Stochastic tractography-based glioblastoma invasion modelling: current stage of Development and future prospects | Martin Kukrál
Stochastic Tractography-based GBM Invasion Model
Authors: Martin Kukrál (1)
Supervisor: Irena Holečková (1)
(1) Department of Neurosurgery, Faculty of Medicine in Pilsen, Charles University and University
Hospital, Pilsen
State-of-the-Art: Glioblastoma (GBM) is the most common and aggressive primary malignant
brain tumor, with the average survival time being only about 15 months. Surgical resection is the
primary therapeutic option for GBM, often followed by adjuvant radiotherapy and chemother
apy. Nevertheless, determining the optimal treatment extent is extremely difficult, as magnetic
resonance imaging (MRI) is unable to detect the full extent of GBM invasion. Therefore, mathe
matical models are used to provide insight into GBM growth patterns below MRI detectability.
The most prominent approaches utilize partial differential equations (PDEs) to describe the in
vasion as a reaction-diffusion system on a 3D voxel grid. These models can be characterized by
various levels of incorporated biological knowledge and MRI data.
Objective: The model aims to provide clinicians with probabilistic patient-specific GBM inva
sion simulations to support surgical planning and targeted radiotherapy. Additionally, the model
should facilitate the analysis of invasion patterns related to the GBM tumor mass localization in
the brain connectome.
Material and Methods: The proposed GBM invasion model builds on the current state-of-the
art approaches in mathematical neuro-oncology by reformulating the reaction-diffusion PDEs
on an undirected graph constructed from structural MRI data. The graph’s edges are weighted
using patient-specific tractography obtained via constrained spherical deconvolution (CSD), en
abling the anisotropic invasion along white matter tracts observed in GBM. The diffusion term
includes further modulation, reflecting changes in GBM growth behavior related to tumor cell
density. The proliferation is expressed using deterministic and stochastic components, allowing
the evaluation of invasion uncertainty introduced by innate biological variability. The model con
tains various parameters that influence the simulation.
Results & Discussion: The simulations indicate that the proposed tractography-based model
expresses the typical finger-like extrusions from the main GBM tumor mass into the surround
ing brain tissue. Although few anisotropic approaches already exist, they do not exhibit nearly
as strong preferential spread along white matter tracts, possibly due to the integration of infor
mation with very limited accuracy about true tract density, such as the diffusion tensor imaging
(DTI), as well as the 3D voxel grid data structure for computations. Planned combination of the
proposed model with machine learning algorithms represents a unique hybrid explainable ar
tificial intelligence (XAI) approach, attempting to combine the interpretability of the baseline
model with modern data-driven methods for improved performance.
Conclusion: Representing the discrete simulation space as a graph with directly integrated pa
tient-specific brain connectomes represents a conceptually novel approach to GBM invasion
modelling. Future work will focus on quantitative validation using clinical cases and integration
with machine learning.
Identification and characterization of Distinct hematopoietic stem and progenitor cells in health and Disease | Safaa Andarawi
Developing a Computational Tool for HSC annotation
Authors: Safaa Andarawi (1,2)
Supervisor: Meritxell Alberich Jordà (2)
(1) Biomedical Center, Faculty of Medicine in Pilsen, Charles University, Czech Republic
(2) Institute of Molecular Genetics of the Czech Academy of Sciences
State-of-the-Art: Hematopoietic stem cells (HSCs) are essential for lifelong blood formation
and are central in rapid regeneration of immune cells following stress. However, defining HSC
states at the molecular level remains difficult due to their heterogeneity and gradual transitions
toward progenitor cells. Single-cell RNA sequencing (scRNA-seq) provides detailed resolution
of these populations but introduces challenges, including overlapping transcriptional profiles,
batch effects, and inconsistent manual annotations. There is a need for a standardized tool that
allows the identification of distinct HSC subsets in a reliable manner.
Objective: To develop robust and reproducible package for accurate annotation of hematopoi
etic stem cells using scRNA-seq data. The framework is designed to move beyond marker-based
labeling by integrating gene program, regulatory network analysis, and trajectory context to
achieve biologically informed cell state.
Material and Methods: Public scRNA-seq datasets of hematopoietic stem and progenitor cells
will be processed using standardized quality control, normalization, and batch correction. Di
mensionality reduction and clustering, followed by identification of cluster-specific gene mod
ules and trajectory inference to define developmental relationships. These integrated features
will be used to develop a supervised classification model for accurate HSC annotation. Model
performance will be evaluated through cross-validation and independent external datasets.
Experimental validation will include FACS isolation and colony-forming assays to confirm func
tional HSC identity.
Results & Discussion: Preliminary analyses suggest that HSC subclusters identified across in
dependent public datasets may share conserved gene modules and gene regulatory network
(GRN) signatures. These shared transcriptional patterns appear to persist despite dataset-spe
cific variation and batch effects, indicating potential biological relevance. Furthermore, several
gene modules and regulatory drivers show possible correlations with trajectory-defined cell
states, implying they may reflect dynamic transitions along the hematopoietic hierarchy. Ongo
ing analyses aim to confirm the consistency and developmental significance of these observed
patterns.
Conclusion: This project establishes a comprehensive and reproducible framework for accu
rate annotation of hematopoietic stem cell states using data. This framework provides a reliable
tool for consistent HSC identification and facilitates deeper insight into hematopoietic develop
ment and functional heterogeneity.
Contribution of cumulus cells to maturation of mtDnA-deficient oocytes | Kateřina Grygarová
Cumulus support in mtDNA-deficient oocytes
Authors: Kateřina Grygarová (1); Filip Tirpák (1), Jan Nevoral (1,2)
Supervisor: Jan Nevoral (1,2)
(1) Biomedical Center, Faculty of Medicine in Pilsen, Charles University
(2) Department of Histology and Embryology, Faculty of Medicine in Pilsen, Charles University
State-of-the-Art: Mitochondrial DNA (mtDNA) is maternally inherited and massively ampli
fied during oocyte growth, reaching >100,000 copies in mature oocytes. This expansion relies
on nuclear regulators, particularly TFAM, which maintains mtDNA and supports mitochondrial
gene expression. Although mtDNA copy number was long considered a proxy for oocyte quality,
mitochondrial silencing and inconsistent predictive value have challenged this view. Notably,
TFAM-deficient oocytes can retain developmental competence despite severe mtDNA deple
tion. During maturation, however, oocytes depend on cumulus cells for metabolic substrates.
In vitro denudation removes this input and may unmask mitochondrial limitations. How mtDNA
depletion affects cumulus-free maturation competence remains insufficiently defined.
Objective: To determine how oocyte-specific Tfam depletion affects oocyte maturation compe
tence and chromosomal stability.
Material and Methods: We used an oocyte-specific Tfam conditional knockout mice (TfamloxP/
loxP; Zp3-Cre) to produce Tfamnull oocytes with depleted mtDNA. Female mice (8–24 weeks)
were hormonally stimulated by i.p. administration of PMSG. Cumulus–oocyte complexes were
collected 44–48 h later, and cumulus cells were removed after 90 min of cultivation by mechan
ical denudation using a fine glass capillary. Denuded oocytes were subjected to in vitro matura
tion at 37 °C in 5% CO₂ for 14–16 h. Maturation was assessed based on first polar body extrusion
and spindle morphology. Oocytes were immunostained with anti-TFAM and anti–acetyl-α-tubu
lin antibodies and visualized by confocal microscopy (Nikon, Japan). Animal experiments were
conducted in accordance with Act No. 246/1992 Coll. (Project No. MSMT-33798/2021-4).
Results & Discussion: Our observations showed that TFAM loss caused a marked reduction in
oocyte mtDNA content and significantly impaired in vitro maturation outcomes (p = 0.0062). In
deed, fewer Tfamnull oocytes matured compared to the wild-type oocytes. Confocal microscopy
and image analysis revealed an increased frequency of spindle abnormalities in Tfamnull oo
cytes. These findings are consistent with a model in which cumulus-derived metabolic support
can buffer compromised oocyte mitochondrial function in intact complexes, whereas denuda
tion during in vitro handling reveals a stronger dependence on mtDNA-supported mitochondrial
capacity for normal meiotic progression.
Conclusion: In denuded oocytes, TFAM loss-driven mtDNA depletion reduces in vitro matura
tion rate and increases the incidence of spindle abnormalities. These findings underscore the
essential role of cumulus cells in oocyte maturation, particularly for oocytes suffering from in
sufficiency of mtDNA content.
Funding: Supported by the Cooperatio Programme (research area MED/DIAG and MED/IMMU),
and SVV 260 773
Feasibility of AtAc-seq analysis for identifying regulatory regions in cD123-high and cD 123-low Aml samples | Eliška Jandová
ATAC-seq analysis of CD123-high/low AML samples
Authors: Eliška Jandová (1,2), Zia Ullah (1,2), Pavel Ostašov (1,2), Valentina S Caputo (3), Monika Holu
bová (2,4)
Supervisor: Monika Holubová (2,4)
(1) Department of Histology and Embryology, Faculty of Medicine in Pilsen, Charles University
(2) Biomedical Center, Faculty of Medicine in Pilsen, Charles University (3) Cancer Biology and
Therapy Laboratory, School of Applied Sciences, London South Bank University, London, UK
(4) Department of Haematology and Oncology, Faculty of Medicine in Pilsen, Charles University
and University Hospital, Pilsen
State-of-the-Art: CD123 appears to be a promising novel therapeutic target in acute myeloid
leukaemia (AML), due to its high expression on AML blasts and low expression on healthy cells.
Several immunotherapeutic strategies targeting CD123 are under development, however, re
sistance in the form of antigen escape is still observed. The mechanisms responsible for CD123
antigen escape are still not well understood, though the ongoing research indicates a substan
tial role of epigenetic and transcriptional regulation. The exact regulatory molecules responsible
for changes in CD123 expression need to be determined. Identifying the molecules responsible
for antigen escape could help establish combined therapy for AML patients and ensure efficacy
of anti-CD123 immunotherapy.
Objective: The study aimed to investigate the feasibility of using ATAC-seq data from AML pa
tients to identify regulatory regions of CD123 gene, employing patient samples that exhibit low
and high CD123 expression levels.
Material and Methods: The relative CD123 expression normalized to GAPDH as the endoge
nous control was established in AML patients using qPCR. Based on the relative expression lev
els, patients were stratified into CD123-high and CD123-low cohorts. AML blasts from 4 CD123
high and 4 CD123-low samples were isolated via FACS using backbone markers CD117, and CD45,
in combination with CD34 (n=4), CD33 (n=2) or CD11b (n=2). The samples were processed using
ATAC-seq kit (Diagenode, USA) and sequenced on NovaSeq 6000 (paired-end, 150 bp) (Illumi
na, USA). A preliminary analysis was conducted on 500 000 reads per sample. The reads were
filtered (MAPQ≥30, paired end), and peaks called via MACS2. Differential accessibility was as
sessed using t-tests on CPM-normalized counts with Benjamini-Hochberg correction.
Results & Discussion: A median of 405K cells was sorted (range 200K-500K), yielding a media
of 65K cells (range 40K-115K) post centrifugation (500xg, 10 min, 4°C). ATAC-seq libraries were
prepared from 50K cells per sample (except the one with 40K cells only). Sequencing generated
~25M reads/sample. Sample 8 had 12M due to library quality issues. QC confirmed high-qual
ity data free of adapter and primer contamination. Preliminary analysis of 500K reads/sample
identified 2,477 consensus peaks (43 on the X chromosome), which were unevenly distributed
among the samples, indicating the presence of different regulatory mechanisms. Initial analysis
did not confirm a trend in the locations or numbers of peaks depending on CD123 expression.
However, deeper analysis is needed to determine the CD123-related region.
Conclusion: Preliminary results confirm that combination of cell sorting with AML-specific
immunophenotype is feasible to detect regulatory regions. Despite 13-23% cell loss during
processing, this approach produces high-quality data. However, higher sequencing depth is re
quired for non-essential gene loci
Funding: The study was supported by the grant of the Ministry of Health of the Czech Republic –
Conceptual Development of Research Organization (Faculty Hospital in Pilsen—FNPl, 00669806)
and by Charles University Research Project No SVV 260 651.
Reduced mitochondrial DnA content in the oocyte and the risk for neurodegenerative diseases in offspring | Věra Vtípilová
Maternal mtDNA and neurodegeneration
Authors: Věra Vtípilová (1,2), Daniel Follprecht (3), Patrícia Karkušová (1,4), Jan Cendelín (4), Michaela
Kohoutová (5), Jana Tůmová (5), Eva Kralovcová (1), Jan Nevoral (1,2)
Supervisor: Jan Nevoral (1)
(1) Biomedical Center, Faculty of Medicine in Pilsen, Charles University, Czech Republic
(2) Department of Histology and Embryology, Faculty of Medicine in Pilsen, Charles University,
Czech Republic (3) Department of Sports Medicine and Active Health Sciences, Faculty of Med
icine in Pilsen, Charles University, Czech Republic (4) Department of Pathological Physiology,
Faculty of Medicine in Pilsen, Charles University, Czech Republic (5) Department of Physiology,
Faculty of Medicine in Pilsen, Charles University, Czech Republic
State-of-the-Art: Mitochondrial transcription factor A (TFAM) is a key regulator of mtDNA rep
lication, transcription, and packaging. Its levels naturally decline with age, leading to impaired
mitochondrial respiration, increased oxidative stress, and cellular senescence. In oocytes, TFAM
is essential for maintaining the mitochondrial pool required for early development. While a sig
nificant reduction in mtDNA content (due to TFAM depletion) can be partially compensated dur
ing embryogenesis, the long-term consequences for offspring remain unclear. It is hypothesized
that impaired mitochondrial quantity in oocytes increases susceptibility to neurodegeneration
in offspring.
Objective: The project aims to investigate how oocyte-specific TFAM depletion and subsequent
mtDNA reduction affect health of offspring, via a) testing behavior and cognitive functions, b)
measurement of mitochondrial respiration, c) assessment of hippocampus histology, and d) ge
netic evaluation of mtDNA.
Material and Methods: We use a transgenic mouse model with oocyte-specific inactivation
of the Tfam gene. Mutant offspring and wild-type control animals in the age of twelve months
undergo a battery of behavioral experiments (e.g., open field, Morris water maze) to assess
cognitive performance. Brain tissue is subsequently collected for detailed analyses. We perform
histological staining to detect neurodegenerative markers, qPCR to quantify mtDNA copy num
ber, and oxygraphy to evaluate mitochondrial respiratory capacity in the cortex. This approach
allows us to correlate maternal mitochondrial deficiency with the functional and structural phe
notype of the offspring‘s brain. All animal experiments were conducted in accordance with Act
No. 246/1992 Coll. (Project No. MSMT-33798/2021-4).
Results & Discussion: Contrary to our hypothesis, data shows no significant differences in cog
nitive tests, hippocampal mtDNA content, or mitochondrial respiration of brain cortex between
wild-type and mutant offspring. Correlation analysis shows relationship between oxygen respi
ration in cortex and failure rate in Morris water maze. These findings suggest the presence of ro
bust compensatory mechanisms during embryogenesis that stabilizes mitochondrial functions
in key brain regions despite maternal deficiency.
Conclusion: Maternal TFAM deficiency does not result in immediate mitochondrial or function
al impairment in the offspring brain, suggesting effective developmental compensation. Ongo
ing mtDNA sequencing and histology analyses aim to clarify the underlying mechanisms.
Funding: Supported by the Cooperatio Programme (research area MED/DIAG and MED/IMMU),
and SVV 260 773.
Differences in astrocyte and microglia activity across cerebellar regions in lurcher and purkinje cell degeneration mouse models | Olena Yakushko
Neuroglial activity in cerebellar degeneration
Authors: Olena Yakushko (1), Jan Cendelín (2), Dana Jelínková (2), Štěpán Kápl (2), Jan Tůma (2), Zden
ka Purkartová (2), Yaroslav Kolinko (1)
Supervisor: Yaroslav Kolinko (1)
(1) Department of Histology and Embryology, Faculty of Medicine in Pilsen, Charles University,
Czech Republic
(2) Department of Pathological Physiology, Faculty of Medicine in Pilsen, Charles University,
Czech Republic
State-of-the-Art: Modern research indicates that during neurodegeneration, astrocytes and
microglia undergo functional reprogramming characterized by the activation of protective and
adaptive mechanisms to maintain tissue homeostasis. Clarifying the activity levels of these glial
cells and correlating their layer-specific responses in cerebellar neurodegenerative disorders
may deepen our understanding of the mechanisms driving cerebellar degeneration and facil
itate the development of targeted therapeutic strategies that support long term recovery and
improve patients’ quality of life.
Objective: The present study aimed to compare astrocyte and microglial activation across dis
tinct cerebellar compartments of B6CBA and B6.BR mouse strains with neurodegeneration.
Material and Methods: Brains from B6CBA Lurcher and B6.BR Purkinje cell degeneration (pcd)
mice with clinical signs of cerebellar degeneration, along with strain matched healthy controls (n
= 6–10 per group), were collected and processed for immunofluorescent labeling using anti-Iba1
antibodies to identify microglia and anti-GFAP antibodies as a marker of astrocytes. To evalu
ate microglial and astrocyte activation, the average staining intensity for Iba1 and GFAP was
quantified in the cerebellum (individual cortical layers, white matter, and cerebellar nuclei) by
measuring signal intensity in each region and comparing it to wild type littermate controls using
software Fiji/ImageJ. Statistical analyses were performed using permutation based ANOVA fol
lowed by post hoc tests with Benjamini–Hochberg correction.
Results & Discussion: GFAP staining intensity was increased in both Lurcher and pcd mice com
pared to healthy controls, indicating astrocyte activation across all examined cerebellar regions.
No differences were observed between the wild type mice strains in any cerebellar compart
ment except the white matter, where the B6CBA mice showed higher signal intensity. Lurcher
mutant exhibited higher GFAP staining than pcd mutant mice across all examined cerebellar
regions except the nuclei, where no significant difference was observed. Iba1 staining intensi
ty was increased in the molecular layer in both mutant groups, as well as in the white matter
of Lurcher mice and the nuclei of pcd mice, compared to their respective healthy controls. No
strain related differences in Iba1 were detected.
Conclusion: The obtained results demonstrated distinct patterns of glial activation in the two
neurodegeneration models. Lurcher mice showed stronger GFAP responses than pcd mice. De
spite regional differences in Iba1 staining intensity in mutant, no strain related effects were
observed in wild type mice.
Funding: The work was supported by the Cooperatio Program, research areas MED/DIAG and
NEUR.
the effect of neuropeptides b and w on cytosolic ca2+ Dynamics in beta cells in Acute mouse pancreatic tissue slices | Tomas Chmelir
NPB/W Effect on β-Cell Ca²⁺ Oscillations
Authors: Tomas Chmelir (1), Xiao Li (3), Hifza Ahmed (3), Shashank Pandey (2), Dagmar Stepanova
Jarkovska (1), Magdalena Chottova Dvorakova (1)
Supervisor: Marjan Slak Rupnik (3)
(1) Department of Physiology, Faculty of Medicine in Pilsen, Charles University (2) Department
of Pharmacology and Toxicology, Faculty of Medicine in Pilsen, Charles University (3) Institute
of Physiology, Center for Physiology and Pharmacology, Medical University of Vienna, Austria
State-of-the-Art: Neuropeptides B (NPB) and W (NPW) are endogenous ligands of the Gi pro
tein-coupled receptors NPBWR1 and NPBWR2 and participate in the central regulation of en
ergy homeostasis. However, their direct role in pancreatic islet physiology and β-cell signaling
remains largely unexplored. Calcium signaling is a key determinant of β-cell excitability and in
sulin secretion, and subtle modulation of intracellular Ca²⁺ dynamics may critically influence
endocrine output. Acute pancreatic tissue slices preserve native cytoarchitecture and paracrine
interactions, providing a physiologically relevant platform for studying β-cell signaling in situ.
Objective: The aim of this study was to determine how NPB and NPW affect cytosolic Ca²⁺
dynamics in pancreatic β cells and to explore the involvement of intracellular Ca²⁺ stores and
cAMP-related signaling.
Material and Methods: Experiments were performed on acute pancreatic tissue slices (130 µm
thick) prepared from C57BL/6J mice (8–11 weeks old, n = 7). Tissue architecture was preserved
by intraductal 1.9 % agarose injection followed by vibratome sectioning. Slices were loaded with
a fluorescent Ca²⁺ indicator Calbryte 520 AM and imaged using confocal microscopy at 37 °C. β
cells were identified based on their characteristic Ca2+ response to stimulation with 8 mM glu
cose. To selectively stimulate oscillatory Ca²⁺ release via IP3 receptors, 100 nM acetylcholine was
added to this solution. Either NPB or NPW was applied in 5 increasing concentrations ranging
from 1 pM to 10 nM, and Ca²⁺ oscillation frequency, duration, and area under the curve were
analyzed using a custom Python-based analysis pipeline.
Results & Discussion: Data indicate that NPB and NPW modulate IP3-dependent Ca²⁺ oscil
lations in glucose-activated β-cells. Under these conditions, neuropeptide application altered
oscillation frequency and waveform characteristics, suggesting an effect on intracellular Ca²⁺
handling. The variability observed between islets highlights the importance of preserved mi
croarchitecture and paracrine signaling in fresh tissue slices. These findings suggest that NPB/W
signaling may fine-tune β-cell excitability rather than directly trigger activity.
Conclusion: NPB and NPW modulate cytosolic Ca²⁺ dynamics in β cells in situ, likely through
mechanisms involving intracellular Ca²⁺ stores. These results provide functional evidence for a
modulatory role of NPB/W signaling in β-cell excitability.
Funding: The research stay at Medical University of Vienna was supported by the AKTION Öster
reich–Tschechien programme, the Charles University Mobility Fund, and the PPSŘ programme
(Programme for the Support of Scientific Research).
Skeletal muscle phenotype in tfAm-deficient mice: integrating high-resolution respirometry, histology, and motor performance | Daniel Follprecht
Skeletal muscle phenotype in TFAM-deficient mice
Authors: Daniel Follprecht (1), Věra Vtípilová (2,3), Patrícia Karkušová (2,4), Jan Cendelín (4), Michaela
Kohoutová (5), Jana Tůmová (5), Eva Kralovcová (2), Kristýna Popelková (2), Jan Nevoral (2,3), Aleš
Kroužecký (1)
Supervisor: Aleš Kroužecký (1)
(1) Department of Sports Medicine and Active Health Sciences, Faculty of Medicine in Pilsen,
Charles University, Czech Republic (2) Biomedical Center, Faculty of Medicine in Pilsen, Charles
University, Czech Republic (3) Department of Histology and Embryology, Faculty of Medicine in
Pilsen, Charles University, Czech Republic (4) Department of Pathological Physiology, Faculty of
Medicine in Pilsen, Charles University, Czech Republic (5) Department of Physiology, Faculty of
Medicine in Pilsen, Charles University, Czech Republic
State-of-the-Art: Mitochondrial transcription factor A (TFAM) is essential for the maintenance
of mitochondrial DNA and mitochondrial gene expression. Altered TFAM dosage in skeletal mus
cle has been linked to mitochondrial remodeling and impaired oxidative phosphorylation. How
ever, the functional consequences for muscle strength and mitochondrial respiratory capacity
during ageing remain insufficiently characterized. We hypothesize that TFAM insufficiency leads
to reduced mitochondrial respiratory capacity in skeletal muscle and may be compensated for
by increased muscle strength.
Objective: To define the contribution of TFAM insufficiency to muscle strength in laboratory
mice and to assess the involvement of TFAM in mitochondrial respiratory capacity in skeletal
muscle.
Material and Methods: A mouse model of TFAM insufficiency (Tfam wt/Δ) and wild-type (WT)
control animals underwent longitudinal motor testing throughout life, with emphasis on mus
cle strength and coordination/locomotor assays (e.g., grip strength, Rotarod, Erasmus ladder,
T-maze). At ~13 months of age, skeletal muscles were collected, with priority given to the quadri
ceps for high-resolution respirometry and parallel molecular profiling. Oxidative capacity was
measured using high-resolution respirometry (Oxygraph-2k). Histological evaluation was per
formed in selected muscles (quadriceps/soleus/gluteus). All animal experiments were conduct
ed in accordance with Act No. 246/1992 Coll. (Project No. MSMT-33798/2021-4).
Results & Discussion: Functional data do not suggest increased muscle strength in Tfam wt/Δ
mice compared with WT controls in grip strength testing. High-resolution respirometry of skel
etal muscle showed that the muscles of Tfam wt/Δ animals preserved substrate-supported res
piratory responses. Surprisingly, we found a negative correlation between grip strength and
mitochondrial respiratory parameters across both Tfam wt/Δ and WT animals.
Conclusion: This study characterizes Tfam-insufficient mice and seeks to identify mitochondrial
determinants of motor performance. The limitation is the small sample size. However, as the
study is ongoing, further inclusion of animals will strengthen validation of the findings.
Funding: Cooperatio – field of science Immunity and Infection
In vitro study of sertoli-leydig system using human primary granulosa cells to better
understand male infertility treatment | Roman Viták
Study of Sertoli-Leydig system
Authors: Roman Viták (1), Giada Mercanile (2,3), Clara Lazzaretti (2), Samantha Sperduti (2,4), Carme
la Perri (2), Manuela Simoni (2,4), Livio Casarini (2,4)
Supervisor: Radek Kučera (1)
(1) Department of Pharmacology and Toxicology, Faculty of Medicine in Pilsen, Charles Universi
ty (2) Unit of Endocrinology. Department of Biomedical, Metabolic and Neural Sciences, Univer
sity of Modena and Reggio Emilia, Modena, Italy (3) Unit of Dermatology, Department of Medical
and Surgical Sciences, University of Modena and Reggio Emilia, Modena, Italy (4) Center for
Genomic Research, University of Modena and Reggio Emilia, Modena, Italy
State-of-the-Art: Male infertility is a health problem with socio-demographic implications; in
~40% of cases the cause remains unknown. Spermatogenesis is regulated by gonadotropins
and testosterone. In hypogonadotropic hypogonadism (HH), treatment with GnRH or exogenous
gonadotropins—most commonly (human chorionic gonadotropin) hCG alone or combined with
FSH—raises intratesticular testosterone and restores spermatogenesis. hCG binds LH receptors
(LHCGR) on Leydig cells and stimulates testosterone production, while FSH acts on Sertoli cells,
probably inducing growth factors that enhance Leydig cell responsiveness to LH/hCG. However,
in idiopathic infertility LH and FSH levels are usually normal, and it remains unclear whether
endogenous FSH sufficiently supports sperm production or quality.
Objective: The aim of the study was to create an in vitro model recapitulating the Sertoli–Leydig
signalling to analyse the effect of FSH and hCG on testosterone production in Leydig cells. To this
purpose, ovarian primary granulosa cells (hGLC) was used as a substitute for Sertoli cells (not
available).
Material and Methods: The production of testosterone was evaluated in the murine Leydig
tumor cell line 1 (mLTC1), expressing mouse LH receptors capable to bind human LH and hCG.
mLTC1 cells were first exposed (pre-treatment) to conditioned medium from cultured hGLC
maintained in the presence or in the absence of FSH. mLTC1 pre-treated with unconditioned
medium served as a control.
Medium was collected and evaluated for testosterone production by Homogenous Time-Re
solved Fluorescence (HTRF) assay.
Results were presented as dose-response curves for testosterone and progesterone production,
as values plotted against hCG and LH concentration in a X-Y graph. The 50% effective concen
trations and other pharmacological parameters will be extrapolated from the dose-response
curves and statistically compared.
Results & Discussion: mLTC1 cells pre-treated with unconditioned control medium showed a
gonadotropin-dependent increase in testosterone. hCG was more potent than LH, inducing a
testosterone plateau about twofold higher. Pre-treatment with hGLC-conditioned medium re
duced LH/hCG potency compared with control medium. The effect was stronger when the con
ditioned medium derived from FSH-treated hGLC, suggesting ovarian cells release unidentified
factors that decrease Leydig cell responsiveness to gonadotropins. This effect may depend on Gs
protein expression, as the mLTC1 response to CTX, but not to forskolin, declined together with
LH/hCG-induced responses.
Conclusion: Results suggest that ovarian cells produce paracrine factors reducing gonad
otropin responses in nearby LHCGR-expressing cells. Thus, hGLC cannot replace Sertoli cell functions that enhance Leydig cell responses to LH/hCG, supporting the existence of sex-specific
regulator of gonadal cell metabolism.
Funding: This work was supported by the Cooperatio Program, research area Pharmaceutical
Science