Estrogen plays an important role both in male and female reproduction. Two estrogen receptor isoforms, Esr1 and Esr2, are expressed in male gonad. In the mouse, Esr1 is expressed in Leydig cells of testis and pituitary. Esr1-/- male mice show enhanced androgen synthesis, spermatogenic defect, and infertility. To evaluate the specific function of Esr1 in Leydig cells, we examined spermatogenesis and steroidogenesis in Esr1f/fCyp17iCre male mice in which Esr1 is deleted specifically in Leydig cells. These mice showed normal spermatogenesis and fertility when compared to wild type from young adulthood to old age. Testosterone synthesis in Esr1f/fCyp17iCre mice at 3-12 months old of age was not different from age-matched wild type mice, while, at 18 months old of age, circulating testosterone concentrations were significantly higher than wild type together with increased levels of Star, Cyp17a1, and Hsd17b3 mRNA and with a hypertropy of Leydig cells. In Esr1f/fCyp17iCre mouse pituitaries, Fshb and Lhb mRNA levels were not different from wild type from young adulthood to old age. Taken together, Esr1 in Leydig cells may be not essential for spermatogenesis and fertility under control of endogenous estrogens and may have a role in aged Leydig cell function.

Estrogen is an important regulator of reproduction in both male and female. The two forms of estrogen receptor (ER) are known, ERα and ERβ. To understand the role of ERα in the testis, we investigated the expression of ERα in the mouse Leydig cells during postnatal development and the effects of estrogen on steroidogenesis and proliferation in progenitor Leydig cells (PLCs). In the testis, ERα mRNA and protein levels were markedly increased from postnatal day (PND) 1 to 14 and decreased thereafter until PND 56. During postnatal development ERα immunoreactivity was strong in the nucleus of Leydig cells at PND 14 when PLCs were abundant in the interstitium and low in the mature adult Leydig cells (ALCs). In fetal Leydig cells (FLCs), ERα immunoreactivity was negligible at birth and became increased at PND 14. This suggests an important role of ERα in Leydig cells during neonatal period. In isolated PLCs, 17β-estradiol (E2) and ERα-selective agonist, PPT suppressed the hCG-induced progesterone production and steroidogenic pathway genes expression. The hCG-induced PLCs proliferation was significantly inhibited by E2 and PPT. In conclusion, estrogen - ERα signaling may negatively regulate functional differentiation and proliferation of PLCs.

Sirt1 belongs to class III histone/protein deacetylase. Sirt1 KO mice have spermatogenic defects. In this study, expression of Sirt1 and acetylated histone 3k9 (H3k9ac) was examined in developing mouse testes. Among two splicing variants of sirt1 mRNA long form was dominant in developing testis. Testicular sirt1 mRNA levels were low at birth, increased until 14 days post partum (pp) and remained constant thereafter. Sirt1 immunoreactivity was weak to negligible in gonocytes, moderate in spermatogonia, absent in preleptotene spermatocytes, moderate in zygotene spermatocytes, strong in pachytene spermatocytes, and weak in diplotene spermatocytes. Round and elongating spermatids were negative for Sirt1. Acetylated histone 3k9 (H3k9ac) immunoreactivity was moderate in spermatogonia and weak to negligible in preleptotene to pachytene spermatocytes but strong in round and elongating spermatids. In Sertoli cells, nuclear Sirt1 immunoreactivity was absent at birth, increased at 14 days pp and markedly increased at 28 days pp onwards. In immature Sertoli cells culture, FSH and testosterone increased sirt1 mRNA, suggesting that Sirt1 participates in protein deacetylation events during the differentiation of Sertoli cells by gonadotropin. In the Leydig cells, nuclear Sirt1 immunoreactivity was weak until 2 weeks pp and decreased in 4 weeks pp onward. suggesting the protein deacetylation by Sirt1 in Leydig cell precursor/progenitor cells. Mutually exclusive expression between Sirt1 and H3k9ac in pachytene spermatocytes in testis suggests that deacetylation of H3K9ac by Sirt1 participates in the gene silencing and/or chromosome behavior in pachytene sspermatocytes.

Water channel proteins, aquaporins (AQPs) contribute to transepithelial water movement in many tissues. To date, 13 mammalian AQPs have been identified. Of these, AQP5 plays an important role in the fluid homeostasis and cell volume control in epithelial cells. In an effort to understand the role of AQP5 in testis, we investigated the expression of AQP5 in developing mouse testis, its regulation by estrogen and LH, and the change of steroidogenesis by AQP5 knockdown. Testes and Leydig cells were isolated from male mice at postnatal day (PND) 1, 7, 14, 28, and 56 and estrogen receptor alpha knockout (ERαKO) mice. In mouse testis, AQP5 immunoreactivity was negligible by PND 14. From PND 28 onward, AQP5 immunoreactivity was found in Leydig cells. In ERαKO mouse Leydig cells, AQP5 mRNA level was significantly lower than wild type. In primary adult Leydig cell culture, the expression of AQP5 mRNA was increased by 17β-estradiol (E2) and human chorionic gonadotropin (hCG), but was not changed in ERαKO Leydig cells. Moreover, the expression of AQP5 mRNA was increased by E2 and ERα-selective agonist PPT, but was not changed by ERβ-selective agonist DPN in primary Leydig cells and mLTC-1. In silico analysis and chromatin immunoprecipitation (ChIP) assay revealed that there are putative estrogen response elements (EREs) and cAMP response elements (CRE) in AQP5 promoter region. Testosterone secretion and steroidogenic pathway genes (StAR, Cyp11a1, Cyp17a1, and 3β-HSD6) expression were decreased by AQP5 siRNA in primary Leydig cells. In conclusion, AQP5 expression was coupled with functional differentiation of adult Leydig cells. AQP5 may play an important role in the fluid homeostasis and cell volume control during development of adult Leydig cells. The expression of AQP5 in Leydig cells could be regulated by ERα and LH signaling and AQP5 may be involved in steroidogenesis.

Amnionless (AMN) is a plasma membrane protein that binds to cubilin and megalin in various epithelia and mediates endocytosis of extracellular ligands. This function has been studied in the kidney where it plays a key role in vitamin B12 and vitamin D homeostasis. Present study aimed to elucidate developmental pattern of expression of AMN during the peri-implantation period in mouse embryos. In an effort to understand functional role of AMN in the histiotropic nutrition in blastocyst, endocytotic function of AMN for apoplipoprotein was examined in blastocyst. Eight-week-old female mice were superovulated by intraperitoneal injection of 5 IU PMSG and 5 IU hCG 48h later. To obtain embryos, females were mated with males. Mouse embryos were collected at 12, 48, 56, 65, 72 and 96 h post-hCG by flushing oviducts and uterus, and we also obtained gestation day 6.5, 7.5 and 8.5 embryos in uterus. All samples were subjected to quantitative RT-PCR, whole-mount immunofluorescence and immunohistochemistry analysis. To analyze endocytotic function of AMN, we examined uptake experiment of FICT labeled apolipoprotein A-I (ApoA-I-FITC) following functional blocking of AMN in blastocysts. AMN and cubilin mRNA was expressed in all developmental stages of mouse embryos. Megalin was the first detected at morula stage. AMN protein was expressed in trophectoderm (TE) and inner cell mass (ICM). AMN and cubilin were expressed in visceral endoderm of GD 6.5 and 7.5 embryos and visceral yolk sec of GD 8.5 embryos. In normal IgG treated embryos, ApoA-I-FITC was detected in intracellular vesicles of TE and ICM. However, in the presence of anti-AMN antibody, ApoA-I-FITC was weakly detected in apical surface of plasma membrane of TE. To date, AMN has been believed to be expressed in visceral endoderm of post implantation embryos. Our results demonstrated that AMN is the important molecular partner of cubilin and megalin in the preimplantation embryos and that AMN mediates endocytosis of apoplipoprotein, which may play a crucial role in embryonic development and normal growth via supporting histiotropic nutrition during peri-implantation period.

Coxsackievirus and adenovirus receptor (CAR) is a member of the Ig-type superfamily of cell adhesion molecules. In polarized epithelial cells CAR is expressed at the tight junction. The mouse CAR gene is composed of at least eight exons, and CAR splice variants that differ at the end of the cytoplasmic tail have been identified in a number of tissues. The present study aimed to examine the expression of (CAR), a TJ protein sealing the muticellular contact point during preimplantation embryos and role of CAR in the formation and integrity of the blastocoel. Eight-week-old female mice were superovulated by intraperitoneal injection of 5 IU PMSG and 5 IU hCG 48h later. Mouse embryos were collected at 12, 48, 56, 65, 72 and 96 h post-hCG in oviducts and uterus, and we also obtained gestation day 5 and 6.5 embryos in uterus. All samples were subjected to RT-PCR, immunofluorescence and immunohistochemistry analysis. To analyze epithelial permeability of CAR, we examined permeability of FITC-labeled dextran (MW 40 kDa) following functional blocking of CAR in blastocysts. Long isoform of CAR mRNA was expressed from throughout the preimplantation development and markedly increased at morulae stage onward. Small amount of short isoform CAR mRNA was expressed at blastocyst stage. On Western blot, 64 kDa protein was detected together with 43 kDa protein corresponding to short and long forms CAR, respectively in blastocysts. CAR immunoreactivity was found in cell contacts between blastomeres from 4-cell stage onward. Under Ca2+ switching condition blocking antibodies for CAR increased the permeability of blastocysts to FITC-dextran, a permeability tracer. At 5 dpc, trophoblasts of the implanting embryos were immunoreactive with anti-CAR IgG. At 6.5 dpc, the egg cylinder stage in mouse, the visceral and parietal endoderm were immunoreactive with anti-CAR IgG. Our results suggest that alternative splicing of CAR transcript is highly dependent on the development of expanding blastocyst. CAR may play a crucial role as a barrier to adenovirus infection and adhesion molecule for epithelial permeability during peri-implantation period.

본 연구는 공원자산을 활용한 공원재정비에 의한 공간구성의 변화가 이용형태 및 이용만족도에 미치는 영향을 파악하여, 향후 한국의 도시공원의 재정비사업의 계획론적인 관점에서의 방향성을 제시하기 위해, 요코하마시 카나자와구의 토미오카 no.5 공원의 재정비사례를 대상으로 2회의 걸친 공원 이용자 설문지 조사와 지역주민, 행정 담당자, 재정비공원 설계자를 대상으로 청취 조사를 실시하였다. 조사를 통해 확인 된 결과를 분석하여 향후 한국의 도시공원 재정비사업의 방향성에 대해 다음과 같이 제시할 수 있다.첫째, 향후 공원재정비 사업의 적극적인 추진이 예측되는 한국에 있어서 공원자산에 관한 이용자의 인식도조사를 통해 파악된 공원자산을, 적극적으로 보존 및 활용한 재정비 수법은 공원의 평가와 만족도향상에 유효하며, 공원 자산을 파악하기 위해 이용자에 대한 인식도 조사가 필요하다고 할 수 있다. 둘째, 재정비를 위한 주민설명회에서 확인 된 공원에 대한 지역주민의 요구와 지자체의 공원재정비의 목적과 과제의 파악을 통해, 이에 맞춰 명확한 설계의도를 설정하는 것은 이용자 만족도 향상에 효과적인 재정비 사업의 프로세스라고 볼 수 있으며, 재정비를 포함한 도시공원정비 사업추진에 시민참여가 활발히 이루어지고 있는 한국에 시사하는 바가 크다고 볼 수 있다.

After vasectomy (VAX), the flux and composition of the epididymal fluid are modified, leading to sequelae to the epididymis. In an effort to understand molecular pathophysiology of the epididymis following VAX, we investigated the changes of gene expression and sperm functions in the epididymis of vasectomized mice. After VAX, the epithelial cell height was significantly decreased in cauda epididymis, resembling the those of vas deference. This suggests that VAX evokes alteration of segmental characteristics of epididymal epithelium. Of note, these was an increase in luminal diameter in corpus and cauda epididymis, indicating the alteration of fluid homeostasis in epididymal lumen and protein synthesis and secretion. Also, the formation of sperm granuloma and infiltration of inflammatory cells were noted in lumen of epididymis at 8 weeks postvasectomy, indicating the activation of immune response in epididymis. The serum TNF-α levels and epididymal TNF-α and IL-1β mRNA levels were significantly increased in VAX mice. Microarray analysis demonstrated that claudin (CLDN) 10 and cystic fibrosis transmembrane conductance regulator (CFTR) were downregulated after vasectomy. In contrast, angiotensin II receptor type 2 (Agtr2) was up-regulated after vasectomy. Taking into account the functional importance of angiotensin system in epididymal epithelia and muscle tissue, VAX may alter the secretory function of epithelia and sperm transport via alteration in angiotensin system. Importantly, the IgG type of anti-sperm antibody (ASA) were markedly increased in the blood of vasectomized mice. Together this indicates that VAX provokes local inflammation in epididymis as well as systemic inflammation, which in turn change the blood epididymal barrier, an important element for epididymal immunological privilege. In addition, the number of sperm in cauda epididymis was increased but the sperm motility was decreased after vasectomy. The spontaneous acrosome reaction was increased by vasectomy after capacitation. This suggests that VAX affects sperm functions as well as sperm maturation. In conclusion, bilateral vasectomy lead to local immune response of epididymis, causing immunologic infertility in men.

The Egr family of zinc finger transcription factors is rapidly induced by various mitogens and regulates cell growth, differentiation, and apoptosis. While it is clear that loss of Egr1 leads to anovulatory infertility due to LHβ deficiency in female mice, molecular function of Egr1 in male reproduction has not been clearly investigated. Here, we demonstrate that Egr1 acts as an intrinsic transcription factor in Leydig cells to regulate their proliferation and steroidogenesis in the testis as well as an extrinsic factor for male reproduction via LHβ transcription in the pituitary. Egr1 is predominantly expressed in spermatogonia and Leydig cells in immature testes and later detected in some of these cell types in mature testes. The fertility potential of Egr1(-/-) male mice is relatively deteriorated even at 2 month-old age and aggravated with aging. The incidence of abnormalities of seminiferous tubules such as Sertoli cell only was dramatically increased with aging. The number and mean size of Leydig cells were significantly reduced in Egr1(-/-) testes. The impairment of Leydig cells is consistent with significant reduction in levels of testosterone and expression of factors critical for steroidogenesis such as StAR in Egr1(-/-) testes. Exogenous administration of hCG rapidly and transiently induced Egr1 expression in Leydig cells culture in vitro. hCG could reinstate reduced mean size of Leydig cells but not reduced number of Leydig cells and aberrantly low StAR expression, suggesting that Egr1 has critical functions for Leydig cell proliferation and their steroidgenesis. In addition, daily sperm production and in vitro fertilization (IVF) competence were significantly reduced, and apoptosis was facilitated in these mice. Furthermore, hCG administration to compensate for relatively low LH levels in Egr1(-/-) males could not restore the compromised reproductive phenotypes such as IVF competence and apoptosis in these mice. Interestingly, expression of Egr2, a member of Egr family, is significantly elevated in Egr1(-/-) Leydig cells suggesting that genetic compensation of Egr2 may alleviate phenotypic aberration of Egr1(-/-) male testes. Collectively, these results suggest that Egr1 act as an intrinsic transcription factor required for proliferation and steroidogenesis of Leydig cells to govern spermatogenesis in the testis.

Estrogen sulfotransferase (EST) is a cytosolic enzyme that catalyzes the sulfo-conjugation of estrogens at the 3-hydroxyl position. Sulfated estrogens lose their ability to interact with the estrogen receptor (ER). Previous studies have reported that testicular expression of EST is under the regulation of LH and androgen. In an effort to understand the biological significance of estrogens in the testis, we analyzed the EST gene expression in the developing mouse testis and Leydig cells and its regulation by estrogen receptor alpha (ERα). Male mice at postnatal day (PND) 1, 7, 14, 28, and 56 and ERα flox/flox Cyp17iCre male mice which show deletion of ERα specifically in Leydig cells were used for this study. Testes and Leydig cells isolated from these mice were subjected to quantitative RT-PCR analysis and immunohistochemistry. In addition, 17β-estradiol (E2), ERα-selective agonist PPT, ERβ -selective agonist DPN, and ER antagonist ICI 182,780 were treated in primary adult Leydig cell culture. These cultured cells were subjected to quantitative RT-PCR analysis. In testis, EST mRNA level was excessively low by PND 14 and markedly increased from puberty (PND 28) onward. In the interstitium, EST mRNA was not detected by PND 14 but considerably expressed from PND 28 onward. EST immunoreactivity was moderate in the interstitium by PND 14. Strong EST immunoreactivity was found in the interstitium from PND 28 onward. In ERαflox/flox Cyp17iCre mouse testis and Leydig cells, EST mRNA level was significantly lower than wild type (ERαflox/flox). In primary adult Leydig cell culture, the expression of EST mRNA was increased by E2 and PPT, but was not changed by DPN. The expression of EST in the testis is developmentally regulated. In adult Leydig cells, EST could play an important role in the steroidogenesis by modulating the activity of estrogens. Estrogen as well as LH and androgen may play a role in the regulation of EST expression in Leydig cells via ERα signaling.

Glycan epitopes of cellular glycoconjugates act as versatile biochemical signals, and this sugar coding plays an important role in cell-to-cell recognition processes. In the present study, our aims were to determine the distribution of sperm receptors with activity for fucosyl- and galactosyl glycans and to address whether mono sugar neoglycoproteins functionally mimic the binding between zona pellucida (ZP) glycoproteins and sperm. In mouse epididymal spermatozoa with intact acrosomes, fucopyranosyl bovine serum albumin (BSA-Fuc) bound to the segment of the acrosome, the equatorial segment, and the postacrosome region of the sperm head. Galactosyl BSA (BSA-Gal) binding activity was similar to that of BSA-Fuc, but was weaker. In acrosome-reacted spermatozoa treated with the Ca2+ ionophore A23187, BSA-Fuc binding was lost in the apical segment of the acrosome but remained in the equatorial segment and postacrosome regions. BSA-Gal binding to the equatorial region was increased. In the presence of 2.5 μg/ml BSA-Fuc, in vitro sperm - ZP binding was significantly decreased, indicating that fucosyl-BSA functionally mimics ZP glycoproteins during sperm-egg ZP interactions. At the same concentration, BSA-Gal was not effective. Fucosyl BSA which efficiently inhibited the sperm-ZP binding can mimic the ZP glycoconjugate and has potential for use as a sperm fertility control agent in mouse.

Development of transgenic plant with desirable traits to cultivated plant is one of the important procedures in plant molecular breeding. However, applicable assessment of transgenic plant in laboratorial scale is not much except cultivating transgenic plant for a whole life in field condition. Here, we analyzed chlorophyll fluorescence in three transgenic soybean lines with AtMYB44 transcription factor for assessment of photosynthetic activity under abiotic stresses such as drought. Soybean varieties used in this study were ‘Bert’ and ‘Bert’ derived three transgenic soybeans, ‘AtMYB44 CM35101’, ‘AtMYB44 CM2471’, and ‘AtMYB44 CM4481’. Analyzed five different chlorophyll fluorescence variables are maximum PSII quantum yield (QY_max), steady state PSII quantum yield (QY_Lss), steady state non-photochemical quenching (NPQ_Lss), coefficient of photochemical quenching in steady-state (Qp_Lss), and fluorescence declineratio in steady-state (Rfd_Lss). To determine main chlorophyll fluorescence variable affected by abiotic stress, principal component analysis (PCA) was conducted with five chlorophyll fluorescence variables measured from four varieties. QY_Lss and NPQ_Lss were main chlorophyll fluorescence variables to evaluate abiotic stress, particularly in drought stress. In comparison with transgenic soybean lines based on chlorophyll fluorescence variables, ‘AtMYB44 CM2471’ and ‘AtMYB44 CM4481’ are more tolerant to drought than the others. Interestingly, three transgenic soybean lines which have a same AtMYB44 gene with different regions of chromosome revealed the quite different responses of chlorophyll fluorescence to drought treatment.