Characterization of human CD4+EOMES+GzmK+ T-cell subsets unveils an uncoupling of suppressive functions from IL-10-producing capacities.

Human CD4+EOMES+ T cells are heterogeneous and contain Th1-cells, Tr1-cells, and CD4+CTL. Tr1- cells and non-classical EOMES+ Th1-cells displayed, respectively, anti- and pro-inflammatory cytokine profiles, but both expressed granzyme-K, produced IFN-γ, and suppressed T-cell proliferation. Diffusion map suggested a progressive CD4+T-cell differentiation from naïve to cytotoxic cells and identified EOMES+Th1-cells as putative Tr1-cell precursors (pre-Tr1).

An Intestinal Th17 Subset is Associated with Inflammation in Crohn's Disease and Activated by Adherent-invasive Escherichia coli.

IFNγ-producing ex-Th17 cells ['Th1/17'] were shown to play a key pathogenic role in experimental colitis and are abundant in the intestine. Here, we identified and characterised a novel, potentially colitogenic subset of Th17 cells in the intestine of patients with Crohn's disease [CD]. Human Th17 cells expressing CCR5 ['pTh17'] co-expressed T-bet and RORC/γt and produced very high levels of IL-17, together with IFN-γ. They had a gene signature of Th17 effector cells and were distinct from established Th1/17 cells. pTh17 cells, but not Th1/17 cells, were associated with intestinal inflammation in CD, and decreased upon successful anti-TNF therapy with infliximab. Conventional CCR5[-]Th17 cells differentiated to pTh17 cells with IL-23 in vitro. Moreover, anti-IL-23 therapy with risankizumab strongly reduced pTh17 cells in the intestine. Importantly, intestinal pTh17 cells were selectively activated by adherent-invasive Escherichia coli [AIEC], but not by a commensal/probiotic E. coli strain. AIEC induced high levels of IL-23 and RANTES from dendritic cells [DC]. Intestinal CCR5+Th1/17 cells responded instead to cytomegalovirus and were reduced in ulcerative colitis [UC], suggesting an unexpected protective role. In conclusion, we identified an IL-23-inducible subset of human intestinal Th17 cells. pTh17 cells produced high levels of pro-inflammatory cytokines, were selectively associated with intestinal inflammation in CD, and responded to CD-associated AIEC, suggesting a key colitogenic role.

The Role of Adhesion Molecules and Extracellular Vesicles in an In Vitro Model of the Blood-Brain Barrier for Metastatic Disease.

Metastatic brain disease (MBD) has seen major advances in clinical management, focal radiation therapy approaches and knowledge of biological factors leading to improved prognosis. Extracellular vesicles (EVs) have been found to play a role in tumor cross-talk with the target organ, contributing to the formation of a premetastatic niche. Human lung and breast cancer cell lines were characterized for adhesion molecule expression and used to evaluate their migration ability in an in vitro model. Conditioned culture media and isolated EVs, characterized by super resolution and electron microscopy, were tested to evaluate their pro-apoptotic properties on human umbilical vein endothelial cells (HUVECs) and human cerebral microvascular endothelial cells (HCMEC/D3) by annexin V binding assay. Our data showed a direct correlation between expression of ICAM1, ICAM2, ß3-integrin and α2-integrin and the ability to firmly adhere to the blood-brain barrier (BBB) model, whereas the same molecules were down-regulated at a later step. Extracellular vesicles released by tumor cell lines were shown to be able to induce apoptosis in HUVEC while brain endothelial cells showed to be more resistant.

Eomesodermin-expressing type 1 regulatory (EOMES+ Tr1)-like T cells: Basic biology and role in immune-mediated diseases.

Type 1 regulatory (Tr1) T cells are currently defined all T cells with regulatory functions that lack FOXP3 expression and produce IL-10. Tr1 cells are heterogeneous, and the different reported properties of Tr1-cell populations have caused some confusion in the field. Moreover, understanding the role of Tr1 cells in immune-mediated diseases has been hampered by the lack of a lineage-defining transcription factor. Several independent studies indicated recently that the transcription factor Eomesodermin (EOMES) could act as a lineage-defining transcription factor in a population of IL-10 and IFN-γ co-producing Tr1-like cells, since EOMES directly induces IFN-γ and cytotoxicity, enhances IL-10, and antagonizes alternative T-cell fates. Here, we review the known properties of EOMES+ Tr1-like cells. They share several key characteristics with other Tr1 cells (i.e., "Tr1-like"), namely high IL-10 production, cytotoxicity, and suppressive capabilities. Notably, they also share some features with FOXP3+ Tregs, like downregulation of IL-7R and CD40L. In addition, they possess several unique, EOMES-dependent features, that is, expression of GzmK and IFN-γ, and downregulation of type-17 cytokines. Published evidence indicates that EOMES+ Tr1-like cells play key roles in graft-versus-host disease, colitis, systemic autoimmunity and in tumors. Thus, EOMES+ Tr1-like cells are key players of the adaptive immune system that are involved in several different immune-mediated diseases.

Deep Phenotyping of T-Cells Derived From the Aneurysm Wall in a Pediatric Case of Subarachnoid Hemorrhage.

Intracranial aneurysms (IAs) are very rare in children, and the characteristics of the T-cells in the IA wall are largely unknown. A comatose 7-years-old child was admitted to our center because of a subarachnoid hemorrhage due to a ruptured giant aneurysm of the right middle cerebral artery. Two days after the aneurysm clipping the patient was fully awake with left hemiparesis. T-cells from the IA wall and from peripheral blood of this patient were analyzed by multi-dimensional flow cytometry. Unbiased analysis, based on the use of FlowSOM clustering and dimensionality reduction technique UMAP, indicated that there was virtually no overlap between circulating and tissue-infiltrating T-cells. Thus, naïve T-cells and canonical memory T-cells were largely restricted to peripheral blood, while CD4-CD8-T-cells were strongly enriched in the IA wall. The unique CD4+, CD8+ and CD4-CD8-T-cell clusters from the IA wall expressed high levels of CCR5, Granzyme B and CD69, displaying thus characteristics of cytotoxic and tissue-resident effector cells. Low Ki67 expression indicated that they were nevertheless in a resting state. Among regulatory T-cell subsets, Eomes+Tr1-like cells were strongly enriched in the IA wall. Finally, analysis of cytokine producing capacities unveiled that the IA wall contained poly-functional T-cells, which expressed predominantly IFN-γ, TNF and IL-2. CD4+T-cells co-expressed also CD40L, and produced some IL-17, GM-CSF and IL-10. This report provides to our knowledge the first detailed characterization of the human T-cell compartment in the IA wall.

Pulmonary Langerhans Cell Histiocytosis and Lymphangioleiomyomatosis Have Circulating Cells With Loss of Heterozygosity of the TSC2 Gene.

BACKGROUND: Lymphangioleiomyomatosis (LAM) and pulmonary Langerhans cell histiocytosis (PLCH) are cystic lung diseases in which a neoplastic cell is thought to be responsible for disease pathogenesis. The neoplastic LAM cell has mutations in the TSC genes, TSC1 or TSC2, whereas the neoplastic PLCH cell may have mutations in several genes (eg, BRAF, NRAS, MAP2K1). These mutations are not specific for PLCH and have been described in multiple cancers. TSC1 or TSC2 mutations and loss of heterozygosity (LOH) have also been described in cancers. RESEARCH QUESTION: Is TSC2 LOH specific to LAM or is it also found in PLCH? STUDY DESIGN AND METHODS: We recruited patients with LAM (n = 53) and healthy volunteers (n = 22) and compared the presence of cells with TSC2 LOH with patients with PLCH (n = 12). Blood and urine samples were collected for analysis. Fluorescence-activated cell sorting (FACS) was used to identify subpopulations of cells from blood and urine samples. We isolated CD45-CD235a-, CD45-CD235a+, and CD45+CD235a- cells from blood after density gradient separation. Cells were screened for TSC2 LOH at five microsatellites markers (ie, kg8, D16S3395, D16S3024, D16S521, D16S291). We obtained four cell subpopulations from urine (ie, CD44v6+CD9+, CD44v6+CD9-, CD44v6-CD9+, CD44v6-CD9-). RESULTS: Using FACS, cells were isolated from blood and urine from patients with PLCH that showed TSC2 LOH. Healthy volunteers did not have cells with TSC2 LOH. As a control, cells isolated from blood and urine from patients with LAM gave results similar to those reported previously. These data show that TSC2 LOH is found in patients with cystic lung diseases with potential neoplastic characteristics, and in patients with cancer. INTERPRETATION: The presence of TSC2 LOH in circulating cells is not specific for LAM. The data suggest that chromosomal abnormalities affecting the TSC2 gene are found in other diseases associated with cells having cancer-like neoplastic cells.

Ex vivo microRNA and gene expression profiling of human Tr1-like cells suggests a role for miR-92a and -125a in the regulation of EOMES and IL-10R.

Ex vivo gene expression and miRNA profiling of Eomes+ Tr1-like cells suggested that they represent a differentiation stage that is intermediate between Th1-cells and cytotoxic CD4+ T-cells. Several microRNAs were downregulated in Eomes+ Tr1-like cells that might inhibit Tr1-cell differentiation. In particular, miR-92a targeted Eomes, while miR-125a inhibited IFN-g and IL-10R expression.

MR-Spectroscopy and Survival in Mice with High Grade Glioma Undergoing Unrestricted Ketogenic Diet.

Glioblastoma multiforme (GBM) is considered the most malignant form of primary brain tumor. Despite multimodal treatment, prognosis remains poor. Ketogenic diet (KD) has been suggested for the treatment of GBM. In this study, the syngenic, orthotopic GL261 mouse glioma model was used to evaluate the effects of KD on the metabolic responses of the tumor using 7T magnetic resonance imaging/spectroscopy. GL261 cells were injected into the caudate nucleus of mice. Following implantation, animals were fed with standard chow or underwent a KD. 18 days after initiating the diet, mice fed with KD displayed significantly higher plasmatic levels of ketone bodies and survived longer than those fed with the standard diet. Decreased concentrations of gamma-aminobutyric acid, N-Acetyl-Aspartate and N-acetylaspartylglutamate were found in tumor tissue after 9 days into the KD, while a huge increase in beta-hydroxybutyrate (bHB) was detected in tumor tissue as compared to normal brain. The accumulation of bHB in the tumor tissue in mice undergoing the KD, may suggest either elevated uptake/release of bHB by tumor cells, or the inability of tumor cells in this context to use it for mitochondrial metabolism.

Adipose-Derived Stem Cells from Fat Tissue of Breast Cancer Microenvironment Present Altered Adipogenic Differentiation Capabilities.

Mesenchymal stem cells (MSCs) are multipotent cells able to differentiate into multiple cell types, including adipocytes, osteoblasts, and chondrocytes. The role of adipose-derived stem cells (ADSCs) in cancers is significantly relevant. They seem to be involved in the promotion of tumour development and progression and relapse processes. For this reason, investigating the effects of breast cancer microenvironment on ADSCs is of high importance in order to understand the relationship between tumour cells and the surrounding stromal cells. With the current study, we aimed to investigate the specific characteristics of human ADSCs isolated from the adipose tissue of breast tumour patients. We compared ADSCs obtained from periumbilical fat (PF) of controls with ADSCs obtained from adipose tissue of breast cancer- (BC-) bearing patients. We analysed the surface antigens and the adipogenic differentiation ability of both ADSC populations. C/EBPδ expression was increased in PF and BC ADSCs induced to differentiate compared to the control while PPARγ and FABP4 expressions were enhanced only in PF ADSCs. Conversely, adiponectin expression was reduced in PF-differentiated ADSCs while it was slightly increased in differentiated BC ADSCs. By means of Oil Red O staining, we further observed an impaired differentiation capability of BC ADSCs. To investigate this aspect more in depth, we evaluated the effect of selective PPARγ activation and nutritional supplementation on the differentiation efficiency of BC ADSCs, noting that it was only with a strong differentiation stimuli that the process took place. Furthermore, we observed no response in BC ADSCs to the PPARγ inhibitor T0070907, showing an impaired activation of this receptor in adipose cells surrounding the breast cancer microenvironment. In conclusion, our study shows an impaired adipogenic differentiation capability in BC ADSCs. This suggests that the tumour microenvironment plays a key role in the modulation of the adipose microenvironment located in the surrounding tissue.

Eomesodermin controls a unique differentiation program in human IL-10 and IFN-γ coproducing regulatory T cells.

Whether human IL-10-producing regulatory T cells ("Tr1") represent a distinct differentiation lineage or an unstable activation stage remains a key unsolved issue. Here, we report that Eomesodermin (Eomes) acted as a lineage-defining transcription factor in human IFN-γ/IL-10 coproducing Tr1-like cells. In vivo occurring Tr1-like cells expressed Eomes, and were clearly distinct from all other CD4+ T-cell subsets, including conventional cytotoxic CD4+ T cells. They expressed Granzyme (Gzm) K, but had lost CD40L and IL-7R expression. Eomes antagonized the Th17 fate, and directly controlled IFN-γ and GzmK expression. However, Eomes binding to the IL-10 promoter was not detectable in human CD4+ T cells, presumably because critical Tbox binding sites of the mouse were not conserved. A precommitment to a Tr1-like fate, i.e. concominant induction of Eomes, GzmK, and IFN-γ, was promoted by IL-4 and IL-12-secreting myeloid dendritic cells. Consistently, Th1 effector memory cells contained precommitted Eomes+ GzmK+ T cells. Stimulation with T-cell receptor (TCR) agonists and IL-27 promoted the generation of Tr1-like effector cells by inducing switching from CD40L to IL-10. Importantly, CD4+ Eomes+ T-cell subsets were present in lymphoid and nonlymphoid tissues, and their frequencies varied systemically in patients with inflammatory bowel disease and graft-versus-host disease. We propose that Eomes+ Tr1-like cells are effector cells of a unique GzmK-expressing CD4+ T-cell subset.

Author Correction: Probing spermiogenesis: a digital strategy for mouse acrosome classification.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Intestinal IFN-γ-producing type 1 regulatory T cells coexpress CCR5 and programmed cell death protein 1 and downregulate IL-10 in the inflamed guts of patients with inflammatory bowel disease.

BACKGROUND: IL-10 is an anti-inflammatory cytokine required for intestinal immune homeostasis. It mediates suppression of T-cell responses by type 1 regulatory T (TR1) cells but is also produced by CD25+ regulatory T (Treg) cells. OBJECTIVE: We aimed to identify and characterize human intestinal TR1 cells and to investigate whether they are a relevant cellular source of IL-10 in patients with inflammatory bowel diseases (IBDs). METHODS: CD4+ T cells isolated from the intestinal lamina propria of human subjects and mice were analyzed for phenotype, cytokine production, and suppressive capacities. Intracellular IL-10 expression by CD4+ T-cell subsets in the inflamed guts of patients with IBD (Crohn disease or ulcerative colitis) was compared with that in cells from noninflamed control subjects. Finally, the effects of proinflammatory cytokines on T-cell IL-10 expression were analyzed, and IL-1ß and IL-23 responsiveness was assessed. RESULTS: Intestinal TR1 cells could be identified by coexpression of CCR5 and programmed cell death protein 1 (PD-1) in human subjects and mice. CCR5+PD-1+ TR1 cells expressed IFN-γ and efficiently suppressed T-cell proliferation and transfer colitis. Intestinal IFN-γ+ TR1 cells, but not IL-7 receptor-positive TH cells or CD25+ Treg cells, showed lower IL-10 expression in patients with IBDs. TR1 cells were responsive to IL-23, and IFN-γ+ TR1 cells downregulated IL-10 with IL-1ß and IL-23. Conversely, CD25+ Treg cells expressed higher levels of IL-1 receptor but showed stable IL-10 expression. CONCLUSIONS: We provide the first ex vivo characterization of human intestinal TR1 cells. Selective downregulation of IL-10 by IFN-γ+ TR1 cells in response to proinflammatory cytokines is likely to drive excessive intestinal inflammation in patients with IBDs.

In vitro antineoplastic effects of brivaracetam and lacosamide on human glioma cells.

BACKGROUND: Epilepsy is a frequent symptom in patients with glioma. Although treatment with antiepileptic drugs is generally effective in controlling seizures, drug-resistant patients are not uncommon. Multidrug resistance proteins (MRPs) and P-gp are over-represented in brain tissue of patients with drug-resistant epilepsy, suggesting their involvement in the clearance of antiepileptic medications. In addition to their anticonvulsant action, some drugs have been documented for cytotoxic effects. Aim of this study was to evaluate possible in vitro cytotoxic effects of two new-generation antiepileptic drugs on a human glioma cell line U87MG. METHODS: Cytotoxicity of brivaracetam and lacosamide was tested on U87MG, SW1783 and T98G by MTS assay. Expression of chemoresistance molecules was evaluated using flow cytometry in U87MG and human umbilical vein endothelial cells (HUVECs). To investigate the putative anti-proliferative effect, apoptosis assay, microRNA expression profile and study of cell cycle were performed. RESULTS: Brivaracetam and lacosamide showed a dose-dependent cytotoxic and anti-migratory effects. Cytotoxicity was not related to apoptosis. The exposure of glioma cells to brivaracetam and lacosamide resulted in the modulation of several microRNAs; particularly, the effect of miR-195-5p modulation seemed to affect cell cycle, while miR-107 seemed to be implicated in the inhibition of cells migration. Moreover, brivaracetam and lacosamide treatment did not modulate the expression of chemoresistance-related molecules MRPs1-3-5, GSTπ, P-gp on U87MG and HUVECs. CONCLUSION: Based on antineoplastic effect of brivaracetam and lacosamide on glioma cells, we assume that patients with glioma could benefit by the treatment with these two molecules, in addition to standard therapeutic options.

Probing spermiogenesis: a digital strategy for mouse acrosome classification.

Classification of morphological features in biological samples is usually performed by a trained eye but the increasing amount of available digital images calls for semi-automatic classification techniques. Here we explore this possibility in the context of acrosome morphological analysis during spermiogenesis. Our method combines feature extraction from three dimensional reconstruction of confocal images with principal component analysis and machine learning. The method could be particularly useful in cases where the amount of data does not allow for a direct inspection by trained eye.

Circulating T regulatory cells migration and phenotype in glioblastoma patients: an in vitro study.

Glioblastoma multiforme (GBM) is the most aggressive primary human brain tumor. The relatively high amount of T regulatory lymphocytes present in the tumor, contributes to the establishment of an immunosuppressive microenvironment. Samples of peripheral blood were collected from GBM patients and healthy controls and a purified population of Treg (CD4(+)/CD25(bright)) was isolated using flow cytometric cell sorting. Treg migrating capacities toward human glioma cell line conditioned medium were evaluated through an in vitro migration test. Our data show that supernatants collected from GBM cell lines were more attractant to Treg when compared to complete standard medium. The addition of an anti-CCL2 antibody to conditioned medium decreased conditioned medium-depending Treg migration, suggesting that CCL2 (also known as Monocyte Chemoattractant Protein, MCP-1) is implicated in the process. The number of circulating CD4(+)/µL or Treg/µL was similar in GBM patients and controls. Specific Treg markers (FOXP3; CD127; Helios; GITR; CTLA4; CD95; CCR2, CCR4; CCR7) were screened in peripheral blood and no differences could be detected between the two populations. These data confirm that the tumor microenvironment is attractive to Treg, which tend to migrate toward the tumor region changing the immunological response. Though we provide evidence that CCL2 is implicated in Treg migration, other factors are needed as well to provide such effect.

Chromosomal speciation in mice: a cytogenetic analysis of recombination.

Within species, populations differing by chromosomal rearrangements ("chromosomal races") may become reproductively isolated in association with reduced hybrid fertility due to meiotic aberrations. Speciation is also possible if hybridizing chromosomal races accumulate genetic differences because of reduced meiotic recombination in the heterozygous configuration in hybrids. Here, we examine recombination in pure races and hybrids within a model system for chromosomal speciation: the hybridization of the Poschiavo (CHPO) and Upper Valtellina (IUVA) chromosomal races of house mouse in Upper Valtellina, Italy. These races differ by Robertsonian fusions/whole-arm reciprocal translocations, such that hybrids produce a pentavalent meiotic configuration. We determined the number and position of the recombination points (using an antibody against the MutL homolog 1 [MLH1] protein) on synaptonemal complexes at pachytene in laboratory-reared CHPO, IUVA, and hybrid males, analyzing at least 112 spermatocytes per karyotypic group, up to a total of 534 spermatocytes. The mean ± standard deviation numbers of MLH1 foci per spermatocyte were 22.2 ± 3.2, 20.1 ± 2.9, 20.7 ± 2.3, and 21.9 ± 2.9 for CHPO, IUVA, CHPO × IUVA, and IUVA × CHPO, respectively. Altogether, 10,146 chromosome arms were examined, allowing multiple comparisons. Overall, recombination events were more frequently distal than proximal or interstitial. The average number of proximal MLH1 foci per chromosome arm decreased going from telocentric to metacentric bivalents to pentavalents (when present), which (together with other factors) influenced the average number of MLH1 foci per cell between CHPO, IUVA, and hybrid mice. The low frequency of proximal recombination in pentavalents of CHPO-IUVA hybrids may promote reproductive isolation between the CHPO and IUVA races, when coupled with reduced hybrid unfitness.

The frequency of heterologous synapsis increases with aging in Robertsonian heterozygous male mice.

The house mouse is characterised by highly variable chromosome number due to the presence of Robertsonian (Rb) chromosomes. During meiosis in Rb heterozygotes, intricated chromosomal figures are produced, and many unsynapsed regions are present during the first prophase, triggering a meiotic silencing of unsynapsed chromatin (MSUC) in a similar mode to the sex chromosome inactivation. The presence of unsynapsed chromosome regions is associated with impaired spermatogenesis. Interestingly, in male mice carrying multiple Rb trivalents, the frequency of germ cell death, defective tubules, and altered sperm morphology decreases during aging. Here, we studied whether synapsis of trivalent chromosomes and MSUC are involved in this improvement. By immunocytochemistry, we analysed the frequency of unsynapsed chromosomes and of those positive to γH2AX (a marker of MSUC) labelling in spermatocytes of 3-, 5- and 7-month-old Rb heterozygotes. With aging, we observed a decrease of the frequency of unsynapsed chromosomes, of spermatocytes bearing them and of trivalents carrying γH2AX-negative unsynapsed regions. Our quantitative results show that both synapsis and MSUC processes are better accomplished during male aging, partially accounting for the improvement of spermatogenesis.

A high incidence of meiotic silencing of unsynapsed chromatin is not associated with substantial pachytene loss in heterozygous male mice carrying multiple simple robertsonian translocations.

Meiosis is a complex type of cell division that involves homologous chromosome pairing, synapsis, recombination, and segregation. When any of these processes is altered, cellular checkpoints arrest meiosis progression and induce cell elimination. Meiotic impairment is particularly frequent in organisms bearing chromosomal translocations. When chromosomal translocations appear in heterozygosis, the chromosomes involved may not correctly complete synapsis, recombination, and/or segregation, thus promoting the activation of checkpoints that lead to the death of the meiocytes. In mammals and other organisms, the unsynapsed chromosomal regions are subject to a process called meiotic silencing of unsynapsed chromatin (MSUC). Different degrees of asynapsis could contribute to disturb the normal loading of MSUC proteins, interfering with autosome and sex chromosome gene expression and triggering a massive pachytene cell death. We report that in mice that are heterozygous for eight multiple simple Robertsonian translocations, most pachytene spermatocytes bear trivalents with unsynapsed regions that incorporate, in a stage-dependent manner, proteins involved in MSUC (e.g., gammaH2AX, ATR, ubiquitinated-H2A, SUMO-1, and XMR). These spermatocytes have a correct MSUC response and are not eliminated during pachytene and most of them proceed into diplotene. However, we found a high incidence of apoptotic spermatocytes at the metaphase stage. These results suggest that in Robertsonian heterozygous mice synapsis defects on most pachytene cells do not trigger a prophase-I checkpoint. Instead, meiotic impairment seems to mainly rely on the action of a checkpoint acting at the metaphase stage. We propose that a low stringency of the pachytene checkpoint could help to increase the chances that spermatocytes with synaptic defects will complete meiotic divisions and differentiate into viable gametes. This scenario, despite a reduction of fertility, allows the spreading of Robertsonian translocations, explaining the multitude of natural Robertsonian populations described in the mouse.

Identification, isolation, and RT-PCR analysis of single stage-specific spermatogenetic cells obtained from portions of seminiferous tubules classified by transillumination microscopy.

The protocol here described allows the analysis of gene expression in single specific mouse spermatogenetic cells. Germ cells were singularly isolated by microdissection of portions of seminiferous tubules classified, based on their transillumination pattern, into four distinct zones along their length. Single portions of a seminiferous tubule, corresponding to specific zones, were mechanically disaggregated into single cells that were (1) identified as spermatogonia, spermatocytes, round or elongated spermatids, (2) isolated using a micromanipulator, and (3) singularly transferred into a test tube for retro-transcription PCR analysis. On each single isolated cell, we have determined the quantitative profile of expression of Gapdh, an endogenous housekeeping gene known to be expressed throughout spermatogenesis. The protocol described allows an accurate analysis of the temporal and quantitative profile of gene expression throughout the whole male gamete differentiation process which so far has mainly been performed on enriched population of cells.