Investigation of the junctional complex at the blood-testis barrier in SCCx43KO mice and establishment and functional characterization of a murine sertoli cell line deficient of connexin43 (2015)

The Sertoli cell specific connexin43
 knockout (SCCx43KO) is a transgenic mouse line in which the gene Gja1,
 coding for connexin43 (Cx43) is knocked out only in the Sertoli cells (SC).
 The protein Cx43 forms a gap junction (GJ) channel. All GJ channels are
 composed of two connexons from neighboring cells, with each connexon being
 composed of six connexins (Cx). A Cx is a four transmembrane protein with two
 extracellular loops, while an additional loop and both N- and C-terminal
 domains are located intracellularly. Within the testes, the GJ channel Cx43
 is located between SC and between SC and germ cells (GC). These channels
 allow for direct cytoplasmic communication between the adjacent cells. Cx43
 is present at the blood-testis barrier (BTB) between two neighboring SC and
 is believed to aid in the dynamic regulation of this barrier. Nevertheless,
 these GJ proteins are structures whose function and dynamic regulation is
 still poorly understood. The BTB is vital for spermatogenesis; its formation
 separates the seminiferous tubules into a basal and adluminal compartment.
 This creates a unique lumen milieu (for haploid GC) which is protected from
 the immune system. While being localized between the SC and GC, Cx43 is believed
 to nurture and aid the GC (spermatogonia and spermatocytes) in their
 proliferation and maturation process during spermatogenesis.
 Recent studies have begun linking
 regulations between GJ and tight junction (TJ) proteins. These TJ proteins
 create the BTB between SC, which initiates formation at the beginning of
 pubertal development. In particular a lack of Cx43 within the SC causes an
 upregulation of the numerous TJ proteins (e.g. occludin and claudin11).
 Hence, the SCCx43KO mouse is an ideal model to study the link between Cx43
 and TJ expression and assembly. It has been well established that Cx43 is the
 key GJ in the SC. Its absence has led to infertility of the male SCCx43KO
 mice. Additionally, Cx43 has been shown to be significantly downregulated in
 some men expressing seminiferous tubules with spermatogonial arrest or
 carcinoma in situ (CIS). Recent publications have stated that the BTB
 is an ideal target for studying male contraceptives. In particular,
 reversible alterations made to the BTB’s junctional complex may act as a
 switch for turning spermatogenesis on or off. Through establishment of two SC
 cultures from wild type (WT) and SCCx43KO mice, researchers could have access
 to a larger experimental field, not be limited by breeding constraints and the
 high cost of maintaining a mouse line. The regulation of TJ compared in mice
 and primary SC cultures with and without Cx43 is the basis for the two
 studies described in this thesis: (1) How is the expression of the TJ protein
 occludin affected at the BTB during puberty in comparing SCCx43KO and WT
 littermates? (2) Are there differences when culturing SC from SCCx43KO mice
 and their WT littermates? How is the expression of the TJ protein
 (specifically claudin11) affected when comparing the SCCx43KO and WT primary
 SC cultures?
 In the first part of the study, the
 expression pattern of the testicular TJ protein occludin was analyzed in SC.
 This was performed using immunohistochemistry (IHC), semi-quantitative
 Western blot (WB) analysis and quantitative real time polymerase chain
 reaction (qRT-PCR) using the established transgenic SCCx43KO mice and their
 WT littermates. Both adolescent to adult SCCx43KO and WT males were compared.
 The goal was to elucidate a possible role of Cx43 on the expression pattern
 and regulation of occludin. The qRT-PCR indicates an increase in occludin and
 claudin11 messenger ribonucleic acid (mRNA) in adult SCCx43KO mice testes,
 while Cx43 was significantly downregulated. These results correspond to
 testicular occludin and claudin11 protein synthesis of adult mice. Yet, no
 significant difference of occludin protein synthesis between WT and SCCx43KO
 were determined between day 8 and 15 postpartum (p.p.) testes using
 semi-quantitative WB. Still, during puberty a one day delayed shift of occludin
 immunoreaction to the BTB region occurred in SCCx43KO males between day 10-12
 p.p. This study demonstrates spatio-temporal alterations in occludin,
 indicating that Cx43 might act as a regulator for BTB formation and function.
 This first study was been published in GERBER et al. 2014.
 In the second part of the study, the
 SCCx43KO mouse line seems ideal to gain further insight into the mechanistic
 GJ (and TJ) formation in SC and the seminiferous epithelium; however
 experiments in the past were limited to an in vivo model. A slightly
 modified method for developing primary SC cultures was established, validated
 and successfully characterized via polymerase chain reaction (PCR), IHC and
 Immunofluorescence (IF). It was evident that both SCCx43KO and WT primary
 cell cultures were similar in morphology and contained ~ 98 % SC. These
 highly pure SC cultures were then subjected to cell viability assays, yet no
 differences in the survival rate were determined between WT and SCCx43KO cell
 cultures on day 4 of culture. Additional measurements of transepithelial
 electrical resistance (TEER) were performed, which indicate significant
 increases in TEER values of the SCCx43KO cells. Using semi-quantitative WB,
 the TJ protein claudin11 was found to be elevated in SCCx43KO cell cultures
 and significantly increased in testes homogenate of adult SCCx43KO mice. Thus
 a SC line deficient of Cx43 may provide a valuable in vitro tool for a
 better understanding of the mechanistic role of Cx43 in spermatogenesis and
 BTB assembly, and this model might aid in finding treatment schemes of male
 infertility due to impaired spermatogenesis.
 Thus, the use of SCCx43KO in ex vivo
 studies can provide supplementary information on the regulation of other TJ
 proteins of the BTB. On the other hand the development of SCCx43KO and WT
 immortalized cell lines (or primary cell cultures) can additionally provide
 further insights into the complex junctional communications between SC (with
 and without Cx43). The ultimate goal is to answer the question: what role
 Cx43 plays within the SC and which role might Cx43 play in impaired
 spermatogenesis. The SCCx43KO mouse model might be suitable to elucidate the
 cause of Cx43 downregulation in men with either impaired spermatogenesis or
 CIS. Finally, it could also be used to study Cx43 as a potential male
 contraceptive target for the reversible regulation of spermatogenesis.