The fluid factor OVGP1 provides a significant oviductal microenvironment for the reproductive process in golden hamster†.

The mammalian oviductal lumen is a specialized chamber that provides an environment that strictly regulates fertilization and early embryogenesis, but the regulatory mechanisms to gametes and zygotes are unclear. We evaluated the oviductal regulation of early embryonic development using Ovgp1 (encoding an oviductal humoral factor, OVGP1)-knockout golden hamsters. The experimental results revealed the following: (1) female Ovgp1-knockout hamsters failed to produce litters; (2) in the oviducts of Ovgp1-knockout animals, fertilized eggs were sometimes identified, but their morphology showed abnormal features; (3) the number of implantations in the Ovgp1-knockout females was low; (4) even if implantations occurred, the embryos developed abnormally and eventually died; and (5) Ovgp1-knockout female ovaries transferred to wild-type females resulted in the production of Ovgp1-knockout egg-derived OVGP1-null litters, but the reverse experiment did not. These results suggest that OVGP1-mediated physiological events are crucial for reproductive process in vivo, from fertilization to early embryonic development. This animal model shows that the fate of the zygote is determined not only genetically, but also by the surrounding oviductal microenvironment.

Targeting of neutrophil activation in the early phase of the disease for prevention of Coronavirus disease-19 severity.

The prevention of the disease severity seems critical for reducing the mortality of Coronavirus (CoV) disease-19. The neutrophils play a key role in the induction of severity. It is proposed here that inhibition of neutrophil activation and/or cascade reactions of complement, leading to this cell activation at the early phase of the disease, is a potential tool to inhibit aggravation of the disease. The need for appropriate timing in intervention is emphasized as follows. (1) Intervention at the very early stage of severe acute respiratory syndrome-CoV-2 infection may harm the defensive host response to the infection because of the critical function of neutrophils in this response, and (2) intervention at too late a stage will not stop the infiltration of fully activated neutrophils that produce large amounts of toxic substances.

Embryos, cancers, and parasites: Potential applications to the study of reproductive biology in view of their similarity as biological phenomena.

Background: At present, there are so many living things on the earth. Most of these organisms have a reproductive strategy called sexual reproduction. Among organisms that reproduce sexually, mammals have an extremely complex and seemingly unnatural method of reproduction, or viviparity. Methods: As an approach to understanding the nature of viviparity, the author have tried to outline the common life phenomena of embryos, cancers, and parasites based on the literature to date, with internal parasites as the keyword. Main findings: Embryo, cancer, and parasite are constituted as a systemic interaction with the host (mother). Based on these facts, the author proposed the hypothesis that in the case of mammals, "the fetus is essentially harmful to the mother", and that the parasitic fetus grows by skillfully evading the mother's foreign body exclusion mechanism. Conclusion: Comparative studies of "embryos", "cancers", and "parasites" as foreign bodies have the potential to produce unexpected discoveries in their respective fields. It is important to consider the evolutionary time axis that the basic structure of our mammalian body arose over 200 million years from the Mesozoic Triassic, the period immediately after the Paleozoic Era, when life on Earth became massively extinct.

Exploration of novel biomarkers for hypertensive disorders of pregnancy by comprehensive analysis of peptide fragments in blood: their potential and technologies supporting quantification.

Among the many complications associated with pregnancy, hypertensive disorders of pregnancy (HDP) constitute one of the most important. Since the pathophysiology of HDP is complex, new disease biomarkers (DBMs) are needed to serve as indicators of disease activity. However, in the current status of laboratory medicine, despite the fact that blood pressure measurement has been used for a long time, not many DBMs contribute adequately to the subsequent diagnosis and treatment. In this article, we discuss studies focusing on peptide fragments in blood identified by comprehensive quantitative methods, among the currently proposed DBM candidates. Furthermore, we describe the basic techniques of peptidomics, especially quantitative proteomics, and outline the current status and challenges of measuring peptides in blood as DBM for HDP.

Cleavage of SPACA1 regulates assembly of sperm-egg membrane fusion machinery in mature spermatozoa†.

The acrosome reaction is a multi-step event essential for physiological fertilization. During the acrosome reaction, gamete fusion-related factor IZUMO1 translocates from the anterior acrosome to the equatorial segment and assembles the gamete fusion machinery. The morphological changes in the acrosome reaction process have been well studied, but little is known about the molecular mechanisms of acrosome reorganization essential for physiological gamete membrane fusion. To elucidate the molecular mechanisms of IZUMO1 translocation, the steps of the acrosome reaction during that process must be clarified. In this study, we established a method to detect the early steps of the acrosome reaction and subdivided the process into seven populations through the use of two epitope-defined antibodies, anti-IZUMO1 and anti-SPACA1, a fertilization-inhibiting antibody. We found that part of the SPACA1 C-terminus in the periacrosomal space was cleaved and had begun to disappear when the vesiculation of the anterior acrosome occurred. The IZUMO1 epitope externalized from the acrosomal lumen before acrosomal vesiculation and phosphorylation of IZUMO1 occurred during the translocation to the equatorial segment. IZUMO1 circumvented the area of the equatorial segment where the SPACA1C-terminus was still localized. We therefore propose an IZUMO1 translocation model and involvement of SPACA1.

Role of the Glycosylphosphatidylinositol-Anchored Protein TEX101 and Its Related Molecules in Spermatogenesis.

Glycosylphosphatidylinositol (GPI)-anchored proteins (APs) on the plasma membrane are involved in several cellular processes, including sperm functions. Thus far, several GPI-APs have been identified in the testicular germ cells, and there is increasing evidence of their biological significance during fertilization. Among GPI-APs identified in the testis, this review focuses on TEX101, a germ cell-specific GPI-AP that belongs to the lymphocyte antigen 6/urokinase-type plasminogen activator receptor superfamily. This molecule was originally identified as a glycoprotein that contained the antigen epitope for a specific monoclonal antibody; it was produced by immunizing female mice with an allogenic testicular homogenate. This review mainly describes the current understanding of the biochemical, morphological, and physiological characteristics of TEX101. Furthermore, future avenues for the investigation of testicular GPI-Aps, including their potential role as regulators of ion channels, are discussed.

Promoting Roles of Embryonic Signals in Embryo Implantation and Placentation in Cooperation with Endocrine and Immune Systems.

Embryo implantation in the uterus is an essential process for successful pregnancy in mammals. In general, the endocrine system induces sufficient embryo receptivity in the endometrium, where adhesion-promoting molecules increase and adhesion-inhibitory molecules decrease. Although the precise mechanisms remain unknown, it is widely accepted that maternal-embryo communications, including embryonic signals, improve the receptive ability of the sex steroid hormone-primed endometrium. The embryo may utilize repulsive forces produced by an Eph-ephrin system for its timely attachment to and subsequent invasion through the endometrial epithelial layer. Importantly, the embryonic signals are considered to act on maternal immune cells to induce immune tolerance. They also elicit local inflammation that promotes endometrial differentiation and maternal tissue remodeling during embryo implantation and placentation. Additional clarification of the immune control mechanisms by embryonic signals, such as human chorionic gonadotropin, pre-implantation factor, zona pellucida degradation products, and laeverin, will aid in the further development of immunotherapy to minimize implantation failure in the future.

NUP62: the target of an anti-sperm auto-monoclonal antibody during testicular development.

Ts4, an autosperm-monoclonal antibody (mAb), reacts with a specific oligosaccharide (OS) of glycoproteins containing bisecting N-acetylglucosamine residues. Ts4 reactivity was observed against epididymal spermatozoa, testicular germ cells, and the early embryo, but not against major organs in adult mice. In mature testis, Ts4 exhibits immunoreactivity with a germ cell-specific glycoprotein, TEX101, whereas the mAb immunoreacts with alpha-N-acetylglucosaminidase in the acrosomal region of cauda epididymal spermatozoa. Thus, Ts4 seems to react against different molecules throughout spermiogenesis via binding to its OS epitope. Since the Ts4-epitope OS is observed only in reproduction-related regions, the Ts4-reactive OS may play a role in the reproductive process. The aim of this study is to investigate the characteristics of the Ts4-reactive molecule(s) during testicular development. Ts4 reactivity was observed in testes from the prenatal period; however, its distribution changed according to the stage of maturation and was identical to that of the adult testes after 29-day-postpartum (dpp). Ts4 immunoreactivity was detected against a protein with 63 kDa in testis from 1 to 29 dpp. In contrast, Ts4 showed reactivity against some other glycoproteins after 29 dpp, including TEX101 at the 5-week-old stage and onward. To identify the Ts4-reactive 63 kDa molecule, we identified NUP62 as the target of Ts4 in 22 dpp testis using liquid chromatography-tandem mass spectrometry analysis. Because NUP62 has been known to play active roles in a variety of cellular processes including mitosis and cell migration, the bisecting GlcNAc recognized by Ts4 on NUP62 may play a role in regulating the early development of germ cells in male gonadal organs.

Expression of TEX101, regulated by ACE, is essential for the production of fertile mouse spermatozoa.

Formation of spermatozoa of normal shape, number, and motility is insufficient for the male siring of pups. The spermatozoa must be accompanied by sound fertilizing ability. We found that males with disrupted testis-expressed gene 101 (Tex101) produce normal-looking but fertilization-incompetent spermatozoa, which were accompanied by a deficiency of a disintegrin and metallopeptidase domain 3 (ADAM3) on sperm plasma membrane. It was also found that the existence of TEX101 on spermatozoa was regulated by angiotensin-converting enzyme (ACE). The removal of GPI-anchored protein TEX101 by ACE was essential to produce fertile spermatozoa, and the function of ACE was not depending on its well-known peptidase activity. The finding of TEX101 as a unique specific substrate for ACE may provide a potential target for the production of an awaited contraceptive medicine for men.

A genetically engineered ovarian cancer mouse model based on fallopian tube transformation mimics human high-grade serous carcinoma development.

Recent evidence suggests that ovarian high-grade serous carcinoma (HGSC) originates from the epithelium of the fallopian tube. However, most mouse models are based on the previous prevailing view that ovarian cancer develops from the transformation of the ovarian surface epithelium. Here, we report the extensive histological and molecular characterization of the mogp-TAg transgenic mouse, which expresses the SV40 large T-antigen (TAg) under the control of the mouse müllerian-specific Ovgp-1 promoter. Histological analysis of the fallopian tubes of mogp-TAg mice identified a variety of neoplastic lesions analogous to those described as precursors to ovarian HGSC. We identified areas of normal-appearing p53-positive epithelium that are similar to 'p53 signatures' in the human fallopian tube. More advanced proliferative lesions with nuclear atypia and epithelial stratification were also identified that were morphologically and immunohistochemically reminiscent of human serous tubal intraepithelial carcinoma (STIC), a potential precursor of ovarian HGSC. Beside these non-invasive precursor lesions, we also identified invasive adenocarcinoma in the ovaries of 56% of the mice. Microarray analysis revealed several genes differentially expressed between the fallopian tube of mogp-TAg and wild-type (WT) C57BL/6. One of these genes, Top2a, which encodes topoisomerase IIα, was shown by immunohistochemistry to be concurrently expressed with elevated p53 and was specifically elevated in mouse STICs but not in the surrounding tissues. TOP2A protein was also found elevated in human STICs, low-grade and high-grade serous carcinoma. The mouse model reported here displays a progression from normal tubal epithelium to invasive HGSC in the ovary, and therefore closely simulates the current emerging model of human ovarian HGSC pathogenesis. This mouse therefore has the potential to be a very useful new model for elucidating the mechanisms of serous ovarian tumourigenesis, as well as for developing novel approaches for the prevention, diagnosis and therapy of this disease.

Eph-ephrin A system regulates human choriocarcinoma-derived JEG-3 cell invasion.

OBJECTIVES: The Eph-ephrin system is a unique system that can induce multiple cellular responses such as cell migration, regulation of angiogenesis, and axonal guidance. Previously, the Eph-ephrin system was reported to regulate human extravillous trophoblast invasion. In this study, we examined the possible involvement of the Eph-ephrin system in the invasion of malignant gestational trophoblastic diseases using a human choriocarcinoma-derived cell line, JEG-3. METHODS: The mRNA expression of class A Ephs and ephrins on JEG-3 cells was examined by reverse transcription-polymerase chain reaction. The effects of recombinant human Eph A1 (r-Eph A1) and r-ephrin A4 on the proliferation and invasion of JEG-3 cells were investigated by cell proliferation and Matrigel invasion assays. The alterations of integrin expression on JEG-3 cells in the presence of r-Eph A1 and r-ephrin A4 were investigated by flow cytometry. The induction of phosphorylation of focal adhesion kinase in JEG-3 cells by r-ephrin A4 was examined by Western blot analysis. RESULTS: By reverse transcription-polymerase chain reaction, mRNAs of Eph A1, A2, and A4 and ephrin A1, A4, and A5 were detected on JEG-3 cells. In Matrigel invasion assay, both r-Eph A1 and r-ephrin A4 promoted the invasion of JEG-3 cells without affecting cell proliferation. During 24-hour culture with r-Eph A1 and r-ephrin A4, the increase in integrin α 5 expression on JEG-3 cells was observed by flow cytometry. Western blotting analysis showed that r-ephrin A4 induced dephosphorylation of focal adhesion kinase in JEG-3 cells. CONCLUSIONS: These findings suggest that Eph-ephrin interaction plays some role in the regulation of choriocarcinoma invasion in cooperation with integrins.

Clinical peptidomic analysis by a one-step direct transfer technology: its potential utility for monitoring of pathophysiological status in female reproductive system disorders.

To date, numerous studies have searched for candidate molecules or clinical examination methods as potential biomarkers for monitoring intractable diseases, such as carcinomas. Evidence accumulated over the past decade shows that many proteolytic peptides appear in human humoral fluids, including peripheral blood, in association with an individual's health condition. Although an analysis of the whole peptide (the 'peptidome') using mass spectrometry is thought to be one of the most powerful and promising experimental approaches, it has failed to identify biomarkers in the clinical blood samples, presumably due to the methodological limitations. In general, commonly used techniques for proteomic analysis of blood require the removal of large amounts of serum/plasma proteins prior to mass spectrometry analysis, and this step seems to have resulted in the overlooking of important biomarkers during the analytical process. Here, we provide a brief overview of a new quantitative peptidomic analysis by a one-step direct transfer technology without depletion of major blood proteins. Using this technology, we herein report experimental data on serum peptidomic analysis for patients with pregnancy-induced hypertension as a clinical model. In addition, we refer to the potential utility of this approach for the monitoring of pathophysiological status in female reproductive system disorders in general.

Peptides associated with hypertensive disorders of pregnancy as possible biomarkers for severity of lower extremity arterial disease.

BACKGROUND AND AIMS: Seven circulating peptides, consisting of 18-28 amino acids, were identified as possible biomarkers of hypertensive disorders of pregnancy (HDP) in our previous study. However, it is unknown whether these peptides are relevant to cardiovascular disease. The purpose of this study was to clarify the relationships between serum levels of these peptides and leg arterial blood flow in patients with lower extremity arterial disease (LEAD). METHODS: The subjects were 165 outpatients with LEAD. Patients with advanced LEAD (stages 5 and 6 of the Rutherford classification) were not included. Leg arterial blood flow was evaluated by ankle brachial pressure index (ABI) and % decrease in ABI after leg exercise induced by a leg loader or treadmill. Concentrations of the seven peptides with m/z 2081 (P-2081), 2091 (P-2091), 2127 (P-2127), 2209 (P-2209), 2378 (P-2378), 2858 (P-2858) and 3156 (P-3156) were measured simultaneously with a mass spectrometer. RESULTS: P-2081, P-2127 and P-2209 levels showed significant positive correlations with leg arterial blood flow, while P-2091, P-2378 and P-2858 levels showed significant inverse correlations with leg arterial blood flow. There was no significant correlation between P-3156 levels and leg arterial blood flow. The above positive and inverse associations between peptide levels and leg arterial blood flow were also found in logistic regression analysis using tertile groups divided by the concentrations of each peptide. CONCLUSIONS: Serum levels of six HDP-related peptides (P-2081, P-2091, P-2127, P-2209, P-2378 and P-2858) were associated with lower extremity arterial blood flow in patients with LEAD, and thus these peptides are possible biomarkers for severity of LEAD.

Role of TGF-beta1 and TNF-alpha1 produced by neoplastic cells in the pathogenesis of fibrosis in patients with hematologic neoplasms.

We conducted this study with the objective of elucidating the mechanism of development of fibrosis in hematologic neoplasms and develop treatments for these patients. Among the suggested mechanisms of development of fibrosis is cases of hematologic neoplasms is the production of TGF-beta1 (transforming growth factor-beta-1) and TNF-alpha1 (tumor necrotizing factor-alpha-1) by the tumor cells, both of which are fibrosis-stimulating cytokines that act on fibroblasts to promote fibrosis. However, there are few reports based on human clinical pathology studies. We conducted an immunohistochemical study on paraffin-embedded formalin-fixed specimens obtained from 104 patients with various pathologic conditions (acute leukemia, malignant lymphoma, inflammation, cancer, etc.). The association of tissue fibrosis with positive immunohistochemistry for TGF- beta1 and/or TNF-alpha1, TGF-beta1 was found to be strongly associated with tissue fibrosis, and in cases with positive immunohistochemistry for TGF-beta1, the odds ratio for fibrosis was 12.8, which was significantly high. Combined positivity for TGF-beta1 and TNF-alpha1 was also associated with a significant odds ratio for fibrosis of 3.4, suggesting that TGF-beta1 expression is an important prerequisite. TGF-beta1 has been suggested as playing a relatively important role in tissue fibrosis. Future clinical application of these cytokines for both diagnosis and treatment is expected.

Possible New Histological Prognostic Index for Large B-Cell Lymphoma.

We conducted a retrospective analysis of GRP94 immunohistochemical (IHC) staining, an ER stress protein, on large B-cell lymphoma (LBCL) cells, intracellular p53, and 15 factors involved in the metabolism of the CHOP regimen: AKR1C3 (HO metabolism), CYP3A4 (CHOP metabolism), and HO efflux pumps (MDR1 and MRP1). The study subjects were 42 patients with LBCL at our hospital. The IHC staining used antibodies against the 17 factors. The odds ratios by logistic regression analysis used a dichotomous variable of CR and non-CR/relapse were statistically significant for MDR1, MRP1, and AKR1C3. The overall survival (OS) after R-CHOP was compared by the log-rank test. The four groups showed that Very good (5-year OS, 100%) consisted of four patients who showed negative IHC staining for both GRP94 and CYP3A4. Very poor (1-year OS, 0%) consisted of three patients who showed positive results in IHC for both GRP94 and CYP3A4. The remaining 35 patients comprised two subgroups: Good (5-year OS 60-80%): 15 patients who showed negative staining for both MDR1 and AKR1C3 and Poor (5-year OS, 10-20%): 20 patients who showed positive staining for either MDR, AKR1C3, MRP1, or p53. The Histological Prognostic Index (HPI) (the four groups: Very poor, Poor, Good, and Very good) is a breakthrough method for stratifying patients based on the factors involved in the development of treatment resistance.

DGKζ is involved in LPS-activated phagocytosis through IQGAP1/Rac1 pathway.

Diacylglycerol kinase (DGK) plays an important role in phosphoinositide signaling cascade by regulating the intracellular level of diacylglycerol and phosphatidic acid. The DGK family is involved in various pathophysiological responses that are mediated through unique binding partners in different tissues and cells. In this study, we identified a small GTPase effector protein, IQGAP1, as a novel DGKζ-associated complex protein. A bacterial endotoxin, lipopolysaccharide (LPS), facilitated the complex formation in macrophages. Both proteins co-localized at the edge and phagocytic cup of the cell. Furthermore, RNA interference-mediated knockdown of DGKζ or IQGAP1 impaired LPS-induced Rac1 activation. Primary macrophages derived from DGKζ(-/-) mice attenuated LPS-induced phagocytosis of bacteria. These results suggest that DGKζ is involved in IQGAP1/Rac1-mediated phagocytosis upon LPS stimulation in macrophages.

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.

Role of Oncostatin M in the Pathogenesis of Vernal Keratoconjunctivitis: Focus on the Barrier Function of the Epithelium and Interleukin-33 Production by Fibroblasts.

Purpose: Vernal keratoconjunctivitis (VKC) is a severe, recurrent allergic conjunctivitis. Previously, we found high concentrations of oncostatin M (OSM) in the tears of patients with VKC. Here, we investigated the role of OSM in VKC by focusing on epithelial barrier function and IL-33 production. Methods: To assess the effect of OSM on the barrier function of human conjunctival epithelial cells (HConEpiCs), we measured transepithelial electrical resistance and dextran permeability. We also assessed expression of tight junction-related proteins such as E-cadherin and ZO-1 in HConEpiCs by Western blotting and immunofluorescence. Then we used immunohistochemistry to evaluate expression of Ki-67, E-cadherin, epithelial-mesenchymal transition-related proteins, and IL-33 in giant papillae (GPs) from patients with VKC. In addition, we used Western blotting, microarray, quantitative real-time polymerase chain reaction, and enzyme-linked immunosorbent assay to examine whether OSM activates signal transducer and activator of transcription 1 (STAT1) or STAT3 and induces the expression of various genes in human conjunctival fibroblasts (HConFs). Results: OSM reduced expression of E-cadherin and ZO-1 in HConEpiCs, indicating barrier dysfunction. In immunohistochemistry, Ki-67 expression was present in the lower epithelial layer of the GPs, and E-cadherin expression was reduced in the superficial and lower layers; double staining revealed that GPs had a high number of fibroblasts expressing IL-33. In addition, in HConFs, OSM phosphorylated both STAT1 and STAT3 and induced IL-33. Conclusions: OSM has important roles in severe, prolonged allergic inflammation by inducing epithelial barrier dysfunction and IL-33 production by conjunctival fibroblasts.

Quantitative peptidomic analysis by a newly developed one-step direct transfer technology without depletion of major blood proteins: its potential utility for monitoring of pathophysiological status in pregnancy-induced hypertension.

We have recently developed a new target plate (BLOTCHIP®) for MALDI-MS. An advantage of this procedure is that it does not require the lowering of protein concentrations in test samples prior to analysis. Accordingly, this new technology enables the detection of peptides present in blood samples, including those that would otherwise be adsorbed to abundant blood proteins and would thus escape detection. Using this technology, we analyzed the peripheral blood of patients with pregnancy-induced hypertension (PIH; the most common serious complication of pregnancy) to test a potential utility of the technology for monitoring of the pathophysiological status. In the present study, we found 23 characteristic peptides for PIH in the blood serum of pregnant women. Offline LC-MALDI MS/MS identified 7 of the 23 peptides as fragments derived from kininogen-1 (three peptides), fibrinogen-α, complement component C4-A/B, α-2-HS-glycoprotein and inter-α-trypsin inhibitor heavy chain H4. 2-D scatter plots with combinations of the peptides found in the present study can be grouped for pregnant women with/without PIH, which would be satisfactory reflected for their status. Additionally, the levels of most of these peptides found were significantly decreased by albumin/IgG depletion prior to BLOTCHIP® analysis in accordance with conventional proteomics procedures. These results indicated that BLOTCHIP® analysis can be applied for discovery study of PIH biomarker candidates.

An overview of sex and reproductive immunity from an evolutionary/anthropological perspective.

Mammalian pregnancy is a curious life phenomenon. Immunologically, the mechanism of pregnancy is difficult to explain because it involves the coexistence of an external foreign body (the embryo) and the host (the mother) for a period of time. How did mammals acquire the ability to become pregnant in parallel with altered immunity? Sex in the evolution of life and its impact on anthropology are major topics of discussion. In this paper, we outline (1) sex and evolution in mammals after the advent of our direct ancestors (apes) up to humans (i.e., the Cenozoic Quaternary), including anthropological aspects such as the development of the central nervous system; (2) the development of reproductive immunity during the Paleozoic era, when biodiversity developed explosively (and many sexually reproducing organisms have emerged); and (3) the characteristic reproductive strategies of mammals, including humans with the immunological aspects of viviparity. We present an overview of mammalian reproductive immunity, which is a heretical aspect of immunology.