Cytoplasmic and nuclear DROSHA in human villous trophoblasts.

In villous trophoblasts, DROSHA is a key ribonuclease III enzyme that processes pri-microRNAs (pri-miRNAs) into pre-miRNAs at the placenta-specific, chromosome 19 miRNA cluster (C19MC) locus. However, little is known of its other functions. We performed formaldehyde crosslinking, immunoprecipitation, and sequencing (fCLIP-seq) analysis of terminal chorionic villi to identify DROSHA-binding RNAs in villous trophoblasts. In villous trophoblasts, DROSHA predominantly generated placenta-specific C19MC pre-miRNAs, including antiviral C19MC pre-miRNAs. The fCLIP-seq analysis also identified non-miRNA transcripts with hairpin structures potentially capable of binding to DROSHA (e.g., SNORD100 and VTRNA1-1). Moreover, in vivo immunohistochemical analysis revealed DROSHA in the cytoplasm of villous trophoblasts. DROSHA was abundant in the cytoplasm of villous trophoblasts, particularly in the apical region of syncytiotrophoblast, in the full-term placenta. Furthermore, in BeWo trophoblasts infected with Sindbis virus (SINV), DROSHA translocated to the cytoplasm and recognized the genomic RNA of SINV. Therefore, in trophoblasts, DROSHA not only regulates RNA metabolism, including the biogenesis of placenta-specific miRNAs, but also recognizes viral RNAs. After SINV infection, BeWo DROSHA-binding VTRNA1-1 was significantly upregulated, and cellular VTRNA1-1 was significantly downregulated, suggesting that DROSHA soaks up VTRNA1-1 in response to viral infection. These results suggest that the DROSHA-mediated recognition of RNAs defends against viral infection in villous trophoblasts. Our data provide insight into the antiviral functions of DROSHA in villous trophoblasts of the human placenta.

Possible transfer of lncRNA H19-derived miRNA miR-675-3p to adjacent H19-non-expressing trophoblast cells in near-term mouse placenta.

LncRNA H19 serves as a regulatory RNA in mouse placental development. However, there is little information available on the in situ expression of H19 in the late-gestation mouse placenta. In this study, we performed quantitative polymerase chain reaction (qPCR) and in situ hybridization (ISH) analyses of lncRNA H19 and its exon 1-derived miRNA miR-675-3p to identify cell types expressing these non-coding RNAs in the mouse placenta during mid-to-late gestation. By qPCR analysis, we confirmed that H19 was highly expressed during mid-to-late gestation (E10.5-E18.5) and that H19-derived miRNA miR-675-3p was remarkably upregulated in the E18.5 placenta. ISH analysis revealed trophoblast cell type-specific expression of lncRNA H19 and miR-675-3p during later stages of gestation. In the junctional zone and decidua of late-gestation placenta, H19 was expressed in trophoblast giant cells and glycogen trophoblast cells; however, H19 was absent in spongiotrophoblast cells. In the labyrinth and chorionic plate, H19 was present in sinusoidal mononuclear trophoblast giant cells, fetal vascular endothelial cells, and basal chorionic trophoblast cells, but not in syncytiotrophoblasts. As expected, these lncRNA H19-expressing cells exhibited miR-675-3p in the E18.5 placenta. Intriguingly, miR-675-3p was also present in H19-negative spongiotrophoblast cells and syncytiotrophoblasts, implying the possible transfer of miR-675-3p from H19-exprssing cells to adjacent H19-non-expressing trophoblast cells. These findings suggest that the mouse placenta expresses lncRNA H19 in a trophoblast cell type-specific fashion during later stages of gestation.

Placenta-specific lncRNA 1600012P17Rik is expressed in spongiotrophoblast and glycogen trophoblast cells of mouse placenta.

A few long noncoding RNAs (long ncRNAs, lncRNAs) exhibit trophoblast cell type-specific expression patterns and functional roles in mouse placenta. However, the cell- and stage-specific expression patterns and functions of most placenta-derived lncRNAs remain unclear. In this study, we explored mouse placenta-associated lncRNAs using a combined bioinformatic and experimental approach. We used the FANTOM5 database to survey lncRNA expression in mouse placenta and found that 1600012P17Rik (MGI: 1919275, designated P17Rik), a long intergenic ncRNA, was the most highly expressed lncRNA at gestational day 17. Polymerase chain reaction analysis confirmed that P17Rik was exclusively expressed in placenta and not in any of the adult organs examined in this study. In situ hybridization analysis revealed that it was highly expressed in spongiotrophoblast cells and to a lesser extent in glycogen trophoblast cells, including migratory glycogen trophoblast cells invading the decidua. Moreover, we found that it is a polyadenylated lncRNA localized mainly to the cytoplasm of these trophoblast cells. As these trophoblast cells also expressed the neighboring protein-coding gene, pappalysin 2 (Pappa2), we investigated the effects of P17Rik on Pappa2 expression using Pappa2-expressing MC3T3-E1 cells and found that P17Rik transfection increased the messenger RNA (mRNA) and protein levels of Pappa2. These results indicate that mouse placenta-specific lncRNA P17Rik modulates the expression of the neighboring protein-coding gene Pappa2 in spongiotrophoblast and glycogen trophoblast cells of mouse placenta during late gestation.

1700108J01Rik and 1700101O22Rik are mouse testis-specific long non-coding RNAs.

Long non-coding RNAs (lncRNAs; > 200 nucleotides in length) have attracted attention as fine-tuners of gene expression. However, little is known about the cell- and stage-specific expression pattern and function of lncRNAs in spermatogenesis. The purpose of this study was to identify mouse testis-associated lncRNAs using a combination of computational and experimental approaches. We first used the FANTOM5 database to survey lncRNA expression in the mouse testis and performed reverse transcription quantitative polymerase chain reaction (real-time PCR) and in situ hybridization (ISH) analyses. In silico analysis showed that most of the highly expressed lncRNAs in the adult mouse testis were testis-specific lncRNAs and were expressed at and following the initiation of spermatogenesis. We selected the antisense lncRNA 1700108J01Rik and long intergenic non-coding RNA 1700101O22Rik from the most highly expressed lncRNAs in the adult testis for further analysis. Real-time PCR analysis confirmed that 1700108J01Rik and 1700101O22Rik were specifically expressed in the testis. ISH analysis revealed that the two mouse-testis-specific lncRNAs were expressed exclusively in testicular germ cells in meiotic prophase and the round spermatid stage, which coincide with the period of transcriptional reactivation during spermatogenesis. The cytoplasmic distribution of these lncRNAs revealed by ISH suggests their involvement in post-transcriptional gene regulation rather than in epigenetic or transcriptional regulation. Our data provide new insight into testis-associated lncRNAs that will be useful in expression and functional studies of spermatogenesis.

Maternal peripheral blood natural killer cells incorporate placenta-associated microRNAs during pregnancy.

Although recent studies have demonstrated that microRNAs (miRNAs or miRs) regulate fundamental natural killer (NK) cellular processes, including cytotoxicity and cytokine production, little is known about the miRNA-gene regulatory relationships in maternal peripheral blood NK (pNK) cells during pregnancy. In the present study, to determine the roles of miRNAs within gene regulatory networks of maternal pNK cells, we performed comprehensive miRNA and gene expression profiling of maternal pNK cells using a combination of reverse transcription quantitative PCR (RT-qPCR)-based miRNA array and DNA microarray analyses and analyzed the differential expression levels between first- and third-trimester pNK cells. Furthermore, we constructed regulatory networks for miRNA-mediated gene expression in pNK cells during pregnancy by Ingenuity Pathway Analysis (IPA). PCR-based array analysis revealed that the placenta-derived miRNAs [chromosome 19 miRNA cluster (C19MC) miRNAs] were detected in pNK cells during pregnancy. Twenty-five miRNAs, including six C19MC miRNAs, were significantly upregulated in the third- compared to first-trimester pNK cells. The rapid clearance of C19MC miRNAs also occurred in the pNK cells following delivery. Nine miRNAs, including eight C19MC miRNAs, were significantly downregulated in the post-delivery pNK cells compared to those of the third-trimester. DNA microarray analysis identified 69 NK cell function-related genes that were differentially expressed between the first- and third-trimester pNK cells. On pathway and network analysis, the observed gene expression changes of pNK cells likely contribute to the increase in the cytotoxicity, as well as the cell cycle progression of third- compared to first-trimester pNK cells. Thirteen of the 69 NK cell function-related genes were significantly downregulated between the first- and third-trimester pNK cells. Nine of the 13 downregulated NK-function-associated genes were in silico target candidates of 12 upregulated miRNAs, including C19MC miRNA miR-512-3p. The results of this study suggest that the transfer of placental C19MC miRNAs into maternal pNK cells occurs during pregnancy. The present study provides new insight into maternal NK cell functions.

Fc gamma receptor IIb participates in maternal IgG trafficking of human placental endothelial cells.

The human placental transfer of maternal IgG is crucial for fetal and newborn immunity. Low-affinity immunoglobulin gamma Fc region receptor IIb2 (FCGR2B2 or FcγRIIb2) is exclusively expressed in an IgG-containing, vesicle-like organelle (the FCGR2B2 compartment) in human placental endothelial cells; thus, we hypothesized that the FCGR2B2 compartment functions as an IgG transporter. In this study, to examine this hypothesis, we performed in vitro bio-imaging analysis of IgG trafficking by FCGR2B2 compartments using human umbilical vein endothelial cells transfected with a plasmid vector containing enhanced GFP-tagged FCGR2B2 (pFCGR2B2-EGFP). FCGR2B2-EGFP signals were detected as intracellular vesicular structures similar to FCGR2B2 compartments in vivo. The internalization and transcytosis of IgG was significantly higher in the pFCGR2B2-EGFP-transfected cells than in the mock-transfected cells, and the majority of the internalized IgG was co-localized with the FCGR2B2-EGFP signals. Furthermore, we isolated FCGR2B2 compartments from the human placenta and found that the Rab family of proteins [RAS-related protein Rab family (RABs)] were associated with FCGR2B2 compartments. Among the RABs, RAB3D was expressed predominantly in placental endothelial cells. The downregulation of RAB3D by small interfering RNA (siRNA) resulted in a marked reduction in the FCGR2B2-EGFP signals at the cell periphery. Taken together, these findings suggest that FCGR2B2 compartments participate in the transcytosis of maternal IgG across the human placental endothelium and that RAB3D plays a role in regulating the intracellular dynamics of FCGR2B2 compartments.

Human exosomal placenta-associated miR-517a-3p modulates the expression of PRKG1 mRNA in Jurkat cells.

During pregnancy, human placenta-associated microRNAs (miRNAs) derived from the miRNA cluster in human chromosome 19 are expressed in villous trophoblasts and secreted into maternal circulation via exosomes; however, little is known about whether circulating placenta-associated miRNAs are transferred into maternal immune cells via exosomes, and modulate expression of target genes in the recipient cells. We employed an in vitro model of trophoblast-immune cell communication using BeWo cells (a human trophoblast cell line) and Jurkat cells (a human leukemic T-cell line) and investigated whether BeWo exosomal placenta-associated miRNAs can suppress expression of target genes in the recipient Jurkat cells. Using this system, we identified PRKG1 as a target gene of placenta-associated miRNA miR-517a-3p. Moreover, we demonstrated that BeWo exosomal miR-517a-3p was internalized into Jurkat cells and subsequently suppressed the expression of PRKG1 in recipient Jurkat cells. Furthermore, using peripheral blood natural killer (NK) cells in vivo, we confirmed that circulating miR-517a-3p was delivered into maternal NK cells as it was into Jurkat cells in vitro. Placenta-associated miR-517a-3p was incorporated into maternal NK cells in the third trimester, and it was rapidly cleared after delivery. Expression levels of miR-517a-3p and its target mRNA PRKG1 were inversely correlated in NK cells before and after delivery. These in vitro and in vivo results suggest that exosome-mediated transfer of placenta-associated miRNAs and subsequent modulation of their target genes occur in maternal NK cells. The present study provides novel insight into our understanding of placenta-maternal communication.

Team-based learning using an audience response system: a possible new strategy for interactive medical education.

Following the "Guidelines for reporting TBL" by Haidet et al, we report on a team-based learning (TBL) course we adopted for our 4th-year students in 2011. Our TBL course is a modified version of the one suggested in the guidelines, but its structure generally follows the core elements described therein. Using an audience response system (ARS), we were able to obtain individual and group readiness assurance test scores immediately and give instant feedback to the students. Instructors were thus able to monitor students' understanding in real time and so appreciated the system, which supports interactive classes even in large classrooms. However, TBL is teacher-oriented, and students were less appreciative of ARS, because they recognized that it could be easily used for grading. Nevertheless, we believe that a combination of TBL, and problem-based learning in a mature design can improve both motivation and understanding among learners.

MiR-21 is enriched in the RNA-induced silencing complex and targets COL4A1 in human granulosa cell lines.

MicroRNAs (miRNAs) are noncoding small RNAs that play important roles in a variety of physiological and pathological events. In this study, we performed large-scale profiling of EIF2C2-bound miRNAs in 3 human granulosa-derived cell lines (ie, KGN, HSOGT, and GC1a) by high-throughput sequencing and found that miR-21 accounted for more than 80% of EIF2C2-bound miRNAs, suggesting that it was enriched in the RNA-induced silencing complex (RISC) and played a functional role in human granulosa cell (GC) lines. We also found high expression levels of miR-21 in primary human GCs. Assuming that miR-21 target mRNAs are enriched in RISC, we performed cDNA cloning of EIF2C2-bound mRNAs in KGN cells. We identified COL4A1 mRNA as a miR-21 target in the GC lines. These data suggest that miR-21 is involved in the regulation of the synthesis of COL4A1, a component of the basement membrane surrounding the GC layer and granulosa-embedded extracellular structure.

Gene suppression of mouse testis in vivo using small interfering RNA derived from plasmid vectors.

We evaluated whether inhibiting gene expression by small interfering RNA (siRNA) can be used for an in vivo model using a germ cell-specific gene (Tex101) as a model target in mouse testis. We generated plasmid-based expression vectors of siRNA targeting the Tex101 gene and transfected them into postnatal day 10 mouse testes by in vivo electroporation. After optimizing the electroporation conditions using a vector transfected into the mouse testis, a combination of high- and low-voltage pulses showed excellent transfection efficiency for the vectors with minimal tissue damage, but gene suppression was transient. Gene suppression by in vivo electroporation may be helpful as an alternative approach when designing experiments to unravel the basic role of testicular molecules.

Knocking-in the R142C mutation in transglutaminase 1 disrupts the stratum corneum barrier and postnatal survival of mice.

BACKGROUND: Mutations in the gene encoding transglutaminase 1 (TG1) are responsible for various types of autosomal recessive congenital ichthyosis (ARCI), such as lamellar ichthyosis (LI), congenital ichthyosiform erythroderma (CIE) and some minor variants of ARCI. A point mutation of R143C in the ß-sandwich domain of TG1 has been often identified in patients with LI or CIE. OBJECTIVE: To elucidate the effect of that point mutation on skin barrier structures and functions, we generated mice with a point mutation of R142C, which corresponds to the R143C mutation in human TG1. METHODS: A mouse line with the R142C point mutation in TG1 was established using a gene targeting technique and the Cre-loxP system. The skin phenotypes were analyzed in homozygous mutant Tgm1(R142C/R142C) mice. RESULTS: In the skin of Tgm1(R142C/R142C) mice, expression of the mutant transcripts was comparable with wild-type or Tgm1(+/R142C) mice. However, the amount of mutated protein in the skin was markedly decreased in Tgm1(R142C/R142C) mice, and the TG1 activity of Tgm1(R142C/R142C) keratinocytes was almost lost. Tgm1(R142C/R142C) mice exhibited morphological and functional skin barrier defects and neonatal lethality. The stratum corneum of those mice lacked cornified envelopes, and loricrin, the major structural component, failed to assemble at the corneocyte cell periphery. Tgm1(R142C/R142C) mice showed a marked increase in transepidermal water loss and their skin was easily permeable to toluidine blue dye. The intercellular lipid lamellar structures of the stratum corneum were irregular and the 13-nm periodic X-ray diffractions from the stratum corneum lipid molecules were lost in vivo. CONCLUSION: From these results, we suggest that the R142C mutation of TG1 reduces the enzyme stability which is indispensable for development of the stratum corneum and skin barrier function and for postnatal survival of mice.

Hydroxysteroid (17-ß) dehydrogenase 1 is dysregulated by miR-210 and miR-518c that are aberrantly expressed in preeclamptic placentas: a novel marker for predicting preeclampsia.

In this study, to search for novel preeclampsia (PE) biomarkers, we focused on microRNA expression and function in the human placenta complicated with PE. By comprehensive analyses of microRNA expression, we identified 22 microRNAs significantly upregulated in preeclamptic placentas, 5 of which were predicted in silico to commonly target the mRNA encoding hydroxysteroid (17-ß) dehydrogenase 1 (HSD17B1), a steroidogenetic enzyme expressed predominantly in the placenta. In vivo HSD17B1 expression, at both the mRNA and protein levels, was significantly decreased in preeclamptic placentas. Of these microRNAs, miR-210 and miR-518c were experimentally validated to target HSD17B1 by luciferase assay, real-time PCR, and ELISA. Furthermore, we found that plasma HSD17B1 protein levels in preeclamptic pregnant women reflected the decrease of its placental expression. Moreover, a prospective cohort study of plasma HSD17B1 revealed a significant reduction of plasma HSD17B1 levels in pregnant women at 20 to 23 and 27 to 30 weeks of gestation before PE onset compared with those with normal pregnancies. The sensitivities/specificities for predicting PE at 20 to 23 and 27 to 30 weeks of gestation were 0.75/0.67 (cutoff value=21.9 ng/mL) and 0.88/0.51 (cutoff value=30.5 ng/mL), and the odds ratios were 6.09 (95% CI: 2.35-15.77) and 7.83 (95% CI: 1.70-36.14), respectively. We conclude that HSD17B1 is dysregulated by miR-210 and miR-518c that are aberrantly expressed in preeclamptic placenta and that reducing plasma level of HSD17B1 precedes the onset of PE and is a potential prognostic factor for PE.

Human villous trophoblasts express and secrete placenta-specific microRNAs into maternal circulation via exosomes.

In this study, we performed small RNA library sequencing using human placental tissues to identify placenta-specific miRNAs. We also tested the hypothesis that human chorionic villi could secrete miRNAs extracellularly via exosomes, which in turn enter into maternal circulation. By small RNA library sequencing, most placenta-specific miRNAs (e.g., MIR517A) were linked to a miRNA cluster on chromosome 19. The miRNA cluster genes were differentially expressed in placental development. Subsequent validation by real-time PCR and in situ hybridization revealed that villous trophoblasts express placenta-specific miRNAs. The analysis of small RNA libraries from the blood plasma showed that the placenta-specific miRNAs are abundant in the plasma of pregnant women. By real-time PCR, we confirmed the rapid clearance of the placenta-specific miRNAs from the plasma after delivery, indicating that such miRNAs enter into maternal circulation. By using the trophoblast cell line BeWo in culture, we demonstrated that miRNAs are indeed extracellularly released via exosomes. Taken together, our findings suggest that miRNAs are exported from the human placental syncytiotrophoblast into maternal circulation, where they could target maternal tissues. Finally, to address the biological functions of placenta-specific miRNAs, we performed a proteome analysis of BeWo cells transfected with MIR517A. Bioinformatic analysis suggests that this miRNA is possibly involved in tumor necrosis factor-mediated signaling. Our data provide important insights into miRNA biology of the human placenta.

MicroRNA (miRNA) cloning analysis reveals sex differences in miRNA expression profiles between adult mouse testis and ovary.

MicroRNAs (miRNAs) are endogenous non-coding small RNAs that can regulate the expression of complementary mRNA targets. Identifying tissue-specific miRNAs is the first step toward understanding the biological functions of miRNAs, which include the regulation of tissue differentiation and the maintenance of tissue identity. In this study, we performed small RNA library sequencing in adult mouse testis and ovary to reveal their characteristic organ- and gender-specific profiles and to elucidate the characteristics of the miRNAs expressed in the reproductive system. We obtained 10,852 and 11 744 small RNA clones from mouse testis and ovary respectively (greater than 10,000 clones per organ), which included 6630 (159 genes) and 10,192 (154 genes) known miRNAs. A high level of efficiency of miRNA library sequencing was achieved: 61% (6630 miRNA clones/10,852 small RNA clones) and 87% (10,192/11,744) for adult mouse testis and ovary respectively. We obtained characteristic miRNA signatures in testis and ovary; 55 miRNAs were detected highly, exclusively, or predominantly in adult mouse testis and ovary, and discovered two novel miRNAs. Male-biased expression of miRNAs occurred on the X-chromosome. Our data provide important information on sex differences in miRNA expression that should facilitate studies of the reproductive organ-specific roles of miRNAs.

RT-PCR-based analysis of microRNA (miR-1 and -124) expression in mouse CNS.

More than 700 microRNAs (miRNAs) have been cloned, and the functions of these molecules in developmental timing, cell proliferation, and cancer have been investigated widely. MiRNAs are analyzed with Northern blot and sequential colony evaluation; however, reverse transcription-polymerase chain reaction (RT-PCR)-based miRNA assay remains to be developed. In this report, we describe improved real-time RT-PCR methods using specific or non-specific RT primer for the semi-quantitative analysis of miRNA expression. The use of the new methods in a model study revealed differential expression of miRNA-1 (miR-1) and miR-124 in mouse organs. Specifically, our methods revealed that miR-124 concentrations in the mouse central nervous system (CNS; cerebral cortex, cerebellum, and spinal cord) were more than 100 times those in other organs. By contrast, miR-1 expression in the CNS was 100-1000 times lower than that in skeletal muscle and heart. Furthermore, we revealed anatomically regional differences in miR-124 expression within the CNS: expression ratios versus the cerebral cortex were 60.7% for the cerebellum and 35.4% for the spinal cord. These results suggest that our RT-PCR-based methods would be a powerful tool for studies of miRNA expression that is associated with various neural events.

[Tutorial session using cross sections of upper and lower limbs in human gross anatomy course].

We performed tutorial education in gross anatomy using thick serial cross sections (3-4 cm) of one of the upper or lower limbs of a cadaver donated for use in the macroscopic dissection course. Group learning (four people) proceeded with the extraction of the point in question from an arbitrary section. Consequently students obtained a deeper understanding of the cross sectional structure of an upper or lower limb and further deepened their understanding of the three-dimensional structure of limbs. Each group collected clinical images of the part corresponding to the selected section to present, thus bringing together the relationship between the anatomical knowledge studied from the specimen and the related disease. A questionnaire survey after the practicum suggested that the practice of using serial cross sections and the presentation of relevant images using Microsoft PowerPoint were effective means of the study. Useful points on the student's understanding were collected with respect to the following three opinions: Fresh aspects, Grasp of the position, and Three-dimensional views. Moreover, the dissection program in gross anatomy combined with thick cross sections was thought to be helpful in that not only was observing the cross structure but also to dissect it if necessary, with the students obtaining a deeper understanding of the structure as a result. In our anatomy practice thereafter, the practicum was done with awareness of the issues faced by the students in mind. It was concluded that the practicum that uses the cross sections is an effective means in gross anatomy education.

[Student evaluation of faculty teaching in molecular anatomy course at Nippon Medical School: results of a questionnaire survey].

For the purpose of improving education, the lecture and practices employed in the Molecular Anatomy Course were evaluated by students. The survey was performed with personal computer connected to a local area network and the results were readily exhibited on our website the following day. The total number of answers was 528 and the reply percentage was 31.2%. The average score (from 1: very bad to 5: very good) was 3.9, with a range of 3.2 to 4.8. Faculty teaching got better grades in the latter half of the period compared to those in the first half, particularly on "preparation" and "understanding". The correlation analysis showed a strong relationship between the understanding of the lecture and the orientation of practice. These findings suggest that constant evaluation by students during the whole period of the instruction is useful for improving the quality of education of our Anatomy Course.

Sexually dimorphic expression of the novel germ cell antigen TEX101 during mouse gonad development.

Prospermatogonia, or gonocytes, are the cells that differentiate from primordial germ cells to the first mature type of spermatogonia in the developing testis. Although prospermatogonia play a central role in this stage (i.e., prespermatogenesis), the details regarding their characterization have not been fully elucidated. Recently, we identified a novel mouse testicular germ cell-specific antigen, TES101 reactive protein (TES101RP), in the adult mouse testis. The protein TES101RP is also designated as protein TEX101. In the present study, we investigated the expression of TEX101 on germ cells in developing mouse gonads using histochemical techniques (i.e., immunohistochemistry, BrdU labeling, and TUNEL staining) and reverse transcription-polymerase chain reaction. TEX101 appeared on germ cells in both male and female gonads after the pregonadal period. In the testis, TEX101 was expressed constitutively on surviving prospermatogonia during prespermatogenesis. After the initiation of spermatogenesis, the prospermatogonia differentiated into spermatogonia. TEX101 expression disappeared from the spermatogonia, but reappeared on spermatocytes and spermatids. In the ovary, TEX101 was expressed on germ cells until the start of folliculogenesis; TEX101 was not detected on oocytes that were surrounded by follicular cells. These findings indicate that TEX101 is a specific marker for both male and female germ cells during gonadal development. Because the on and off switching of TEX101 expression in germ cells almost parallels the kinetics of gametogenesis, TEX101 may play an important physiological role in germ cell development.

TEX101 is shed from the surface of sperm located in the caput epididymidis of the mouse.

It is generally believed that cell-to-cell cross-talk and signal transduction are mediated by cell surface molecules that play diverse and important regulatory roles in spermatogenesis and fertilization. Recently, we identified a novel plasma membrane-associated protein, TES101-reactive protein (TES101RP, or TEX101), on mouse testicular germ cells. In this study, we investigate Tex101 mRNA expression in the adult mouse testis using in situ hybridization, and we examine the fate of TEX101 during sperm transport by immunohistochemical and Western blot analyses. Tex101 mRNA was expressed in a stage-specific manner in spermatocytes and in step 1-9 spermatids of the testis, but not in spermatogonia. Although the TEX101 protein remained on the cell surfaces of step 10-16 spermatids and testicular sperm, it was shed from epididymal sperm located in the caput epididymidis. The results of this study provide additional information on germ cell-specific TEX101 expression during spermatogenesis and post-testicular sperm maturation.

Development of ichthyosiform skin compensates for defective permeability barrier function in mice lacking transglutaminase 1.

Transglutaminase 1 (TGase 1) is one of the genes implicated in autosomal recessive congenital ichthyosis. Skin from TGase 1(-/-) mice, which die as neonates, lacks the normal insoluble cornified envelope and has impaired barrier function. Characterization of in situ dye permeability and transepidermal water loss revealed defects in the development of the skin permeability barrier in TGase 1(-/-) mice. In the stratum corneum of the skin, tongue, and forestomach, intercellular lipid lamellae were disorganized, and the corneocyte lipid envelope and cornified envelope were lacking. Neonatal TGase 1(-/-) mouse skin was taut and erythrodermic, but transplanted TGase 1(-/-) mouse skin resembled that seen in severe ichthyosis, with epidermal hyperplasia and marked hyperkeratosis. Abnormalities in those barrier structures remained, but transepidermal water loss was improved to control levels in the ichthyosiform skin. From these results, we conclude that TGase 1 is essential to the assembly and organization of the barrier structures in stratified squamous epithelia. We suggest that the ichthyosiform skin phenotype in TGase 1 deficiency develops the massive hyperkeratosis as a physical compensation for the defective cutaneous permeability barrier required for survival in a terrestrial environment.