Two distinct host-parasite associations mediate seasonal ecosystem linkages.

Nematomorph parasites manipulate terrestrial arthropods to enter streams where the parasites reproduce. These manipulated arthropods become a substantial prey subsidy for stream salmonids, causing cross-ecosystem energy flow. Diverse nematomorph-arthropod associations underlie the energy flow, but it remains unknown whether they can mediate the magnitude and temporal attributes of the energy flow. Here, we investigated whether distinct phylogenetic groups of nematomorphs manipulate different arthropod hosts and mediate seasonal prey subsidy for stream salmonids. The results of our molecular-based diagnoses show that Gordionus and Gordius nematomorphs infected ground beetle and orthopteran hosts, respectively. The presumable ground beetle hosts subsidized salmonid individuals in spring, whereas the presumable orthopteran hosts did so in autumn. Maintaining the two distinct nematomorph-arthropod associations thus resulted in the parasite-mediated prey subsidy in both spring and autumn in the study streams. Manipulative parasites are common, and often associated with a range of host lineages, suggesting that similar effects of phylogenetic variation in host-parasite associations on energy flow might be widespread in nature.

Improvements in in vitro spermatogenesis: oxygen concentration, antioxidants, tissue-form design, and space control.

Incorporation of bovine serum-derived albumin formulation (AlbuMAX) into a basic culture medium, MEMα, enables the completion of in vitro spermatogenesis through testicular tissue culture in mice. However, this medium was not effective in other animals. Therefore, we sought an alternative approach for in vitro spermatogenesis using a synthetic medium without AlbuMAX and aimed to identify its essential components. In addition to factors known to be important for spermatogenesis, such as retinoic acid and reproductive hormones, we found that antioxidants (vitamin E, vitamin C, and glutathione) and lysophospholipids are vital for in vitro spermatogenesis. Moreover, based on our experience with microfluidic devices (MFD), we developed an alternative approach, the PDMS-ceiling method (PC method), which involves simply covering the tissue with a flat chip made of PDMS, a silicone resin material used in MFD. The PC method, while straightforward, integrates the advantages of MFD, enabling improved and uniform oxygen and nutrient supply via tissue flattening. Furthermore, our studies underscored the significance of lowering the oxygen concentration to 10-15%. Using an integrated cultivation method based on these findings, we successfully achieved in vitro spermatogenesis in rats, which has been a long-standing challenge. Further improvements in culture conditions would pave the way for spermatogenesis completion in diverse animal species.

A novel alternative method for long-term evaluation of male reproductive toxicity and its recovery using a pre-pubertal mouse testis organ culture system.

Successful treatment of pediatric cancers often results in long-term health complications, including potential effects on fertility. Therefore, assessing the male reproductive toxicity of anti-cancer drug treatments and the potential for recovery is of paramount importance. However, in vivo evaluations are time-intensive and require large numbers of animals. To overcome these constraints, we utilized an innovative organ culture system that supports long-term spermatogenesis by placing the testis tissue between a base agarose gel and a polydimethylsiloxane ceiling, effectively mirroring the in vivo testicular environment. The present study aimed to determine the efficacy of this organ culture system for accurately assessing testicular toxicity induced by cisplatin, using acrosin-green fluorescent protein (GFP) transgenic neonatal mouse testes. The testis fragments were treated with different concentrations of cisplatin-containing medium for 24 h and incubated in fresh medium for up to 70 days. The changes in tissue volume and GFP fluorescence over time were evaluated to monitor the progression of spermatogenesis, in addition to the corresponding histopathology. Cisplatin treatment caused tissue volume shrinkage and reduced GFP fluorescence in a concentration-dependent manner. Recovery from testicular toxicity was also dependent on the concentration of cisplatin received. The results demonstrated that this novel in vitro system can be a faithful replacement for animal experiments to assess the testicular toxicity of anti-cancer drugs and their reversibility, providing a useful method for drug development.

Seasonal ecosystem linkages contribute to the maintenance of migratory polymorphism in a salmonid population.

The influence of resource subsidies on animal growth, survival and reproduction is well understood, but their ultimate effects on life history have been less explored. Some wild species have a partially migratory life history, wherein migration is dictated based upon threshold traits regulated in part by the seasonal availability of resources. We conducted a large-scale field manipulation experiment where we provided a terrestrial invertebrate subsidy to red-spotted masu salmon. Individuals in stream reaches that received a subsidy had, on average, a 53% increase in growth rate relative to those in control reaches. This increased growth resulted in a greater proportion of individuals reaching the threshold body size and smolting in the autumn. Consequently, 19-55% of females in subsidized reaches became migratory, whereas 0-14% became migratory in the control reaches. Our findings highlight seasonal ecosystem linkage as a key ecosystem property for maintaining migratory polymorphism in partially migratory animals.

NMDA Receptor Enhances Correlation of Spontaneous Activity in Neonatal Barrel Cortex.

Correlated spontaneous activity plays critical role in the organization of neocortical circuits during development. However, cortical mechanisms regulating activity correlation are still elusive. In this study, using two-photon calcium imaging of the barrel cortex layer 4 (L4) in living neonatal mice, we found that NMDA receptors (NMDARs) in L4 neurons are important for enhancement of spontaneous activity correlation. Disruption of GluN1 (Grin1), an obligatory NMDAR subunit, in a sparse population of L4 neurons reduced activity correlation between GluN1 knock-out (GluN1KO) neuron pairs within a barrel. This reduction in activity correlation was even detected in L4 neuron pairs in neighboring barrels and most evident when either or both of neurons are located on the barrel edge. Our results provide evidence for the involvement of L4 neuron NMDARs in spatial organization of the spontaneous firing activity of L4 neurons in the neonatal barrel cortex.SIGNIFICANCE STATEMENT Precise wiring of the thalamocortical circuits is necessary for proper sensory information processing, and thalamus-derived correlated spontaneous activity is important for thalamocortical circuit formation. The molecular mechanisms involved in the correlated activity transfer from the thalamus to the neocortex are largely unknown. In vivo two-photon calcium imaging of the neonatal barrel cortex revealed that correlated spontaneous activity between layer four neurons is reduced by mosaic knock-out (KO) of the NMDA receptor (NMDAR) obligatory subunit GluN1. Our results suggest that the function of NMDARs in layer four neurons is necessary for the communication between presynaptic and postsynaptic partners during thalamocortical circuit formation.

The Relationship between Vector Species Richness and the Risk of Vector-Borne Infectious Diseases.

AbstractInfectious diseases can impact human welfare and impede wildlife management. Much recent research explores whether biodiversity increases or decreases infectious disease risk. Here, we theoretically study the relationship between vector species richness and the risk of vector-borne diseases using an epidemiological model of a single host and multiple vectors. The model considers that vectors are involved in interspecific feeding interference that causes transmission interference and in interspecific recruitment competition that mediates susceptible vector regulation. The model reveals three possible shapes of the vector richness-disease risk relationship: monotonic amplification, hump-shaped, and monotonic dilution patterns. The monotonic amplification pattern occurs across a wide parameter region. The hump-shaped and monotonic dilution patterns are found when transmission interference is strong and recruitment competition is weak. Unexpectedly, susceptible vector regulation not only promotes dilution but can strengthen amplification if coupled with strong transmission interference. Our results suggest that vector richness might be more likely to cause amplification rather than dilution, and shifts in the community mean trait values of vectors could also affect disease risk along the vector richness gradient.

LDL uptake-dependent phosphatidylethanolamine translocation to the cell surface promotes fusion of osteoclast-like cells.

Osteoporosis is associated with vessel diseases attributed to hyperlipidemia, and bone resorption by multinucleated osteoclasts is related to lipid metabolism. In this study, we generated low-density lipoprotein receptor (LDLR)/lectin-like oxidized LDL receptor-1 (LOX-1, also known as Olr1) double knockout (dKO) mice. We found that, like LDLR single KO (sKO), LDLR/LOX-1 dKO impaired cell-cell fusion of osteoclast-like cells (OCLs). LDLR/LOX-1 dKO and LDLR sKO preosteoclasts exhibited decreased uptake of LDL. The cell surface cholesterol levels of both LDLR/LOX-1 dKO and LDLR sKO osteoclasts were lower than the levels of wild-type OCLs. Additionally, the amount of phosphatidylethanolamine (PE) on the cell surface was attenuated in LDLR/LOX-1 dKO and LDLR sKO preosteoclasts, whereas the PE distribution in wild-type OCLs was concentrated on the filopodia in contact with neighboring cells. Abrogation of the ATP binding cassette G1 (ABCG1) transporter, which transfers PE to the cell surface, caused decreased PE translocation to the cell surface and subsequent cell-cell fusion. The findings of this study indicate the involvement of a novel cascade (LDLR∼ABCG1∼PE translocation to cell surface∼cell-cell fusion) in multinucleation of OCLs.

Age-associated changes in miRNA profile of bovine follicular fluid.

In brief: This study shows that ageing affects miRNA profiles in follicular fluid, and an miRNA that is highly abundant in the follicular fluid of young cows supports the growth of oocytes derived from early antral follicles. Abstract: We examined age-associated changes in miRNA profiles in the follicular fluid (FF) of cows. The role of miR-19b, which is abundant in the FF of young cows, in in vitro growth of early antral follicles (EAFs)-derived oocytes was assessed. FF was collected from the antral follicles of young (20-40 months) and aged (>120 months) cows. The miRNA profiles were similar between the FF of both age groups, whereas the abundance of some miRNAs differed between these samples. The miRNA profiles in granulosa cells (GCs) and the spent culture medium of oocyte-GC complexes (OGCs) derived from EAFs were distinct. Some miRNA groups overlapped among the GCs, culture media, and FFs. miR-19b was highly abundant in the FF of young cows, GCs, and culture medium. The supplementation of OGC culture medium with miR-19b increased the diameter, acetylation levels, and fertilisation ability of the oocytes. To assess whether miR-19b was functional in the GCs, a dual-luciferase assay, suppression of target protein, and RNA-sequencing of the GCs followed by functional annotation of the differentially expressed genes (DEGs) were conducted. Functional annotation of the DEGs suggested that miR-19b influences genes associated with FoxO signalling, endocytosis, and NR3C1 in GCs. These results suggest that in FFs, ageing affects the abundance of miRNAs that have important roles in oocyte development.

Direct and indirect effects of amphidromous shrimps on nutrient mineralization in streams in Japan.

Animals affect element cycling in ecosystems by consumption and excretion. Amphidromous shrimps frequently dominate low-mid altitude streams, where downstream connectivity to oceans is sustained. Although shrimps' direct influence on benthic communities has been studied, little is known about their influences on nutrient cycling. Here, we hypothesized that the dominance of shrimps alters nutrient mineralization by benthic macroinvertebrates in streams due to the difference in the quality and quantity of excretion between shrimps and aquatic insects. We tested this hypothesis through a field manipulative experiment, excretion measurements of animals, and field surveys. In the field manipulative experiment, the presence of shrimps slightly decreased the biomass of aquatic insects but tripled total benthic macroinvertebrate biomass directly through their own biomass. The mass-specific NH4+ excretion rate by shrimps was similar to aquatic insects, and the areal NH4+ excretion by benthic macroinvertebrates was increased by 2.5 times in the presence of shrimps. In contrast, shrimps excreted significantly less soluble reactive phosphorus (SRP) than aquatic insects, and the presence of shrimps did not affect areal SRP excretion by benthic macroinvertebrates. The field survey showed a positive correlation of NO3- concentration with the shrimp density, inferring the excess NH4+ was nitrified. Although the nutrient concentration of stream water is frequently attributed to watershed conditions, the results of this study indicate that downstream connectivity to oceans may also influence nutrient dynamics of the stream through the density of amphidromous shrimps.

Sertoli cell replacement in explanted mouse testis tissue supporting host spermatogenesis†.

Spermatogenesis takes place in the seminiferous tubules, starting from the spermatogonial stem cell and maturing into sperm through multiple stages of cell differentiation. Sertoli cells, the main somatic cell constituting the seminiferous tubule, are in close contact with every germ cell and play pivotal roles in the progression of spermatogenesis. In this study, we developed an in vitro Sertoli cell replacement method by combining an organ culture technique and a toxin receptor-mediated cell knockout system. We used Amh-diphtheria toxin receptor transgenic mice, whose Sertoli cells specifically express human diphtheria toxin receptor, which renders them sensitive to diphtheria toxin. An immature Amh-diphtheria toxin receptor testis was transplanted with the donor testis cells followed by culturing in a medium containing diphtheria toxin. This procedure successfully replaced the original Sertoli cells with the transplanted Sertoli cells, and spermatogenesis originating from resident germ cells was confirmed. In addition, Sertoli cells in the mouse testis tissues were replaced by transplanted rat Sertoli cells within culture conditions without requiring immunosuppressive treatments. This method works as a functional assay system, making it possible to evaluate any cells that might function as Sertoli cells. It would also be possible to investigate interactions between Sertoli and germ cells more closely, providing a new platform for the study of spermatogenesis and its impairments.

Antioxidant vitamins and lysophospholipids are critical for inducing mouse spermatogenesis under organ culture conditions.

In vitro mouse spermatogenesis using a classical organ culture method became possible by supplementing basal culture medium with only the product of bovine serum albumin purified by chromatography (AlbuMAX), which indicated that AlbuMAX contained every chemical factor necessary for mouse spermatogenesis. However, since the identity of these factors was unclear, improvements in culture media and our understanding of the nutritional and signal substances required for spermatogenesis were hindered. In the present study, chemically defined media (CDM) without AlbuMAX was used to evaluate each supplementary factor and their combinations for the induction of spermatogenesis. Similar to in vivo conditions, retinoic acid, triiodothyronine (T3 ), and testosterone (T) were needed. Based on differences in spermatogenic competence between AlbuMAX, fetal bovine serum, and adult bovine serum, we identified α-tocopherol, which strongly promoted spermatogenesis when combined with ascorbic acid and glutathione. Differences were also observed in the abilities of lipids extracted from AlbuMAX using two different methods to induce spermatogenesis. This led to the identification of lysophospholipids, particularly lysophosphatidylcholine, lysophosphatidic acid, and lysophosphatidylserine, as important molecules for spermatogenesis. New CDM formulated based on these results induced and promoted spermatogenesis as efficiently as AlbuMAX-containing medium. In vitro spermatogenesis with CDM may provide a unique experimental system for research on spermatogenesis that cannot be performed in in vivo experiments.

The effects of resource subsidy duration in a detritus-based stream ecosystem: A mesocosm experiment.

Most resource subsidies are temporally variable, dynamically affecting the consumer populations, community structures and ecosystem functions of recipient ecosystems. Temporally variable resource subsidies are characterized by the duration, magnitude, timing and frequency of resource subsidy inputs. These different characteristics may have different mechanisms by which to affect recipient ecosystems. Few studies have examined the duration of resource subsidy inputs on recipient ecosystems, although there exist previous studies focusing on magnitude, timing and frequency. We provide the first experimental test of the effects of subsidy duration on a stream ecosystem by using an outdoor mesocosm experiment, in which we directly manipulated the subsidy duration (pulsed vs. prolonged) of terrestrial invertebrate input into the mesocosm. Given the same overall amount of terrestrial invertebrate subsidy was added, a prolonged subsidy allowed large-stage fish to effectively monopolize the subsidy over small-stage fish, which led small-stage fish to maintain their predation pressure on in-situ prey, that is, benthic invertebrates. On the other hand, a pulsed subsidy allowed small-stage fish to increase their feeding rate of the subsidy and to become away from foraging in-situ prey. Consequently, weaker indirect positive effects on in-situ benthic prey and leaf break-down rate were found with the prolonged versus pulsed subsidy. However, these indirect effects varied by the dominant benthic prey species, which differed in edibility for fish. Such predator-specific vulnerability of benthic prey can be important in mediating trophic cascades in detritus-based stream food webs. Phenological events that generate temporal subsidies (e.g. salmon spawning run and arthropod emergence) can be synchronized (pulsed) or desynchronized (prolonged) within and among species, depending on the degree of spatial and temporal environmental heterogeneity. The effects of subsidy duration would thus be important to better understand ecological processes in spatially and temporally coupled ecosystems.

Captive-bred populations of a partially migratory salmonid fish are unlikely to maintain migratory polymorphism in natural habitats.

Variation in life history is fundamental to the long-term persistence of populations and species. Partial migration, in which both migratory and resident individuals are maintained in a population, is commonly found across animal taxa. However, human-induced habitat fragmentation continues to cause a rapid decline in the migratory phenotype in many natural populations. Using field and hatchery experiments, we demonstrated that despite both migrants and residents being maintained in captive environments, few individuals of the red-spotted masu salmon, Oncorhynchus masou ishikawae, became migrants in natural streams when released prior to the migration decision. Released fish rarely reached the threshold body size necessary to become migrants in natural streams, presumably owing to lower growth rates in natural than in captive environments. The decision to migrate is often considered a threshold trait in salmonids and other animal taxa. Our findings highlight the need for management programmes that acknowledge the effects of the environment on the determination of the migratory phenotypes of partially migratory species when releasing captive-bred individuals prior to their migratory decisions.

Anadromous red-spotted masu salmon (Oncorhynchus masou ishikawae), a southernmost sea-migration form of salmonid, displays low variation in both age at seaward migration and sea age.

We examined variations in age at seaward migration and sea age for the anadromous form of red-spotted masu salmon (Oncorhynchus masou ishikawae) in two Japanese rivers. The anadromous form of red-spotted masu salmon expressed only two sea migration patterns in the two rivers: (a) the majority of the salmon (95%, n = 81) were of age-0, and age-1 migrants were rare (n = 4); and (b) all the salmon examined (n = 22) made a return migration within a year, with 23% of the salmon exhibiting potamodromy in the river. Owing to low variation in their sea migratory patterns, the anadromous form of red-spotted masu salmon is likely vulnerable to environmental fluctuations.

CD147 Is Essential for the Development of Psoriasis via the Induction of Th17 Cell Differentiation.

Th17 cells play an important role in psoriasis. The differentiation of naïve CD4+ T cells into Th17 cells depends on glycolysis as the energy source. CD147/basigin, an integral transmembrane protein belonging to the immunoglobulin superfamily, regulates glycolysis in association with monocarboxylate transporters (MCTs)-1 and -4 in cancer cells and T cells. We examined whether CD147/basigin is involved in the pathogenesis of psoriasis in humans and psoriasis-model mice. The serum level of CD147 was increased in patients with psoriasis, and the expression of CD147 and MCT-1 was elevated in their dermal CD4+ RORγt+ T cells. In vitro, the potential of naïve CD4+ T cells to differentiate into Th17 cells was abrogated in CD147-/- T cells. Imiquimod (IMQ)-induced psoriatic dermatitis was significantly milder in CD147-/- mice and bone marrow chimeric mice lacking CD147 in the hematopoietic cells of myeloid lineage. These findings demonstrate that CD147 is essential for the development of psoriasis via the induction of Th17 cell differentiation.

Transcriptome analysis reveals inadequate spermatogenesis and immediate radical immune reactions during organ culture in vitro spermatogenesis.

Cultivation of neonatal mouse testis tissue can induce spermatogenesis and produce fertile sperms. However, in vitro spermatogenesis mediated by the current organ culture method comes short in fully mimicking the in vivo counterpart, partly due to a lack of knowledge underlying molecular phenotypes of in vitro spermatogenesis. In this study, we investigated transcriptome of cultured testis tissues using microarray method. Principle component analysis of the transcriptome data revealed delay and/or arrest of spermatogenesis and immediate radical immune reactions in the cultured testis tissues. The delay/arrest of spermatogenesis occurred before and during early meiotic phase, resulting in inefficient progression of meiosis. The immune reaction, on the other hand, was drastic and overwhelming, in which TLR4-NF-kB signaling was speculated to be involved. Notably, treatment with TAK242, an inhibitor of TLR4-NF-kB signaling pathway, ameliorated the macrophage activation which otherwise would exacerbate the inflammation. Thus, the present study revealed for the first time at molecular level that the deficiency of germ cell differentiation and the immense immune reaction are major abnormalities in the cultured testis tissues.

GRP78 antibodies damage the blood-brain barrier and relate to cerebellar degeneration in Lambert-Eaton myasthenic syndrome.

Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune disease of the neuromuscular junction caused by autoantibodies binding to P/Q-type voltage-gated calcium channels. Breakdown of the blood-brain barrier and diffusion of cerebellar granule/Purkinje cell-reactive autoantibodies into the CNS are critical for the pathogenesis of paraneoplastic cerebellar degeneration (PCD) with Lambert-Eaton myasthenic syndrome. We recently found evidence that glucose-regulated protein 78 (GRP78) autoantibodies in the plasma of patients with neuromyelitis optica promote the CNS access of AQP4 autoantibodies. In the present study, we investigated whether the GRP78 autoantibodies in PCD-LEMS IgG boost the brain uptake of cerebellar cell-reactive antibodies across the blood-brain barrier and facilitate cerebellar dysfunction. We first evaluated the effects of purified IgG from PCD-LEMS or PCD patients on the blood-brain barrier function in human brain microvascular endothelial cells using a high content imaging system with nuclear factor κB p65 and intracellular adhesion molecule 1 (ICAM1) immunostaining. Next, we identified GRP78 autoantibodies causing blood-brain barrier permeability in PCD-LEMS IgG by co-immunoprecipitation and the living cell-based antibody binding assays. Exposure of brain microvascular endothelial cells to IgG from PCD-LEMS patients induced nuclear factor κB p65 nuclear translocation, ICAM1 upregulation, reduced claudin-5 expression, increased permeability and increased autocrine IL-1ß and IL-8 secretion; the IgG from patients with Lambert-Eaton myasthenic syndrome did not have these effects. We detected GRP78 autoantibodies in the IgG of LEMS-PCD (83.3%, n = 18), but observed fewer in patients with LEMS (6.6%, n = 15) and none were observed in the control subjects (n = 8). The depletion of GRP78 autoantibodies reduced the biological effect of LEMS-PCD IgG on brain microvascular endothelial cells. These findings suggest that GRP78 autoantibodies play a role beyond neuromyelitis optica and that they have direct implications in the phenotypic differences between PCD-LEMS and LEMS.

Daily growth increments in otoliths of wild-caught honmoroko Gnathopogon caerulescens.

Otolith growth increments in wild-caught alizarin complex one (ALC)-marked honmoroko Gnathopogon caerulescens were examined to verify the veracity of the age determination method in cyprinids. ALC-marked G. caerulescens recaptured from their natural environment had lapilli increment counts outside the ALC ring mark that had formed on a daily basis during the juvenile stage. This apparently being the first direct evidence of daily periodicity of otolith increment formation in wild-caught cyprinids.

In vitro spermatogenesis in two-dimensionally spread mouse testis tissues.

PURPOSE: Mouse in vitro spermatogenesis is possible with classical organ culture methods, by placing the testis tissue at the interphase between culture medium and air. In this condition, however, a tissue piece tends to round up to be compact, whose central region suffers from shortage of nutrients and oxygen. In this study, the authors improved the culture condition by spreading each tissue thin and flat, by which they were able to get better access to the oxygen and nutrients. METHODS: Immature mouse testis tissues placed on agarose gel block were forced to spread flat by covering with a polydimethylsiloxane (PDMS) ceiling chip (PC chip). They were then cultured for weeks and evaluated by the transgene expression of Acr-Gfp, which reflects the progression of spermatogenesis. RESULTS: Testis tissues covered with PC chip initiated and maintained spermatogenesis in its wider region than those without PC chip covering. Flow cytometric analysis demonstrated that the PC method yielded more numerous meiotic germ cells than those without PC. Immunohistochemical examination confirmed the authentic histological figure of spermatogenesis from spermatogonia up to round or elongating spermatids. CONCLUSIONS: The PC chip method is simple and effective to improve the efficiency of in vitro spermatogenesis in the organ culture system.

A monolayer microfluidic device supporting mouse spermatogenesis with improved visibility.

In our previous study, we produced a microfluidic device (MFD) which successfully maintained spermatogenesis for over 6 months in mouse testis tissues loaded in the device. In the present study, we developed a new MFD, a monolayer device (ML-D) with a barrier structure consisting of pillars and slits, which is simpler in design and easier to make. This ML-D was also effective for inducing mouse spermatogenesis and maintained it for a longer period than the conventional culture method. In addition, we devised a way of introducing sample tissue into the device during its production, just before bonding the upper layer of polydimethylsiloxane (PDMS) and bottom glass slide. The tissue can obtain nutrients horizontally from the medium running beside it and oxygen vertically from above through PDMS. In addition, the glass slide set at the bottom improved the visibility of the sample tissue with an inverted microscope. When we took photos of cultured tissue of the Acr-Gfp transgenic mouse testis in ML-D sequentially every day, morphological changes of the acrosome during spermiogenesis were successfully recorded. The ML-D is simple in design and useful for culturing testis tissue for inducing and maintaining spermatogenesis with clearer visibility. Due to the new method of sample loading, tissues other than testis should also be applicable.