Exploring Subcellular Cerebellar Fractions with the Electron Microscope.

Differential ultracentrifugation and subcellular fractionation historically helped to study the components of the cell, to discover new cellular organelles, and to decipher their morphological and molecular properties. In neuroscience, the technique has yielded important results on neuron biochemistry and the mechanisms of synaptic transmission. This Cerebellar Classic is devoted to the pioneering work of Manuel del Cerro, Ray S. Snider, and Mary Lou Oster-Granite, who isolated purified fractions after successive centrifugations of the rat cerebellum from birth to adulthood and studied them under the electron microscope.

Commentary on "Subcellular Fractions of Adult and Developing Rat Cerebellum, by M. del Cerro, R. S. Snider and M. L. Oster, July 1969".

Subcellular fractionation by differential ultracentrifugation has allowed the study of the cell and its organelles from a morphological, physiological, and biochemical perspective. Combined with electron microscopy, and by using animals at different stages of postnatal development, these methods yielded useful results concerning the ontogeny of synaptosomes, mitochondria, and myelin and broadened the possibilities to investigate the molecular underpinnings of cerebellar histogenesis.

Cryogenic Super-Resolution Fluorescence and Electron Microscopy Correlated at the Nanoscale.

We review the emerging method of super-resolved cryogenic correlative light and electron microscopy (srCryoCLEM). Super-resolution (SR) fluorescence microscopy and cryogenic electron tomography (CET) are both powerful techniques for observing subcellular organization, but each approach has unique limitations. The combination of the two brings the single-molecule sensitivity and specificity of SR to the detailed cellular context and molecular scale resolution of CET. The resulting correlative data is more informative than the sum of its parts. The correlative images can be used to pinpoint the positions of fluorescently labeled proteins in the high-resolution context of CET with nanometer-scale precision and/or to identify proteins in electron-dense structures. The execution of srCryoCLEM is challenging and the approach is best described as a method that is still in its infancy with numerous technical challenges. In this review, we describe state-of-the-art srCryoCLEM experiments, discuss the most pressing challenges, and give a brief outlook on future applications.

Visualization of cell-type dependent effects of anti-E2 antibody and interferon-gamma treatments on localization and expression of Broccoli aptamer-tagged alphavirus RNAs.

Sindbis virus (SINV) is an alphavirus that causes age-dependent encephalomyelitis in mice. Within 7-8 days after infection infectious virus is cleared from neurons through the antiviral effects of antibody and interferon-gamma (IFNγ), but RNA persists. To better understand changes in viral RNA associated with immune-mediated clearance we developed recombinant strains of SINV that have genomic and subgenomic viral RNAs tagged with the Broccoli RNA aptamer that binds and activates a conditional fluorophore for live cell imaging of RNA. Treatment of SINV-Broccoli-infected cells with antibody to the SINV E2 glycoprotein had cell type-specific effects. In BHK cells, antibody increased levels of intracellular viral RNA and changed the primary location of genomic RNA from the perinuclear region to the plasma membrane without improving cell viability. In undifferentiated and differentiated AP7 (dAP7) neuronal cells, antibody treatment decreased levels of viral RNA. Occasional dAP7 cells escaped antibody-mediated clearance by not expressing cell surface E2 or binding antibody to the plasma membrane. IFNγ decreased viral RNA levels only in dAP7 cells and synergized with antibody for RNA clearance and improved cell survival. Therefore, analysis of aptamer-tagged SINV RNAs identified cell type- and neuronal maturation-dependent responses to immune mediators of virus clearance.

Three-dimensional imaging of podocyte ultrastructure using FE-SEM and FIB-SEM tomography.

Podocytes are specialized epithelial cells used for glomerular filtration in the kidney. They can be divided into the cell body, primary process and foot process. Here, we describe two useful methods for the three-dimensional(3D) visualization of these subcellular compartments in rodent podocytes. The first method, field-emission scanning electron microscopy (FE-SEM) with conductive staining, is used to visualize the luminal surface of numerous podocytes simultaneously. The second method, focused-ion beam SEM (FIB-SEM) tomography, allows the user to obtain serial images from different depths of field, or Z-stacks, of the glomerulus. This allows for the 3D reconstruction of podocyte ultrastructure, which can be viewed from all angles, from a single image set. This is not possible with conventional FE-SEM. The different advantages and disadvantages of FE-SEM and FIB-SEM tomography compensate for the weaknesses of the other. The combination renders a powerful approach for the 3D analysis of podocyte ultrastructure. As a result, we were able to identify a new subcellular compartment of podocytes, "ridge-like prominences" (RLPs).

Subcellular distribution and tolerance of cadmium in Canna indica L.

Canna indica L. is a promising species for heavy metal phytoremediation due to its fast growth rate and large biomass. However, few studies have investigated cadmium (Cd) tolerance mechanisms. In the present study, Canna plants were cultivated under hydroponic conditions with increasing Cd concentrations (0, 5, 10, 15 mg/L). We found that the plants performed well under 5 mg/L Cd2+ stress, but damage was observed under higher Cd exposure, such as leaf chlorosis, growth inhibition, a decreased chlorophyll content, and destruction of the ultrastructure of leaf cells. Additionally, Canna alleviated Cd toxicity to a certain extent. After Canna was exposed to 5, 10 and 15 mg/L Cd2+ for 45 d, the highest Cd concentration was exhibited in roots, which was almost 17-47 times the Cd concentration in leaves and 8-20 times that in stems. At the subcellular level, cellular debris and heat-stable proteins (HSPs) were the main binding sites for Cd, and the proportion of Cd in the two subcellular fractions accounted for 71.4-94.2% of the total Cd. Furthermore, we found that granules could participate in the detoxification process when Cd stress was enhanced. Our results indicated that Canna indica L. can tolerate Cd toxicity by sequestering heavy metals in root tissues, fencing out by cell wall, and binding with biologically detoxified fractions (granules and HSPs).

Live-cell imaging of subcellular structures for quantitative evaluation of pluripotent stem cells.

Pluripotent stem cells (PSCs) have various degrees of pluripotency, which necessitates selection of PSCs with high pluripotency before their application to regenerative medicine. However, the quality control processes for PSCs are costly and time-consuming, and it is essential to develop inexpensive and less laborious selection methods for translation of PSCs into clinical applications. Here we developed an imaging system, termed Phase Distribution (PD) imaging system, which visualizes subcellular structures quantitatively in unstained and unlabeled cells. The PD image and its derived PD index reflected the mitochondrial content, enabling quantitative evaluation of the degrees of somatic cell reprogramming and PSC differentiation. Moreover, the PD index allowed unbiased grouping of PSC colonies into those with high or low pluripotency without the aid of invasive methods. Finally, the PD imaging system produced three-dimensional images of PSC colonies, providing further criteria to evaluate pluripotency of PSCs. Thus, the PD imaging system may be utilized for screening of live PSCs with potentially high pluripotency prior to more rigorous quality control processes.

Release of astroglial vimentin by extracellular vesicles: Modulation of binding and internalization of C3 transferase in astrocytes and neurons.

Clostridium botulinum C3 transferase (C3bot) ADP-ribosylates rho proteins to change cellular functions in a variety of cell types including astrocytes and neurons. The intermediate filament protein vimentin as well as transmembrane integrins are involved in internalization of C3bot into cells. The exact contribution, however, of these proteins to binding of C3bot to the cell surface and subsequent cellular uptake remains to be unraveled. By comparing primary astrocyte cultures derived from wild-type with Vim-/- mice, we demonstrate that astrocytes lacking vimentin exhibited a delayed ADP-ribosylation of rhoA concurrent with a blunted morphological response. This functional impairment was rescued by the extracellular excess of recombinant vimentin. Binding assays using C3bot harboring a mutated integrin-binding RGD motif (C3bot-G89I) revealed the involvement of integrins in astrocyte binding of C3bot. Axonotrophic effects of C3bot are vimentin dependent and postulate an underlying mechanism entertaining a molecular cross-talk between astrocytes and neurons. We present functional evidence for astrocytic release of vimentin by exosomes using an in vitro scratch wound model. Exosomal vimentin+ particles released from wild-type astrocytes promote the interaction of C3bot with neuronal membranes. This effect vanished when culturing Vim-/- astrocytes. Specificity of these findings was confirmed by recombinant vimentin propagating enhanced binding of C3bot to synaptosomes from rat spinal cord and mouse brain. We hypothesize that vimentin+ exosomes released by reactive astrocytes provide a novel molecular mechanism constituting axonotrophic (neuroprotective) and plasticity augmenting effects of C3bot after spinal cord injury.

Deep learning enables cross-modality super-resolution in fluorescence microscopy.

We present deep-learning-enabled super-resolution across different fluorescence microscopy modalities. This data-driven approach does not require numerical modeling of the imaging process or the estimation of a point-spread-function, and is based on training a generative adversarial network (GAN) to transform diffraction-limited input images into super-resolved ones. Using this framework, we improve the resolution of wide-field images acquired with low-numerical-aperture objectives, matching the resolution that is acquired using high-numerical-aperture objectives. We also demonstrate cross-modality super-resolution, transforming confocal microscopy images to match the resolution acquired with a stimulated emission depletion (STED) microscope. We further demonstrate that total internal reflection fluorescence (TIRF) microscopy images of subcellular structures within cells and tissues can be transformed to match the results obtained with a TIRF-based structured illumination microscope. The deep network rapidly outputs these super-resolved images, without any iterations or parameter search, and could serve to democratize super-resolution imaging.

Obesity modifies the stoichiometry of mitochondrial proteins in a way that is distinct to the subcellular localization of the mitochondria in skeletal muscle.

BACKGROUND: Skeletal muscle mitochondrial content and function appear to be altered in obesity. Mitochondria in muscle are found in well-defined regions within cells, and they are arranged in a way that form distinct subpopulations of subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria. We sought to investigate differences in the proteomes of SS and IMF mitochondria between lean subjects and subjects with obesity. METHODS: We performed comparative proteomic analyses on SS and IMF mitochondria isolated from muscle samples obtained from lean subjects and subjects with obesity. Mitochondria were isolated using differential centrifugation, and proteins were subjected to label-free quantitative tandem mass spectrometry analyses. Collected data were evaluated for abundance of mitochondrial proteins using spectral counting. The Reactome pathway database was used to determine metabolic pathways that are altered in obesity. RESULTS: Among proteins, 73 and 41 proteins showed different (mostly lower) expression in subjects with obesity in the SS and IMF mitochondria, respectively (false discovery rate-adjusted P ≤ 0.05). We specifically found an increase in proteins forming the tricarboxylic acid cycle and electron transport chain (ETC) complex II, but a decrease in proteins forming protein complexes I and III of the ETC and adenosine triphosphate (ATP) synthase in subjects with obesity in the IMF, but not SS, mitochondria. Obesity was associated with differential effects on metabolic pathways linked to protein translation in the SS mitochondria and ATP formation in the IMF mitochondria. CONCLUSIONS: Obesity alters the expression of mitochondrial proteins regulating key metabolic processes in skeletal muscle, and these effects are distinct to mitochondrial subpopulations located in different regions of the muscle fibers. TRIAL REGISTRATION: ClinicalTrials.gov (NCT01824173).

Acute hepatologic and nephrologic effects of calcitriol in Syrian golden hamster (Mesocricetus auratus).

Although vitamin D is included in the group of fat-soluble vitamins, it must be considered as a prohormone. Its active forms, including calcitriol, have pleiotropic effects and play an important role in the regulation of cell proliferation, differentiation and apoptosis, as well as in hormone secretion, and they demonstrate anti-cancer properties. Since calcitriol delivery can be beneficial for the organism, and Syrian golden hamsters represent a unique experimental model, we decided to investigate its toxicity in this species. In this study, we injected calcitriol intraperitoneally at doses 0 (control), 0.180±0.009 µg/kg and 0.717±0.032 µg/kg. Animal behavior was observed for 72 hrs after injection, and afterwards blood, liver and kidneys were collected for post-mortem examination, electron microscopy, and hematology analyses. The highest dose of calcitriol induced a change in animal behavior from calm to aggressive, and the liver surface showed morphological signs of damage. Following injection of calcitriol, ultrastructural changes were also observed in the liver and kidneys, e.g. vacuolization and increased number of mitochondria. There was also a trend for increased serum levels of aspartate aminotransferase (AST), but not of alanine aminotransferase (ALT) or GGTP (gamma-glutamyl transpeptidase). There was no change in Ca, Mg and P levels, as well as in blood morphology between experimental and control groups. These results indicate that calcitriol at 0.717, but not at 0.180 µg/kg, may induce acute damage to the liver and kidneys, without inducing calcemia. We propose that the hepatotoxic effect of calcitriol in hamster constitutes the primary cause of behavioral changes.

Subcellular origin of mitochondrial DNA deletions in human skeletal muscle.

OBJECTIVE: In patients with mitochondrial DNA (mtDNA) maintenance disorders and with aging, mtDNA deletions sporadically form and clonally expand within individual muscle fibers, causing respiratory chain deficiency. This study aimed to identify the sub-cellular origin and potential mechanisms underlying this process. METHODS: Serial skeletal muscle cryosections from patients with multiple mtDNA deletions were subjected to subcellular immunofluorescent, histochemical, and genetic analysis. RESULTS: We report respiratory chain-deficient perinuclear foci containing mtDNA deletions, which show local elevations of both mitochondrial mass and mtDNA copy number. These subcellular foci of respiratory chain deficiency are associated with a local increase in mitochondrial biogenesis and unfolded protein response signaling pathways. We also find that the commonly reported segmental pattern of mitochondrial deficiency is consistent with the three-dimensional organization of the human skeletal muscle mitochondrial network. INTERPRETATION: We propose that mtDNA deletions first exceed the biochemical threshold causing biochemical deficiency in focal regions adjacent to the myonuclei, and induce mitochondrial biogenesis before spreading across the muscle fiber. These subcellular resolution data provide new insights into the possible origin of mitochondrial respiratory chain deficiency in mitochondrial myopathy. Ann Neurol 2018;84:289-301.

Cellular and Subcellular Localization of Endoplasmic Reticulum Chaperone GRP78 Following Transient Focal Cerebral Ischemia in Rats.

The 78-kDa glucose-regulated protein (GRP78), a chaperone protein located in the endoplasmic reticulum (ER), has been reported to have neuroprotective effects in the injured central nervous system. Our aim was to examine the expression profiles and subcellular distributions of GRP78 and its association with the neuroglial reaction in the rat striatum after transient, focal cerebral ischemia. In sham-operated rats, constitutive, specific immunoreactivity for GRP78 was almost exclusively localized to the rough ER of striatal neurons, with none in the resting, ramified microglia or astrocytes. At 1 day post reperfusion, increased expression was observed in ischemia-resistant cholinergic interneurons, when most striatal neurons had lost GRP78 expression (this occurred earlier than the loss of other neuronal markers). By 3 days post reperfusion, GRP78 expression had re-emerged in association with the activation of glial cells in both infarct and peri-infarct areas but showed different patterns in the two regions. Most of the expression induced in the infarct area could be attributed to brain macrophages, while expression in the peri-infarct area predominantly occurred in neurons and reactive astrocytes. A gradual, sustained induction of GRP78 immunoreactivity occurred in reactive astrocytes localized to the astroglial scar, lasting for at least 28 days post reperfusion. Using correlative light- and electron-microscopy, we found conspicuous GRP78 protein localized to abnormally prominent, dilated rough ER in both glial cell types. Thus, our data indicate a link between GRP78 expression and the activated functional status of neuroglial cells, predominantly microglia/macrophages and astrocytes, occurring in response to ischemia-induced ER stress.

Differential regional and subcellular localization patterns of afadin splice variants in the mouse central nervous system.

AF6/afadin is an F-actin scaffold protein that plays essential roles in the organization of cell-cell junctions of polarized epithelia. Afadin comprises two major isoforms produced by alternative splicing - a longer isoform l-afadin, having the F-actin-binding domain, and a shorter isoform s-afadin, harboring the amino acid sequences unique to the isoform but lacking the F-actin-binding domain. We recently identified functional differences between l- and s-afadin isoforms in the regulation of axon branching in primary cultured cortical neurons; the former potentiates and the latter blocks axon branching. Previous biochemical reports indicate differences in tissue and cell-type distributions of isoforms, and it was shown that l-afadin is ubiquitously expressed in various tissues and cell-types, while s-afadin is predominantly expressed in neuronal tissues and cultured neurons. However, the spatial expression pattern of s-afadin across neuronal tissues or within neurons has not been revealed because no antibody specific for s-afadin is yet available. In this study, we report the generation and characterization of an antibody that specifically distinguishes s-afadin from l-afadin, and its application to investigate the expression profile of s-afadin in primary cultured neurons and tissue cryosections of adult mouse brain and retina. We describe differential regional and subcellular localization patterns of l- and s-afadin isoforms in the mouse central nervous system.

Anatomical characterization of PDF-tri neurons and peptidergic neurons associated with eclosion behavior in Drosophila.

The peptidergic Pigment-dispersing factor (PDF)-Tri neurons are a group of non-clock neurons that appear transiently around the time of adult ecdysis (=eclosion) in the fruit fly Drosophila melanogaster. This specific developmental pattern points to a function of these neurons in eclosion or other processes that are active around pupal-adult transition. As a first step to understand the role of these neurons, we here characterize the anatomy of the PDF-Tri neurons. In addition, we describe a further set of peptidergic neurons that have been associated with eclosion behavior, eclosion hormone (EH), and crustacean cardioactive peptide (CCAP) neurons, to single cell level in the pharate adult brain. PDF-Tri neurons as well as CCAP neurons co-express a classical transmitter indicated by the occurrence of small clear vesicles in addition to dense-core vesicles containing the peptides. In the tritocerebrum, gnathal ganglion and the superior protocerebrum PDF-Tri neurites contain peptidergic varicosities and both pre- and postsynaptic sites, suggesting that the PDF-Tri neurons represent modulatory rather than pure interneurons that connect the subesophageal zone with the superior protocerebrum. The extensive overlap of PDF-Tri arborizations with neurites of CCAP- and EH-expressing neurons in distinct brain regions provides anatomical evidence for a possible function of the PDF-Tri neurons in eclosion behavior.

Metabolomics and Lipidomics Profiling of a Combined Mitochondrial Plus Endoplasmic Reticulum Fraction of Human Fibroblasts: A Robust Tool for Clinical Studies.

Mitochondria and endoplasmic reticulum (ER) are physically and functionally connected. This close interaction, via mitochondria-associated membranes, is increasingly explored and supports the importance of studying these two organelles as a whole. Metabolomics and lipidomics are powerful approaches for the exploration of metabolic pathways that may be useful to provide deeper information on these organelles' functions, dysfunctions, and interactions. We developed a quick and simple experimental procedure for the purification of a mitochondria-ER fraction from human fibroblasts. We applied combined metabolomics and lipidomics analyses by mass spectrometry with excellent reproducibility. Seventy-two metabolites and 418 complex lipids were detected with a mean coefficient of variation around 12%, among which many were specific to the mitochondrial metabolism. Thus this strategy based on robust mitochondria-ER extraction and "omics" combination will be useful for investigating the pathophysiology of complex diseases.

Localization of endogenous amyloid-ß to the coeruleo-cortical pathway: consequences of noradrenergic depletion.

The locus coeruleus (LC)-norepinephrine (NE) system is an understudied circuit in the context of Alzheimer's disease (AD), and is thought to play an important role in neurodegenerative and neuropsychiatric diseases involving catecholamine neurotransmitters. Understanding the expression and distribution of the amyloid beta (Aß) peptide, a primary component of AD, under basal conditions and under conditions of NE perturbation within the coeruleo-cortical pathway may be important for understanding its putative role in pathological states. Thus, the goal of this study is to define expression levels and the subcellular distribution of endogenous Aß with respect to noradrenergic profiles in the rodent LC and medial prefrontal cortex (mPFC) and, further, to determine the functional relevance of NE in modulating endogenous Aß42 levels. We report that endogenous Aß42 is localized to tyrosine hydroxylase (TH) immunoreactive somatodendritic profiles of the LC and dopamine-ß-hydroxylase (DßH) immunoreactive axon terminals of the infralimbic mPFC (ILmPFC). Male and female naïve rats have similar levels of amyloid precursor protein (APP) cleavage products demonstrated by western blot, as well as similar levels of endogenous Aß42 as determined by enzyme-linked immunosorbent assay. Two models of NE depletion, DSP-4 lesion and DßH knockout (KO) mice, were used to assess the functional relevance of NE on endogenous Aß42 levels. DSP-4 lesioned rats and DßH-KO mice show significantly lower levels of endogenous Aß42. Noradrenergic depletion did not change APP-cleavage products resulting from ß-secretase processing. Thus, resultant decreases in endogenous Aß42 may be due to decreased neuronal activity of noradrenergic neurons, or, by decreased stimulation of adrenergic receptors which are known to contribute to Aß42 production by enhancing γ-secretase processing under normal physiological conditions.

Solubilization of human cells by the styrene-maleic acid copolymer: Insights from fluorescence microscopy.

Extracting membrane proteins from biological membranes by styrene-maleic acid copolymers (SMAs) in the form of nanodiscs has developed into a powerful tool in membrane research. However, the mode of action of membrane (protein) solubilization in a cellular context is still poorly understood and potential specificity for cellular compartments has not been investigated. Here, we use fluorescence microscopy to visualize the process of SMA solubilization of human cells, exemplified by the immortalized human HeLa cell line. Using fluorescent protein fusion constructs that mark distinct subcellular compartments, we found that SMA solubilizes membranes in a concentration-dependent multi-stage process. While all major intracellular compartments were affected without a strong preference, plasma membrane solubilization was found to be generally slower than the solubilization of organelle membranes. Interestingly, some plasma membrane-localized proteins were more resistant against solubilization than others, which might be explained by their presence in specific membrane domains with differing properties. Our results support the general applicability of SMA for the isolation of membrane proteins from different types of (sub)cellular membranes.

Neocortical Chandelier Cells Developmentally Shape Axonal Arbors through Reorganization but Establish Subcellular Synapse Specificity without Refinement.

Diverse types of cortical interneurons (INs) mediate various kinds of inhibitory control mechanisms to balance and shape network activity. Distinct IN subtypes develop uniquely organized axonal arbors that innervate different subcellular compartments of excitatory principal neurons (PNs), which critically contribute to determining their output properties. However, it remains poorly understood how they establish this peculiar axonal organization and synaptic connectivity during development. Here, taking advantage of genetic labeling of IN progenitors, we examined developmental processes of axonal arbors and synaptic connections formed by murine chandelier cells (ChCs), which innervate axon initial segments (AISs) of PNs and thus powerfully regulate their spike generation. Our quantitative analysis by light microscopy revealed that ChCs overgrow and subsequently refine axonal branches as well as varicosities. Interestingly, we found that although a significant number of axonal varicosities are formed off AISs in addition to on AISs, presynaptic markers are predominantly colocalized with those on AISs throughout development. Immunoelectron microscopic (IEM) analysis also demonstrated that only varicosities apposed to AISs contain presynaptic profiles. These results suggest that subcellular synapse specificity of ChCs is genetically predetermined while axonal geometry is shaped through remodeling. Molecular cues localized at AISs may regulate target recognition and synapse formation by ChCs.

Subcellular distribution of calcium during spermatogenesis of zebrafish, Danio rerio.

Calcium plays a variety of vital regulatory functions in many physiological and biochemical events in the cell. The aim of this study was to describe the ultrastructural distribution of calcium during different developmental stages of spermatogenesis in a model organism, the zebrafish (Danio rerio), using a combined oxalate-pyroantimonate technique. Samples were treated by potassium oxalate and potassium pyroantimonate during two fixation stages and examined using transmission electron microscopy to detect electron dense intracellular calcium. The subcellular distribution of intracellular calcium was characterized in spermatogonium, spermatocyte, spermatid, and spermatozoon stages. The area which is covered by intracellular calcium in different stages was quantified and compared using software. Isolated calcium deposits were mainly detectable in the cytoplasm and the nucleus of the spermatogonium and spermatocyte. In the spermatid, calcium was partially localized in the cytoplasm as isolated deposits. However, most calcium was transformed from isolated deposits into an unbound pool (free calcium) within the nucleus of the spermatid and the spermatozoon. Interestingly, in the spermatozoon, calcium was mainly localized in a form of an unbound pool which was detectable as an electron-dense mass within the nucleus. Also, sporadic calcium deposits were scattered in the midpiece and flagellum. The proportional area which was covered by intracellular calcium increased significantly from early to late stages of spermatogenesis. The extent of the area which was covered by intracellular calcium in the spermatozoon was the highest compared to earlier stages. Calcium deposits were also observed in the somatic cells (Sertoli, myoid, Leydig) of zebrafish testis. The notable changes in the distribution of intracellular calcium of germ cells during different developmental stages of zebrafish spermatogenesis suggest its different homeostasis and physiological functions during the process of male gamete development.