Hormone induced differential transcriptome analysis of Sertoli cells during postnatal maturation of rat testes
Hormone induced differential transcriptome analysis of Sertoli cells during postnatal maturation of rat testes
Mukesh Gautam 0 1 2
Indrashis Bhattacharya 0 2
Umesh Rai 0 1 2
Subeer S. Majumdar 0 2
0 a Current address: The Ken & Ruth Davee Department of Neurology, Northwestern University , Chicago , Illinois, United States of America ¤b Current address: Department of Zoology, Hemvati Nandan Bahuguna Garhwal University , Srinagar, Garhwal, Uttarakhand , India
1 Department of Zoology, University of Delhi , Delhi , India , 2 Cellular Endocrinology Laboratory , National Institute of Immunology , New Delhi , India , 3 National Institute of Animal Biotechnology , Hyderabad , India
2 Editor: Suresh Yenugu, University of Hyderabad , INDIA
Data Availability Statement: Relevant data are
found within the paper. The raw microarray data
has been submitted to NCBI GEO under the
accession number GSE48795.
Funding: Funded by Indian Council of Medical
Research. Grant number: 5/10/FR/8/2010-RHN.
Tata Innovation Award.Grant number:BT/HRD/35/
01/01/2010. SSM received the funding. The
funders had no role in study design, data collection
and analysis, decision to publish, or preparation of
Sertoli cells (Sc) are unique somatic cells of testis that are the target of both FSH and
testosterone (T) and regulate spermatogenesis. Although Sc of neonatal rat testes are exposed to
high levels of FSH and T, robust differentiation of spermatogonial cells becomes
conspicuous only after 11-days of postnatal age. We have demonstrated earlier that a developmental
switch in terms of hormonal responsiveness occurs in rat Sc at around 12 days of postnatal
age during the rapid transition of spermatogonia A to B. Therefore, such ªfunctional
maturationº of Sc, during pubertal development becomes prerequisite for the onset of
spermatogenesis. However, a conspicuous difference in robust hormone (both T and FSH) induced
gene expression during the different phases of Sc maturation restricts our understanding
about molecular events necessary for the spermatogenic onset and maintenance. Here,
using microarray technology, we for the first time have compared the differential
transcriptional profile of Sc isolated and cultured from immature (5 days old), maturing (12 days old)
and mature (60 days old) rat testes. Our data revealed that immature Sc express genes
involved in cellular growth, metabolism, chemokines, cell division, MAPK and Wnt
pathways, while mature Sc are more specialized expressing genes involved in glucose
metabolism, phagocytosis, insulin signaling and cytoskeleton structuring. Taken together, this
differential transcriptome data provide an important resource to reveal the molecular
network of Sc maturation which is necessary to govern male germ cell differentiation, hence,
will improve our current understanding of the etiology of some forms of idiopathic male
Spermatogenesis is a complex process where every step of male Germ cell (Gc) development
are essentially supported by somatic Sertoli cells (Sc) [
]. In response to various hormonal and
biochemical stimulation Sc produce important factors that regulate Gc division and
]. The functions of Sc are largely governed by the synergistic effect of Follicle
Stimulating Hormone (FSH) and testosterone (T) as Sc bear receptors for both the hormones .
Although Sc of infant primates and rodents are exposed to sufficient levels of FSH and T,
robust onset of spermatogenesis is not seen during infancy but is discernible only during the
onset of puberty [
]. We have demonstrated earlier that a developmental switch, in terms of
hormonal responsiveness, of Sc occurs in rats at around 12 days of postnatal age coinciding
with the rapid transition of spermatogonia A to B . During pubertal development, Sc
become mature further with the changing need of differentiating Gc to support
spermatogenesis. Therefore, such a pubertal maturation of testicular Sc is considered to be a prerequisite of
male fertility [
In rodents, upto 3±5 days after birth, Sc keep proliferating and attract the gonocytes
towards the basement membrane for establishing the spermatogonial stem cell (SSC) niche
]. Neonatal Sc continue to proliferate and thereby directly regulating the numbers of SSC
niches in the developing testes [
]. During second week of postnatal life, as the Gc enter into
meiosis, Sc stop proliferating and become enlarged to hold the growing number of Gc within
their cytoplasmic extensions. The Sc-Sc tight junctions are formed to establish the
blood-testis-barrier (BTB) at the age of 15±16 days after birth [
]. Sc prominently expresses tight
junction and adherent junction proteins to hold various stages from spermatocyte to elongated
spermatids. Besides nurturing, mature Sc also maintain a finite number of Gc by regulating
apoptosis, scavenging dead cells, and changing adherent junctions to release mature sperm
into lumen. By the age of 55±60 days, fully mature sperm are seen in epididymis of rat
suggesting that by this time, adult Sc are fully differentiated to extend support to all stages of Gc for
maintaining spermatogenesis [
To understand the regulatory mechanism of spermatogenesis, it is imperative to explore
the changing landscape of gene expression during various phases of Sc maturation.
Microarray is a powerful technique to study differentially expressed genes in large numbers [
Differential gene expression during spermatogenesis has been studied by various researchers
using microarray technology studied by us [
] and others [14±16]. These gene
expression profiling experiments reveal candidate genes for the regulation of spermatogenesis and
fertility as well as targets for innovative contraceptives that act on gene products absent in
other somatic tissues. Here, using microarray technology, we have compared the hormone
stimulated (FSH and T in combination) differential transcriptome profile of Sc purified and
cultured from immature (5 days old), maturing (12 days old) and mature (60 days old) rat
testes. Our results suggest that transcriptome of Sc during different maturation stages differ
significantly and this information can help advance our understanding of regulatory
mechanisms of spermatogenesis.
Material and methods
Wistar outbred rats (Rattus norvegicus) were procured from colony maintained by Small
Animal Facility, National Institute of Immunology, New Delhi, India. Animals were maintained
in a standard day night cycle with stable temperature and humidity and provided food and
water ad libitum. All the animal experimentations were approved by Institutional Animal
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Ethics Committee (IAEC) and performed following standard guidelines of ``Committee for the
Purpose of Control and Supervision of Experiment on Animals (CPCSEA),º Government of
Isolation and culture of Sertoli cells from 5 days and 12 days old rat testes
Sc cultures were prepared from 5 days and 12 days old rat testes as described earlier [
testes were decapsulated and chopped finely before sequential digestion with collagenase and
pancreatin enzyme at 34ÊC. Clusters of Sc were separated from mixture of cell suspension by
differential centrifugation. Cultures were maintained in DMEM-F12 HAM media containing
1% FBS for first 24 hrs before replacing with growth factor media (GF media) containing 5 μg/
ml sodium selenite, 10 μg/ml insulin, 5μg/ml transferrin, and 2.5 ng/ml epidermal growth
factor. Sc cultures were maintained in defined GF media for 4 days. On day 3, cells were treated
with 20 mMTris HCl (pH 7.4) for 3±5 min to get rid of contaminating Gc [
]. In vitro
hormone treatment was performed on day 4.
Isolation and culture of Sertoli cells from 60 days old rat
Sc from mature rat testes (60 days old) were isolated and cultured as described earlier [
Briefly, testes were decapuslated and seminiferous tubules were chopped, washed and
repeatedly digested with 1mg/ml collagenase until most of Sc clusters were released. Sc cells were
cultured and maintained for 4 days before initiation of experiments as described previously [
On day 3, cells were treated with 20 mMTris HCl (pH 7.4) for 5 min to get rid of
contaminating Gc [
Purity of Sc culture
On day 4, Gc contamination were found to be less than 5% in Sc cultures of all age groups.
Purity of Sc in culture were identified by vimentin staining (Abcam, USA, Ab8978) whereas
Peritubular Cell (PTc) or Leydig Cell (Lc) contaminations were determined by the alkaline
phosphatase or the 3β-HSD activity respectively, as described by us before [
In vitro hormone treatment
On day 4 of culture, cells of all age groups were incubated with GF media containing
hormones (50ng/ml o-FSH and 10−7M T in combination = FT media) in pulsatile manner (30min
of FT pulse/3hr)The doses of both o-FSH and T were previously found bioactive in cultured Sc
obtained from all three age groups studied [
]. Sc were exposed for ½ hour (hr) to FT
media and then the FT media was removed and replenished with fresh GF media for 2½ hr.
This was repeated upto 11 hr. Please note that since the treatment was terminated at 11thhr i.e.
11/2 after receiving the 4th pulse of FT media, Sc were in GF media at the time of termination.
This experiment was repeated at least three times in each age group of Sc cultures prepared on
different calendar dates. At the end of the experiments, cells were dislodged, washed and then
suspended in RNA-later and stored at -80ÊC until RNA extraction.
RNA extraction, labeling and microarray hybridization
Total RNA was isolated from Sc using Qiagen (Valencia, CA) RNAEasy Mini kit according to
the instructions of the manufacturer. Purity of Total RNA was assessed by the NanoDrop1
ND-1000 UV-Vis Spectrophotometer (Nanodrop technologies, Rockland, USA). Total RNA
with OD260/OD280>1.8 and OD260/OD270 1.3 was used for microarray experiments.
RNA integrity was assessed using RNA 6000 Nano Lab Chip on the 2100 Bioanalyzer (Agilent,
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Palo Alto, CA). For the assessment of total RNA quality, the Agilent 2100 Expert Software
used to determine RNA Integrity Number (RIN) which provided a quantitative value for RNA
integrity. RNA having RIN 9.0 out of maximum scoring of 10 was used for microarray
hybridization. The RNA which passed quality control parameters was labeled with Cy3 dye
and hybridized on rat whole genome 4X44K gene chip (Agilent Technology Inc.) as described
by us earlier [
Microarray scanning, feature extraction and array analysis
Hybridized arrays were scanned at 5μm resolution on an Agilent DNA Microarray Scanner,
Model G2565BA. Data extraction from images was done using Feature Extraction software of
Agilent. Hybridization signals were quantified using GeneSpringGx v 11.0.1 software from
Agilent Technologies. Pearson's correlation coefficients were computed to assess the reliability
of data obtained from RNA preparation for 3 samples each from 5 day, 12 day and 60 day Sc.
The data retrieved from separate membranes with the same RNA samples yielded QC statistics
highly concordant with that of the manufacturer, and it revealed more than 95% confidence
level. In order to identify biological variation, integrated signal analysis for a given membrane
was performed and signal spots that were low after averaging, as compared to average
background plus 2SD values, were removed. Within each hybridization panel, the 50th percentile
of all measurements was used as a positive control for normalization for each gene. Data
normalization, averaging, calculation of relative abundance of transcripts, ratio analysis, and fold
changes were performed on log transformed data using GeneSpringGx v.11.0.1 (Agilent
Technologies, Santa Clara, CA, USA). Per-membrane and per-gene normalization were conducted
using GeneSpringGx v 11.0.1 normalization algorithms. Principal Component Analysis (PCA)
was performed for all annotated 5 day, 12 day and 60 day Sc samples for all expressed genes to
assess the similarity in gene expression patterns on the basis of underlying variability and
cluster structures using algorithm in Gene- Spring11.0.1 [
K-means cluster analysis and unsupervised hierarchic clustering analysis (HCA) was
performed using GeneSpringGx v.11.0.1 software. In K-means cluster analysis, gene expression
levels were randomly assigned into distinct clusters and the average expression vector was
computed for each cluster. For every gene, the algorithm then computed the distance to all
expression vectors, and moved the gene to the cluster whose expression vector was closest to it.
The entire process was repeated iteratively until no gene products could be reassigned to a
different cluster. To further evaluate the patterns in gene expression profiles, unsupervised
hierarchic clustering (HCA) of data in pairwise comparisons among samples using a standardized
Pearson's uncentered correlation vector with average linkage for distance measures, and their
visualization in the form of a heat map and dendrogram were performed.
Normalized data were used in pairwise overall comparisons between 5 day, 12 day and 60
day samples. The resulting gene lists from each pairwise comparison only included the genes
that showed a fold change of 2.0 or higher and a p< 0.05 by using a parametric Welch t test
with Benjamini-Hochberg multiple testing corrections for false discovery rate (FDR). All
statistical analyses were performed using GeneSpring v.11.0.1 software. Identification of
differentially enriched gene ontology (GO) terms for genes in 4 clusters in K-means analysis, as well
as, those identified in differential expression analysis was carried out using a gene ontology
(GO) tree machine and GeneSpring v.11.0.1. Further, functional analysis of differentially
expressed genes was performed using GeneGo Metacore software (Thermo scientific,
St. Joseph, MI, USA), DAVID online data analysis tool and the Kyoto Encyclopedia of Genes
and Genomes (KEGG) platform (http://www.genome.jp/kegg/) and STRING database for
pathways analysis to link genomic information with higher order functional information. For
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preparation of heat maps of some important differentially expressed genes, a web based
application matrix2png was used [
]. For making interacting network of genes, STRING database
tool was used [
Quantitative RT-PCR (qRT-PCR) amplifications were performed in additional sets of Sc
cultures obtained from all 3 age groups with uniform hormonal treatments used in microarray to
validate expression level of various genes as per described by us earlier [
curve analysis was performed immediately after amplification to ensure that there was only
one (gene specific) amplification peak. For each sample, the calculated quantity of each gene
was then normalized with relation to the quantity found for Ppia (Cyclophilin A). The relative
quantities of mRNA for target genes were determined by 2-ΔΔCt method. The means (±SEMs)
of 3 individual experiments were determined for each treatment group for the target gene. The
list of primers used for Real Time PCR is given in Table 1.
The differential expression of genes was determined by pairwise comparison of the genes that
showed a fold change of 2.0 or higher and a p< 0.05 by using a parametric Welch t test with
Benjamini-Hochberg multiple testing corrections for false discovery rate (FDR). The statistical
analyses to generate gene expression list were performed using GeneSpring v.11.0.1 software
(Agilent Technologies, Santa Clara, CA, USA). For qRT-PCR experiments, one treatment
group comprised of 3±4 wells of Sc within one culture set. At least 3 such sets of cultures
(performed on different calendar dates) were used to interpret the data. Data was expressed as
mean ± SEM. Statistical analyses of data were performed by non-parametric student's t-test for
comparison between two groups. Statistical tests were done using GraphPad Prism 5.01
(GraphPad Software Inc., La Jolla, CA, USA). P values < 0.05 were considered as statistically
Results and discussion
This study was undertaken to determine the comprehensive gene expression profile of Sc
obtained from different stages of postnatal testicular development. Sc drastically transform
during pubertal maturation to fulfill the changing need of developing Gc. Therefore, the
knowledge of differentially expressed genes by Sc during different postnatal ages will provide a
deeper insight into Sc mediated regulation of spermatogenic onset at puberty and its
maintenance in adulthood. Here, we have compared the differential gene expression of maturing (12
days old rats i.e. 12d) and mature (60 days old rats i.e. 60d) Sc, which have been normalized
against that of the immature (5 days old rats i.e. 5d) Sc. Five-days-old (5d) neonatal rats
represent proliferative Sc with the establishment of the spermatogonial stem cell niche on the
basement membrane within the seminiferous epithelium; 12-days-old (12d) rats represent
maturing Sc facilitating the robust generation of spermatogonia B [
] and 60-day-old rats
(60d) represent fully mature, non-proliferative adult Sc where the blood testis barrier (BTB) is
completely established with the presence of all stages of Gc upto sperm [
Microarray analyses have been employed by different laboratories to identify independent
action of either FSH [25±27] or T [28±32] on testicular gene expression. Whereas both FSH
and T act synergistically to regulate Sc gene expression for supporting Gc differentiation [
Therefore, major aim of the present study was to investigate the age related change in Sc
transcriptome that has similar hormonal background. The doses of o-FSH and T were previously
reported to be bioactive in terms of gene expression in Sc of all the age groups studied [
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Product Size (bp)
Similar to previous attempts [
], recently, Zimmermann and coworkers have reported the
transcriptional dynamics of murine Sc revealing many candidate genes essential for the
progression of first spermatogenic wave . To understand if there are some common conserved
genes between developing mouse and rat Sertoli cells, we have compared 394 differentially
expressed genes obtained from Zimmermann's data with 4395 genes found by us. We have
identified 98 common genes which are accounted mostly for cell-cell communication,
metabolism, energy production and cell growth (S1 Fig). However, it is important to note here that
Zimmerman et.al. have reported Sc transcriptome at different postnatal ages in mouse,
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whereas our study distinctly illustrated rat Sc transcriptome under the influence of uniform
Rat is considered a better model (than mice) for understanding the regulation of testicular
]. Due to larger body size and higher blood volume and litter size, rats are more
favored for surgical, physiological and pharmacological experiments [
]. Moreover, in
terms of genetic distance, it is 5 million years closer to human than mice [
]. Keeping this
in mind, rat Sc were used in the present study.
Previously flow sorted Sc were directly used for their RNA expression analyses [
Therefore, there is a probability that these cells may not be free from the proteolytic shock generated
by the prolonged enzymatic exposure during the isolation process [
]. Such shock may
induce transcriptional changes in the sorted cells contributing the final readout [
Therefore, in this study, instead of direct use of the isolated cells, Sc of all age groups were uniformly
cultured for 4 days, a time period that was enough for stabilization the cells from the shock of
the isolation process.
Pulsatile pattern of hormone release plays a critical role in optimal hormonal action in the
target tissue as suggested by many in vivo studies [
]. The importance of pulsatile hormone
treatment to endocrine cells has also been well-characterized in cultured pituitary cells 
Administration of pulsatile GnRH (by changing both the amplitude and frequency) to cultured
pituitary cells has been practiced for determining the differential transcriptional regulation of
LH and FSH β subunits [
]. High and slow frequency pulses favor LH and FSH-β mRNAs
expression, respectively [
]. Pulsatile release of gonadotropins is also known to regulate
testicular function in vivo [
]. For example, pulsatile LH-releasing hormonal-therapy was found to
be effective over constant GnRH in restoring the normal gonadotropin secretion inducing
fertility in men with hypogonadotropic eunuchoidism [
]. We have recently demonstrated that
for hormone induced gene expression study, pulsatile treatment of hormones (both FSH and
T in combination) to cultured Sc from 18 days-old pre-pubertal rats' shows better readouts as
compare to that of the conventional, constant stimulation with hormones [
]. Although the
establishment of hypothalamo-hypophyseal testicular axis steadily improves with age in
rodents, GnRH pulse generator activity initiates during fetal development . Moreover,
neonatal Sc obtained from 5 days old rats do express both Androgen Receptor and
]. Therefore, for the present study, cultured Sc of all three age groups were uniformly
stimulated with pulsatile hormones (FSH and T in combination for ½ hr after every 3hr) on
day 4 of culture to determine age specific, hormone induced differential transcriptome profile.
RNA isolated from Sc was labeled with Cy3 and hybridized on Agilent's rat whole genome
4x44K Gene Chips (Agilent Technologies, Santa Clara, USA). The results of unsupervised
principal component analysis (PCA) of all samples and unsupervised hierarchic clustering
analysis (HCA) revealed a high order of sample homogeneity. Fig 1A shows the results of the
unsupervised HCA of the gene expression profiles of the 3 samples each of 5d, 12d and 60d Sc.
The principal component analysis (PCA) shows that all the three biological replicates for 5d,
12d and 60d are falling in their respective dimensions (Fig 1B). Volcano plots of genes
differentially expressed between 5d and 12d (Fig 1C) and 5d and 60d (Fig 1D) provides a visual
presentation of fold change of genes is respect to p-value.
To identify the clusters of genes whose expression is regulated in a similar way throughout
the samples, K-means clustering tool of GeneSpring software was applied to differentially
expressed genes across the 3 samples each of 5d, 12d and 60d Sc. As shown in S2A Fig, 4
major groups (named as clusters 1, 2, 3 and 4) were identified. Gene Ontology (GO) based
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Fig 1. Analysis of microarray data for homogeneity of biological replicate samples. (A) Unsupervised hierarchical clustering analysis (B) Principal
component analysis (C) Volcano plot analysis of differentially regulated genes between 5 days and 12 days and (D) Volcano plot analysis of
differentially regulated genes between 5 days and 60 days.
functionality analysis of K-mean clusters shows distribution of genes among cellular
component (CC), molecular function (MF) and biological (BP) (S2B Fig). The raw microarray data
has been submitted to NCBI GEO under the accession number GSE48795.
Differential gene expression analysis
The resulting gene lists from each pairwise comparison included the genes that showed a
fold change of 2 (log2) or higher and a P < 0.05 by using a parametric Welch t test with
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Fig 2. Commonality analysis of differentially regulated genes between 12 days and 60 days Sc in comparison with
5 days Sc.
Benjamini-Hochberg multiple testing corrections for false discovery rate (FDR). We
compared the gene expression data obtained from12d and 60dSc against 5dSc to find
differentially expressed genes. Venn diagram in Fig 2 shows that a total of 5074 genes were
differentially regulated of which 3746 were exclusive in 12d and 548 were only in 60d. Out of
3746 total regulated genes in 12d, 1945 were upregulated and 1801 were downregulated (S1
and S2 Tables). Likewise, out of 548 genes regulated in 60d, 360 genes were upregulated and
188 genes were downregulated (S3 and S4 Tables). A possible explanation for this difference
in the number of transcripts differentially regulated in 12d Sc and 60d Sc could be due to
their respective developmental state. We have reported earlier that rat Sc acquire necessary
developmental competence in terms of FSH responsiveness at 12 days of postnatal age
coinciding with the rapid transition of spermatogonia A to B [
]. On the other hand, we have also
reported recently that by 60 days of age Sc are fully differentiated and physiologically stable
in terms of responsiveness towards T for supporting the spermatogenesis [
]. Therefore, it
is reasonable to assume that such difference in the Sc physiology might be responsible for
their differential gene expression pattern.
Some of the important and highly regulated genes upregulated in 12d, upregulated in 60d,
downregulated in 12d, downregulated in 60d and differentially regulated in 12d and 60d are
listed in Tables 2, 3, 4, 5 and 6, respectively.
Gene Ontology (GO) terms analysis was performed using DAVID online resource. GO
term analysis showed enrichment of relevant biological processes such as ªGerm cell
developmentº, ªchemokine pathwayº, ªwater transportº, ªcell adhesion moleculesº, ªgrowth factorsº,
ªgrowth factors binding proteinsº, ªinsulin signalingº, ªcarbohydrate metabolismº, ªlipid
metabolic pathwayº, ªprogrammed cell deathº, ªcell divisionº, ªdifferentiationº being the most
prominent terms (Table 7).
% of DR refers to the percent of differentially regulated transcripts falling under the term;
p-value is the raw p-value from Fishers exact test. GO term analysis was done using DAVID
bioinformatics tool for functional analysis (http://david.abcc.ncifcrf.gov/).
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Gene bank Accession
Testis expressed gene 101
Synaptonemal complex protein 2
Synaptonemal complex protein 3
Cyclin B1 interacting protein 1
Synaptonemal complex protein 1
DEAD (Asp-Glu-Ala-Asp) box polypeptide 4
Coagulation factor II
Solute carrier family 30 (zinc transporter), member 2
Nuclear receptor subfamily 4, group A, member 3
ELL associated factor 2
Lectin, galactose binding, soluble 4
From the differential gene expression profile list, our main focus was on the set of genes
which might be important during postnatal maturation of Sc and therefore play crucial role in
spermatogenesis. The gene clusters were selected on the basis of their respective relevance in
spermatogenesis. After initial data analysis, a thorough literature search was done to
Gene bank Accession
Gene bank Accession
Lactate dehydrogenase C
Fatty acid binding protein 9, testis
Synaptonemal complex protein 3
Retinitis pigmentosaGTPase regulator interacting protein 1
Testis expressed gene 101
Synaptonemal complex protein 1
Synaptonemal complex protein 2
Cysteine-rich secretory protein 2
Similar to alpha-1 major acute phase protein prepeptide
Angiotensin II receptor, type 2
Flavin containing monooxygenase 2
Homeobox protein Hox-A5
Integral membrane protein 2A
Matrix metallopeptidase 7
understand how these gene clusters obtained in our microarray data were important in the
process of spermatogenesis. Thereafter, the clusters of genes selected were used for further
Genes potentially involved in some of the important biological functions related to
spermatogenesis are discussed below.
Chemokines are small cytokines important for chemotactic migration of cells including
primordial germ cells. The chemokines that are important for homing of Gc, were downregulated
in 12d and 60d Sc when Gc have already established in their respective niche. Since 5d Sc are
actively involved in attracting Gc and creating germinal stem cell niche, higher expression of
chemokine related genes by 5d Sc indicates important role of chemokine in immature testis
(Fig 3A). Interacting network of these chemokine molecules reveals that chemokines and their
receptors are closely related to each other. CXCL12, also known as stromal cell derived
factor1 (SDF-1), is expressed by Sc and CXCR4 is expressed by the Gc population of the adult
human testes [
]. Interaction between the chemokine CXCL12 and its receptor CXCR4 is
responsible for the maintenance of adult stem cell niches.
Mitogen Activated Protein Kinase (MAPK) pathway
The MAPK pathway acts to integrate diverse signals to regulate a variety of cellular functions
such as cell cycle progression, cell adherence, motility and metabolism and thereby influence a
number of developmental processes [
]. MAPK pathway is pre-dominant in immature Sc
and downregulate upon maturation of Sc [
]. MAPK pathway genes were differentially
expressed in microarray data. Majority of these genes were downregulated in both 12d and
60d Sc. All of these genes were directly interacting with each other forming strong network
(Fig 3B). Genes which were downregulated in 60d but induced in 12d Sc were dual specificity
phosphatases (Dusp1&7), brain derived neurotrophic factor (Bdnf), nuclear receptor
subfamily 4, group A, member 1 (Nr4a1) and neurotrophin 3 (Ntf3). MAP3K1 act to regulate or
integrate signals during testis development as Map3k1-deficient mice are non-viable [
MAPK signaling initiates G1 mitotic arrest in a variety of terminally differentiated cell types
]. DUSP and JIK were upregulated in 12d and 60d Sc. [
]. These two genes are negative
feedback regulator of MAPK signaling, suggesting predominant role of MAPK signaling is
immature Sc but not upon their differentiation .
Cytokines are potent growth factors and their differential expression is important for
regulating different functions in infant and adult testis [
]. Sc produce cytokines such as Interleukins
(ILs), Interferons (IFNs), Tumor necrosis factor (TNF), transforming growth factor (TGF) etc.
which play important role in spermatogenesis [
]. Interleukins were the majority of cytokines
found differentially expressed in our microarray data. With exception of few, most of these IL,
their isoforms and their receptors were downregulated in both 12d and 60d Sc. (Fig 3C). The
Transforming Growth Factor beta (TGFβ) superfamily of ligands, including TGFBs, activins,
bone morphogenetic proteins (BMPs), nodal, and growth and differentiation factors (GDFs)
govern Gc development [
]. TGF-b3 regulates the permeability of Sc tight junctions helping
spermatogonia to cross BTB during progression of spermatogenesis and release of mature
sperm while spermiation [
]. IL-1α is the most abundant Sc growth factor in the
pubertal and adult testis while IL-1β is a potent growth factor for immature rat Sc [
regulate number of Gc in the fetal testis and Gc maturation at the onset of spermatogenesis,
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Fig 3. An overview of some important pathways differentially regulated in 12d and 60d Sc as compared to 5d Sc and their interactome map.
Chemokine signaling (3A), MAPK signaling (3B), Cytokine signaling (3C) and Growth factors (3D). The heatmap shows differential expression of
some selected pathways genes across three biological replicates in 12d and 60d Sc. Interacting network analysis of these genes shows how these genes
interact with each other and form a network.
contributing to the complex signaling networks that govern normal testis development [
Network analysis shows that interleukins and transforming growth factor molecules make two
distinct networks which are connected to each other.
A number of growth factors are expressed in testis which control migration, differentiation,
and proliferation of primordial germ cells and Sc [
]. Majority of these growth factors are
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Fig 4. An overview of some important pathways differentially regulated in 12d and 60d Sc as compared to 5d Sc and their interactome
map. Cytoskeleton and small GTPases (4A), Apoptosis (4B), Spermatogenesis (4C), Neuronal processes (4D), Metabolism (4E) and Wnt
signaling (4F). The heatmap shows differential expression of some selected pathways genes across three biological replicates in 12d and 60d Sc.
Interacting network analysis of these genes shows how these genes interact with each other and form a network.
Insulin-like growth factors, Fibroblast growth factors and their receptors and binding proteins.
Growth factors provide signals for developing Sc and hence their expression is increased in
12d Sc. Elevated expression of these genes up to 12d of age in Sc signifies their role in
migration of Gc in SSC niche, proliferation of spermatogonia, entry of spermatogonia in meiosis
and Sc-Gc interaction. Some genes i.e. PDGFa, Mst1, KitL, GDNF, TFF2 and GDF15 were
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induced in 12d Sc and were persistently expressed in 60d Sc also (Fig 3D). These genes interact
with each other and forms strong network. These genes are implicated in proper germ-line
development and regulation of proliferation and differentiation of Gc during spermatogenesis
]. Genes of the IGF signaling pathway has been shown to express in testis, especially in
Sc. They act as strong mitogenic and anti-apoptotic signal during proliferation of Sc. IGF1
inhibits differentiation of Gc . FGFs are involved in the proliferation and differentiation of
testicular cells and involved in the regulation of spermatogenesis [
]. In contrast to other
growth factors, FGF18 was upregulated in both 12d and 60d Sc. FGF18 plays role in pre-Sc
functions but its role after birth has yet not been assigned.
Small GTPases and cytoskeleton maintenance
Small GTpases are involved in regulating Sc-Gc tight junctions, Gc movement, and cell
]. Small GTPase which are upregulated in both 12d and 60d Sc were guanine
nucleotide binding protein, alpha 13 (Gna13), muscle and microspikes RAS (Mras), Ras-related
protein Rab-1B (Rab1b), ras homolog gene family, member V (Rhov), disabled homolog 1
(Dab1) and Rho GTPase activating protein 5 (Arhgap5). Members of Rho GTPases are
important part of the signaling pathway that is involved in inducing cytoskeletal changes,
lamellipodia and filopodia formation and the promotion of cell motility [
of RhoBGTPases modulates the actin cytoskeleton network resulting in adjacent junction
disruption and germ cell loss from the seminiferous epithelium [
]. Sc have a highly
developed membrane trafficking system that might be regulated by small GTPases [
(Fig 4A). Network analysis reveals that these genes make two networks which are
Many genes involved in cell-cell junctions were found well expressed in 12d and 60d Sc.
These genes such as Integrin alpha 6 subchain (Itga6), vinculin (Vcl), V-raf murine sarcoma
viral oncogene B1-like protein (Braf), laminin, alpha 1 (Lama1), Collagen alpha 2 (Cola2) and
reelin (Reln) forms important interacting component of Sc-Sc and Sc-Gc junctions. We found
Integrin alpha 4 subchain (Itga4) and Integrin alpha 5 subchain (Itga5) downregulated in both
12d and 60d Sc. These two genes are reported to be highly expressed in embryonic and infant
testis as they are crucial for making spermatogonial stem cell niches [
Apoptosis and phagocytosis
Many of the genes involved in programmed cell death were induced in 12d Sc. The genes
upregulated in both 12d and 60d were Microphthalmia-associated transcription factor (Mitf), Cbp/
p300-interacting transactivator, with Glu/Asp-rich carboxy-terminal domain, 2 (Cited2),
Bcl2-related ovarian killer protein (Bok), crystallin, alpha B (Cryab). Fos-like antigen 1 (Fosl1)
and cyclin-dependent kinase 5, regulatory subunit 1 (p35) (Cdk5r1) were upregulated in 12d
but remain unchanged in 60d Sc. The genes which were upregulated in 12d but downregulated
in 60d were hormone-regulated proliferation associated protein 20 (Hrpap20), Fas ligand
(TNF superfamily, member 6) (Faslg), endothelin receptor type B (Ednrb), glutamate receptor,
ionotropic, delta 2 (Grid2) and similar to zinc finger protein mRit1 beta (Bcl11b) (Fig 4B).
Many of the genes in this pathway are connected with each other in a network. However, this
network is not very strong as compared to other networks.
Genes involved in phagocytosis were dynein, cytoplasmic, heavy polypeptide 2 (Dnch2),
cartilage oligomeric matrix protein (Comp), mannose receptor, C type 1 (Mrc1) and
phospholipase A2 receptor (Pla2r1). These genes were upregulated in 60d Sc. These genes were either
slightly downregulated or remained unaltered in 12d Sc.
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Genes involved in homeostasis
Another major group of genes conspicuously expressed by mature Sc are the genes which are
important for maintenance of homeostatic environment inside seminiferous tubule. This
included genes from sodium and potassium ion channels, water transporters, voltage gated
ion channels, voltage dependent chloride channels etc. Sc play a key role in the establishment
of an adequate luminal environment in the seminiferous tubules of the male reproductive
tract. Secretion of the seminiferous tubular fluid (STF) is important for successful completion
of spermatogenesis as it provides the medium of transport to maturing spermatozoa [
release begins during sexual development and is dependent on FSH [
]. Sc regulate the
passage of ions and the selective flow of water, steroids and carbohydrates into the tubular
The genes which are important for maturation of sperm in adult were found to upregulated in
60d Sc. The example of some of these genes are Spermatogenesis Associated 20 (Spata20),
Capping Actin Protein Of Muscle Z-Line Alpha Subunit 3 (Capza3), Phosphoglycerate Mutase 2
(Pgam2), DnaJ Heat Shock Protein Family (Hsp40) Member B13 (Dnajb13), Heat Shock
Protein Family A (Hsp70) Member 2 (Hspa2), Thioredoxin Domain Containing 8 (Txndc8),
P-element Induced WImpy testis in Drosophila 11 (Piwi11), glycerol kinase-like 1 (GK-rs1)
and Gametogenetin Binding Protein 1 (Ggnbp1) (Fig 4C).
Genes expressed in nervous system
The genes which are normally expressed in neuronal processes were found to be expressed by
Sc. Interestingly, genes which are prominently expressed in nervous system were found
upregulated in 12d and 60d Sc. Sortilin1 (Sort1) is expressed in brain, spinal cord and muscles but
also has roles in embryogenesis [76±78]. It acts as receptor for neurotensin and co-receptor for
Nerve Growth Factor (NGF) and Brain Derived Neurotrophic Factor (BDNF) mediating cell
survival and apoptosis. Nerve growth factors have been shown to play important role in Sc-Sc
]. Caveolae are small invaginations of the plasma membrane which plays
important roles in signal transduction, cholesterol transport, and endocytosis [
Caveolin2 are more abundant in terminally differentiated cells [
]. Most of such genes were
upregulated in both 12d and 60d Sc. (Fig 4D).
Sc possess specialized metabolic mechanisms to meet specific energy demand of developing
germ cells [
]. Sertoli cells convert glucose into lactate which is preferentially uptake up by
germ cells. In our microarray results, most of the genes which are associated with glucose
metabolism were induced in 60 day Sc. These genes were either remained unaltered or slightly
downregulated in 12d Sc. The example of such genes are aminoadipate-semialdehyde synthase
(Aass), betaine-homocysteine methyltransferase (Bhmt), glucokinase activity, related sequence
2 (Gk-rs2), hypoxanthine guanine phosphoribosyl transferase (Hprt), inositol
1,4,5-trisphosphate 3-kinase A (Itpka), Pyruvate carboxylase (Pc) and phosphoglycerate mutase 2 (Pgam2).
fructose-1,6- biphosphatase 1 (Fbp1) was downregulated in 12dSc (Fig 4E).
The wingless-related MMTV integration site (WNT) gene family encodes a large number of
secreted signaling glycoproteins that are involved in many biological processes including
16 / 25
embryonic development, adult tissue homeostasis, maintenance of progenitor cell types and
cell fate determination and differentiation [
]. Differential expression of Wnt signaling is
important for proper functioning as Sc specific constitutive activation of WNT/CTNNB
pathway causing sterility. Wnt ligands (Wnt4, Wnt5a) were upregulated and Wnt pathway
inhibitors (sFRP) were downregulated in 12d and 60d Sc. Wnt 4 is important for proper functioning
of Sc after birth as differentiation of Sc becomes compromised in Wnt4 mutant testes [
There are no reports of Wnt 2 expression in Sc hence upregulation of Wnt 2 in only 60d Sc
may be intriguing. WNT5a has been shown to promote SSC self-renewal [
Frizzled-related proteins (sFRPs) are secreted glycoproteins that can antagonize WNT mediated
signaling by direct competitive interaction with WNT ligands [
]. sFRP1 has been shown
to downregulate in mature testis showing its importance in regulating spermiation in adult rat
] (Fig 4F). Network analysis reveals that all the Wnt ligands and receptors form strong
network and are closely related. The other members of the pathway form another network and
both these networks are connected together.
qRT-PCR validation of genes identified by microarray
In order to authenticate the array data the expression patterns of some of the genes were
validated by quantitative reverse transcription PCR (q-RT-PCR) with additional sets of Sc culture
obtained from all three age groups with consistent hormonal treatments (pulsatile FSH and T
in combination for 11hr) and were compared with that of the array data. The genes considered
were divided in five groups: 1) FSH responsive genes like SCF, GDNF, ABP, Transferrin and
Dmrt1 in both 5d and 12d as reported earlier either by us or others [2±4]. T responsive genes
like Claudin11, Occludin and Connexin43 in both 5d and 12d as reported earlier either by us
or others [
]. Genes downregulated in both 12d and 60d [UNC-5 family of netrin
receptors (Unc5c), Mesothelin (Msln), FAT Tumor Suppressor Homolog 3 (Fat3), Roundabout
homolog 1 (Robo1) and WNT1-inducible-signaling pathway protein 2 (Wisp)]; 2) genes
downregulated in 12d but upregulated in 60d [Alpha-aminoadipic semialdehyde synthase
(Aass), Chemokine (C-C motif) ligand 5 (Ccl5)], and 3) genes upregulated in both 12d and
60d [Hepatoma derived growth factor 1 (Pwwp1), spermatogenic zip 1 (Spz 1) and Testin].
For each sample, the calculated quantity of each gene was normalized with the endogenous
control Ppia (Cyclophilin A). The relative quantities of mRNAs for all target genes were
determined by 2-ΔΔCt method as reported by us before [
]. It is important to note that,
like the array data, the gene expression values obtained from 12d and 60d Sc by q-RT-PCR
were normalized against that found in 5d Sc. Expression patterns of both FSH or T responsive
genes (Fig 5A and 5B) and some other selective genes (Fig 6) generated by qRT-PCR analyses
were consistent with their expression pattern found in microarray. This observation further
strengthens the authenticity of our array data.
Postnatal maturation of testicular Sc is critical in regulating male fertility. Therefore, it
becomes essential to investigate the genes expressed by Sc during this phase of development.
Our microarray results have indicated differential expression of genes associated with
cytoarchitecture, metabolism, cytokines, chemokines, growth factors, MAPK signaling and Wnt
signaling among others. Taken together, this differential transcriptome data provide an
important resource database to reveal the molecular network of Sc maturation which is necessary to
govern male germ cell differentiation. This information will improve our current
understanding of the etiology of some forms of idiopathic male infertility with persistent immature Sc
even in adulthood.
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Fig 5. Fold rise in FSH (A) and T (B) responsive genes compared between microarray data and Quantitative real
time PCR. Data represented as mean ± SEM.
18 / 25
Fig 6. Validation of some selected genes from microarray data using Quantitative real time PCR. Data represented
as mean ± SEM.
PLOS ONE | https://doi.org/10.1371/journal.pone.0191201
19 / 25
S1 Fig. Comparison of gene sets between the present study and the data published by Zim
merman et.al. There are 96 genes common between our data and Zimmerman's data and
these genes are mainly involved in signal transduction, cell-cell communication, energy and
metabolism and cell growth.
S2 Fig. K-mean analysis of co-expressed genes. (A) Entity based clustering analysis of co
expressed genes identified four clusters of co-expressed genes. (B) Gene Ontology (GO) based
functional analysis of genes grouped under four clusters. Functional analysis was performed
for molecular function (MF), cellular components (CC) and biological processes (BP).
S1 Table. Genes upregulated in 12d Sc.
S2 Table. Genes downregulated in 12d Sc.
S3 Table. Genes upregulated in 60d Sc.
S4 Table. Genes downregulated in 60d Sc.
We are thankful to staff of the Small Animal Facility. Thanks are due to Ram Singh, Dharamvir
Singh, and Birendra Nath Roy for technical assistance. We also thank Dr. A. F. Parlow (NHPP,
NIH) for providing the hormones used in this study. We are grateful to the present and past
Directors of NII for valuable support. We are thankful to Genotypic Technology Private
Limited, Bengaluru, India for microarray hybridizations and data generation.
We thank the Department of Biotechnology (DBT) and Indian Council of Medical Research
(ICMR), Government of India, for financial support.
Conceptualization: Mukesh Gautam, Indrashis Bhattacharya, Umesh Rai, Subeer S.
Data curation: Mukesh Gautam, Indrashis Bhattacharya, Subeer S. Majumdar.
Formal analysis: Mukesh Gautam, Subeer S. Majumdar.
Funding acquisition: Subeer S. Majumdar.
Methodology: Mukesh Gautam, Indrashis Bhattacharya, Subeer S. Majumdar.
Supervision: Umesh Rai, Subeer S. Majumdar.
Validation: Mukesh Gautam.
Writing ± original draft: Mukesh Gautam.
20 / 25
Writing ± review & editing: Indrashis Bhattacharya, Umesh Rai, Subeer S. Majumdar.
21 / 25
22 / 25
49. Plant TM 60 YEARS OF NEUROENDOCRINOLOGY: The hypothalamo-pituitary-gonadal axis. J
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