CD34 is Expressed in Endothelial Cells in Embryonic Testes and is Additionally Expressed in Non-Endothelial Cells in Postnatal Mouse Testes.

CD34 is expressed in various cell types in various tissues/organs, and has been regarded as being expressed in progenitors in various differentiation pathways. On the other hand, morphological studies have reported the presence of a special type of interstitial cells, telocytes, which generally express CD34, and have extremely long moniliform prolongations in various tissues/organs in vertebrates. We have recently reported the successful reconstruction of testicular structures by 3-D re-aggregation culture of dissociated prepubertal mouse testicular cells, and the roles of CD34 + cells in the reconstruction. However, it was unknown whether CD34 is expressed in embryonic through adult testes, and if so, in what cell type it is expressed. In order to clarify the expression of CD34 and behavior of CD34 + cells during development of mouse testes, we performed immunohistochemical studies. The results show that CD34 is expressed in two cell types in testes; one is endothelial cells which co-express CD31, VE-cadherin, and integrin ß1, but barely express PDGFRα and integrin α4 and α9, throughout development, while the other one is non-endothelial cells in which CD34 expression is initiated after birth, and which co-express PDGFRα and integrin α4, α9, and ß1. The latter corresponds to telocytes. The present findings will lead to clarifying the roles of these two types of CD34 + cells in spermatogenesis.

Beneficial Effects of Receiving Johrei on General Health or Hypothermia Tendency.

OBJECTIVES: Johrei is a type of biofield therapy that is said to bring physical and mental well-being to the recipient. This study sought to measure changes in body temperature and circulation resulting from Johrei treatment, for generally healthy subjects and for individuals with a tendency toward hypothermia. PARTICIPANTS: A total of 199 qualified Johrei practitioners and 144 non-qualified operators provided Johrei and placebo treatments, respectively. Volunteer subjects -186 in general health and 39 with a hypothermia tendency - participated in this study to receive either or both of these treatments. METHODS: Each subject was given a 10 min treatment daily by either a qualified practitioner or a non-qualified operator. The effects on subjects of receiving each treatment were compared by observing quantitative changes in blood flow and surface body temperature after a course of treatment. RESULTS: A total of 107 healthy subjects were randomly assigned to the qualified-practitioner group or the non-qualified operator group. Treatment by qualified practitioners significantly enhanced blood flow and surface body temperature in the subjects' designated neck area compared to that in treatment by non-qualified operators. This finding was further corroborated by a comparative experiment in which each healthy subject was treated by both a qualified practitioner and a non-qualified operator. These results indicate that only the qualified-practitioner treatment increased the subject's-blood flow and surface body temperature. Similarly, in a comparative study of qualified-practitioner treatment against non-qualified-operator treatment, subjects tending toward hypothermia showed increased blood flow and elevated body temperature with only the authentic Johrei treatment.

VCAM1-α4ß1 integrin interaction mediates interstitial tissue reconstruction in 3-D re-aggregate culture of dissociated prepubertal mouse testicular cells.

Roles of interstitial tissue in morphogenesis of testicular structures remain less well understood. To analyze the roles of CD34+ cells in the reconstruction of interstitial tissue containing Leydig cells (LCs), and testicular structures, we used 3D-reaggregate culture of dissociated testicular cells from prepubertal mouse. After a week of culture, adult Leydig cells (ALCs) were preferentially incorporated within CD34+ cell-aggregates, but fetal LCs (FLCs) were not. Immunofluorescence studies showed that integrins α4, α9 and ß1, and VCAM1, one of the ligands for integrins α4ß1 and α9ß1, are expressed mainly in CD34+ cells and ALCs, but not in FLCs. Addition of function-blocking antibodies against each integrin and VCAM1 to the culture disturbed the reconstruction of testicular structures. Antibodies against α4 and ß1 integrins and VCAM1 robustly inhibited cell-to-cell adhesion between testicular cells and between CD34+ cells. Cell-adhesion assays indicated that CD34+ cells adhere to VCAM1 through the interaction with α4ß1 integrin. Live cell imaging showed that CD34+ cells adhered around ALC-aggregates. CD34+ cells on the dish moved toward the aggregates, extending filopodia, and entered into them, which was disturbed by VCAM1 antibody. These results indicate that VCAM1-α4ß1 integrin interaction plays pivotal roles in formation of testicular interstitial tissues in vitro and also in vivo.

Roles of CD34+ cells and ALK5 signaling in the reconstruction of seminiferous tubule-like structures in 3-D re-aggregate culture of dissociated cells from neonatal mouse testes.

Tissue reconstruction in vitro can provide, if successful, a refined and simple system to analyze the underlying mechanisms that drive the morphogenesis and maintain the ordered structure. We have recently succeeded in reconstruction of seminiferous cord-like and tubule-like structures using 3-D re-aggregate culture of dissociated testicular cells. In testis formation, endothelial cells that migrated from mesonephroi to embryonic gonads have been shown to be critical for development of testis cords, but how endothelial cells contribute to testis cord formation remains unknown. To decipher the roles of endothelial and peritubular cells in the reconstruction of cord-like and tubule-like structures, we investigated the behavior of CD34+ endothelial and p75+ cells, and peritubular myoid cells (PTMCs) in 3-D re-aggregate cultures of testicular cells. The results showed that these 3 types of cells had the capacity of re-aggregation on their own and with each other, and of segregation into 3 layers in a re-aggregate, which were very similar to interstitial and peritubular tissues in vivo. Observation of behaviors of fluorescent Sertoli cells and other non-fluorescent types of cells using testes from Sox9-EGFP transgenic mice showed dynamic cell movement and segregation in re-aggregate cultures. Cultures of testicular cells deprived of interstitial and peritubular cells resulted in dysmorphic structures, but re-addition of them restored tubule-like structures. Purified CD34+ cells in culture differentiated into p75+ cells and PTMCs. These results indicate that CD34+ cells differentiate into p75+ cells, which then differentiate into PTMCs. TGFß signaling inhibitors, SB431542 and ALK5i, disturbed the reconstruction of cord-like and tubule-like structures, and the latter compromised re-construction of interstitial-like and peritubular-like structures, as well as the proliferation of CD34+, p75+, PTMCs, and Sertoli cells, and their movement and differentiation. These results indicate that CD34+ cells and signaling through ALK5 play pivotal roles in the morphogenesis of interstitial-like, peritubular-like and cord-like structures.

Effect of a Japanese energy healing method known as Johrei on viability and proliferation of cultured cancer cells in vitro.

OBJECTIVES: The objective was to explore the effect of a Japanese energy healing method known as Johrei on the viability and proliferation of cultured human cancer cells in vitro. DESIGN: A randomly selected 96-well plate or a culture dish of various types of human cancer cell lines in culture were exposed to Johrei treatment. For comparison purpose, an equal number of untreated or volunteer-treated cultures were chosen as the control group. Johrei treatment was repeatedly performed at appropriate time intervals over the course of the experiments. Cell viability was examined by a colorimetric assay with a Cell Counting kit. Morphological changes were analyzed by phase-contrast and time-lapse microscopy. Cell proliferation and early and late stages of cell death were also determined with the use of a bromodeoxyuridine (BrdU) cell proliferation assay kit and an Annexin V-FLUOS Staining kit, respectively. OUTCOME MEASURES: Quantitative data were presented as means±standard deviation. The outcome measures were the differences in viable cell numbers that remained under healing practice versus control conditions, and the statistical significance of differences in their mean values was assessed. RESULTS: The viability loss of cultured human cancer cells in the Johrei group was significantly higher than that of either of the control groups, despite the fact that the responsiveness to Johrei varied with different cancer cell types. The proliferation rate of gastric cancer cells exposed to Johrei treatments for 72 hours was more significantly decreased compared with that of the untreated cells, whereas the extent of dying and/or dead cells in the Johrei group was more profound than that of the untreated cells. CONCLUSIONS: These results provide evidence that Johrei treatment induces the viability loss of various cancer cells in vitro, mainly due to the increased cell death and the decreased proliferation.

Neuromuscular junction integrity after chronic nerve compression injury.

Chronic nerve compression injuries (CNC) are progressive demyelinating disorders characterized by a gradual decline of the nerve conduction velocity (NCV) in the affected nerve region. CNC injury induces a robust Schwann cell response with axonal sprouting, but without morphologic evidence of axonal injury. We hypothesize that early CNC injury occurs without damage to neuromuscular junction of motor axons. A well-established animal model was used to assess for damage to motor axons. As sprouting is considered a hallmark of regeneration during and after axonal degeneration and sprouting was confirmed visually at 2 weeks in CNC animals, we assessed for axonal degeneration in motor nerves after CNC by evaluating the integrity of the neuromuscular junction. NCV exhibited a gradual progressive decline consistent with the human condition. Compound motor action potential amplitudes decreased slightly immediately and plateaued, indicating that there was not sustained and increasing axonal loss. Sprouting was confirmed using immunofluorescence and by an increase in number of unmyelinated axons and Remak bundles. Blind analysis of the neuromuscular junction showed no difference between control and CNC images, indicating that there was no evidence for end-unit axonal loss in the soleus muscle. Because the progressive decline in NCV was not paired with a similar progressive decline in amplitude, it is likely that axonal loss is not responsible for slowing of action potentials. Blind analysis of the neuromuscular junction provides further evidence that the axonal sprouting seen early after CNC injury is not a consequence of axonal degeneration in the motor nerves.

Effect of insulin-like growth factor-1 and 2,3,7,8-tetrachlorodibenzo-p-dioxin on the expression of luteinizing hormone receptors in cultured granulosa cells.

In this study, we attempted to assess the effects of insulin-like growth factor-1 (IGF-1) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on follicle-stimulating hormone (FSH)-induced luteinizing hormone (LH) receptor expression in rat granulosa cells to understand the effects of these factors on normal reproductive function. Treatment with FSH, as expected, produced a substantial increase in LH receptor mRNA expression level, and cotreatment with an increasing concentration of IGF-1 resulted in a dose-dependent increase in FSH-induced LH receptor mRNA expression level. On the other hand, the cotreatment with FSH and TCDD (10 pM) resulted in a significant decrease in LH receptor mRNA expression level after 24 h. The decay curves for the LH receptor mRNA transcript showed a significant increase in half-life after the addition of IGF-1 and a significant decrease after the addition of TCDD. These data suggest a possible role for changes in LH receptor mRNA stability in the IGF-1- and TCDD-induced regulation of the LH receptor in rat granulosa cells. The rates of LH receptor mRNA gene transcription, assessed by nuclear run-on transcription assay, did not increase after the addition of IGF-1, but decreased after the addition of TCDD. The data for IGF-1 indicate that the interface between circulating hormones and paracrine/autocrine systems could provide an important mechanism for amplifying the effects of gonadotropin hormones at the local level. In addition, the endocrine-disrupting effects of TCDD are, at least in part, caused by the direct action on the LH receptor expression on granulosa cells.

Mechanisms of action of transforming growth factor beta on the expression of follicle-stimulating hormone receptor messenger ribonucleic acid levels in rat granulosa cells.

The present study was undertaken to identify the mechanisms underlying the effect of transforming growth factor (TGF) beta on FSH receptor (FSH-R) in rat granulosa cells. Compared to the control, the treatment of granulosa cells with TGFbeta (10 ng/ml) increased FSH-R mRNA transcripts (5.5 and 2.4 kilobases) in a time-dependent manner, with a maximum increase of approximately 2-fold at 48 h. We then investigated whether the effect of TGFbeta on FSH-R mRNA levels was the result of increased transcription and/or altered mRNA stability. To determine whether the FSH-R 5'-flanking region plays a role in directing FSH-R mRNA expression, the proximal area of the FSH-R 5'-flanking regions were inserted into an expression vector, pGL-Basic, which contains luciferase as the receptor gene, and the resulting plasmids were transiently transfected into rat granulosa cells. The FSH (30 ng/ml) significantly enhanced the activity of 1862 base pairs of the FSH-R 5'-flanking region, but treatment with TGFbeta did not significantly influence the activity induced by FSH. On the other hand, the decay curves for FSH-R mRNA transcript in primary granulosa cells showed a significant increase in half-life after the addition of TGFbeta. Transforming growth factor beta stimulates the expression of follistatin mRNA accumulation in a dose- and time-dependent manner. Treatment with activin produced a substantial increase in FSH-R mRNA level. Concurrent treatment with follistatin neutralized this activin effect on FSH-R mRNA, as reported, although concurrent treatment with follistatin did not affect TGFbeta-induced FSH-R mRNA. Therefore, the profile of the TGFbeta effect on FSH-R mRNA granulosa cells may be caused by the increased stability of FSH-R mRNA and insensitivity to the follistatin.

Effect of IGF-1 and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the expression of LH receptors during cell differentiation in cultured granulosa cells.

Ovarian granulosa cells undergo a complex differentiation process during the growth and maturation of ovarian follicle. This process includes the acquisition of cell surface LH receptor, which mediates the granulosa cell's ability to respond to circulating LH. The results of the actions of LH on the mature granulosa cell include steroidogenesis, luteinization, and ovulation. As such, induction of the LH receptor in granulosa cells is a critical step in reproductive physiology. In the present study, we attempted to assess the effects of IGF-1 and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on FSH-induced LH receptor expression in rat granulosa cells to understand the actions of these factors on normal reproductive function. Treatment with FSH, as expected, produced a substantial increase in LH receptor mRNA level, and concurrent treatment with increasing concentrations of IGF-1 brought about dose-dependent increases in FSH-induced LH receptor mRNA. On the other hand, the concurrent treatment of TCDD (10 pM) resulted in a significant decrease in LH receptor after 24 h. The decay curves for LH receptor mRNA transcript showed a significant increase in the half-life after the addition of IGF-1 and a significant decrease after addition of TCDD. These data suggests a possible role for changes in LH receptor mRNA stability in the IGF-1 and TCDD induced regulation of LH receptor in rat granulosa cells. The rates of LH receptor mRNA gene transcription, assessed by nuclear run-on transcription assay, were not increased by the addition of IGF-1, but decreased by the addition of TCDD. The data of IGF-1 present that the interface between circulating hormones and paracrine/autocrine systems could provide an important mechanism to amplify the effects of gonadotropin hormones at the local level. In addition, the endocrine-disrupting effects of TCDD are, at least in part, caused by direct action on the expression of LH receptor expression in granulosa cells.

Modeling the production and uses of biological resources from the viewpoint of energy flow in a rural village in Sichuan, China.

This study aimed at clarifying the impact of deforestation and afforestation on the quality of life in a village in Sichuan Province, China. We devised a conceptual model of bioresource production and use based on quantified energy flow. The basic structure of the model has three sectors: production, use, and externals. We developed comprehensive methodology to quantify the model. Bioresource use per person in 1997 was 3.7 GJ for food, 10.2 GJ for fodder, 0.2-0.4 GJ for building material, 12.8 GJ for fuel, and 1.8 GJ for fertilizer, totaling 28.6-28.8 GJ. We used four environmental indicators to evaluate bioresource production and use: a biological productivity indicator, a use-efficiency indicator, a supply-demand balance indicator, and a self-sufficiency indicator. Use of these indicators showed that supply-demand balance of fuel was dramatically improved from 30% to 85% by afforestation, but 99% of bioresource use still depends on domestic products. Thus, it is necessary to improve biological productivity and promote the efficient use of bioresources to achieve sustainable living in the area. Massive deforestation in the 1950s caused a direct shortage of building material and fuel wood. The shortage of wood led to a stagnation in the rebuilding of houses, and fuel wood was substituted with crop residues. Because crop residues had been used for fertilizer and fodder, their use as fuel caused a shortage of fertilizer and fodder. This was an indirect impact of deforestation on people's quality of life.

Effect of transforming growth factor beta on the expression of luteinizing hormone receptor in cultured rat granulosa cells.

The present study was undertaken in order to identify the mechanism underlying the effect of transforming growth factor-beta (TGFbeta) on LH receptor (LH-R) expression in rat granulosa cells. Treatment with FSH produced a substantial increase in LH-R mRNA level, and concurrent treatment with increasing concentrations of TGFbeta brought about dose-dependent increases in FSH-induced LH receptor mRNA. TGFbeta, either alone or in combination with FSH, did not affect intracellular cAMP levels. We then investigated whether the effect of TGFbeta and FSH on LH-R mRNA levels results in increased transcription and/or altered mRNA stability. To determine whether the LH receptor 5'-flanking region plays a role in directing LH receptor mRNA expression, the proximal area of the LH receptor 5'-flanking regions were inserted into an expression vector, pGL-Basic, which contains luciferase as the receptor gene, and the resulting plasmids were transiently transfected into rat granulosa cells. FSH (30 ng/ml) significantly enhanced the activity of 1389 base pairs of the LH receptor 5'-flanking region, but treatment with TGFbeta did not significantly influence the activity induced by FSH. On the other hand, the decay curves for LH-R mRNA transcript in primary granulosa cells showed a significant increase in half-life after the addition of TGFbeta.