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REPRODUCTION

REVIEW

Bloodtestis barrier and Sertoli cell function: lessons from


SCCx43KO mice
Jonathan Gerber, Julia Heinrich and Ralph Brehm
Institute of Anatomy, University of Veterinary Medicine Hannover, Foundation, Bischofsholer Damm 15, 30173
Hannover, Germany
Correspondence should be addressed to R Brehm; Email: Ralph.Brehm@tiho-hannover.de

Abstract
The gap junction protein connexin43 (CX43) plays a vital role in mammalian spermatogenesis by allowing for direct cytoplasmic
communication between neighbouring testicular cells. In addition, different publications suggest that CX43 in Sertoli cells (SC) might be
important for bloodtestis barrier (BTB) formation and BTB homeostasis. Thus, through the use of the Cre-LoxP recombination system,
a transgenic mouse line was developed in which only SC are deficient of the gap junction protein, alpha 1 (Gja1) gene. Gja1 codes for the
protein CX43. This transgenic mouse line has been commonly defined as the SC specific CX43 knockout (SCCx43KO) mouse line. Within
the seminiferous tubule, SC aid in spermatogenesis by nurturing germ cells and help them to proliferate and mature. Owing to the
absence of CX43 within the SC, homozygous KO mice are infertile, have reduced testis size, and mainly exhibit spermatogenesis arrest at
the level of spermatogonia, seminiferous tubules containing only SC (SC-only syndrome) and intratubular SC-clusters. Although the SC
specific KO of CX43 does not seem to have an adverse effect on BTB integrity, CX43 influences BTB composition as the expression pattern
of different BTB proteins (like OCCLUDIN, b-CATENIN, N-CADHERIN, and CLAUDIN11) is altered in mutant males. The supposed roles
of CX43 in dynamic BTB regulation, BTB assembly and/or disassembly and its possible interaction with other junctional proteins
composing this unique barrier are discussed. Data collectively indicate that CX43 might represent an important regulator of dynamic
BTB formation, composition and function.
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Sertoli cells
Within the seminiferous epithelium of the testis, somatic
Sertoli cells (SC) are known as the mother or nurturing
cells by aiding germ cells (GC) in their development,
proliferation and maturation during spermatogenesis.
Adult SC (Fig. 1) stretch from the basal lamina (BL) to the
lumen of the seminiferous epithelium. They separate the
seminiferous epithelium into a basal and an adluminal
compartment through the formation of the bloodtestis
barrier (BTB) that is located around the basal third of the
seminiferous tubule. This barrier is established through
tight, adherens and gap junction proteins that form the
so called SCSC junctional complex with gap junctions
being believed to play a vital role in its formation
and dynamic regulation (Pelletier 1995, Cyr et al. 1999,
Segretain et al. 2004, Carette et al. 2010, 2013, Mok
et al. 2011, Gerber et al. 2014, Jiang et al. 2014, Gerber
2015, Rode et al. 2015).
This SC formed BTB specifically prevents the intercellular diffusion of substances/molecules and is
essential for establishing a unique adluminal compartment. This barrier is also vital to protect haploid GC from
the innate immune system. Additionally, once the GC
q 2016 Society for Reproduction and Fertility
ISSN 14701626 (paper) 17417899 (online)

enter the adluminal compartment their nutritional


supply is cut off from the blood stream. Hence, these
GC-populations are then solely dependent upon the
nutritional supply from SC (Mruk & Cheng 2004,
Bergmann 2006, Cheng & Mruk 2012).
Fetal SC are accountable for sex determination during
testicular development through the expression and
synthesis of e.g. anti-Mullerian hormone (AMH) and
SOX9. In particular, SOX9 activates AMH, which is
responsible for the regression of the Mullerian ducts
during early stages of male development (Kent et al.
1996, De Santa Barbara et al. 1998, Graves 1998,
Frojdman et al. 2000, Hemendinger et al. 2002).
The differentiation and maturation of a pre-SC into an
adult SC may be called functional maturation (Sharpe
et al. 2003). SC transition from an immature cell to a
differentiated and functional adult cell occurs during
puberty. Nevertheless, it is uncertain if the functional
maturation of SC involves a rapid or gradual change
(Sharpe et al. 2003). The commencement of spermatogenesis is a possible source for final functional
maturation, but it is not vital. It has been shown in
both, animals and men, that the absence of GC does not
necessarily terminate SC maturation (Sharpe et al. 2003,
DOI: 10.1530/REP-15-0366
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J Gerber and others

Figure 1 Seminiferous epithelium of adult WT mice and BTB


composition. (A) Toluidine blue stained semi-thin section of an adult
WT testis. A nucleus of a SC (arrow) and GC in various stages of
spermatogenesis (arrowheads) are marked. Scale bar 25 mm.
(B) Schematic drawing of an adult SC with GC (arrowheads). The SC sits
on the basal lamina (BL) and stretches into the lumen of the seminiferous
tubule. The BTB (boxes) separates the seminiferous tubule into a basal
and adluminal compartment. The SC surrounds GC with its cell
membrane and aid in GC nourishment and migration towards the
lumen. Finally, the GC are released into the lumen (spermiation;
modified from Clermont (1993)). (C) The junctional complex at the
region of the BTB containing gap junctions (1), adherens junctions (2),
and tight junctions (3); modified from Pelletier & Byers (1992).

Brehm & Steger 2005). But the absenteeism of meiotic


and post meiotic GC has also been found to create
functional alterations of mature SC, which resemble
those of pre-SC (Jegou & Sharpe 1993, Sharpe et al.
1993, Boujrad et al. 1995, Guitton et al. 2000, Brehm &
Steger 2005).
Adult (mature) SC are sustentacular within the
seminiferous epithelium (Fig. 1). They provide an
extreme cytoplasmic ramification by surrounding the
adjacent developing GC. Furthermore, SC also aid in GC
migration towards the lumen since the GC themselves
lack specific architectural characteristics of migrating
cells (Bergmann 2006). In addition, adult SC are (so far)
thought to be post-mitotic cells, whose final total cell
number is dependent upon two proliferation periods.
Generally in mice, these initial proliferation time points
consist of the fetal, neonatal and peri-pubertal period
(Sharpe et al. 2003, Brehm & Steger 2005). Nevertheless,
little is known about the factors which constitute the
ultimate SC number, but the number is alleged to be
regulated by genes and hormones. In particular it is
known that the follicle-stimulating hormone (FSH)
stimulates SC proliferation (Jegou & Sharpe 1993,
Brehm & Steger 2005).

SC, BTB, and junctional proteins


In the testis, cellcell interactions involve physical and
dynamic organization mediated by distant signals
through hormones, secreted growth factors and signaling
molecules, as well as direct junctions. Within the
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seminiferous epithelium, communication and contact


is mainly mediated by the somatic SC (Byers et al. 1993,
Enders 1993, Jegou 1993, Russel 1993, Sharpe 1993,
Risley et al. 2002). For this purpose, mature SC form a
complex network of specific intercellular junctions with
individual adjacent GC populations and each other. This
SCSCjunctional complex constitutes the anatomical
basis of the BTB (Dym & Fawcett 1970, Bergmann et al.
1989, Pelletier & Byers 1992, Pelletier 1995, 2011).
During normal spermatogenesis, the BTB must open
(and then close again) to allow developing GC to pass
and enter the adluminal compartment. Yet the actual
dynamic restructuring of the BTB is still unclear.
However, a nice model for the successive steps of GC
migration across the murine SC tight junctions was
recently published (Smith & Braun 2012; Fig. 2). In
another study, tubulobulbar complexes have been
shown to be involved in basal junction remodeling and
consequently in translocation of spermatocytes through
the BTB (Du et al. 2013).
BTB-development initiates during puberty concomitant with the first wave of spermatogenesis. This process
is believed to coincide with the termination of SC
proliferation and their functional maturation. The direct
cellcell interactions within the seminiferous tubules are
mainly dependent upon tight, adherens, and gap
junctions as these different junction types have been
identified and located between SC (Cyr et al. 1999, Mruk
& Cheng 2004, Brehm et al. 2007, Sridharan et al. 2007,
Carette et al. 2010, Weider et al. 2011a, Cheng &
Mruk 2012, Gerber et al. 2014, Gerber 2015, Noelke
et al. 2015).
Furthermore, it has been verified in numerous
publications that the lack of even a single junctional
protein localised at the BTB results in distinct testicular
phenotypes and mostly causes sterility; see also Table 1
for more details (Reaume et al. 1995, Gow et al. 1999,
Juneja et al. 1999, Plum et al. 2000, Saitou et al. 2000,
Brehm et al. 2007, Sridharan et al. 2007, Winterhager
et al. 2007, Carette et al. 2010, Weider et al. 2011a,b,
Chojnacka et al. 2012, Giese et al. 2012, Gerber et al.
2014, Jiang et al. 2014, 2015, Gerber 2015, Noelke et al.
2015). It may also be mentionable in this context that it
was reported in different studies that BTB components
may act as early targets of environmental and reproductive toxicants mostly leading to testicular disorders
(Defamie et al. 2001, Fiorini et al. 2004, Sobarzo et al.
2006, 2009, Li et al. 2009a, Salian et al. 2009, Pointis
et al. 2011).
Interestingly, it has been shown in many studies, that
proteins forming gap junction channels and proteins
fulfilling cell attachment or forming tight junctions
mutually influence expression and functions of one
another (Derangeon et al. 2009, Pelletier 2011). At the
BTB level, the possibility that gap junctions exert
such a control on the other components of the barrier
has been postulated (Yan et al. 2008, Lee et al. 2009,
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Figure 2 Schematic drawing of supposed BTB dynamics during germ cell migration at stages VIIIIX of spermatogenesis in adult WT mice.
The images show two neighbouring SC with their nuclei (1) and the SC junctional complex at the region of BTB (2). (A) The preleptotene (3)
spermatocytes have lost contact to the basal lamina (BL) moving towards the adluminal compartment, the spermatogonia (4) are still adhered to the
BL. (B) To secure the integrity of the BTB, new SC TJs (6) are formed just below the preleptotene/leptotene spermatocytes (3), so that these cells are
enclosed in an intermediate compartment. (C) In the last step the old SC TJs are removed and leptotene spermatocytes (3) are released into the
adluminal compartment. Figure is based on data from Smith & Braun (2012).

Carette et al. 2010). This hypothesis is in agreement with


the previous morphological studies in continual (guinea
pig) and seasonal breeder (mink) testes, which suggest
that connexin43 (CX43) between SC participates in the
formation and regulation of the BTB (Pelletier 1995).
Recently, it has been reported that the blockage of CX, by
using a pan-CX peptide that recognizes all characterized
CX within the testis, concomitantly leads to diminished
OCCLUDIN and upregulated N-CADHERIN expression
(Lee et al. 2006).
Tight junction formation allows for an impermeable
intercellular barrier between adjacent cells. These tight
junctions are characteristically present in epithelial cells
and create a cellular polarity with an apical and basal

side. In the seminiferous tubules, this is particularly


evident between SC and their BTB (Mruk & Cheng 2004,
Brehm & Steger 2005, Bergmann 2006, Carette et al.
2010, Cheng & Mruk 2012, Li et al. 2012). Ocln codes
for the tight junction protein OCCLUDIN (Furuse et al.
1998) and is believed to be the initiator for BTB
formation (Nagano & Suzuki 1976), although it is yet
detectable in fetal murine SC (Cyr et al. 1999). BTB
formation due to OCCLUDIN expression begins at
around day 10 postpartum and correlates with the
initiation of spermatogenesis (Cyr et al. 1999, Gerber
et al. 2014). Interestingly, mice lacking the Ocln gene
show a complex (testicular) phenotype as in young
homozygous mutants (6 weeks old), the testis was found

Table 1 A brief description of major published findings of specific murine junctional protein KOs (and KIs), their effect on male fertility and detected
defects of the BTB formation.
Protein

Mouse line

Fertile

BTB

Details

CLAUDIN11

Osp/claudin-11 null

No

Yes

Global KO

CX43

CX43KO

NA

NA

CX43
CX43
CX43
CX43
CX43

CX43KI26
CX43KI32
CX43KI40
GCCx43KO
SCCX43KO

No
No
No
Yes
No

NA
NA
NA
NA
Yes

CX46
CX50
OCCLUDIN
ZO2
CADHERIN2
CATENIN
b-CATENIN

CX46K/K
CX50K/K
OccludinK/K
ZO2K/K
SCCdh2KO
SCCtnnb1KO
Ctnnb1tm1Mmt/C;
Amhr2tm3(cre)Bhr/C

Yes
Yes
Yes/no
No
Yes
Yes
No

NA
NA
NA
Yes
Yes
NA
NA

References

Gow et al. (1999) and


Mazaud-Guittot et al. (2010)
Global KO
Reaume et al. (1995) and Juneja
et al. (1999)
Global KO with knockin CX26
Winterhager et al. (2007)
Global KO with knockin CX32
Plum et al. (2000)
Global KO with knockin CX40
Plum et al. (2000)
Knockout in germ cells
Gunther et al. (2013)
Knockout in Sertoli cells, AMH-Cre
Brehm et al. (2007), Sridharan et al.
(2007) and Carette et al. (2010)
Global KO, altered BTB composition
Pelletier et al. (2015)
Global KO, altered BTB composition
Pelletier et al. (2015)
Global KO, progressive infertility
Saitou et al. (2000)
ZO2-chimera
Xu et al. (2009)
Knockout in Sertoli cells, subfertility
Jiang et al. (2015)
Knockout in Sertoli cells, AMH-R2-Cre Tanwar et al. (2011)
Activation of b-CATENIN in Sertoli
Boyer et al. (2008) and Tanwar
cells, AMH-R2-Cre
et al. (2010)

NA, not applicable.


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to be normally developed showing spermatogenesis, but


in older KO mice (60 weeks old) spermatogenesis was
dramatically altered (e.g. atrophied tubules containing
only SC (Saitou et al. 2000)).
Nevertheless, BTB establishment is not solely dependent upon OCCLUDIN indicating a much more
complex composition and function (Schulzke et al.
2005). As of 2011 (Mineta et al. 2011), 23 different
CLAUDIN proteins have been discovered so far.
These various CLAUDINs are considered to be seal or
barrier specific (Gunzel & Yu 2013), with CLAUDIN3
(Meng et al. 2005), CLAUDIN5 (Morrow et al. 2009),
and CLAUDIN11 (Gow et al. 1999, Morita et al. 1999)
being associated with murine BTB formation (Morrow
et al. 2010).
In murine testis, it was determined that CLAUDIN11
mRNA expression peaks between days 6 and 15
postpartum, CLAUDIN3 begins at day 15 postpartum
and CLAUDIN5 increases at days 620 postpartum,
which correlates with pubertal BTB formation (Hellani
et al. 2000, Meng et al. 2005, Morrow et al. 2009). It is
not until day 13 postpartum that CLAUDIN11 protein is
detectable in the SC, CLAUDIN3 is first detectable at
day 13 postpartum at all levels of the seminiferous epithelium and CLAUDIN5 is first visible in the seminiferous
epithelium at day 8 postpartum surrounding all GC
(Meng et al. 2005, Morrow et al. 2009, Mazaud-Guittot
et al. 2010). Thereafter CLAUDIN11 is present at all
phases of the murine seminiferous epithelial cycle
(Morrow et al. 2010). Contrarily, CLAUDIN3 and
CLAUDIN5 are stage specific in adult mice during the
stages VIIIX, when preleptotene/leptotene spermatocytes pass the BTB. Thus CLAUDIN3 and CLAUDIN5
might aid in the dynamic restructuring of the BTB (Meng
et al. 2005, Morrow et al. 2009, Chihara et al. 2010,
Smith & Braun 2012). So far, CLAUDIN11 has only been
identified within SC and not in GC (Morrow et al. 2010),
while CLAUDIN3 and CLAUDIN5 have been found
in both SC and GC (Morrow et al. 2009, Chihara
et al. 2013). It is proposed that CLAUDIN11 plays a
vital role in the maintenance of SC epithelial differentiation (Mazaud-Guittot et al. 2010) and may modulate
cell migration and invasion (Morrow et al. 2010).
CLAUDIN11 deficiency also results in the loss of the
SC epithelial phenotype and male sterility in mice (Gow
et al. 1999, Mazaud-Guittot et al. 2010). Also in men,
impaired spermatogenesis can be associated with an
altered BTB composition and function (Cavicchia et al.
1996). In men with testicular carcinoma-in-situ (CIS),
CLAUDIN11 has been found to be overexpressed in SC
(Fink et al. 2009), associated with a downregulation
of CX43 (Brehm et al. 2002, 2006). In addition,
CLAUDIN11 and CX43 were found to have altered
spatial patterns of organization in men with primary
seminiferous tubule failures (meiotic arrest and SC-only
syndrome (SCO); Haverfield et al. 2013).
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Testicular OCCLUDIN as well as CLAUDIN transcription, translation and cellular localization can be
regulated by e.g. testosterone, transcription factors,
FSH and/or GC (Mruk & Cheng 2004, Yan et al. 2008,
Lie et al. 2009, Morrow et al. 2010). Interestingly,
postmeiotic GC have been shown to have an inhibitory
effect on CLAUDIN11 expression in rat seminiferous
epithelium, whereas spermatogonia and premeiotic
spermatocytes have no effect (Florin et al. 2005).
As mentioned before, also localised at the BTB are gap
junctions, which allow for the direct communication
between two adjacent SC. The most predominant gap
junction protein within the testis of different species so
far is CX43 (e.g. Steger et al. 1999, St-Pierre et al. 2003,
Brehm & Steger 2005, Bergmann 2006, Brehm et al.
2006, 2007, Sridharan et al. 2007, Carette et al. 2010,
Weider et al. 2011a, Almeida et al. 2012, Gerber et al.
2014, Gerber 2015, Noelke et al. 2015, Rode et al.
2015). As found in most animal cell types, gap junction
channels allow for direct cytoplasmic connection
between adjacent cells. A gap junction channel,
composed of two hemichannels called connexons, is
responsible for direct intercellular communication
between neighbouring cells. Each cell contains a single
connexon composed of six CX proteins. A CX is a four
transmembrane protein with two extracellular loops,
one intracellular loop and both N- and C-terminal ends
located intracellularly. These channels allow the
passage for small molecules and ions (!1 kDa; Kumar
& Gilula 1996). They transport peptides, second
messengers and nucleotides, which facilitate metabolic
coupling and synchronized responses to hormones
(Brehm & Steger 2005). Additional gap junction and
CX43 functions include a dynamic cell modulation of
the cytoskeletal structure as well as extrinsic guidance to
promote cellcell adhesion (Stout et al. 2004). Single
connexons might regulate physiological roles by controlling ATP, Ca2C and NADC wave signalling within
cells (Stout et al. 2004, Spray et al. 2006, Pointis et al.
2010). Gap junction channels have also been linked to
cellular growth and differentiation (Kumar & Gilula
1996). A total of twenty different CX genes coding for gap
junction channels in mice have been discovered since
2004 (Sohl & Willecke 2004).

CX43 and SC
Within the SC and seminiferous epithelium, CX43
appears to be the most dominant gap junction protein,
yet numerous other CX-proteins have also been identified within the testis, e.g. CX26, CX32, CX33, CX46, and
CX50 (Brehm & Steger 2005, Pelletier et al. 2015). Like
all CX, CX43s name originates from its molecular weight
of 43 kDa (Kumar & Gilula 1996). The SC, as the mother
cell, is considered to be the central mediator for
communication within the seminiferous tubule (Brehm
& Steger 2005). Thus, gap junctions between SC
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SCCx43KO mice and bloodtestis barrier

containing CX43 may form an intercellular communication network corresponding to the initial syncytiumlike organisation within the seminiferous epithelium
allowing the coordination of SC metabolism and
indirectly via metabolic and signalling coupling the
synchronization of GC proliferation and differentiation
(Risley et al. 2002, Decrouy et al. 2004).
CX43 has been further shown to be critical to maintain
the homeostasis of the BTB via its effects on tight junction
reassembly (Li et al. 2010) however a knockdown of
CX43 alone by RNAi did not affect the barrier integrity (Li
et al. 2009b). In the latter study, a surge in the protein
level of CLAUDIN11 was observed but its levels at the
cell surface remained unchanged. In another study, the
effects of the reproductive toxicant di(2-ethylhexyl)
phthalate (DEHP) on testicular gap and tight junction
protein expression were investigated in prepubertal rats
(Sobarzo et al. 2009). There it was shown that DEHP did
not impair BTB permeability but induced GC sloughing
that was accompanied by a downregulation of CX43 and
ZO1 and alterations in other junctional proteins
(Sobarzo et al. 2009).
CX43 importance within SC has become predominantly evident in men with impaired spermatogenesis
(i.e. arrest at the level of spermatogonia) and testicular
CIS. In both occurrences it was demonstrated that there
was a downregulation of CX43 (and Cx43 mRNA) within
the SC or tubule (Steger et al. 1999, Brehm et al. 2002,
2006, Brehm & Steger 2005). Thus, the central role of
the SC within the testis, specifically the seminiferous
epithelium, is as apparent as the prominence of CX43
within the SC.

CX43 and mouse models


Initially a global KO of the gap junction protein, alpha 1
(Gja1) gene (CX43KO) was established to study the
importance and function of murine CX43. However,
these global KO mice were not suitable to study
spermatogenesis, since they died at birth due to an
obstruction of the right ventricular outflow tract (Reaume
et al. 1995). Interestingly, it was noticed at birth that the
gonadal size of both mutant sexes were abnormally
small. Hence an assumption was drawn that the absence
of CX43 might affect the number of GC that populate the
fetal gonads (Juneja et al. 1999).
Since CX43 is crucial for survival after birth in mice,
two knockin (KI) mouse lines were developed using
CX32 and CX40, CX43KI32 and CX43KI40 to replace
CX43. Both KI-lines overcame death at birth, which
implements that CX32 and CX40 can replace CX43 for
mice vitality. Nevertheless, both KI strains still indicated
increased ventricular susceptibility. However, male KI
mice were all sterile, signifying that these added CX were
not adequate for fertility, hence further demonstrating
the unique importance of CX43 (Plum et al. 2000).
Comparable outcomes were noticed using a CX43KI26
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mouse line: these mice were viable, but the males were
again infertile (Winterhager et al. 2007; Table 1).

SC specific CX43 knockout mice


In 2007, two independent research groups developed SC
specific CX43 knockout (SCCx43KO; i.e. SC-Cx43 KO
as used by Sridharan et al. (2007)) mice strains, deficient
of CX43 only in SC, using the Cre-LoxP recombination
system (Brehm et al. 2007, Sridharan et al. 2007). A
conditional SC-specific KO of the Gja1 gene exposed
that CX43 expression in SC is an absolute necessity
for normal testicular development and initiation of
spermatogenesis. Furthermore, no obvious phenotypical
alterations were detected, and homozygous female
knockout SCCx43KOK/K mice showed no loss in fertility
(Brehm et al. 2007, Sridharan et al. 2007). However and
in contrast, a GC-specific KO of the Gja1 gene
(GCCx43KO) did not imply the significance of CX43
within the GC, since these mice were still fertile. These
findings indicate that CX43 expressed in nurturing SC is
probably crucial for uni-directional cross talk between
SC to GC. Whereas, in GC to SC communication, this CX
might be substituted by other CX expressed in GC like
CX45 (Gunther et al. 2013).
The SCCx43KO mouse line is established by crossing
transgenic homozygous-CX43flox-LacZ mice (Theis
et al. 2000, 2001) with homozygous AMH-Cre transgenic mice (Lecureuil et al. 2002). A unique feature of
the Cx43flox-LacZ mouse is the encoded silent LacZreporter gene (coding for b-GALACTOSIDASE), which is
only activated once Gja1 has been deleted (Theis et al.
2000, 2001). The expression of the Cre enzyme is
regulated under the control of an Amh promoter, which
is specific to SC. This enzyme then cleaves the targeted
flox gene Gja1, which encodes for CX43. Thus, only
in SC with at least one allele lacking Gja1,
b-GALACTOSIDASE is detectable and can be used as
one validation method of a successful Gja1 gene
deletion (Theis et al. 2000, 2001, Brehm et al. 2007,
Sridharan et al. 2007, Gerber et al. 2014, Gerber 2015).
Through multigenerational breeding it is feasible to
accomplish 50% of the male offspring to be WT and the
other 50% to be KO (Brehm et al. 2007, Sridharan et al.
2007). In the past 8 years numerous publications
concerning the SCCx43KO mice have been published:
Brehm et al. (2007), Sridharan et al. (2007), Carette
et al. (2010), Weider et al. (2011b), Chojnacka et al.
(2012), Giese et al. (2012), Gerber et al. (2014) and
Noelke et al. (2015).

Discoveries using the SCCx43KO mouse line


Macroscopically, adult KO mice showed descended
gonads, but testes volumes and masses were drastically
lower when compared with WT littermates (Fig. 3).
Compared with WT littermates, histological analysis of
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Figure 3 Macroscopic images and haematoxylin and eosin (HE)


stainings from adult SCCx43KO mice. (A) Half of the urogenital system
from a WT (left side) and from a KO (right side) mouse.
(B) Representative testis from a WT (left side) and a KO (right side)
mouse. The KO testes are dramatically smaller in comparison to the
WT. (C and D) Images are HE stained tissue sections from WT (C) and
KO (D) mice. Note the morphological and histological alterations in the
KO in comparison with the WT. The scale bar represents 1 cm for (A),
0.5 cm for (B), and 100 mm for (C and D).

the KO testes revealed impaired spermatogenesis (Fig. 3)


with tubules showing an arrest of spermatogenesis at the
level of spermatogonia and/or SCO, intratubular SC cell
clusters and abnormal SC cytoplasmic vacuoles (Fig. 4).
In addition, SC numbers per seminiferous tubule
were increased and a reduced number of spermatogonia
per tubule was detected (Brehm et al. 2007, Sridharan
et al. 2007).
SC of adult KO males were found to still be
proliferating (Sridharan et al. 2007), when typically
normal SC proliferation terminates around 2 weeks after
birth. This was determined using immunohistochemistry
and serial sections at days 20 and 60 postpartum with the
SC-specific marker WT1 and the proliferation marker
MKI67. It was also demonstrated that adult KO testes
contain 73% more SC than their WT littermates despite
the decrease in testes size. Furthermore the gene
expression of Thra, a SC maturation marker, was also
found to be significantly upregulated in KO mice at both
days 20 and 60 postpartum. It was hypothesized that the
absence of CX43 in SC caused these somatic cells to
remain in an apparently intermediate and permanent
proliferative state (Sridharan et al. 2007).
These proliferative SC characteristics were supported
through an additional investigation by Weider et al.
(2011b), in which it was shown that gene expression and
protein synthesis of AMH was prolonged in SC of KO
mice during puberty. Thus the conclusion was drawn
that CX43-deficient SC are in a partially altered or in an
intermediate state of differentiation. Furthermore, these
mutant SC also showed a permanent and uniform
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expression of GATA1, a stage specific SC maturation


marker, at protein and mRNA level. Typically, GATA1
synthesis in adult WT mice is stage dependent. It was
hypothesized that this permanent and uniform
expression was due to the lack of maturing GC.
Furthermore, in KO males VIMENTIN (a SC intermediate
filament) and ANDROGEN RECEPTOR (a SC maturation
marker) were not altered in their protein synthesis during
postpartal development. Using histology and transmission electron-microscopy it was noticed that basally
located SC in the KO mice still depicted the typical
mature tripartite nucleolus. While the intratubular SC
cell clusters did not express this mature feature. These
clusters initiated during puberty and increased in
number and size with aging, while occasionally
containing also single GC (Weider et al. 2011b). These
results emphasise the critical involvement of CX43 to
the normal maturational process and progression of SC,
which typically results in the termination of SC
mitogenesis during the pubertal period (Sridharan et al.
2007, Weider et al. 2011b).
A recent publication from Noelke et al. (2015),
investigated the supposed Leydig cell (LC) hyperplasia
of the KO mice (Brehm et al. 2007). LC hyperplasia was
confirmed through counting these interstitial cells in
adult KO and WT mice using the optical dissector
method. Through the use of laser microdissection,
western blot (WB) and PCR analysis it was shown that
LC of adult KO mice synthesised no CX43 (unlike in the
WT). The gene expression of Gja1 and Gjc1 (CX45) was
also significantly downregulated. Furthermore, one of
the known primary functions of the LC is the steroid
synthesis, which was also investigated via immunohistochemistry and PCR. In both adult WT and KO testes
steroidogenesis seems not to be impaired (Noelke et al.
2015) supporting results investigating male CX43KO
mice (Kahiri et al. 2006).

Figure 4 Seminiferous epithelium of adult KO mice. (A) Toluidine blue


stained semi-thin section of an adult KO testis. In the box, the nucleus
of a SC (arrow) and a spermatogonium (arrowhead) are marked. The
cytoplasm of the SC includes numerous vacuoles. Scale bar 25 mm.
(B) Schematic drawing of a SC of an adult KO mouse. The SC sits on the
BL, its cytoplasm includes numerous vacuoles (V) and it is lower than
in WT mice. The BTB (boxes) is formed between adjacent SC.
Spermatogonia (arrowheads) are the only GC that are found in KO mice.
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SCCx43KO mice and bloodtestis barrier

R21

In addition to these qualitative, histological and


proliferative dissimilarities in KO males, a total of 658
genes exhibited an altered regulation (135 upregulated
and 523 downregulated) in a microarray study
comparing KO and WT mice at the age of 8 days
postpartum. The majority of the downregulated genes
were fundamental for spermatogenesis (Giese et al.
2012), but Ocln gene expression was not (yet) significantly altered at this age point.

SCCx43KO mice and BTB


Recently, regulation of tight junction expression and/or
synthesis through CX43 in SC has become one focal
point of the SCCx43KO studies. This is indicated through
recent reviews: Weider et al. (2011a) and Cheng & Mruk
(2012). Carette et al. (2010) established that the synthesis
of CX43 was significantly reduced in adult KO mice
using semi-quantitative WB analysis and immunofluorescence (IF). In addition, the permeability of the BTB in
the SCCx43KO mice was investigated, since the KO
mice exhibited an arrest of spermatogenesis at the level
of spermatogonia. Unexpectedly, the KO mice contained
a functionally intact BTB just as the WT mice (Fig. 5),
but associated with an increased expression of tight
and adherens junctions (e.g. b-CATENIN, N-CADHERIN,
and OCCLUDIN). The formation of the BTB in both adult
WT and KO mice was confirmed through the use of two
different experimental assays: an electron opaque tracer
(e.g. lanthanum nitrate) and a hypertonic fixation solution
(e.g. glucose, Fig. 5).
Using a rat SC line, SerW3 and CX43 siRNA similar
results were achieved in vitro as those of the KO mice.
The SerW3 cells subjected to CX43-siRNA also
depicted an increase in tight and adherens junction
(b-CATENIN, N-CADHERIN, and OCCLUDIN) synthesis
(Carette et al. 2010).
In adult KO mice communication via CX43 channels
was investigated to ensure that no other CX (e.g. CX32)
could compensate its function. Functional compensation of CX43 could not be detected in seminiferous
tubular fragments of mutant mice using fluorescence
recovery after photo bleaching (FRAP). This supports the
theory that gap junction channels containing CX43 are
especially vital for direct cytoplasmic communication
between neighbouring SC. Via semi-quantitative WB,
IF and electron microscope analysis, it was further
established that numerous junctional proteins (e.g.
N-CADHERIN, b-CATENIN, and OCCLUDIN) were
upregulated in the adult KO mice when compared with
their WT littermates. Conversely, ZO1 was shown to be
downregulated in both WB and IF (Carette et al. 2010).
Concluding, CX43 is not crucial for establishing a
functional BTB, but appears to interfere more likely with
barrier dynamics (assembly/disassembly). And loss of
CX43 leads to alterations in tight and adherens junction
expression and localization (Carette et al. 2010).
www.reproduction-online.org

Figure 5 Toluidine blue stained semi-thin sections (A and B) and


transmission electron micrographs (C and D) from adult WT and
SCCx43KO testes. (A) Stems from a WT and images B, C and D stem
from KO mice. (A and B) These images show testes subjected to a
hypertonic fixation solution containing 10% glucose. The arrows
indicate shrinkage artefacts only in spermatogonia in both genotypes.
It is also evident that the SC are not affected. (C) Representative
example of a fixation solution using a 2% lanthanum nitrate tracer.
This tracer (coming from the interstitial compartment of the testis)
attempts to enter the lumen of the seminiferous tubule between two
neighbouring SC. Owing to the presence of an intact BTB the tracer
cannot pass this barrier. (D) This image is an enlargement of image C
and the arrow indicates that the tracer diffusion decreases at the height
of the BTB. The scale bar represents 50 mm for (A), 25 mm for (B),
1.7 mm for (C) and 0.4 mm for (D).

These results were supported in a recent study through


a RT real-time PCR analysis of adult mice, in which the
tight junction genes Cldn11 (CLAUDIN11) and Ocln
(OCCLUDIN) were significantly upregulated in adult
KO males. As expected, in the same study, the Gja1 gene
was significantly downregulated (Gerber et al. 2014).
Owing to the unexpected formation and function of the
BTB in adult KO mice (Carette et al. 2010) a time study on
the pubertal establishment of this barrier was performed
(Gerber et al. 2014) to elucidate possible spatiotemporal alterations in BTB formation. It is believed that
OCCLUDIN initially forms the BTB at the beginning of
spermatogenesis (Nagano & Suzuki 1976) and it was
significantly upregulated in the adult KO mice (Carette
et al. 2010). Gerber et al. (2014) demonstrated that
pubertal KO mice exhibit a 1 day delay in OCCLUDIN
localisation towards the BTB region when compared with
their WT littermates (Fig. 6). However, WB revealed that
until the age of 15 postpartum no significant increase in
OCCLUDIN synthesis was detectable in the KO mice,
as seen for adult KO mice (Gerber et al. 2014). Data
obtained from Mammalian Reproductive Genetics
(mrgd.org, accessed on 21st April 2015) showed that
Olcn expression begins to decrease at around day 10
postpartum and remains low throughout adulthood
(Shima et al. 2004). Taken together that the adult KO
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J Gerber and others

Figure 6 Immunohistochemical staining of OCCLUDIN at ages 10


(A and B) and 11 (C and D) days postpartum. Images A and C originate
from WT, while images B and D originate from KO testes. The arrows
indicate the localization of OCCLUDIN at the height of the BTB region.
It is evident that the move of OCCLUDIN immunolocalisation to the
BTB is slightly delayed in the KO mice. The scale bar represents 50 mm.

mice express and synthesize more tight junctions, it may


be proposed that the lack of CX43 in the KO mice
prevents the downregulation of OCCLUDIN. In other
words, CX43 normally seems to be able to (down)regulate OCCLUDIN (and other tight junctions) in adult
WT mice (Gerber et al. 2014, Gerber 2015).
Noteworthy, preliminary in vitro data from isolated
primary SC derived from SCCx43KO mice showed that
transepithelial electrical resistance (TEER) values of
primary CX43-deficient SC cultures were significantly
higher than those of WT cultures. It has been well
established that as tight junctions between cells in cell
culture increase this results in an increase in TEER values
and vice versa. Thus primary CX43-deficient SC cultures
might contain more tight junctions and thus establish a
tighter barrier, just as shown by the increased synthesis of
additional tight junctions (e.g. CLAUDIN11) of ex vivo
experiments of KO and WT mice (Gerber 2015).

(Carette et al. 2010, Gerber et al. 2014, Gerber 2015).


Although the gap junction channel CX43 between two
SC is not vital for the establishment of the BTB it may
play a key role in this barriers dynamic process (i.e.
opening and closing for GC migration (Fig. 7)).
In this context, a new hypothesis on the dynamic
process of BTB formation might be proposed that is based
upon the calcium switch model: it has been observed, via
TEER analysis, that MDCK (MadinDarby canine kidney)
cells, lacking Ca2C in their culture medium, showed a
decrease of tight junction formation. On the other hand,
the addition of Ca2C to the culture medium caused an
increase in TEER indicating an increase in tight junction
assembly between cells (Gumbiner & Simons 1986,
Kartenbeck et al. 1991, Cereijido et al. 1993, Denker &
Nigam 1998, Li et al. 2010). In SC cultures, tight junction
strands are disrupted within 15 min after incubation in
medium absent of Ca2C, and reformation occurs within
90 min after addition of Ca2C (Grima et al. 1998). Li et al.
(2010) examined the calcium switch theory in relation
to CX43 and the BTB. As previously stated, single
connexons composed of CX43 may also regulate
physiological roles by controlling wave signalling
(e.g. Ca2C; Stout et al. 2004, Spray et al. 2006, Pointis
et al. 2010). These CX43connexons may allow a direct
connection between SC, between SC and the adluminal
and/or basal compartment of the seminiferous tubules
allowing for Ca2C diffusion. A depletion of Ca2C through
the seminiferous epithelial cycle regulation may cause
a simultaneous depletion of Ca2C within the SC.
This depletion of Ca2C within the SC causes a decrease

SCCx43KO mice, BTB and possible hypothesis


Owing to the evident link between CX43 and regulation
of tight junctions, it is proposed that adult mutant males
might be infertile due to the overexpression of tight
junctions (e.g. OCCLUDIN) and a dysregulation of the
BTB assembly/disassembly. It is suggested that this
overexpression causes a tighter BTB with GC not being
able to pass through the barrier. Thus GC development
terminates at the level of the spermatogonia, which is
histologically evident in adult KO mice. This impenetrable BTB hypothesis might be verified by understanding the mechanistic link between CX43 and the
establishment and maintenance of tight junctions
Reproduction (2016) 151 R15R27

Figure 7 Supposed impaired BTB dynamics in adult SCCx43KO mice.


The schematic drawing shows two neighbouring SC with their nuclei
(1), the junctional complex at the region of the BTB (2), and a
spermatogonium (3). Adult KO mice seem to express more tight
junctions and establish a tighter barrier. GC are not able to migrate
through this barrier resulting in an arrest of spermatogenesis at the level
of spermatogonia. BL, basal lamina.
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SCCx43KO mice and bloodtestis barrier

in tight junction expression at the SCSC interface, as


shown through TEER experiments and IF experiments
of rat primary SC cultures (Li et al. 2010). Hence these
alterations in Ca2C concentrations during the seminiferous epithelial cycle might be a key signal in
regulating BTB dynamics. Leaving the question open
what is causing the cyclic regulation of Ca2C
concentrations within the seminiferous tubules.
A recent study, from Sanchez-Cardenas et al. (2012),
showed that co-localized GC from a sliced seminiferous
tubule exhibited a synchronous Ca2C concentration
oscillation pattern. This synchronous oscillation pattern
was proposed to be controlled through intercellular
bridges. Additionally, this study stated that it was
possible to record Ca2C responses of cells which
functionally and anatomically resemble SC. It was
suggested that the SC response is mediated through the
diffusion of Ca2C via gap junction between neighbouring SC. Furthermore, the authors also hypothesised that
this Ca2C diffusion through a tubule might participate in
a synchronous signal during spermatogenesis (SanchezCardenas et al. 2012). In summary, it might be possible
that the Ca2C oscillation of GC is synchronous to the
seminiferous epithelial cycle. This in return might cause
for a synchronous alteration within the tubules via the
uptake of Ca2C into the GC from the tubules. When
Ca2C concentration is decreased in the tubule it may
also decrease within the SC via gradient diffusion
through a CX43 connexon (i.e. calcium switch). This
decrease in the SC thus causes a relocalisation (away
from the SCSC interface) of tight junctions and a
disassembly of the BTB. It is possible that in the KO mice,
which lack CX43 (connexon) in SC, never experience
such an internal decrease in Ca2C concentration. This
then causes the BTB to remain intact. Nevertheless it is
still unclear if the Ca2C signal stems from the basal and/
or adluminal compartments of the seminiferous tubules.

Further implications for the SCCx43KO


animal model
As of 2010, there are w48.5 million infertile couples
worldwide (Mascarenhas et al. 2012), from which
w39% are due to female impediments, 20% are linked
to male impediments, 26% stem from couple incompatibility and the remaining 15% are unknown (Nieschlag
2009). With w1/3 of infertile men are being diagnosed
with idiopathic infertility (Schatzl & Schmidbauer 2009).
In this context, the SCCx43KO mouse model might
supply background information or mechanistic insights
into unknown causes for human male infertility as it has
been shown in different studies that CX43 is downregulated in men associated with different histological
phenotypes of spermatogenic impairment (Steger et al.
1999, Brehm et al. 2002) and associated with BTBalterations (Fink et al. 2009, Haverfield et al. 2013).
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R23

A potential young male reproductive issue is testicular


cancer, where in Europe the incidence is 63/100 000 per
year. This rate is doubling every 20 years. The potential
precursor of testicular GC-tumours is known as CIS or
testicular intraepithelial neoplasia, which develops into
seminoma and/or non-seminoma (Schmoll et al. 2010).
In this regard, it has been demonstrated that CX43
is downregulated in human seminiferous tubules
containing CIS-cells, and in seminoma (Steger et al.
1999, Brehm et al. 2002, 2006). Even though no
occurrence of testicular cancer has been detected within
the SCCx43KO mice so far, this model might still be ideal
to uncover regulations of CX43 within the life cycle of SC
(and SC tumours). As stated above, studies with the
SCCx43KO mice have shown that a lack of CX43 in SC
has an effect on SC proliferation and maturation
(Sridharan et al. 2007, Weider et al. 2011b).
Amongst male vasectomy and condoms, the female
oral contraceptive is one of the most prevalent methods
used in family planning. Many female contraceptive
types have been developed since the 1950s and made
publicly accessible. However, no male pharmaceutical
has been as successful as the female counterpart so far
(Poston & Bouvier 2010). Recent reviews state the
significance for developing a male contraceptive and
designate the BTB as a key structure and its (reversible)
regulation for this development (Cheng & Mruk 2012).
CX43 has been linked as an important regulator of BTB
assembly and function as shown in the SCCx43KO
model (Carette et al. 2010, Gerber et al. 2014, Gerber
2015). Thus, through the establishment and use of
SC-lines (with and without CX43, derived from
SCCx43KO mice), it would be interesting to test various
potential pharmacons and to analyze their effects on the
interactions between SC and the BTB.
Finally, in recent publications it was elucidated that
SC might help to establish extra-testicular long term
immuno-privileged allogenic and xenogeneic transplants (Korbutt et al. 1997, Mital et al. 2010, 2014).
The production of immunomodulatory factors by SC and
the protective barrier capacity of SC could be used in
transplants (e.g. diabetes: b cells for insulin production)
and allow for a long term survival and protection from
the patients immune system. On the other hand, specific
substances (e.g. insulin) could transverse through the SC
and from there be distributed throughout the patient
(Mital et al. 2010). One advantage for implementing the
CX43-deficient SC for allogenic and xenogeneic transplants would be the over-synthesis of tight junction
proteins causing a possible tighter and more protective
immunological barrier.

Conclusion
Since it could be shown that in cases of impaired
spermatogenesis, CX43 expression is altered/downregulated in men and several animal species, the SCCx43KO
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J Gerber and others

mouse model represents an interesting tool to investigate male infertility. During the last years, the BTB has
become one important objective in this field of research.
Although the SC specific KO of CX43 does not have an
adverse effect on BTB integrity, it seems to have an effect
on BTB dynamics and composition. Male SCCx43KO
mice are infertile due to e.g. an arrest of spermatogenesis
at the level of spermatogonia and the expression
pattern of different BTB proteins (e.g. OCCLUDIN,
CLAUDIN11, b-CATENIN, and N-CADHERIN) is altered
in mutant males. These data might be interpreted as
an evidence for an altered BTB assembly and/or
disassembly.
To further investigate junctions in/between SC in the
absence or presence of CX43, primary KO and WT SC
cultures have been established and are being investigated for functional differences. The ultimate goal is to
determine the mechanistic regulation of CX43 and other
junction types within the SC and the seminiferous
epithelium. This might help to better understand
unknown causes of human male infertility and testicular
cancer, as well as to develop new strategies in male
contraceptive development and improve the efficiency
of immuno-privileged transplants.

Declaration of interest
The authors declare that there is no conflict of interest that
could be perceived as prejudicing the impartiality of the
review.

Funding
Studies mentioned in this review were supported by grants from
the Deutsche Forschungsgemeinschaft (DFG of Germany,
especially BR3365/1-1 and BR3365/2-1) and the LOEWE
Focus Group Male Infertility and Urogenital Infections
(MIBIE) funded by the state of Hessen, Germany. In addition,
Jonathan Gerber was a recipient of a scholarship of the KonradAdenauer-Stiftung, Bonn, Germany.

Acknowledgements
The authors are grateful to Caren-Imme von Stemm for her
excellent schematic drawings and Marion Langeheine and
Gudrun Wirth for their excellent technical assistance.

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Received 13 August 2015


First decision 8 September 2015
Revised manuscript received 5 November 2015
Accepted 10 November 2015

Reproduction (2016) 151 R15R27