Differential genome-wide gene expression profiling of bovine largest and second-largest follicles: identification of genes associated with growth of dominant follicles

<p>Abstract</p> <p>Background</p> <p>Bovine follicular development is regulated by numerous molecular mechanisms and biological pathways. In this study, we tried to identify differentially expressed genes between largest (F1) and second-largest follicles (F2), and classify them by global gene expression profiling using a combination of microarray and quantitative real-time PCR (QPCR) analysis. The follicular status of F1 and F2 were further evaluated in terms of healthy and atretic conditions by investigating mRNA localization of identified genes.</p> <p>Methods</p> <p>Global gene expression profiles of F1 (10.7 +/- 0.7 mm) and F2 (7.8 +/- 0.2 mm) were analyzed by hierarchical cluster analysis and expression profiles of 16 representative genes were confirmed by QPCR analysis. In addition, localization of six identified transcripts was investigated in healthy and atretic follicles using in situ hybridization. The healthy or atretic condition of examined follicles was classified by progesterone and estradiol concentrations in follicular fluid.</p> <p>Results</p> <p>Hierarchical cluster analysis of microarray data classified the follicles into two clusters. Cluster A was composed of only F2 and was characterized by high expression of 31 genes including IGFBP5, whereas cluster B contained only F1 and predominantly expressed 45 genes including CYP19 and FSHR. QPCR analysis confirmed AMH, CYP19, FSHR, GPX3, PlGF, PLA2G1B, SCD and TRB2 were greater in F1 than F2, while CCL2, GADD45A, IGFBP5, PLAUR, SELP, SPP1, TIMP1 and TSP2 were greater in F2 than in F1. In situ hybridization showed that AMH and CYP19 were detected in granulosa cells (GC) of healthy as well as atretic follicles. PlGF was localized in GC and in the theca layer (TL) of healthy follicles. IGFBP5 was detected in both GC and TL of atretic follicles. GADD45A and TSP2 were localized in both GC and TL of atretic follicles, whereas healthy follicles expressed them only in GC.</p> <p>Conclusion</p> <p>We demonstrated that global gene expression profiling of F1 and F2 clearly reflected a difference in their follicular status. Expression of stage-specific genes in follicles may be closely associated with their growth or atresia. Several genes identified in this study will provide intriguing candidates for the determination of follicular growth.</p

Cloning and expression of two new prolactin-related proteins, prolactin-related protein-VIII and -IX, in bovine placenta

BACKGROUND: Prolactin-related proteins (PRPs) are specific proteins of the growth hormone/prolactin (GH/PRL) family in bovine placenta. This study reports the identification and sequencing of a full-length cDNA for two new members of bovine PRPs, bPRP-VIII and -IX, and their localization and quantitative expression in bovine placenta. METHODS: New bPRP-VIII and -IX were identified from bovine placentome. Localization and quantitative gene expression in the placenta were respectively investigated by in situ hybridization and real-time RT-PCR methods. Recombinant proteins of these genes were produced by a mammalian HEK293 cell expression system. RESULTS: Full-length bPRP-VIII and -IX cDNA were respectively cloned with 909 and 910 nucleotide open-reading-frames corresponding to proteins of 236 and 238 amino acids. The predicted bPRP-VIII amino acid sequence shared about 40 to 70% homology with other bPRPs, and bPRP-IX had about 50 to 80 % homology of others. The two new bPRPs were detected only in the placenta by RT-PCR. mRNA was primarily expressed in the cotyledon and intercotyledonary tissues throughout gestation. An in situ hybridization analysis revealed the presence of bPRP-VIII and -IX mRNA in the trophoblastic binucleate and/or trinucleate cells. bPRP-VIII mRNA was observed in the extra-embryonic membrane on Day 27 of gestation, however, no bPRP-IX mRNA was observed in the extra-embryonic membrane in the same stage of pregnancy by quantitative real-time RT-PCR analysis. Both new bPRP genes were possible to translate a mature protein in a mammalian cell expression system with approximately 28 kDa in bPRP-VIII and 38 kDa in bPRP-IX. CONCLUSION: We identified the new members of bovine prolactin-related protein, bPRP-VIII and -IX. Localization and quantitative expression were confirmed in bovine placenta by in situ hybridization or real-time PCR. Their different temporal and spatial expressions suggest a different role for these genes in bovine placenta during gestation

Cloning and expression of SOLD1 in ovine and caprine placenta, and their expected roles during the development of placentomes

<p>Abstract</p> <p>Background</p> <p>The Ly-6 (Ly-6/uPAR) superfamily members share the Ly-6 domain defined by distinct disulfide bonding patterns between 8 or 10 cysteine residues. They comprise membrane- and secretory-type proteins. We recently reported the gene and protein characterization of the bovine secreted protein of Ly-6 domain 1 (SOLD1). Bovine SOLD1 is expressed in trophoblast mononucleate cells (TMCs) and is localized in the cotyledonary mesenchyme. Here, we compared the expression and functionality of SOLD1 among the ruminants. We examined mRNA expression by chorionic fibroblasts as a measure of one of the SOLD1 functions.</p> <p>Results</p> <p>Ovine and caprine SOLD1 mRNAs have 303 bp open reading frames and encode for deduced SOLD1 proteins with 100 amino acids, including a 22-aa-long signal peptide at the N-terminal. Both of the SOLD1 amino acid sequences have high similarities with the bovine sequence. Both SOLD1 mRNAs were also expressed in TMCs of cotyledons and intercotyledonary membranes. The mature SOLD1 proteins were localized in the mesenchymal villi of cotyledons after secretion. Bovine, ovine and caprine SOLD1 affected gene expression in mesenchymal fibroblasts <it>in vitro</it>; nucleoredoxin expression was upregulated and BCL2-like 13 was downregulated. Thus, we suggest that SOLD1 acts as a modulator of cell proliferation and apoptosis.</p> <p>Conclusion</p> <p>Expressing cells and protein localization of SOLD1 coincided among the three ruminants. SOLD1 participated in regulating nucleoredoxin and BCL2-like 13 expression in chorionic fibroblasts. SOLD1 is produced specifically in the cotyledons and intercotyledonary membranes in ruminants and appears to be involved in the construction of the ruminant placenta.</p

Expression and characterization of novel ovine orthologs of bovine placental prolactin-related proteins

<p>Abstract</p> <p>Background</p> <p>The prolactin-related proteins (PRPs) are non-classical placental-specific members of the prolactin/growth hormone family. Among ruminants, they are expressed in the cotyledonary villi of cattle and goat. We investigated placental PRP in sheep in order to gain a comprehensive understanding of the function and evolution of these molecules. We also examined the sequence properties, expression and lactogenic activation of the cloned genes.</p> <p>Results</p> <p>We cloned two novel ovine <it>PRPs</it>, named <it>oPRP1 </it>and <it>oPRP2</it>. <it>oPRP2 </it>had a typical <it>PRP </it>sequence similar to bovine <it>PRP1 </it>(<it>bPRP1</it>). <it>oPRP1 </it>had a short sequence identical with bovine or caprine type <it>PRP </it>but the reading frame was shifted. Both <it>oPRPs </it>were expressed in trophoblast giant binucleate cells (BNC) as in cattle and goat. <it>oPRP1 </it>expression declined from the early to the middle stage of gestation. In contrast, <it>oPRP2 </it>expression remained constant throughout the gestation period. <it>oPRP2 </it>was translated to form a mature protein in a mammalian cell expression system. Western blotting showed a molecular mass of 35 kDa for the FLAG-tag fusion <it>oPRP2</it> protein. This recombinant protein and <it>bPRP1</it> were bioassayed using Nb2 lymphoma cells; it was confirmed that neither ruminant <it>PRP</it> had lactogenic activity because the Nb2 lymphoma cells did not proliferate.</p> <p>Conclusion</p> <p>We have identified two novel <it>PRPs</it>, <it>oPRP1 </it>and <it>oPRP2</it>, in ovine placenta. Both these ovine <it>PRPs</it> were localized and quantitatively expressed in BNC. Absence of lactogenic activity was confirmed for the <it>oPRP2</it> molecule. It is anticipated that novel and known ruminant <it>PRPs</it> have common functions, except for lactogenic activity.</p

Quantitative analysis of bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) gene expression in calf and adult bovine ovaries

<p>Abstract</p> <p>Background</p> <p>It has been reported that calf oocytes are less developmentally competent than oocytes obtained from adult cows. Bone morphogenetic protein 15 (BMP15) and growth and differentiation factor 9 (GDF9) play critical roles in folliculogenesis, follicular development and ovulation in mammalian ovaries. In the present study, we attempted to compare the expression patterns of <it>BMP15 </it>and <it>GDF9 </it>in the cells of calf and cow ovaries to determine a relationship between the level of these genes and the low developmental competence of calf oocytes.</p> <p>Methods</p> <p>Bovine tissues were collected from 9-11 months-old calves and from 4-6 years-old cows. We characterized the gene expression of BMP15 and GDF9 in calf and adult bovine oocytes and cumulus cells using quantitative real-time reverse transcriptase polymerase chain reaction (QPCR) and <it>in situ </it>hybridization. Immunohistochemical analysis was also performed.</p> <p>Results</p> <p>The expression of <it>BMP15 </it>and <it>GDF9 </it>in cumulus cells of adult ovaries was significantly higher than that in calf ovaries, as revealed by QPCR. <it>GDF9 </it>expression in the oocytes of calf ovaries was significantly higher than in those of the adult ovaries. In contrast, <it>BMP15 </it>expression in the oocytes of calf and adult ovaries was not significantly different. The localization of gene expression and protein were ascertained by histochemistry.</p> <p>Conclusions</p> <p>Our result showed for the first time BMP15 and GDF9 expression in bovine cumulus cells. <it>BMP15 </it>and <it>GDF9 </it>mRNA expression in oocytes and cumulus cells was different in calves and cows.</p

Correlation Between the Qualities and Abilities and their Adaptability to School, and Developmental Changes as Seen in Elementary and Junior High School Children: Focusing on the Factors, “Subjectivity and Activeness,” “Pride,” and “Cooperation in Labor”

本研究では，小学3年生から中学3年生までの児童生徒計1,775名を対象に，「主体性・積極性」，「自尊心」，「協働する力」から構成される「資質・能力質問紙」（若松・部谷岡ら，2017）を用いて，多面的に学校適応との関連を検討するとともに，学年ごとの資質・能力の量的差異について分析を行った。まず，重回帰分析の結果では，「自尊心」と「協働する力」の標準偏回帰係数（β）が有意であり，学校適応への弱い正の影響が示された。次に，学年（7）×下位尺度（3）の分散分析を行った結果，資質・能力の学年進行による量的差異は，「主体性・積極性」，「自尊心」，「協働する力」の間で相互に異なることが明らかになった。これらの資質・能力を育成することは，学校生活のみならず，生涯にわたり社会生活を営む上での基盤となる力の向上につながるものであると考えられる。今後は，学年進行による縦断的変化についての更なる検討が必要である。This study considers the diverse aspects of the correlation between outcomes of the survey, “Qualities and Abilities Questionnaire” (Wakamatsu, Hiyaoka et.al. , 2017) covering qualities such as “subjectivity and activeness,” “pride,” and “cooperation in labor,” conducted among 1,775 students from 3rd grade in elementary school to 3rd year in junior high school, and their ability to adapt to school. The study also analyzed the quantitative differences seen among the qualities and abilities of the different grades. Firstly, multiple regression analysis showed that the standard partial regression coefficient β was significant for “pride” and “cooperation in labor,” showing slight positive influence to adaptability to school. Secondly, the analysis of variance on grade (7) x subscale (3) showed that the quantitative difference in qualities and abilities depending on the advancement of grades are different among “subjectivity and activeness,” “pride,” and “cooperation in labor.” Nurturing these qualities and abilities will result in improving the skills that will become the foundation for the students’life-long social life, and there is need for further consideration of changes that occur according to advancement of grades

Characterization and Expression Analysis of SOLD1, a Novel Member of the Retrotransposon-Derived Ly-6 Superfamily, in Bovine Placental Villi

BACKGROUND:Ly-6 superfamily members have a conserved Ly-6 domain that is defined by a distinct disulfide bonding pattern between eight or ten cysteine residues. These members are divided into membrane-type and secretory-type proteins. In the present study, we report the identification of a novel Ly-6 domain protein, secreted protein of Ly-6 domain 1 (SOLD1), from bovine placenta. PRINCIPAL FINDINGS:SOLD1 mRNA was expressed in trophoblast mononucleate cells and the protein was secreted into and localized in the extracellular matrix of the mesenchyme in cotyledonary villi. SOLD1 bound mainly with type I collagen telopeptide. We confirmed secretion of SOLD1 from the basolateral surface of a bovine trophoblast cell line (BT-1). It may be related to the organization of the extra-cellular matrix in the mesenchyme of fetal villi. Since trophoblast mononucleate cells are epithelial cells, their polar organization is expected to have a crucial role in the SOLD1 secretion system. We established that SOLD1 is an intronless bovine gene containing the Alu retrotransposon, which was integrated via cytoplasmic reverse transcription. CONCLUSION:We identified a novel retrotransposon-like Ly-6 domain protein in bovine placenta. SOLD1 is a crucial secreted protein that is involved in the organization of the mesenchyme of the cotyledonary villi. Furthermore, the gene encoding SOLD1 has an interesting genomic structure