Cryopreservation and Transplantation of Spermatogonial Stem Cells.

Cryopreservation as a method that enables long-term storage of biological material has long been used for the conservation of valuable zebrafish genetic resources. However, currently, only spermatozoa of zebrafish can be successfully cryopreserved, while protocols for cryopreservation of eggs and embryos have not yet been fully developed. Transplantation of germline stem cells (GSCs) has risen as a favorable method that can bypass the current problem in cryopreservation of female genetic resources and can lead to reconstitution of fish species and lines through surrogate production. Here, we describe essential steps needed for the cryopreservation of spermatogonial stem cells (SSCs) and their utilization in the conservation of zebrafish genetic resources through SSC transplantation and surrogate production.

Type I Angiotensin II Receptor Blockade Reduces Uremia-Induced Deterioration of Bone Material Properties.

Chronic kidney disease (CKD) is associated with a high incidence of fractures. However, the pathophysiology of this disease is not fully understood, and limited therapeutic interventions are available. This study aimed to determine the impact of type 1 angiotensin II receptor blockade (AT-1RB) on preventing CKD-related fragility fractures and elucidate its pharmacological mechanisms. AT-1RB use was associated with a lower risk of hospitalization due to fractures in 3276 patients undergoing maintenance hemodialysis. In nephrectomized rats, administration of olmesartan suppressed osteocyte apoptosis, skeletal pentosidine accumulation, and apatite disorientation, and partially inhibited the progression of the bone elastic mechanical properties, while the bone mass was unchanged. Olmesartan suppressed angiotensin II-dependent oxidation stress and apoptosis in primary cultured osteocytes in vitro. In conclusion, angiotensin II-dependent intraskeletal oxidation stress deteriorated the bone elastic mechanical properties by promoting osteocyte apoptosis and pentosidine accumulation. Thus, AT-1RB contributes to the underlying pathogenesis of abnormal bone quality in the setting of CKD, possibly by oxidative stress. © 2020 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Characteristics of autologous protein solution and leucocyte-poor platelet-rich plasma for the treatment of osteoarthritis of the knee.

Recently, platelet-rich plasma (PRP) has received attention as a treatment for patients with osteoarthritis of the knee (OAK), a chronic degenerative disease, to bridge the gap between conservative and surgical treatments. Here, we investigated the differences in the humoral factors present in two types of PRP purified using the Autologous Protein Solution (APS) kit (group Z; leucocyte-rich PRP) or the Cellaid Serum Collection Set P type (group J; leucocyte-poor [LP]-PRP). Differences in humoral factors between healthy subjects (n = 10) and OAK patients (n = 12; group Z = 6, group J = 6), and the relationship between humoral factors and clinical outcome scores were investigated. Both anti-inflammatory and inflammatory cytokines were highly enriched in APS. The concentrations of tumour necrosis factor (TNF)-α, platelet-derived growth factor, fibroblast growth factor, soluble TNF-receptor 2, soluble Fas and transforming growth factor-ß1 were higher in group Z, while the total amounts were higher in group J. The concentration of interleukin-1 receptor antagonist was positively correlated with the magnitude of change in the clinical outcome score and may contribute to improving knee-joint function. This is the first description of the humoral factors in APS and LP-PRP prepared from healthy subjects or OAK patients of Asian descent.

Preservation of zebrafish genetic resources through testis cryopreservation and spermatogonia transplantation.

Zebrafish is one of the most commonly used model organisms in biomedical, developmental and genetic research. The production of several thousands of transgenic lines is leading to difficulties in maintaining valuable genetic resources as cryopreservation protocols for eggs and embryos are not yet developed. In this study, we utilized testis cryopreservation (through both slow-rate freezing and vitrification) and spermatogonia transplantation as effective methods for long-term storage and line reconstitution in zebrafish. During freezing, utilization of 1.3 M of dimethyl sulfoxide (Me2SO) displayed the highest spermatogonia viability (~60%), while sugar and protein supplementation had no effects. Needle-immersed vitrification also yielded high spermatogonia viability rates (~50%). Both optimal slow-rate freezing and vitrification protocols proved to be reproducible in six tested zebrafish lines after displaying viability rates of >50% in all lines. Both fresh and cryopreserved spermatogonia retained their ability to colonize the recipient gonads after intraperitoneal transplantation of vasa::egfp and actb:yfp spermatogonia into wild-type AB recipient larvae. Colonization rate was significantly higher in dnd-morpholino sterilized recipients than in non-sterilized recipients. Lastly, wild-type recipients produced donor-derived sperm and donor-derived offspring through natural spawning. The method demonstrated in this study can be used for long-term storage of valuable zebrafish genetic resources and for reconstitution of whole zebrafish lines which will greatly improve the current preservation practices.

Specific visualization of live type A spermatogonia of Pacific bluefin tuna using fluorescent dye-conjugated antibodies†.

During our previous work toward establishing surrogate broodstock that can produce donor-derived gametes by germ cell transplantation, we found that only type A spermatogonia (ASGs) have the potency to colonize recipient gonads. Therefore, the ability to visualize ASGs specifically would allow the sequential analysis of donor cell behavior in the recipient gonads. Here we produced monoclonal antibodies that could recognize the cell surface antigens of ASGs in Pacific bluefin tuna (Thunnus orientalis), with the aim of visualizing live ASGs. We generated monoclonal antibodies by inoculating Pacific bluefin tuna testicular cells containing ASGs into mice and then screened them using cell-based enzyme-linked immunosorbent assay (ELISA), immunocytochemistry, flow cytometry (FCM), and immunohistochemistry, which resulted in the selection of two antibodies (Nos. 152 and 180) from a pool of 1152 antibodies. We directly labeled these antibodies with fluorescent dye, which allowed ASG-like cells to be visualized in a one-step procedure using immunocytochemistry. Molecular marker analyses against the FCM-sorted fluorescent cells confirmed that ASGs were highly enriched in the antibody-positive fraction. To evaluate the migratory capability of the ASGs, we transplanted visualized cells into the peritoneal cavity of nibe croaker (Nibea mitsukurii) larvae. This resulted in incorporated fluorescent cells labeled with antibody No. 152 being detected in the recipient gonads, suggesting that the visualized ASGs possessed migratory and incorporation capabilities. Thus, the donor germ cell visualization method that was developed in this study will facilitate and simplify Pacific bluefin tuna germ cell transplantation.

Semiquantitative analysis of virtual histology derived from intravascular ultrasound images at vascular access stenosis.

Vascular access failure, such as recurrent stenosis and thrombosis, is a major concern in patients with end-stage kidney disease. Neointimal hyperplasia development at the anastomosis site of outflow vessels is a primal cause for recurrent vascular access failure. We previously shed some lights into a role of vitamin D, which exerts a protective effect against neointimal hyperplasia formation. Virtual histology, derived from intravascular ultrasound technology, provides novel insights into plaque composition analysis in atherosclerotic diseases. However, there is so far a lack of evidence on the relation between virtual histology and pathophysiological findings. To elucidate this missing link, we comprehensively reviewed 10 chronic hemodialysis patients who underwent repeated intravascular ultrasound-guided balloon angioplasty. Their age, dialysis vintage, and follow-up period were 75.0 ± 4.24, 20.5 ± 2.12, and 11.5 ± 0.71 (mean ± standard deviation) years, respectively. Pathological cross-sectional analyses were performed using specimens from vascular access surgeries during the follow-up period. Interestingly, positive relation is found between virtual histology-constructed fibrous tissue and pathological neointimal hyperplasia. Strikingly, immunohistological analysis revealed that vitamin D receptor-positive myofibroblasts were abundantly distributed in the equivalent area to virtual histology fibrous tissue. Our 10-year follow-up data of resistant vascular access stenosis indicates strong correlation between vitamin D receptor-rich neointimal vessel hypertrophy and intravascular ultrasound-assisted virtual histological analysis. Intravascular ultrasound technology is one of the minimally invasive diagnostic tools to provide histologically relevant tissue structure information and help determine target vessel stenosis on vascular access.

TGF-Beta Signaling in Bone with Chronic Kidney Disease.

Transforming growth factor (TGF)-ß signaling is not only important in skeletal development, but also essential in bone remodeling in adult bone. The bone remodeling process involves integrated cell activities induced by multiple stimuli to balance bone resorption and bone formation. TGF-ß plays a role in bone remodeling by coordinating cell activities to maintain bone homeostasis. However, mineral metabolism disturbance in chronic kidney disease (CKD) results in abnormal bone remodeling, which leads to ectopic calcification in CKD. High circulating levels of humoral factors such as parathyroid hormone, fibroblast growth factor 23, and Wnt inhibitors modulate bone remodeling in CKD. Several reports have revealed that TGF-ß is involved in the production and functions of these factors in bone. TGF-ß may act as a factor that mediates abnormal bone remodeling in CKD.

[Fragility Fractures in Hemodialysis Patients. Abnormalities of osteocyte functions in chronic kidney disease.]

Increased fracture incidence and ectopic calcifications which are accompanied with mineral metabolic disorders are well seen in dialysis patients. The role of bone in chronic kidney disease patients including dialysis has been investigated in clinical findings and experimental examinations. The abnormalities of osteocyte function have been pointed to as an active inducer of pathology of a deregulated endocrine organ.

Molecular Abnormalities Underlying Bone Fragility in Chronic Kidney Disease.

Prevention of bone fractures is one goal of therapy for patients with chronic kidney disease-mineral and bone disorder (CKD-MBD), as indicated by the Kidney Disease: Improving Global Outcomes guidelines. CKD patients, including those on hemodialysis, are at higher risk for fractures and fracture-related death compared to people with normal kidney function. However, few clinicians focus on this issue as it is very difficult to estimate bone fragility. Additionally, uremia-related bone fragility has a more complicated pathological process compared to osteoporosis. There are many uremia-associated factors that contribute to bone fragility, including severe secondary hyperparathyroidism, skeletal resistance to parathyroid hormone, and bone mineralization disorders. Uremia also aggravates bone volume loss, disarranges microarchitecture, and increases the deterioration of material properties of bone through abnormal bone cells or excess oxidative stress. In this review, we outline the prevalence of fractures, the interaction of CKD-MBD with osteoporosis in CKD patients, and discuss possible factors that exacerbate the mechanical properties of bone.

Production of the medaka derived from vitrified whole testes by germ cell transplantation.

The medaka (Oryzias latipes) is a teleost model distinguished from other model organisms by the presence of inbred strains, wild stocks, and related species. Cryopreservation guarantees preservation of these unique biological resources. However, because of their large size, cryopreservation techniques for their eggs and embryos have not been established. In the present study, we established a methodology to produce functional gametes from cryopreserved testicular cells (TCs). Whole testes taken from medaka were cryopreserved by vitrification. After thawing, the cells dissociated from cryopreserved testicular tissues were intraperitoneally transplanted into sterile triploid hatchlings. Some cells, presumably spermatogonial stem cells, migrated into the genital ridges of recipients and resulted in the production of eggs or sperm, based on sex of the recipient. Mating of recipients resulted in successful production of cryopreserved TC-derived offspring. We successfully produced individuals from the Kaga inbred line, an endangered wild population in Tokyo, and a sub-fertile mutant (wnt4b-/-) from cryopreserved their TCs. This methodology facilitates semi-permanent preservation of various medaka strains.

[Several factors related with bone quality abnormalities exist in patients with renal failure].

Recently, several papers revealed that bone pathophysiological abnormalities related with end-stage renal disease(ESRD)contain a multiplicity of injured bone quality. Although deteriorated bone quality in ESRD patients is understood as a risk factor of fracture in these patients, actual conditions still remain unclear. Here, I will provide a short review on both the deteriorated bone quality in ESRD patients and animal models, and also specific conditions which is providing abnormality. I also will discuss the problem points of abnormal bone quality in ESRD patients.

Long-term (5 years) cryopreserved spermatogonia have high capacity to generate functional gametes via interspecies transplantation in salmonids.

Although sperm cryopreservation is a powerful tool widely applicable in biodiversity conservation and broodstock management, cryopreservation of teleost eggs and embryos remains challenging. In the present study, we demonstrated that spermatogonia of rainbow trout (Oncorhynchus mykiss) cryopreserved for 5 years possessed the ability to differentiate into functional eggs or sperm in the gonads of triploid recipient masu salmon (Oncorhynchus masou). After cryopreservation for 5 years in liquid nitrogen, intraperitoneally transplanted spermatogonia migrated toward, and incorporated into, the gonads of xenogeneic recipients. The transplanted spermatogonia resumed spermatogenesis and oogenesis in male and female recipients, respectively, and differentiated into sperm or eggs within the gonads of male and female recipients at 2 years posttransplantation. The differentiated sperm and eggs generated normal rainbow trout representative of donor phenotypes. Thus, cryopreservation of spermatogonia is a powerful and reliable method for long-term preservation of fish genetic resources.

An efficacy of intensive vitamin D delivery to neointimal hyperplasia in recurrent vascular access stenosis.

PURPOSE: Neointimal hyperplasia (NH) causes vascular access (VA) stenosis, which results in serious under-dialytic morbidity in hemodialysis patients. We sought to assess whether a vitamin D intervention to NH lesions leads to better VA patency and examined clinical and in vitro studies. METHODS: A pilot clinical study of six hemodialysis patients was conducted to elucidate whether 0.5 µg calcitriol injection to stenotic lesion after balloon angioplasty (PTA) maintains better vessel patency until the next follow-up angiography. Localized vitamin D exposure was utilized by delivering and fixing calcitriol intensively at the stenotic lesion through a side-hole catheter with balloon clamping. We also performed vascular smooth muscle cell (VSMC) culture to detect both apoptosis (cell death detection assay) and cell viability (5-Bromo-2'-deoxy-uridine incorporation), and explored the efficacy of vitamin D to inhibit VSMC proliferation. Additionally, immunohistochemistry (IHC) was conducted to examine vitamin D receptor (VDR) expression at NH lesion, obtained from VA surgery. RESULTS: Percent patency, the proportion between stenotic and non-stenotic vessel diameters, increased significantly (p = 0.03) after directly catheter-delivered 0.5 µg calcitriol administration. In vitro VSMC studies, 0.1 nM calcitriol significantly (p<0.05) enhanced apoptosis and cell-cycle inhibition for two different calcitriol exposure times (15 minutes and 24 hours). IHC staining revealed that VDR-positive hyperplastic cells were observed at NH lesion. CONCLUSIONS: Intensive vitamin D exposure at NH lesion has an ability to inhibit further VSMC proliferation, and presumably leads to greater patency rate for recurrent VA stenosis. Further studies are needed to clarify whether its unique property is exhibited through VDR-mediated mechanism.

Altered material properties are responsible for bone fragility in rats with chronic kidney injury.

Chronic kidney disease (CKD) is associated with an increased risk of fragility fractures, but the underlying pathophysiological mechanism remains obscure. We performed an in vivo experimental study to examine the roles of uremia and abnormal mineral/parathyroid metabolism in the development of bone metabolic abnormalities in uremic rats. Male Sprague-Dawley rats were divided into four groups, comprising sham operation (high turnover bone control=HTB-Cont), 5/6-nephrectomy (high turnover bone nephrectomized=HTB-Nx), thyroparathyroidectomy (low turnover bone control=LTB-Cont), and thyroparathyroidectomy plus 5/6 nephrectomy (low turnover bone nephrectomized=LTB-Nx), and maintained for 16 weeks. Uremia was successfully created in the LTB-Nx and HTB-Nx groups, while hyperparathyroidism was only found in the HTB-Nx group. Cancellous bone histomorphometry revealed significantly higher bone turnover in the HTB-Nx group than in the LTB-Nx group. Storage modulus at 1 Hz and tan delta in cortical bone of the femur, which represent the viscoelastic mechanical properties, were significantly lower in both Nx groups than in the Cont groups regardless of bone metabolism. Pentosidine-to-matrix ratio was increased and crystallinity was decreased in both Nx groups regardless of bone turnover. Mineral-to-matrix ratio was significantly decreased in the HTB-Nx group, but increased in the LTB-Nx group. Enzymatic collagen crosslinks were decreased in the HTB-Nx group. The degree of orientation of the c-axis in carbonated hydroxyapatite (biological apatite=BAp) crystallites was decreased in both Nx groups regardless of bone metabolism. Stepwise multivariate regression revealed that pentosodine-to-matrix ratio and BAp preferential c-axis orientation were significantly associated with storage modulus and tan delta. In conclusion, bone elastic mechanical properties deteriorated regardless of bone metabolism or bone mass in rats with chronic kidney injury. Various changes in bone mineral properties were associated with CKD, including abnormal parathyroid function, impaired bone turnover, and uremia associated with the accumulation of uremic toxins, were responsible for these changes. Pentosidine-to-matrix ratio and BAp orientation at position 5 were the two meaningful determinants of elastic bone mechanical strength, and both factors were associated with the severity of uremia, but not parathyroid function or bone metabolism. These two factors may account for the increased bone fragility among CKD patients.

Chronic kidney disease and bone metabolism.

Chronic kidney disease-related mineral and bone disease (CKD-MBD) is a syndrome defined as a systemic mineral metabolic disorder associated with CKD, and the term renal osteodystrophy indicates a pathomorphological concept of bone lesions associated with CKD-MBD. Cortical bone thinning, abnormalities in bone turnover and primary/secondary mineralization, elevated levels of circulating sclerostin, increased apoptosis in osteoblasts and osteocytes, disturbance of the coupling phenomenon, iatrogenic factors, accumulated micro-crackles, crystal/collagen disorientation, and chemical modification of collagen crosslinks are all possible candidates found in CKD that could promote osteopenia and/or bone fragility. Some of above factors are the consequences of abnormal systemic mineral metabolism but for others it seem unlikely. We have used the term uremic osteoporosis to describe the uremia-induced bone fragility which is not derived from abnormal systemic mineral metabolism. Interestingly, the disease aspect of uremic osteoporosis appears to be similar to that of senile osteoporosis.

[New Developments in CKD-MBD. Bone quality and fracture in patients with chronic kidney disease].

Chronic kidney disease (CKD) patients have an extremely increased risk of fragility fractures, but the underling pathophysiological mechanisms remain obscure. Recently, the progresses of analysis technology have revealed the changes of bone quality in CKD condition. In particular, we can observe the characteristic changes of bone microarchitecture and bone chemical compositions in both human bone biopsy samples and experimental animal bones. Here, I will provide a short review on these bone quality factors and discuss on the relationship between bone quality and fracture in CKD patients.

Accumulated uremic toxins attenuate bone mechanical properties in rats with chronic kidney disease.

The prevalence of hip fracture is very high among patients with chronic kidney disease (CKD); however, the reason for this is unclear. We examined the effects of accumulated uremic toxins on bone chemical composition and elastic mechanical properties. Rats underwent thyroparathyroidectomy and progressive partial nephrectomy (TPTx-Nx), and were administered with vehicle or AST-120 to reduce serum indoxyl sulfate (IS) levels. Bone mechanical properties, bone mineral density (BMD), cortical bone chemical composition, and histomorphometry were determined. Storage modulus was reduced in TPTx-Nx rats compared with rats that underwent TPTx alone. BMD and histomorphometric parameters did not differ between the groups. In terms of cortical bone chemical composition, the mineral/matrix ratio and carbonate substitution was increased, whereas crystallinity was decreased in TPTx-Nx rats. The enzymatic crosslink ratio and pentosidine:matrix ratio were increased in TPTx-Nx rats. AST-120 abolished the effects of TPTx-Nx and decreased the serum IS concentration. Stepwise multiple regression analysis revealed that the pentosidine:matrix and mineral:matrix ratios were independent contributors to the storage modulus. In conclusion, the accumulated uremic toxins, including IS, seem to play an important role in deteriorating bone mechanical properties by altering the chemical composition of bone. This mechanism may account for the increased prevalence of hip fracture among patients with CKD.

p-Cresyl sulfate induces osteoblast dysfunction through activating JNK and p38 MAPK pathways.

Recent data suggest that several uremic toxins may contribute to the development of bone abnormalities in chronic kidney disease. p-Cresyl sulfate (PCS), the sulfate conjugate of p-cresol, is a protein-bound uremic toxin associated with the progression of chronic kidney disease, cardiovascular risk, and mortality. However, the effects of PCS on bone metabolism remain unclear. In the present study, we evaluated the toxic effects of PCS on primary mouse osteoblasts, compared with an extensively studied uremic toxin indoxyl sulfate (IS). Pre-treatment of osteoblasts with PCS at 0.125 mM and above significantly decreased parathyroid hormone (PTH)-induced cAMP production in a dose-dependent manner. PCS also induced a significant increase in intracellular production of reactive oxygen species (ROS) at 0.25 mM and above, but not at lower concentrations. PCS at 0.125 mM (a concentration that did not induce significant ROS increase) decreased cell viability by augmenting DNA fragmentation and reducing cell proliferation. Inhibition of JNK and p38 mitogen-activated protein kinase (MAPK) abolished the PCS-induced increase in DNA fragmentation and decrease in cAMP production in osteoblastic cells. Compared with PCS, IS induced ROS production at 0.05 mM but did not reduce cAMP production from 0.05 to 0.5 mM. IS induced decrease in cell viability and increase in DNA fragmentation at 0.5mM only. These results suggest that PCS damages osteoblastic cells through not only increasing ROS production but also activating JNK/p38 MAPKs, which is different from the mechanism of injury by IS. These damages of osteoblasts induced by PCS may play a critical role in impairing bone metabolism in patients with chronic kidney disease in whom PCS accumulates.

Short-term in vitro culturing improves transplantability of type A spermatogonia in rainbow trout (Oncorhynchus mykiss).

Continuous production of sperm within the testes is supported by spermatogonial stem cells capable of both self-renewal and the production of numerous differentiated germ cells. We previously demonstrated that a subpopulation of trout type A spermatogonia transplanted into the body cavity of a recipient embryo incorporated into the genital ridge, where they produced functional gametes within the gonads. Various cell-surface proteins could have played a role in the incorporation of spermatogonia into recipient genital ridges. During the preparation of cell suspensions for transplantation in our experimental protocol, however, dissociation of testis by strong proteases was unavoidable. This was problematic as cell-surface proteins may have been at least partially digested by protease activity. In the present study, recovery of spermatogonial surface proteins using short-term culture prior to transplantation was attempted. It was found that spermatogonia cultured in vitro could be harvested by ethylenediaminetetraacetic acid (EDTA) instead of protease treatment. Furthermore, when cultured spermatogonia collected by EDTA treatment were maintained for 24 hr in vitro, they exhibited high adhesiveness. These cultured spermatogonia also possessed higher survival of transplantation compared to spermatogonia newly dispersed by trypsin treatment. These results indicated that spermatogonia possess a reduced ability to migrate toward, adhere to, and/or be incorporated into the recipient genital ridge immediately after protease treatment. Short-term in vitro culturing, however, could allow spermatogonia to recover the surface proteins required for successful incorporation into the recipient genital ridge.

Generation of functional eggs and sperm from cryopreserved whole testes.

The conservation of endangered fish is of critical importance. Cryobanking could provide an effective backup measure for use in conjunction with the conservation of natural populations; however, methodology for cryopreservation of fish eggs and embryos has not yet been developed. The present study established a methodology capable of deriving functional eggs and sperm from frozen type A spermatogonia (ASGs). Whole testes taken from rainbow trout were slowly frozen in a cryomedium, and the viability of ASGs within these testes did not decrease over a 728-d freezing period. Frozen-thawed ASGs that were intraperitoneally transplanted into sterile triploid hatchlings migrated toward, and were incorporated into, recipient genital ridges. Transplantability of ASGs did not decrease after as much as 939 d of cryopreservation. Nearly half of triploid recipients produced functional eggs or sperm derived from the frozen ASGs and displayed high fecundity. Fertilization of resultant gametes resulted in the successful production of normal, frozen ASG-derived offspring. Feasibility and simplicity of this methodology will call for an immediate application for real conservation of endangered wild salmonids.