Cellular and Molecular Life Sciences (2021) 78:3621–3635

https://doi.org/10.1007/s00018-021-03757-2 Cellular and Molecular Life Sciences

ORIGINAL ARTICLE

Targeted disruption of galectin 3 in mice delays the first wave

of spermatogenesis and increases germ cell apoptosis
Tao Lei1,8 · Sandra M. Blois2,3 · Nancy Freitag2,3,4 · Martin Bergmann5 · Sudhanshu Bhushan1 · Eva Wahle1 ·
Annie Chi‑Chun Huang6 · Hung‑Lin Chen6 · Michaela F. Hartmann7 · Stefan A. Wudy7 · Fu‑Tong Liu6 ·
Andreas Meinhardt1 · Monika Fijak1

Received: 15 October 2020 / Revised: 22 December 2020 / Accepted: 6 January 2021 / Published online: 28 January 2021
© The Author(s), under exclusive licence to Springer Nature Switzerland AG part of Springer Nature 2021

Abstract
Galectin 3 is a multifunctional lectin implicated in cellular proliferation, differentiation, adhesion, and apoptosis. This lectin is
broadly expressed in testicular somatic cells and germ cells, and is upregulated during testicular development. Since the role
of galectin 3 in testicular function remains elusive, we aimed to characterize the role of galectin 3 in testicular physiology. We
found that galectin 3 transgenic mice (Lgals3−/−) exhibited significantly decreased testicular weight in adulthood compared
to controls. The transgenic mice also exhibited a delay to the first wave of spermatogenesis, a decrease in the number of germ
cells at postnatal day 5 (P5) and P15, and defective Sertoli cell maturation. Mechanistically, we found that Insulin-like-3 (a
Leydig cell marker) and enzymes involved in steroid biosynthesis were significantly upregulated in adult Lgals3−/− testes.
These observations were accompanied by increased serum testosterone levels. To determine the underlying causes of the
testicular atrophy, we monitored cellular apoptosis. Indeed, adult Lgals3−/− testicular cells exhibited an elevated apoptosis
rate that is likely driven by downregulated Bcl-2 and upregulated Bax and Bak expression, molecules responsible for live/
death cell balance. Moreover, the percentage of testicular macrophages within C ­ D45+ cells was decreased in Lgals3−/− mice.
These data suggest that galectin 3 regulates spermatogenesis initiation and Sertoli cell maturation in part, by preventing
germ cells from undergoing apoptosis and regulating testosterone biosynthesis. Going forward, understanding the role of
galectin 3 in testicular physiology will add important insights into the factors governing the development of germ cells and
steroidogenesis and delineate novel biomarkers of testicular function.

Keywords Galectin 3 · Testis · Spermatogenesis · Sertoli cell maturation · Leydig cells · Apoptosis

Supplementary Information The online version contains

supplementary material available at https​://doi.org/10.1007/s0001​
8-021-03757​-2.

* Monika Fijak Berlin, Corporate Member of Freie Universität Berlin,

monika.fijak@anatomie.med.uni‑giessen.de Humboldt Universität zu Berlin, Berlin, Germany
5
1 Institute of Veterinary Anatomy, Histology, and Embryology,
Department of Anatomy and Cell Biology, Justus-Liebig-
Justus-Liebig-University of Giessen, Giessen, Germany
University of Giessen, Aulweg 123, 35385 Giessen,
6
Germany Institute of Biomedical Sciences, Academia Sinica, Taipei,
2 Taiwan
Department of Obstetrics and Fetal Medicine, AG
7
Glycoimmunology, University Medical Center Steroid Research and Mass Spectrometry Unit, Pediatric
Hamburg-Eppendorf, Martinistrasse 52, 20251 Hamburg, Endocrinology and Diabetology, Center of Child
Germany and Adolescent Medicine, Justus-Liebig-University
3 of Giessen, Giessen, Germany
Experimental and Clinical Research Center, A Cooperation
8
between the Max Delbrück Center for Molecular Present Address: Department of Obstetrics and Gynecology,
Medicine in the Helmholtz Association, The Charité Union Hospital, Tongji Medical College, Huazhong
Universitätsmedizin Berlin, Berlin, Germany University of Science and Technology, Wuhan, China
4
Division of General Internal and Psychosomatic Medicine,
Berlin Institute of Health, Charité Universitätsmedizin

13
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3622
Introduction
Mammalian spermatogenesis is a complex process com-
prised of pre-meiotic, meiotic, and spermiogenic stages
[1–3]. During the pre-meiotic stage, spermatogonia prolif-
erate and differentiate by mitosis before transforming into
meiotic spermatocytes [2]. Meiosis is a unique process of
cell division, taking place only in germ cells and leading
to the formation of haploid gametes [4]. After two rounds
of cell division, the spermatocytes form round spermatids,
which later—after undergoing several morphological and
structural changes—transform into elongated spermatids
[3, 5]. In mice, the first wave of spermatogenesis is ini-
tiated in a few days after birth, and then continues in a
synchronized manner, up to post-natal day (P) 35 [6, 7].
Germ cell types arise in a sequential pattern during the
first wave of spermatogenesis in all cords [7]. At P5 the
seminiferous tubules are mainly comprised of spermato-
gonia and immature Sertoli cells [8]. Then, early sper-
matocytes emerge at P9, pachytene spermatocytes at P14
as meiosis is initiated, and round spermatids at P20 [9].
During this period, the Sertoli cells mature, morphologi-
cally diversify and become functional—as evidenced by
the secretion of testicular fluid to the lumen [10, 11]. The
precise regulation of the proliferation and differentiation
of these somatic and germ cells during the first wave of
spermatogenesis is essential to ensuring normal fertility.
Galectin 3 is a 30-kDa lectin that binds to β-galactoside
[12]. Galectin 3 is involved in various cellular processes,
including proliferation, differentiation, macrophage activa-
tion, apoptosis regulation, and angiogenesis. Functionally,
galectin 3 is best characterized for its role in driving inflam-
mation, fibrosis, and immune responses [13–15]. This lectin
is widely expressed in different tissues and developmentally
regulated. In human, murine and porcine testes galectin 3
expression is up-regulated during testicular development
[16–18]. Galectin 3 is expressed in the testis and epididymis,
in ejaculated spermatozoa and extracellular vesicles in
human seminal plasma, which serve as vehicles to transfer
the lectin to the sperm surface during post-testicular matu-
ration. Sperm-bound galectin-3 then plays a critical role in
spermatozoa—zona pellucida binding [16, 19–21].
Furthermore, galectin 3 expression can be upregulated
upon treating cultured Sertoli cells with follicle stimulat-
ing hormone (FSH) or epidermal growth factor (EGF) [16].
Galectin 3 expression also increases in the atrophied semi-
niferous tubules after vasovasostomy in mice, which could
result from testicular inflammation [17]. Indeed, we have
observed galectin 3 upregulation in rat testes from animals
with testicular inflammation (unpublished observations).
In a broader context of reproduction galectin 3 play
a role in embryo implantation, immune regulation,
13
T. Lei et al.
trophoblast-matrix interaction and in placental angiogen-
esis [22]. Deficiency in galectin 3 during murine preg-
nancy leads to placental dysfunction, higher abortion rate
and fetal growth restriction [23].
Since galectin 3 is often secreted into biological fluids,
its role as an important biomarker of heart or autoimmune
diseases is frequently discussed [24].
Taken together, it seems that galectin 3 is an important
lectin involved in testicular development and inflammation.
Yet despite these observations, the testicular function of
galectin 3 is unknown. In this study, we aimed to investigate
the influence of galectin 3 depletion on key parameters of
testicular function such as germ cell proliferation, differen-
tiation and apoptosis; Sertoli cell maturation; Leydig cell
steroidogenesis; and testicular macrophage numbers using
whole body galectin 3 knockout mice Lgals3−/−.
Materials and methods
Animals
Homozygous galectin-3 knockout mice (Lgals3−/−) were
bred on a C57/BL6 genetic background, as described previ-
ously [25]. Both Lgals3−/− and Lgals3+/+ mice were main-
tained under standard specific-pathogen-free conditions.
Organ collection in this study was approved by the local
Animal Ethics Committee (Regierungspräsidium Giessen
GI Nr. 646_M). For euthanasia, animals were deeply anaes-
thetized by inhalation of 5% isoflurane and sacrificed by
cervical dislocation.
Testes were collected from neonatal (P5), pre-pubertal
(P15), pubertal (P20), and adult (P35, P50, P100) mice.
Both testes were removed, weighed, and either snap frozen
in liquid nitrogen for RNA extraction and protein isolation
or fixed in Bouin’s solution for paraffin embedding. Paraffin
tissue Sects. (5 µm) were subjected to histological analysis
(hematoxylin–eosin staining) and immunostaining.
To measure steroid hormone concentrations, serum sam-
ples were collected from mice at P50.
Gas chromatography tandem mass spectrometry
(GC–MS/MS) measurement of steroid hormones
Up to 100 µL serum was spiked with a cocktail of inter-
nal standards containing [16,16,17-2H3] testosterone (d3-
T), [16,16,17-2H3] 5α-androstanediol-3α,17β (d3-AD),
[16,16,17-2H3] dihydrotestosterone (d3-DHT) [26]. After
equilibration, the samples were extracted with ethyl ace-
tate and then purified by gel chromatography on Sephadex
LH-20 mini columns. Heptafluorobutyric anhydride (Sigma-
Aldrich) was used for derivatization. Gas chromatography
was performed on an Optima® 1-MS capillary column
Targeted disruption of galectin 3 in mice delays the first wave of spermatogenesis and increases… 3623

(25 m × 0.2 mm I.D., df 0.1 µm, Macherey–Nagel, Düren,
Germany) housed in a Thermo Scientific Trace 1310 Gas
Chromatograph with a TriPlus RSH Autosampler cou-
pled to a TSQ 800 triple quadrupole MS (Thermo Scien-
tific, Dreieich, Germany). Helium (1.0 mL/min) was used
as the carrier gas. The steroids of interest were eluted at
a rate of 3 °C/min until the column temperature reached
250 °C. Quantification was performed in the multiple reac-
tion monitoring mode (MRM). The following MRM or
m/z ratios were measured for the analytes and their corre-
sponding internal standards: m/z 665.1 (668.1) for T (d3-
T), m/z 455.3/241.3 (458.3/244.4) for AD (d3-AD) and m/z
414.1/185.2 (417.2/188.2) for DHT (d3-DHT). The limits
of the quantifications were 0.1 ng/ml for T and AD, and
0.03 ng/ml for DHT.
Immunofluorescence staining
Testicular paraffin-embedded sections were dewaxed in
xylene, rehydrated in decreasing concentrations of ethanol,
and boiled for antigen retrieval for 20 min in 10 mM sodium
citrate buffer. After blocking with 5% goat serum in TBS
containing 0.1% Tween 20 (TBST), the sections were incu-
bated overnight with the indicated primary antibody at 4 ℃
(Table 1). The sections were then washed in TBST, and incu-
bated with the appropriate secondary antibody at room tem-
perature (RT) for 1 h. The nuclei were counterstained with
Topro 3 (Life Technologies, Darmstadt, Germany). Images
were captured under a conventional fluorescence (Axioplan
2 Zeiss microscope, Carl Zeiss, Göttingen, Germany) and
confocal (Leica TCS2, Wetzlar, Germany) microscope.
Histology, immunohistochemistry,
and morphometry
Tissue sections were deparaffinized in xylene, hydrated in
serial dilutions of ethanol, and treated for antigen retrieval
by boiling for 20 min in 10 mM sodium citrate buffer. To
block endogenous peroxidase activity, the sections were
incubated in 1.2% ­H2O2 in methanol for 30 min at RT.
Then, the sections were blocked with 5% goat serum (Vec-
tor Laboratories, Burlingame, USA) in TBST, and incubated
with the indicated primary antibody (Table 1) overnight at
4 °C. An Envision System (DAKO, Hamburg, Germany) for
PCNA or Vectastain ABC Elite Kit (Vector Laboratories)
for HSD11b1 was used to detect primary antibody bind-
ing, according to the manufacturer`s instructions, combined
with DAB staining. The Sects. (5 µm) were stained with
hematoxylin and eosin (H&E) and evaluated as previously
described [1].
For comparative analyses, age-matched animals were
chosen. Sections were always processed simultaneously. All
images were obtained under a Leica DM LB microscope
(Leica, Wetzlar, Germany).
Quantitative microscopic analyses were performed using
a Leica DM LB microscope (Leica, Wetzlar, Germany).
For each mouse, 100 seminiferous tubules in Lgals3−/− and
Lgals3+/+ testicular sections at P15 and P20 were analyzed.
Each tubule was classified according to lumen formation and

Table 1  Detailed list of antibodies and antibody dilutions used in this study
Primary antibodies Manufacturer Catalogue no Dilution

Rabbit monoclonal anti-Bak Cell signaling technology, Germany 12,105 1:1000*

Rabbit polyclonal anti-Bax Cell signaling technology, Germany 2772 1:1000*
Rabbit monoclonal anti-Bcl-2 Cell signaling technology, Germany 2870 1:1000*
Mouse monoclonal anti-β-actin Ac-15 Sigma-Aldrich, Germany A5441 1:5000*
Rabbit monoclonal anti-galectin 3 GeneTex, USA GTX62084 1:500**
Rat monoclonal anti-germ cell specific antigen (TRA98) Abcam, UK ab82527 1:1000**
Rabbit polyclonal anti-HSD11B1 Abcam, UK ab39364 1:100**
Rabbit polyclonal anti-Insl3 Biorbyt Ltd, UK orb648755 1:100*
Rabbit polyclonal anti-PCNA Abcam, UK ab18197 1:500**
Rabbit polyclonal anti-phospho-histone H3 (Ser10) Cell signaling technology, Germany 9701 1:200**
Rabbit monoclonal anti-Sox9 Abcam, UK ab185230 1:250**
Rabbit monoclonal anti—StAR Cell signaling technology, Germany 8449 1:1000*
Secondary antibodies Manufacturer Catalogue No Dilution
Alexa Fluor 448 goat anti-rabbit IgG Thermo fisher scientific, USA A11008 1:1000**
Alexa Fluor 546 goat anti-rat IgG Thermo fisher scientific, USA A11081 1:1000**
HRP-labelled goat anti-rabbit IgG MP biomedicals, USA 855,676 1:10,000*
HRP-labelled sheep anti-mouse IgG Sigma-Aldrich, Germany A5906 1:10,000*

Antibody dilution used for *Western blotting or **Immunofluorescence/Immunohistochemistry

13
3624 T. Lei et al.

the most differentiated germ cell stage it contained (Sper—
spermatogonia, L/Z—leptotene/zygotene spermatocyte,
P—pachytene spermatocyte, RS—round spermatid). The
percentage of seminiferous tubules of each class was calcu-
lated for each mouse and then plotted against age.
TUNEL assay
Apoptosis was quantified by the TUNEL method (ApopTag
7100 Kit, Chemicon, Hofheim, Germany) according to the
manufacturer’s instructions. Counterstaining was carried
out with hematoxylin. Counting was performed in a blinded
fashion. All tubules of one testicle cross-section were ana-
lyzed by counting the total number of tubules as well as
the total number of tubules with at least one TUNEL-posi-
tive nucleus. Results are presented as percentage (positive
tubules/all tubules × 100) (n = 3 per group).
Western blotting
Testes or cells were homogenized in ice-cold RIPA buffer
(50 mM Tris pH 8.0, 1% Nonidet P 40, 0.5% deoxycho-
late, 0.1% SDS, 150 mM NaCl) containing a 1% protease
inhibitor cocktail (Sigma-Aldrich, Steinheim, Germany).
Equal amounts of protein were separated by 10% SDS–pol-
yacrylamide gel electrophoresis and electro-blotted onto
nitrocellulose membranes (GE Healthcare, Freiburg, Ger-
many). The efficiency of the transfer was monitored by
Ponceau S staining. The membranes were blocked with 5%
BSA in TBST for 1 h before incubation overnight with the
appropriate primary antibody (Table 1). Then, the mem-
branes were washed and incubated with the appropriate
secondary HRP-conjugated antibody (Table 1). The sig-
nals were visualized using ECL (Millipore Corporation,
Billerica, USA) and analyzed by densitometry (Fusion FX,
Witec AG, Luzern, Switzerland).
RNA extraction and quantitative PCR
Total RNA was isolated using an RNeasy Mini kit (Qiagen,
Hilden, Germany) according to the manufacturer’s instruc-
tions. Quantitative PCR was performed on a CFX96 Touch
thermal cycler (Bio-Rad, Munich, Germany) using iTaq Uni-
versal SYBR Green Supermix (Bio-Rad, Munich, Germany).
The primer sequences and amplicon sizes are provided in
Table 2. Relative gene expression was calculated by using
the ΔΔCt method [27].

Table 2  List of PCR primers Gene Primer (5` → 3`) Entrez gene ID Amplicon
used in this study size (bp)

Actb F: TGA​CAG​GAT​GCA​GAA​GGA​GAT​ 11,461 156

R: TAC​TCC​TGC​TTG​CTG​ATC​CAC​
Amh F: CGA​GCT​CTT​GCT​GAA​GTT​CC 11,705 302
R: TGA​AAC​AGC​GGG​AAT​CAG​AG
Cyp11a1 F: CCA​GTG​TCC​CCA​TGC​TCA​AC 13,070 74
R: TGC​ATG​GTC​CTT​CCA​GGT​CT
Cyp17a1 F: GCC​CAA​GTC​AAA​GAC​ACC​TAAT​ 13,074 159
R: GTA​CCC​AGG​CGA​AGA​GAA​TA
Ddx4 F: GGT​CCA​AAA​GTG​ACA​TAT​ATA​CCC​ 13,206 140
R: TTG​GTT​GAT​CAG​TTC​TCG​AGT​
Gapdh F: TGA​CGT​GCC​GCC​TGG​AGA​AA 14,433 98
R: AGT​GTA​GCC​CAA​GAT​GCC​CTT​CAG​
Hprt F: CTG​GTG​AAA​AGG​ACCTC​ 15,452 110
R: CTG​AAG​TAC​TCA​TTA​TAG​TCAAG​
Hsd3b1 F: TGG​ACA​AAG​TAT​TCC​GAC​CAGA​ 15,492 250
R: GGC​ACA​CTT​GCT​TGA​ACA​CAG​
Insl3 F: TGC​AGT​GGC​TAG​AGC​AGA​GA 16,336 151
R: GTG​CAG​CCA​GTA​AGA​CAG​CA
Lgals3 F: GAT​CAC​AAT​CAT​GGG​CAC​AG 16,854 100
R: ATT​GAA​GCG​GGG​GTT​AAA​GT
Star F: TGC​CCA​TCA​TTT​CAT​TCA​TCCTT​ 76,205 232
R: AAA​AGC​GGT​TTC​TCA​CTC​TCC​
Wt1 F: ACC​CAG​GCT​GCA​ATA​AGA​GA 22,431 230
R: CCT​GGT​GTG​GGT​CTT​CAG​AT

13
Targeted disruption of galectin 3 in mice delays the first wave of spermatogenesis and increases…
Transfection of MLTC‑1 cell line with Lgals3 siRNA
A mouse Leydig tumor cell line (MLTC-1) was cultured
in ATCC-formulated RPMI-1640 (Gibco, Darmstadt, Ger-
many) supplemented with 10% fetal bovine serum, 100 U/
mL penicillin, and 0.1 mg/mL streptomycin. Cell cultures
were maintained in a 5% ­CO2 humidified atmosphere at
37 °C. The cell line was kindly provided by Prof. Thomas
Linn and Dr. Qingkui Jiang (Clinical Research Unit, Cen-
tre of Internal Medicine, Justus-Liebig-University, Giessen,
Germany). A commercially available, pre-designed short
interfering RNA (siRNA) against Lgals3 and Silencer®
select negative control siRNA was purchased from Life
Technologies GmbH (Darmstadt, Germany). Transient trans-
fection was performed using L ­ ipofectamine® RNAiMAX
Transfection Reagent (Thermo Fisher Scientific, Waltham,
USA), according to the manufacturer’s instructions. Briefly,
1 × 105 MLTC-1 cells were seeded in a 12-well plate and
transfected at 60–80% confluency with 15 pmol siRNA and
4.5 µl Lipofectamine® RNAiMAX per well. After 48 h incu-
bation, the cells were treated with 1 IU/ml hCG (Predalon
5000 I.E. Organon, Oberschleissheim, Germany) for 24 h.
The supernatants and cell lysates were collected to measure
testosterone levels and Western blot analyses, respectively.
Testosterone measurement
The levels of testosterone in the cell culture supernatants
were measured using a testosterone ELISA kit (IBL Interna-
tional, Hamburg, Germany), according to the manufacturer’s
protocol.
Flow cytometry
Decapsulated testes were incubated with 1.2 mg/ml col-
lagenase A and 15 U/ml DNase (Roche Diagnostics, Man-
nheim, Germany) in PBS in a shaking water bath at 34 °C
for 15 min. The enzymes were inactivated by adding ice-cold
PBS, and the tubule fragments were allowed to settle for
4 min. Then, the supernatant was filtered and centrifuged at
300 × g for 10 min at 4 °C. The pellet was washed with PBS
and the erythrocytes were depleted by osmotic lysis using
red blood cell lysis buffer (Qiagen, Hilden, Germany) for
5 min at RT. The final cell suspension was washed in wash-
ing buffer (0.5% BSA and 2 mM EDTA in PBS) and then
processed directly for flow cytometric macrophage staining.
Interstitial cells (1 × 106) were incubated with the following
fluorochrome conjugated recombinant REAfinity antibod-
ies: CD45 -VioGreen (clone REA737), CD11b -VioBright
FITC (clone REA592), Ly6C -PE (clone REA796), and
F4/80 -APC (clone REA126) (all from Miltenyi Biotech,
Bergisch-Gladbach, Germany). Background staining was
evaluated using the appropriate REA control antibodies
3625
(clone REA293; Miltenyi Biotech). Finally, the cells were
washed in washing buffer. Data were collected for 700,000
events using a MACSQuant Analyzer 10 flow cytometer
and analyzed using MACSQuantify software version 2.11
(Miltenyi Biotech).
Statistical analyses
The data represent the means ± SEM. Comparisons between
two groups were made by two-tailed t test. P values < 0.05
were considered statistically significant. All tests were per-
formed using GraphPad Prism 5 software (GraphPad Soft-
ware, San Diego, USA).
Results
Loss of galectin 3 delays the first wave
of spermatogenesis in Lgals3−/− mice
To investigate the effects of endogenous galectin 3 on tes-
ticular development, we harnessed Lgals3−/− mice [25].
These mice have been previously extensively studied in the
context of fibrosis, inflammation, heart failure, atheroscle-
rosis or cancer but not for the effects of galectin 3 deficiency
on male reproduction and fertility [24]. In our first analy-
ses, testis weights were determined from mice at different
time points after birth and in adulthood. Testicular weights
increased progressively with age in mice of all genotypes
but were significantly decreased in adult Lgals3 −/− mice
(P50 and P100) compared to age-matched Lgals3+/+ con-
trols (Fig. 1a).
To characterize testicular development and the onset of
spermatogenesis in Lgals3−/− testes, we performed a his-
tological analysis of the testes at P5, P15 and P20. At P5,
the seminiferous tubules from Lgals3+/+ and Lgals3−/− tes-
tes were mainly composed of Sertoli cells and spermato-
gonia (Fig. 1b i–ii). We also observed gonocytes (G) in
both genotypes (Fig. 1b i–ii). By P15, meiosis progressed
such that pachytene spermatocytes (P) were now present
in the Lgals3+/+ testes. However, most of the differentiated
germ cells in Lgals3−/− mice were composed of zygotene
spermatocytes (Z), with numerous tubules containing only
spermatogonia (Fig. 1b iii–iv). Finally, by P20, we observed
signs that the next step of spermatogenesis had occurred
in Lgals3+/+ mice, as evidenced by the presence of round
spermatids (RS) in the seminiferous tubules (Fig. 1b v)
indicating complete passage through meiosis. By contrast,
most tubules in age-matched Lgals3−/− testes contained only
pachytene spermatocytes, a mid-meiotic stage (Fig. 1b vi)
As such, it seems that loss of galectin 3 delays the first wave
of spermatogenesis in Lgals3−/− mice.
13
3626
Fig. 1  The first wave of spermatogenesis is delayed in Lgals3−/−
mice. a Testicular weight (mg) collected from Lgals3+/+ and
Lgals3−/− mice at postnatal (P) days 5, 15, 20, 35, 50 and 100;
(n = 4, * P < 0.05). b Representative histology of mouse testes from
Lgals3+/+ (i, iii, v) and Lgals3−/− mice (ii, iv, vi) at P5 (i, ii), P15 (iii,
iv) and P20 (v, vi). (C and D) The percentage of tubule cross sections
To confirm these findings, we aimed to quantify sper-
matogenesis in testes from Lgals3+/+ and Lgals3−/− animals
at P15 and P20 (Fig. 1c, d). To do so, we determined the
percentage of seminiferous tubules containing the most
advanced stage of spermatogenesis in each section. At P15,
we found that the percentage of tubules containing pachy-
tene spermatocytes was significantly lower in Lgals3−/− tes-
tes as compared to Lgals3+/+ testes (9% vs. 23%, respec-
tively; Fig. 1c). Subsequently, at P20, the percentage of
tubules containing round spermatids and pachytene sper-
matocytes in Lgals3−/− testes was also significantly lower
compared with Lgals3+/+ testes (3.6% vs. 17% and 53% vs.
82%, respectively; Fig. 1d). Conversely, the percentage of
13
T. Lei et al.
was quantified according to the most differentiated germ cell stage
visible (G gonocytes, L leptotene spermatocyte, P pachytene sper-
matocyte, RS round spermatid, S Sertoli cell, Sper spermatogonia,
Z zygotene spermatocyte) at P15 (c) and P20 (d); (n = 3, *P < 0.05,
**P < 0.01, ***P < 0.001). The data represent the means ± SEM
tubules containing less-differentiated stages of spermato-
genesis (such as leptotene/zygotene spermatocytes or sper-
matogonia) was increased in Lgals3−/− testes compared to
Lgals3+/+ testes (14% vs. 3.7% and 18% vs. 0%, respectively;
Fig. 1d). Taken together, these data illustrate that the first
wave of spermatogenesis in Lgals3−/− testes is delayed.
Germ cell number is reduced in neonatal Lgals3−/−
testes, independently of cellular proliferation
We next wanted to investigate the underlying causes of
this apparent delay of the first wave of spermatogenesis in
Lgals3−/− testes. To do so, we assayed germ cell numbers
Targeted disruption of galectin 3 in mice delays the first wave of spermatogenesis and increases…
and germ and somatic cell proliferation by immunostaining
with the germ cell marker Tra98 and the mitosis marker
phospho-histone H3 Ser10 (PH3) during early postnatal tes-
tis development. We found that Lgals3−/− testes had nearly
a twofold lower number of germ cells at P5 compared to
Lgals3+/+ control testes (Fig. 2a–g).
We also monitored Ddx4 (VASA) expression, which
serves as a germ cell marker during germ cell differen-
tiation [28]. Here we found that Ddx4 mRNA expression
was downregulated in neonatal (P5) and prepubescent
(P15) Lgals3−/− testes compared to control Lgals3+/+ tes-
tes (Fig. 2h), which strongly supports a reduction in germ
cell numbers in Lgals3−/− testes. By contrast, the number of
dividing germ cells (PH3- and Tra98-positive) and somatic
cells (PH3-positive) in Lgals3−/− testes was unaffected
(Fig. 2f, g).
Fig. 2  Testicular germ cell numbers are reduced in neonatal
Lgals3−/− testes. a–d Identification of germ cell numbers in Lgals3+/+
and Lgals3−/− testes using Tra98 immunofluorescence staining (red)
at P5 (a–d). Phosphorylated histone 3 staining (PH3, green; b, c, d)
and Tra98 staining were used to distinguish between dividing germ
and somatic cells (a, c, d). Part d shows the magnified areas iden-
tified by the white boxes in part (c). The thin arrow indicates germ
cells; the thick arrow indicates somatic cells, both positive for PH3
3627
Next we studied the expression of proliferating cell
nuclear antigen (PCNA)—a marker of cell proliferation.
At P5, PCNA staining revealed no overt differences in
the proliferation of testicular cells between Lgals3+/+ and
Lgals3−/− mice (Supplementary Fig. 1). By contrast, several
seminiferous tubules were found without proliferating cells
in Lgals3−/− testes at P15 and P20 (Supplementary Fig. 1).
Together, these findings point to a reduced number of gono-
cytes and spermatogonia that is independent of proliferation,
in the neonatal Lgals3−/− testis.
Germ cells are less frequent in the seminiferous
tubules of neonatal and pubertal ­Lgals3−/− testes
The PCNA staining results described above showed in sev-
eral tubules no sign of cell proliferation in P15 and P20
(a cell proliferation marker). Nuclei were counterstained with Topro
3 (blue). e–g The numbers of germ cells (e), PH3-positive germ cells
(f) and somatic cells (g) were normalized to the total cross-section
area ­(mm2) in both Lgals3+/+ and Lgals3−/− testes. h Ddx4 mRNA
expression (germ cell marker) of P5 and P15 testes was normal-
ized to Actb and Gapdh; (n = 3–4, *P < 0.05). The data represent the
means ± SEM
13
3628
Lgals3−/− testes. To decipher a possible influence of endog-
enous galectin 3 on germ cell development, we stained testes
from neonatal (P5), pre-pubertal (P15) and pubertal (P20)
Lgals3+/+ and Lgals3−/− mice with the Sertoli cell marker
Sox9 and the germ cell marker Tra98. Our quantitative
analyses revealed that in P5 Lgals3−/− testes, the number of
seminiferous tubules without germ cells was significantly
increased as compared to Lgals3+/+ testes (46% vs. 13%,
respectively). In Lgals3+/+ testes some tubules were entirely
free of germ cells (Fig. 3a–e), an observation not seen from
P15 onwards when all seminiferous tubules showed germ
cells. Conversely, 8% of tubules at P15 and ~ 15% of tubules
at P20 in Lgals3−/− mice had no germ cells (Fig. 3e). By
P35, however, all seminiferous tubules in Lgals3−/− tes-
tes contained germ cells. These results indicate that the
number of seminiferous tubules containing germ cells in
Lgals3−/− mice is reduced during the neonatal and prepu-
bescent periods.
Lgals3−/− testes display defects in Sertoli cell
maturation
Sertoli cell maturation and concomitant fluid secretion
results in the formation of the lumen of the seminiferous
tubules during testicular development [29]. To characterize
the effects of Lgals3 gene disruption on Sertoli cell matura-
tion, we analyzed lumen formation at P15 and performed a
quantitative analysis of the Sertoli cell maturation marker
anti-Müllerian hormone (Amh) [11]. The percentage of
tubules containing a lumen in Lgals3−/− testes was two-fold
lower compared to in Lgals3+/+ control tubules (Fig. 3f–g).
Moreover, our results showed that Amh mRNA was highly
expressed in the neonatal period (P5) in testes from both
Lgals3+/+ and Lgals−/− mice (Fig. 3h) and then decreased at
P15 before reaching extremely low levels at P20 and P35 in
both strains. These findings confirm the progressive matura-
tion process of Sertoli cells (Fig. 3h). Interestingly, although
slightly decreased compared to P5, the levels of Amh mRNA
expression in Lgals3−/− testes at P15 were three-fold higher
as compared to Lgals3+/+ control testes, indicating a delay to
Sertoli cell maturation in Lgals3−/− testes (Fig. 3h).
We next studied the effects of galectin 3 on Sertoli cell
number, and the ratio between Sertoli and germ cells in the
developing testes. To do so, we performed an immunostain-
ing using Sox9 and Tra98 antibodies at P15. Although not
quantified, the immunostaining revealed no obvious differ-
ence in Sox9 expression between Lgals3−/− and Lgals3+/+
testes at P15 (Supplementary Fig. 2).
Wilms’ tumor gene (Wt1) is considered a stable marker
of Sertoli cells during testicular development [11]. Our
analyses of Wt1 mRNA expression showed that the expres-
sion levels in Lgals3−/− testes were comparable to those in
Lgals3+/+ testes at P15 (Supplementary Fig. 2). This finding
13
T. Lei et al.
suggests that the number of Sertoli cells is unaffected in
Lgals3−/− mice. As such, while Lgals3−/− testes display
altered Sertoli cell maturation, the overall number of Sertoli
cells is unaffected.
Lgals3−/− testicular cells exhibit an increased rate
of apoptosis accompanied by mild atrophy
To determine the underlying causes of testicular atrophy
in adult Lgals3−/− mice, we performed TUNEL staining
to detect the level of cellular apoptosis. Quantifications of
TUNEL-positive cells revealed higher numbers of apoptotic
cells in adult Lgals3−/− testes compared to Lgals3+/+ testes at
P5, P35, P50 and P100 (Fig. 4a–c). Due to the fact that ~ 15%
of tubules at P20 in Lgals3−/− mice contained no germ cells
analysis of TUNEL positive cells had a bias and was conse-
quently not included.
Galectin 3 can stabilize the mitochondrial membrane by
binding to the anti-apoptotic protein Bcl-2; it can also inhibit
cytochrome c release by blocking the activity of the pro-
apoptotic proteins Bak and Bax [30–32] (Fig. 4d). We thus
investigated the expression of these pro- and anti-apoptotic
proteins in Lgals3−/− and Lgals3+/+ testes at P50. Bcl-2 was
downregulated, while Bak and Bax were upregulated in
Lgals3−/− testes, as compared to Lgals3+/+ testes (Fig. 4e–h).
These data indicate that an increase in the number of apop-
totic cells in adult Lgals3−/− mouse testes results from an
imbalance in anti-/pro-apoptotic protein expression. This
perturbation might contribute to mild testicular atrophy in
Lgals3−/− mice.
Gonadal Insl3 and steroidogenic enzymes
and circulating testosterone levels are upregulated
in Lgals3−/− mice
Production of galectin 3 in granulosa cells is regulated by
luteotropic hormone (LH), which indicates an involvement
of galectin 3 in steroidogenesis [33]. Similar to granulosa
cells, the predominant function of testicular Leydig cells
is sex hormone production (testosterone) through a steroi-
dogenic process [34]. To investigate whether Lgals3 gene
ablation affects Leydig cell function, we analyzed the gene
and protein expression of Insulin-like factor 3 (Insl3) and
steroidogenic enzymes (Cyp11a1, Hsd3b1 and Cyp17a1) in
adult Lgals3−/− and Lgals3+/+ mice by quantitative RT-PCR
and Western blotting.
Compared to Lgals3+/+ mice, the Cyp11a1 and Hsd3b1
mRNA expression levels were significantly upregu-
lated in adult Lgals3−/− mice at P35 and P100 (Fig. 5a);
Cyp17a1 mRNA expression was significantly increased in
Lgals3−/− mice testes at P50 and P100 (Fig. 5a).
Targeted disruption of galectin 3 in mice delays the first wave of spermatogenesis and increases…
Fig. 3  Testes from Lgals3−/− mice exhibit a higher number of semi-
niferous tubules without germ cells and altered maturation of Sertoli
cells. a–d Representative immunofluorescent labeling of germ cells
with Tra98 (red; a, c, d) and Sertoli cells with Sox9 (green; b–d)
in testes from Lgals3+/+ and Lgals3−/− mice at P5. Part d shows the
magnified areas identified by the white boxes in part (c). The white
stars indicate the seminiferous tubules without germ cells. The enu-
meration of tubules containing germ cells was performed on P5, P15
and P20 (i). At least 100 seminiferous tubules were counted in each
testis; (n = 3, *P < 0.05). e Representative HE staining of testes from
Expression of Insl3 in adult testes is an indicator of the
number and differentiation status of the Leydig cells [35].
In Lgals3 −/− mice testes, Insl3 mRNA expression was
increased at P35, P50 and P100 (Fig. 5b). In agreement
with the mRNA data, Insl3 and the steroidogenic acute
3629
Lgals3+/+ and Lgals3−/− mice at P15. The lower panel (iii, iv, v, vi)
shows the magnified areas from (i) and (ii) containing seminiferous
tubules with (iii, v) or without (iv, vi) a lumen in Lgals3+/+ (i) and
Lgals3−/− (ii) testes, respectively. f–g The percentage of seminiferous
tubules without (f) and with (g) a lumen was quantified by counting at
least 100 seminiferous tubule cross sections in each testis. (H) Rela-
tive Amh mRNA expression in testes from Lgals3+/+ and Lgals3−/−
mice at P5, P15, P20 and P35 was normalized to Actb and Gapdh;
(n = 3, *P < 0.05, **P < 0.01). The data represent the means ± SEM
regulatory protein StAR were also elevated in Lgals3−/− tes-
tes compared to control Lgals3+/+ testes at P50 (Fig. 5c).
By using Leydig cell staining with an antibody against
11β-hydroxysteroid dehydrogenase type 1 (HSD11B1) no
13
3630
Fig. 4  Testicular cells in Lgals3−/− mice experience increased apop-
tosis due to an imbalance of anti- and pro-apoptotic proteins. a, b
TUNEL staining (arrows) of testicular sections from Lgals3+/+ mice
and Lgals3−/− mice at P50. c The numbers of apoptotic germ cells
were quantified in 100 tubular cross sections in testes from Lgals3+/+
and Lgals3−/− mice at P5, P15, P35, P50 and P100. d A scheme is
illustrating how galectin 3 can stabilize the mitochondrial membrane
differences in the cell numbers between transgenic and WT
animal groups at P50 were apparent (Supplementary Fig. 3).
Next, we investigated whether the increased expression of
steroidogenic enzymes affected the concentration of steroids
in the serum. To do so, we used a GC–MS/MS method to
measure testosterone, androstandiol-3a, 17b (AD) and dihy-
drotestosterone (DHT) levels in sera taken from Lgals3+/+
and Lgals3−/− mice. Here, testosterone and AD levels were
increased in adult Lgals3−/− mice as compared to Lgals3+/+
mice (Fig. 5d); no significant change was found in DHT sera
levels (Fig. 5d). Taken together, these results suggest that sex
hormone production is increased in Lgals3−/− mice.
Lgals3 depletion in cultured Leydig tumor cells
upregulates StAR and testosterone production
Our data thus far suggest that galectin 3 affects the steroido-
genic activity of Leydig cells. To understand how galectin 3
affects Leydig cell function, we determined the level of tes-
tosterone secretion in a cultured Leydig cell line (MLTC-1)
13
T. Lei et al.
by binding to Bcl-2 and inhibiting cytochrome c release. Bcl-2 inhib-
its activity of pro-apoptotic proteins Bak and Bax. Western blot e and
densitometric analysis of Bcl-2 f Bak g and Bax h expression in testes
from Lgals3+/+ and Lgals3−/− mice at P50 is shown; (n = 3, *P < 0.05;
**P < 0.01; ***P < 0.001). β-actin was used as a loading control. The
data represent the means ± SEM
in which we depleted Lgals3 with siRNA. The efficiency of
Lgals3 siRNA silencing is shown in Supplementary Fig. 4.
We stimulated testosterone production by incubating the
cells with hCG [36], and found that StAR protein levels
and testosterone secretion were upregulated in both Lgals3-
depleted and control cells (Fig. 5e–f). However, Lgals3
silencing led to a stronger increase in StAR expression and
testosterone production under hCG stimulation compared to
control cells (Fig. 5e–f). These results indicate an inhibitory
effect of endogenous galectin 3 on testicular steroidogenesis.
Loss of Lgals3 results in a depletion of testicular
macrophages
Galectin 3 is considered a chemoattractant for mac-
rophages under physiological conditions, and can also
accelerate M2 macrophage infiltration in tumor environ-
ments [37, 38]. Moreover, the macrophages possess a
unique function in the maintenance of testicular immune
privilege and support spermatogonial differentiation and
Targeted disruption of galectin 3 in mice delays the first wave of spermatogenesis and increases…
Fig. 5  Expression of steroidogenic enzymes and Insl3 is upregulated
in Lgals3−/− testes. a–b The relative mRNA expression of Cyp11a1,
Hsd3b1, Cyp17a1 (A) and Insl3 b in Lgals3+/+ and Lgals3−/− testes
was analyzed by real-time RT-PCR at P35, P50 and P100. The right
panel shows the main pathway of steroidogenesis in rodents, with
the corresponding enzymes highlighted by a dotted frame. c West-
ern blot analysis of StAR and Insl3 protein expression in testes from
Lgals3+/+ and Lgals3−/− mice at P50; (n = 3, *P < 0.05, ***P < 0.001,
****P < 0.0001). β-actin was used as a loading control. (D) Testoster-
Leydig cell steroidogenesis [39, 40]. Importantly for our
research, testicular macrophages express galectin 3 [18].
In our final analyses, we thus investigated whether the
macrophage population was affected in Lgals3−/− testes.
To do so, we performed flow cytometry using an extended
antibody panel (CD45, Ly6C, F4/80 and CD11b) to target
testicular macrophages (Supplementary Fig. 5). Within the
­ D45+ leukocytes, we defined res-
population of testicular C
ident macrophages as ­Ly6C−F4/80+CD11b+. The percent-
age of testicular macrophages was significantly decreased
3631
one, androstandiol-3a, 17b, and dihydrotestosterone (DHT) concen-
trations in the sera from Lgals3+/+ and Lgals3−/− mice at P50, deter-
mined by GC–MS/MS; (n = 3–4, *P < 0.05, **P < 0.01). e–f StAR
expression and testosterone levels in galectin 3-depleted MLTC-1
cells stimulated with 1 IU/ml hCG for 24 h. StAR protein expression
in the cell lysates e and the testosterone concentrations in conditioned
media (F) were determined by Western blotting and ELISA, respec-
tively. Representative data are shown; (n = 3–4, *P < 0.05, **P < 0.01,
***P < 0.001, ****P < 0.0001). The data represent the means ± SEM
by 11% in adult Lgals3 −/− mice compared to control
Lgals3+/+ (Fig. 6a–b). In sum, these data support that the
endogenous galectin 3 regulates the number of testicular
macrophages and their depletion may lead to disturbances
in the spermatogenesis (via affecting spermatogonia) and
steroidogenesis.
Overall, our results indicate that endogenous galectin
3 plays an important regulatory role in the development
of germ cells, maturation of Sertoli cells, steroidogenesis
13
3632
Fig. 6  The percentage of testicular macrophages in the population
of ­CD45+ leukocytes is decreased in Lgals3−/− mice. a–b Testicular
macrophage frequency was assessed by flow cytometry in Lgals3+/+
and maintenance of the macrophages niche, all comprising
essential testicular functions.
Discussion
Galectin 3 is widely expressed in testicular cells includ-
ing Sertoli, peritubular, Leydig, and germ cells and its
upregulation is associated with testicular maturation [16,
18]. However, the role of endogenous galectin 3 in male
gonadal development is unknown. Our results show for the
first time that mice lacking galectin 3 expression experience
a delay to the first wave of spermatogenesis, as evidenced by
a decrease in the number of germ cells during the neonatal
period and defects in Sertoli cell maturation. Moreover, we
found evidence for increased Leydig cell marker and steroi-
dogenic enzyme expression, elevated testosterone produc-
tion, an increased rate of germ cell apoptosis, and lower
numbers of testicular macrophages in adult Lgals3−/− testes.
Overall, these changes are accompanied by mild testicular
atrophy. Together, these findings all point to an important
role of endogenous galectin 3 in testicular physiology.
Our initial analyses identified a significant reduction in
testicular weight in adult Lgals3−/− mice compared to age-
matched WT controls. We propose that the delay to the first
wave of spermatogenesis in these transgenic animals may be
responsible for this decrease in organ mass. Similar observa-
tions have been shown for regulatory genes, such as Rhox13,
TR4, Rnf138, and Tap73 where a delay of the first wave
of spermatogenesis is accompanied by a reduced testicular
weight that persists into adulthood in mice [41–44].
In addition, Lgals3−/− mice produce lower numbers of
germ cells during the neonatal period. This is similar to mice
lacking a functional Taf4b gene that are also characterized
by reduced germ cell numbers during late embryogenesis
and the neonatal period, which subsequently led to a delay
to meiotic initiation [45]. Beside its influence on germ cell
development or spermatogenic differentiation, galectin 3
13
T. Lei et al.
and Lgals3−/− testes at P50. A representative dot plot a and the cor-
responding quantification b is shown; (n = 4–6, *P < 0.05). The data
represent the means ± SEM
seems to have a more complex multilayered influence on
reproduction. In this regard, in human seminal plasma
galectin 3 is associated with extracellular vesicles, which
are responsible for immunosuppression and regulation of
sperm function in the female reproductive tract [19]. Fur-
thermore, the acrosomal region of capacitated human sper-
matozoa shows strong galectin 3 labelling, which mediates
zona pellucida binding and affects fertilization in vitro [21].
Moreover, in our study we observed altered Sertoli cell
maturation kinetics in Lgals3−/− testes, whereby the lumen
formation and the loss of Amh mRNA expression was
delayed compared to WT testes. In maturating Sertoli cells
AMH expression is progressively reduced reaching almost
undetectable levels during transition to adulthood [46].
Galectin 3 expression in Sertoli cells is regulated by several
factors, including EGF, TNF, FSH, and adult germ cells,
which suggests that galectin 3 expression may affect Sertoli
cell function [16]. Our data support that loss of galectin 3 in
Sertoli cells could cause a delay to Sertoli cell maturation
during the neonatal period, followed by a decrease in the
number of germ cells. Abnormal development, proliferation
and/or maturation of Sertoli cells in early life could lead to
testicular dysfunction in adult life [11]. Such disruption in
the Sertoli cell maturation has been associated with testicu-
lar dysgenesis syndrome [11, 46]. Conversely, the absence of
germ cells in animal models and in human patients results in
a delay to Sertoli cell maturation, but has no effect on their
final maturation (reviewed in [11]).
We also observed an increase in the number of
tubules without germ cells in both neonatal and pubertal
Lgals3−/− testes, which mirrors the reduced number of
germ cells in the neonatal period. Chiarini-Garcia et al.
reported that undifferentiated spermatogonia can migrate
to ensure the uniform distribution of germ cell progeny at
the basement of the seminiferous tubules [47]. A study by
Jahnukainen et al. showed that after irradiation, spermato-
genesis can recover in single testicular lobules in primate
testes, indicating that spermatogenic stem cells are capable
Targeted disruption of galectin 3 in mice delays the first wave of spermatogenesis and increases…
of re-colonizing their respective loop of the seminiferous
tubules [48]. Therefore, we hypothesize that re-colonization
by undifferentiated spermatogonia may explain our obser-
vation that many tubules were seen without germ cells in
neonatal and prepubescent Lgals3−/− testes, while they were
eventually replenished in adulthood. However, testicular
weight is affected in a long term and persists into adulthood,
an observation that can be explained by the higher number
of apoptotic germ cell in Lgals3−/− testis.
In female mice, galectin 3 expression is increased dur-
ing luteolysis and is associated with a loss of progesterone
synthesis in vivo, which can be reversed by administration
of hCG [33]. In this line, galectin 3 is downregulated in
hCG-stimulated human granulosa cells [33]. In male mice,
galectin 3 is expressed in Leydig cells [49]. Interestingly, we
found an upregulated expression of the Leydig cell marker
Insl3 and steroidogenic enzymes (Cyp11a1, Hsd3b1 and
Cyp17a1) in adult Lgals3−/− testes with concomitant ele-
vated serum levels of testosterone and DHT. To determine
the relationship between galectin 3 and testosterone produc-
tion, we knocked down galectin 3 in the MLTC-1 Leydig
cell line using Lgals3 siRNA. Following hCG stimulation,
StAR expression and testosterone secretion were found
increased in these cells. These data indicate that Lgals3 has
an inhibitory effect on hCG-stimulated testosterone produc-
tion in Leydig cells, although in our study we cannot exclude
that high levels of LH may also play a role in the increased
androgen levels in Lgals3−/− mice.
We also revealed an increased number of apoptotic cells
in adult Lgals3−/− testes that is likely due to a perturbed
Bcl-2, Bax and Bak expression ratio. Data from previous
studies have suggested that galectin 3 can form heterodimers
with Bcl-2 and thus stabilize the mitochondrial membrane
[31, 50]. Bcl-2 also inhibits the oligomerization of Bax/Bak,
which is associated with mitochondrial membrane permea-
bilization and cell apoptosis [51, 52]. Diao et al. recently
reported that Lgals3 downregulation by siRNA in pituitary
tumor cells leads to an increase in Bax and caspase 3 expres-
sion, and thus an elevated apoptotic rate [53]. Moreover,
increased galectin 3 expression levels serve as a poor prog-
nosis marker in hepatocellular carcinoma, bladder cancer,
and pancreatic carcinoma — all related to the anti-apoptotic
properties of galectin 3 [54–58]. Thus, the increased num-
bers of apoptotic cells in Lgals3−/− testes in this study seems
to be the result of an imbalanced anti- and pro-apoptotic
protein expression due to the absence of endogenous galectin
3, an effect leading to testis shrinkage and lower number of
germ cells in adulthood.
Sano et al. found that galectin 3 acts as a chemoattractant
for macrophages under physiological conditions [38]. In con-
trast, the galectin 3 inhibitor lactose can restrain the infiltra-
tion of macrophages and neutrophils and induce pancreatic
edema in acute pancreatitis [59]. Here, we found a decreased
3633
percentage of testicular macrophages within the population
of ­CD45+ leukocytes in adult Lgals3−/− mice. Interestingly,
subpopulations of testicular macrophages called “peritubular
macrophages” due to their close proximity to peritubular
cells near the seminiferous tubule wall, express factors such
as CSF1 which are involved in spermatogonial proliferation
and differentiation, a finding that can explain reduced num-
bers of spermatogonia in macrophage depleted testis [39]. It
can therefore be hypothesized that the reduced numbers of
testicular macrophages in Lgals3−/− testis at least partially
contribute to the mild testicular atrophy seen in adulthood
in these mice. On the other hand, interstitial macrophages,
which are intimately connected to Leydig cells produce fac-
tors, e.g. 25-hydroxycholesterol, which could influence the
steroidogenesis of Leydig cells [60]. 25-hydroxycholesterol
from testicular macrophages is involved in the differentia-
tion of Leydig cells to fully functional steroidogenic cells
that occurs during post-natal maturation of the testis [61], a
process in which macrophage numbers increase [62]. Here,
we investigated the total number of macrophages in adult
testis without further division into subpopulations. How-
ever, based on our in vitro results using a galectin-3 depleted
MLTC-1 cell line, the increased expression of StAR protein
followed by an enhanced secretion of testosterone points
to a direct influence of this lectin on Leydig cell steroido-
genesis, independent of an interaction with macrophages.
Furthermore, the testosterone concentration in the serum
is increased in Lgals3−/− mice which also suggests a com-
pensatory mechanism to maintain normal spermatogenesis.
Furthermore, these findings are in line with other studies
demonstrating an increased expression of Star, Cyp11a1 and
Cyp11b1 in the thymus followed by higher levels of corticos-
terone in sera and thymus of galectin 3 deficient mice [63].
Critically, it needs to be noted that our approach to utilize
a whole body knockout of Lgals3−/−does not fully allow to
determine an impact of galectin 3 at the local testicular level.
Yet, the broad expression of galectin-3 in most testicular
cells would render it almost very difficult to deplete this
lectin from the male gonad using cell-specific knock outs.
In conclusion, our data show that Lgals3−/− mice expe-
rience a delay of the first wave of spermatogenesis. The
resulting impairment to testicular development ultimately
causes mild testicular atrophy due to an increased rate
of germ cell apoptosis and a decreased number of mac-
rophages in adult testes. Increased expression of steroi-
dogenic enzymes in Lgals3−/− testes, as well as functional
in vitro data indicate that galectin 3 is involved in andro-
gen biosynthesis. Our results thus underline an essential
role for galectin 3 in archetypical testicular functions.
Acknowledgements The authors would like to thank Prof. Ralf Mid-
dendorff and Sabine Tasch for providing the PCNA antibody and
acknowledge Prof. Thomas Linn and Dr. Qingkui Jiang for offering the
13
3634
MLTC-1 cell line. We are grateful to Yahia Almousa for assistance with
the fixation protocol and tissue collection. We appreciate the support
from our research assistants Petra Moschansky, Maria Daniltchenko
and Gudrun Koch.
Funding The financial support of the China Scholarship Council to Tao
Lei and of the Medical Faculty of Justus-Liebig University is gratefully
acknowledged. This work was supported by grants from the Deutsche
Forschungsgemeinschaft (DFG) BL1115/2-1 and Heisenberg Program
(BL1115/3-1-BL1115/7-1) to S.M.B.
Data availability The datasets generated during and analysed during
the current study are available from the corresponding author on rea-
sonable request.
Compliance with ethical standards
Conflict of interests The author(s) declare(s) that they have no com-
peting interests.
Ethics approval All procedures involving animals were carried out in
strict accordance with guidelines for care and use of experimental ani-
mals of the German law of welfare. Organ collection from mice was
approved by the local Animal Ethics Committee (Regierungspräsidium
Giessen GI Nr. 646_M).
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