of pregnancy; 2) discovery of steroids and proteins required for pregnancy recognition signaling and their mechanisms of action in pigs and ruminant species; 3) investigation of fetal-placental development and placental transport of nutrients, including water and electrolytes; 4) identification of linkages between nutrition and fetal-placental development; 5) defining aspects of the endocrinology of pregnancy; and 6) contributing to efforts to exploit the therapeutic value of interferon tau, particularly for treatment of autoimmune diseases. My current studies are focused on the role of select nutrients in the uterine lumen, specifically amino acids and glucose, that affect development and survival of the conceptus and translation of mRNAs and, with colleagues at Seoul National University, gene expression by the avian reproductive tract at key periods postovulation. Another goal is to understand stromal-epithelial cell signaling, whereby progesterone and estrogen act via uterine stromal cells that express receptors for sex steroids to stimulate secretion of growth factors (e.g., fibroblast growth factors and hepatocyte growth factor) that, in turn, regulate functions of uterine epithelial cells and conceptus trophectoderm.</P>

Expression and Relationship of Male Reproductive ADAMs in Mouse1

Kim, Taewan,Oh, Jungsu,Woo, Jong-Min,Choi, Eunyoung,Im, Sin Hyeog,Yoo, Yung Joon,Kim, Do Han,Nishimura, Hitoshi,Cho, Chunghee Society for the Study of Reproduction 2006 BIOLOGY OF REPRODUCTION Vol.74 No.4

A number of a disintegrin and metalloprotease (ADAM) family members are expressed in mammalian male reproductive organs such as testis and epididymis. These reproductive ADAMs are divided phylogenically into three major groups: ADAMs 1, 4, 6, 20, 21, 24, 25, 26, 29, 30, and 34 (the first group); ADAMs 2, 3, 5, 27, and 32 (the second group); and ADAMs 7 and 28 (the third group). Previous mouse knockout studies indicate that ADAM1, ADAM2, and ADAM3 have intricate expressional relationships, playing critical roles in fertilization. In the present study, we analyzed processing, biochemical characteristics, localization, and expressional relationship of the previously-unexplored, second-group ADAMs (ADAM5, ADAM27, and ADAM32). We found that all of the three ADAMs are made as precursors in the testis and processed during epididymal maturation, and that ADAM5 and ADAM32, but not ADAM27, are located on the sperm surface. Using sperm from Adam2??/?? and Adam3??/?? mice, we found that, among the three ADAMs, the level of ADAM5 is modestly and severely reduced in Adam3 and Adam2 knockout sperm, respectively. Further, we analyzed ADAM7, an epididymis-derived sperm surface ADAM from the separate phylogenetic group, in the knockout sperm. We found that the level of ADAM7 is also significantly reduced in both Adam2 and Adam3-null sperm. Taken together, our results suggest a novel expressional relationship of ADAM5 and ADAM7 with ADAM2 and ADAM3, which play critical roles in fertilization.

Comprehensive analysis of prostaglandin metabolic enzyme expression during pregnancy and the characterization of AKR1B1 as a prostaglandin F synthase at the maternal-conceptus interface in pigs.

Seo, Heewon,Choi, Yohan,Shim, Jangsoo,Yoo, Inkyu,Ka, Hakhyun Society for the Study of Reproduction [etc.] 2014 BIOLOGY OF REPRODUCTION Vol.90 No.5

<P>Prostaglandins (PGs) are important lipid mediators regulating various reproductive processes in many species. In pigs, the expression pattern of PGE2 and PGF2α metabolic enzymes and the regulatory mechanism controlling PGE2 and PGF2α levels in the uterus during pregnancy are not completely understood. This study determined endometrial expression of the genes (PLA2G4A, PTGS1, PTGS2, PTGES, PTGES2, PTGES3, AKR1B1, CBR1, and HPGD) involved in PGE2 and PGF2α metabolism during the estrous cycle and pregnancy and measured levels of PGE2 and PGF2α in uterine endometrial tissues and uterine flushings at the time of conceptus implantation in pigs. Except PTGES3, expression of the genes studied changed in a pregnancy-stage-specific manner, and localization of PTGES, AKR1B1, CBR1, and HPGD mRNAs were cell-type specific in the uterine endometrium. Levels of both PGE2 and PGF2α in uterine endometrial tissues and uterine lumen were higher on Day 12 of pregnancy than those of the estrous cycle and affected by different morphology of spherical and filamentous conceptuses. Furthermore, we determined that endometrial expression of AKR1B1, known to encode a PGF2α synthase in other species, was increased by estrogen and interleukin-1beta and that AKR1B1 exhibited PGF2α synthase activity in the porcine uterine endometrium. These results in pigs indicate that the PGE2 and PGF2α metabolic enzymes are expressed stage specifically in the endometrium during pregnancy and regulate the abundance of PGE2 and PGF2α in the uterus at the time of implantation and that AKR1B1 may act as a major PGF synthase in the endometrium during early pregnancy.</P>

Change of Genes in Calcium Transport Channels Caused by Hypoxic Stress in the Placenta, Duodenum, and Kidney of Pregnant Rats

Yang, Hyun,An, Beum-Soo,Choi, Kyung-Chul,Jeung, Eui-Bae Society for the Study of Reproduction, Inc. 2013 BIOLOGY OF REPRODUCTION Vol.88 No.2

<P>Preeclampsia is a pregnancy-specific disease characterized by concurrent development of hypertension, proteinuria, and oxidative stress in the placenta. In this study, we induced hypoxic stress in rats during pregnancy to reproduce physiological conditions associated with preeclampsia. The maternal weight of hypoxic pregnant rats was lower than that of normoxic animals. The level of calcium ions were also increased in urine collected from the hypoxic animals. In contrast, urinary concentrations of sodium, chloride, and potassium ions declined in hypoxic rats, and developed to proteinuria. The expression of genes known as two biomarkers, sFLT1 (for preeclampsia) and HIF-1alpha (for hypoxia), were highly induced in the placenta, duodenum, and kidney by hypoxic stress. The overexpression of sFLT1 and HIF-1alpha demonstrated that our experimental conditions closely mimicked ones that are associated with preeclampsia. In the present study, we measured the expression of calcium transporters (TRPV5, TRPV6, PMCA1, NCKX3, NCX1, and CaBP-9k) in the placenta, duodenum, and kidney under hypoxic conditions on Gestational Day 19.5 in rats. Placental TRPV5, TRPV6, and PMCA1 expression was up-regulated in the hypoxic rats, whereas the levels of NCX1 and CaBP-9k were unchanged. In addition, NCKX3 expression was increased in the placenta of hypoxic rats. Duodenal expression of CaBP-9k, TRPV5, TRPV 6, and PMCA1 was decreased in the hypoxic rats, whereas levels of NCXs were not altered. Renal expression of NCKX3 and TRPV6 was increased, whereas NCX1 was decreased in the hypoxic rats compared to the normoxic controls. Taken together, these results indicate that physiological changes observed in the hypoxic rats were similar to ones associated with preeclampsia. Expression of calcium transport genes in the placenta, duodenum, and kidney perturbed by hypoxic stress during pregnancy may cause calcium loss in the urine, and thereby induce calcium-deficient characteristics of preeclampsia.</P>

Chitosan nanoparticles cause pre- and postimplantation embryo complications in mice.

Park, Mi-Ryung,Gurunathan, Sangiliyandi,Choi, Yun-Jung,Kwon, Deug-Nam,Han, Jae-Woong,Cho, Ssang-Goo,Park, Chankyu,Seo, Han Geuk,Kim, Jin-Hoi Society for the Study of Reproduction [etc.] 2013 BIOLOGY OF REPRODUCTION Vol.88 No.4

<P>Embryo development is a complex and tightly controlled process. Nanoparticle injury can affect normal development and lead to malformation or miscarriage of the embryo. However, the risk that these nanoparticles may pose to reproduction is not clear. In this study, chitosan nanoparticles (CSNP) of near uniform size, in the range of 100 nm, were synthesized and confirmed by a particle size analyzer and transmission electron microscopy. Morulae-stage embryo exposure to CSNP during in vitro culture caused blastocyst complications that had either no cavity or a small cavity. Furthermore, CSNP-treated embryos showed lower expression of not only trophectoderm-associated genes but also pluripotent marker genes. When blastocysts developed in both media with and without CSNP were transferred to recipients, the percentage of blastocysts resulting in viable pups was significantly reduced. These detrimental effects are linked to the reduction of total cell numbers, enhanced apoptosis, and abnormal blastocoels forming at the blastocyst stage, indicating that CSNP treatment might have long-term adverse biological effects in view of pregnancy outcome.</P>