Beruflich Dokumente
Kultur Dokumente
REVIEW
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.
Reproduction (2016) 151 R15R27
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)
R16
R17
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).
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
R18
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
www.reproduction-online.org
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.
R19
mouse line: these mice were viable, but the males were
again infertile (Winterhager et al. 2007; Table 1).
R20
R21
R22
R23
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
Reproduction (2016) 151 R15R27
R24
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.
References
Almeida J, Conley AJ, Mathewson L & Ball BA 2012 Expression of
anti-Mullerian hormone, cyclin-dependent kinase inhibitor (CDKN1B),
androgen receptor, and connexin 43 in equine testes during puberty.
Theriogenology 77 847857. (doi:10.1016/j.theriogenology.2011.09.007)
Bergmann M 2006 Physiology of spermatogenesis. In Andrology for the
Clinician, pp 272281. Eds WB Schill, FH Comhaire& TB Hargreave.
Berlin, Heidelberg, Germany: Springer.
Reproduction (2016) 151 R15R27
R25
correlates with the disruption of Sertoligerm cell junctions but not the
interSertoli tight junction. Journal of Chemical Biology 273
2104021053. (doi:10.1074/jbc.273.33.21040)
Guitton N, Touzalin AM, Sharpe RM, Cheng CY, Pinon-Lataillade G,
Meritte H, Chenal C & Jegou B 2000 Regulatory influence of germ cells
on sertoli cell function in the pre-pubertal rat after acute irradiation of the
testis. International Journal of Andrology 23 332339. (doi:10.1046/
j.1365-2605.2000.00248.x)
Gumbiner B & Simons K 1986 A functional assay for proteins involved
in establishing an epithelial occluding barrier: identification of a
uvomorulin-like polypeptide. Journal of Cell Biology 102 457468.
(doi:10.1083/jcb.102.2.457)
Gunther S, Fietz D, Weider K, Bergmann M & Brehm R 2013 Effects of a
murine germ cell-specific knockout of Connexin 43 on Connexin
expression in testis and fertility. Transgenic Research 22 631641.
(doi:10.1007/s11248-012-9668-1)
Gunzel D & Yu AS 2013 Claudins and the modulation of tight junction
permeability. Physiological Reviews 93 525569. (doi:10.1152/physrev.
00019.2012)
Haverfield JT, Meachem SJ, OBryan MK, McLachlan RI & Stanton PG 2013
Claudin-11 and connexin-43 display altered spatial patterns of
organization in men with primary seminiferous tubule failure compared
with controls. Fertility and Sterility 100 658666. (doi:10.1016/
j.fertnstert.2013.04.034)
Hellani A, Ji J, Mauduit C, Deschildre C, Tabone E & Benahmed M 2000
Developmental and hormonal regulation of the expression of oligodendrocyte-specific protein/claudin 11 in mouse testis. Endocrinology 141
30123019. (doi:10.1210/endo.141.8.7625)
Hemendinger RA, Gores P, Blacksten L, Harley V & Halberstadt C 2002
Identification of a specific Sertoli cell marker, Sox9, for use in
transplantation. Cell Transplantation 11 499505.
Jegou B 1993 The Sertoligerm cell communication network in mammals.
International Review of Cytology 147 2596.
Jegou B & Sharpe RM 1993 Paracrine mechanisms in testicular control. In
Molecular Biology of the Male Reproductive System, pp 271310. Ed.
DM De Kretser. New York, NY, USA: Academic Press.
Jiang XH, Bukhari I, Zheng W, Yin S, Wang Z, Cooke HJ & Shi QH 2014
Bloodtestis barrier and spermatogenesis: lessons from geneticallymodified mice. Asian Journal of Andrology 16 572580. (doi:10.4103/
1008-682X.125401)
Jiang X, Ma T, Zhang Y, Zhang H, Yin S, Zheng W, Wang L, Wang Z,
Khan M, Sheikh SW et al. 2015 Specific deletion of Cdh2 in Sertoli cells
leads to altered meiotic progression and subfertility of mice. Biology of
Reproduction 92 79. (doi:10.1095/biolreprod.114.126334)
Juneja SC, Barr KJ, Enders GC & Kidder GM 1999 Defects in the germ line
and gonads of mice lacking connexin43. Biology of Reproduction 60
12631270. (doi:10.1095/biolreprod60.5.1263)
Kahiri CN, Khalil MW, Tekpetey F & Kidder GM 2006 Leydig cell function
in mice lacking connexin43. Reproduction 132 607616. (doi:10.1530/
rep.1.01234)
Kartenbeck J, Schmelz M, Franke WW & Geiger B 1991 Endocytosis
of junctional cadherins in bovine kidney epithelial (MDBK) cells
cultured in low Ca2C ion medium. Journal of Cell Biology 113
881892. (doi:10.1083/jcb.113.4.881)
Kent J, Wheatley SC, Andrews JE, Sinclair AH & Koopman P 1996 A malespecific role for SOX9 in vertebrate sex determination. Development 122
28132822.
Korbutt GS, Elliott JF & Rajotte RV 1997 Cotransplantation of allogeneic
islets with allogeneic testicular cell aggregates allows long-term graft
survival without systemic immunosuppression. Diabetes 46 317322.
(doi:10.2337/diab.46.2.317)
Kumar NM & Gilula NB 1996 The gap junction communication channel.
Cell 84 381388. (doi:10.1016/S0092-8674(00)81282-9)
Lecureuil C, Fontaine I, Crepieux P & Guillou F 2002 Sertoli and granulosa
cell-specific Cre recombinase activity in transgenic mice. Genesis 33
114118. (doi:10.1002/gene.10100)
Lee NP, Leung KW, Wo JY, Tam PC, Yeung WS & Luk JM 2006 Blockage of
testicular connexins induced apoptosis in rat seminiferous epithelium.
Apoptosis 11 12151229. (doi:10.1007/s10495-006-6981-2)
Lee NP, Yeung WS & Luk JM 2009 Junction interaction in the seminiferous
epithelium: regulatory roles of connexin-based gap junctions. Frontiers
in Bioscience 12 15521562. (doi:10.2741/2168)
Reproduction (2016) 151 R15R27
R26
www.reproduction-online.org
R27
43 in the testis and messenger RNA levels in the epididymis of the rat.
Biology of Reproduction 68 12321240. (doi:10.1095/biolreprod.102.
010504)
Tanwar PS, Kaneko-Tarui T, Zhang L, Rani P, Taketo MM & Teixeira J 2010
Constitutive WNT/b-catenin signaling in murine Sertoli cells disrupts
their differentiation and ability to support spermatogenesis. Biology of
Reproduction 82 422432. (doi:10.1095/biolreprod.109.079335)
Tanwar PS, Zhang L & Teixeira JM 2011 Adenomatous polyposis coli (APC)
is essential for maintaining the integrity of the seminiferous epithelium.
Molecular Endocrinology 25 17251739. (doi:10.1210/me.2011-0057)
Theis M, Magin TM, Plum A & Willecke K 2000 General or cell typespecific deletion and replacement of connexin-coding DNA in the
mouse. Methods 20 205218. (doi:10.1006/meth.1999.0938)
Theis M, de Wit C, Schlaeger TM, Eckardt D, Kruger O, Doring B, Risau W,
Deutsch U, Pohl U & Willecke K 2001 Endothelium-specific replacement of the connexin43 coding region by a lacZ reporter gene. Genesis
29 113. (doi:10.1002/1526-968X(200101)29:1!1::AID-GENE1000O
3.0.CO;2-0)
Weider K, Bergmann M & Brehm R 2011a Connexin 43: its regulatory role
in testicular junction dynamics and spermatogenesis. Histology and
Histopathology 26 13431352.
Weider K, Bergmann M, Giese S, Guillou F, Failing K & Brehm R 2011b
Altered differentiation and clustering of Sertoli cells in transgenic mice
showing a Sertoli cell specific knockout of the connexin 43 gene.
Differentiation 82 3849. (doi:10.1016/j.diff.2011.03.001)
Winterhager E, Pielensticker N, Freyer J, Ghanem A, Schrickel JW, Kim JS,
Behr R, Grummer R, Maass K, rschel S et al. 2007 Replacement of
connexin43 by connexin26 in transgenic mice leads to dysfunctional
reproductive organs and slowed ventricular conduction in the heart.
BMC Developmental Biology 7 26. (doi:10.1186/1471-213X-7-26)
Xu J, Anuar F, Ali SM, Ng MY, Phua DC & Hunziker W 2009 Zona occludens-2
is critical for bloodtestis barrier integrity and male fertility. Molecular
Biology of the Cell 20 42684277. (doi:10.1091/mbc.E08-12-1236)
Yan HH, Mruk DD, Lee WM & Cheng CY 2008 Cross-talk between tight and
anchoring junctions lessons from the testis. Advances in Experimental
Medicine and Biology 636 234254. (doi:10.1007/978-0-387-09597-4_13)