BACKGROUND
connexins form intercellular conduits permitting diffusional exchange of ions, secondary messenger molecules and metabolites up to  <dig> dalton.
these exchange properties regulate and coordinate several cell biological functions such as cell growth, differentiation and developmental processes  <cit> .
six connexin protein subunits form a hemichannel, also called connexon.
two docking connexons, contributed by two adjacent cells, form a gap junctional channel.
to date,  <dig> different connexin genes have been identified in the mouse genome  <cit> .
connexins can assemble into homomeric or heteromeric hemichannels, which in turn can dock to hemichannels of the same or different connexin composition in the plasma membrane of contacting cells to form homotypic or heterotypic channels  <cit> .
gap junction channels composed of different connexin isoforms differ from each other in unitary conductance and permeability to second messenger molecules  <cit> .
this raised the question to which extent the different connexin isoforms limit or support functional specialization of different cell types.
of the known connexin  genes, cx <dig> is most abundantly expressed in many cell types.
in mouse heart, cx <dig> protein is found in the working myocardium and in purkinje fibers  <cit> .
cx <dig> deficient mice die shortly after birth due to obstruction of right ventricular outflow tract of the heart  <cit> .
in reproductive organs cx <dig> has been shown to control proliferation of granulosa cells  <cit> .
in the testis, cx <dig> containing gap junction channels connect the leydig as well as the sertoli cells and loss of cx <dig> seems to impair spermatogenesis  <cit> .
it was described that postnatal lethality of cx <dig> deficient mice could be rescued by cx <dig> or cx <dig>  indicating that cx <dig>  cx <dig> and cx <dig> share some common functions  <cit> .
nevertheless, cx <dig> knock-in cx <dig>  and cx <dig> knock-in cx <dig>  mice showed functional and morphological differences, compared to wild-type mice.
cx <dig> is one of the two major gap junction proteins expressed in hepatocytes.
it was also found in placenta  <cit> , in the epidermis, and in several other organs, including support cells of the inner ear  <cit> , and in the uterine epithelium during pregnancy in response to embryo recognition  <cit> .
in mammary tissue, cx <dig> and cx <dig> are present in the secretory epithelium and cx <dig> is localized in the myoepithelium of the mammary gland  <cit>  as well as in several cell lines suggesting that these connexins might be important for development during pregnancy and function during lactation  <cit> .
cx <dig> and cx <dig> have been co-localized in gap junctions of mouse mammary epithelium during lactation and both homomeric and heteromeric connexins have been identified  <cit> .
the unique expression patterns of cx <dig> and cx <dig> suggest distinct and overlapping roles in mammary development and function.
cx <dig> mrna and protein were first detected in mouse mammary tissue on day  <dig> of pregnancy which was followed by a steady increase and maximal expression during lactation  <cit> .
in contrast, a significant induction of cx <dig> expression occurred within a few hours after parturition  <cit> .
the analysis of connexin knock-in mice is a useful strategy to identify unique features of different connexins and to distinguish them from properties shared by several connexin channels.
especially the cx <dig> locus was used several times for the generation of knock-in mice.
the phenotypes of cx43ki <dig>  cx43ki <dig> and cx43ki <dig> mice have been investigated  <cit> .
the regulatory consequences of cx <dig> phosphorylation were investigated in several laboratories  <cit> .
here we have replaced the coding region of cx <dig> by that of cx <dig>  the non-phosphorylated cx <dig> protein is of special interest, since it is possible to gain insights into the function of connexin channels independent of the regulation by phosphorylation.
furthermore, we wanted to clarify whether the replacement of cx <dig> by cx <dig> causes a similar phenotype as observed with homozygous cx43ki <dig> mice, i.e.
severe impairment of spermatogenesis.
in addition, heterozygous cx43ki <dig> females exhibited problems with lactation.
it had been hypothesized that this phenotype could be due to defects in cooperation between myoepithelial and glandular cells  <cit> .
RESULTS
generation and transgenic expression analysis of cx43ki26neo mice
in order to generate cx4326/ <dig> mice, we replaced the cx <dig> coding dna by cx <dig> coding dna followed by a neomycin resistance cassette in hm- <dig> embryonic stem  cells.
the coding region of cx <dig> was cloned behind the translational start codon of cx <dig>  for this purpose an additional ncoi restriction site was created by insertion of an additional cytosine residue using modified primers .
three correctly recombined es cell clones gave rise to chimeras that transmitted the mutant allele through the germ line when injected into blastocysts.
heterozygous, male cx4343/ <dig> mice were crossed with female flp recombinase expressing mice.
the flp-mediated recombination led to excision of the pgk-neomycin cassette.
the heterozygous mice gave birth to homozygous mutant offspring  when intercrossed, as demonstrated by southern blot and pcr analyses .
a multiplex pcr was established for genotyping using a common reverse primer directed to the 3' utr of cx <dig>  a forward primer directed to the coding region of cx <dig> and another one to the coding region of cx <dig> were employed.
a  <dig> amplicon was derived from the wild-type allele and a  <dig> bp amplicon was derived from the cx <dig> knock-in cx <dig> allele .
in the southern blot hybridization, the cx <dig> external probe recognized a  <dig> kb wild-type fragment and a  <dig>  kb knock-in fragment .
intron spanning rt-pcr analyses were used to demonstrate spliced isoforms, transcribed from the different cx <dig> alleles.
the mrna from brain, heart, liver and testis was transcribed in cdna, and the pcrs were carried out with different primers for cx <dig>  cx <dig> and β-actin .
as expected, the primers yielded an amplicon of  <dig> bp for cx <dig>  an amplicon of  <dig> bp for cx <dig> and an amplicon of  <dig> bp for β-actin .
the cx <dig> amplicon was found in brain, heart and testis of wild-type and heterozygous mice , but only weakly in liver where ito cells and cholangiocytes but not hepatocytes express cx <dig>  cx <dig> was expressed in brain, liver as well as testis of all mice due to endogenous expression and in particular in hearts of heterozygous cx4343/ <dig> as well as homozygous cx4326/ <dig> mice.
as expected, wild-type mice cx4343/ <dig> did not express cx <dig> mrna in their hearts.
expression of the cx <dig> protein was confirmed by western blot analysis  in the hearts of heterozygous cx4343/ <dig> and homozygous cx4326/ <dig> mice.
no cx <dig> protein was detected in hearts of homozygous cx4343/ <dig> mice.
dominant impairment of mammary function in cx4343/ <dig> heterozygous mice
only about 17% of the homozygous cx4326/ <dig> mice instead of 25%, expected according to mendelian inheritance, were born and only 6%  survived to day  <dig> post partum and longer .
preferential loss of homozygous cx4326/ <dig> animals coincided with general high mortality in the offspring of heterozygous cx4343/ <dig> parents .
this high mortality correlated with the maternal heterozygous cx4343/ <dig> phenotype.
the surviving heterozygous adult mice  were indistinguishable in their appearance from wild type litter mates, whereas homozygous males and females weighed about 30% less.
the generally high mortality among the pups of heterozygous cx4343/ <dig> mothers was accompanied by decreased postnatal growth, and continuous loss of the pups, suggesting that the pups died from starvation this phenomenon was already observed with heterozygous cx4343/ <dig> mothers, but was not reported for heterozygous cx43-deficient  mice  <cit> , confirming the dominant and specific mode of inheritance of the cx4343/ <dig> allele.
maternal behaviour of heterozygous cx4343/ <dig> mothers did not differ from controls.
when newborn litters of cx4343/ <dig> mothers were raised by wild type cx4343/ <dig> foster mothers, survival of the pups was highly improved.
this procedure had to be carried out, in order to expand the cx4343/ <dig> mouse colony but it was not quantitated due to occasional loss of pups not accepted by the foster mothers.
these observations suggested an insufficient nutrition due to an impaired mammary gland function during lactation.
in comparison to wild-type mice, histological analysis of heterozygous cx4343/ <dig> mothers  revealed that the glandular alveoli were less dilated and the ductuli were obviously less branched resulting in lower amount of secretory alveoli.
instead, extended areas of adipose tissue were observed between the glandular units .
higher magnification confirmed secretory transformation of alveolar epithelium with dilated alveoli and the typical apocrine protrusions as well as milk droplets in the alveoli .
both the presence of milk droplets found in alveoli and the histological features suggested that reduced development of the glands rather than milk production was impaired in heterozygous cx4343/ <dig> mothers.
infertility of homozygous cx4326/ <dig> mice
histological analysis of lung, thymus, pancreas, spleen, gastrointestinal tract, liver, kidney and brain did not reveal any pathological differences between heterozygotes and homozygotes when compared to wild-type controls .
however, the testes of homozygous cx4326/ <dig> mice weighed only about one fourth of those from wild-type males  and cx4326/ <dig> male as well as female mice were infertile as demonstrated by mating them with wild-type animals.
histological investigations revealed that, compared to the wild-type controls , the differentiated stages of spermatogenesis were absent in knock-in mice, although tubuli seminiferi were developed .
spermatogonia were still present, and sporadically primary spermatocytes were observed but neither spermatozoa nor other stages of spermatogenesis were found .
as a consequence, no sperms were detected in the lumen of the ductuli epididymidis .
since it was shown that the leydig cells are connected by cx <dig>  <cit> , and impaired spermatogenesis could be due to a decreased number of testosterone producing cells and the presence of the androgen receptor.
immunolabeling of the androgen receptor revealed that specifically stained leydig cells were present in a similar amount as in the controls .
most of the seminiferous tubules were only lined by sertoli cells  but like in the wild-type controls , sertoli cells of the cx4343/ <dig> testes  expressed the androgen receptor.
finally, intratesticular testosterone levels were normal in homozygous cx4326/ <dig> mice as compared to controls .
compared to the ovaries of wild-type animals  the ovaries of homozygous cx4326/ <dig> mice were obviously smaller and exhibited only few follicles .
the full range of follicle stages was missing and corpora lutea were absent.
the maturation of the few follicles appeared to be arrested at the early secondary stage .
in some cases the morphology of the follicles was impaired, i.e.
in most follicles the oocytes were missing, and instead of an oocyte and follicle cells, a cyst like structure was observed .
in an extreme case, the complete ovary was displaced by one big cyst .
since this effect could be ascribed to an impaired release of follicle stimulating hormone  of the anterior pituitary gland, we investigated the fsh levels in homogenates of the pituitary gland.
fsh hormone levels showed high interindividual variations but did not significantly differ between pituitary glands of cx4326/ <dig> mice when compared with heterozygous or wild-type animals .
cardiac effects of replacing cx <dig> with cx26
cx <dig> is the most abundantly expressed connexin isoform in atrial and ventricular working myocardium of mice.
its loss in cx43-deficient mice resulted in dysmorphogenesis and obstruction of the right ventricular outflow tract, leading to early postnatal death  <cit> .
the hearts of adult heterozygous and homozygous cx4326/ <dig> mice showed no structural defects like those in cx <dig> deficient mice except of the small size of homozygous cx4326/ <dig> hearts, in comparison to control animals .
cx <dig> deficient neonates revealed significantly reduced heart rates as compared to littermates .
ecg parameters revealed no significant differences.
p durations were  <dig> ±  <dig> ms ,  <dig> ±  <dig> ms  and  <dig> ±  <dig> ms , respectively.
pr intervals were  <dig> ±  <dig> ms ,  <dig> ±  <dig> ms  and  <dig> ±  <dig> ms , respectively.
qrs durations were  <dig> ±  <dig> ms ,  <dig> ±  <dig> ms  and  <dig> ±  <dig> ms .
qt and qtc intervals were  <dig> ±  <dig> ms and  <dig> ±  <dig> ms ,  <dig> ±  <dig> ms and  <dig> ±  <dig> ms ,  <dig> ±  <dig> ms and  <dig> ±  <dig> ms , respectively.
no significant differences were obtained for left-ventricular mass ,  <dig> ±  <dig> mg ,  <dig> ±  <dig> mg , respectively) and ejection fraction ,  <dig> ±  <dig> % ,  <dig> ±  <dig> % , respectively) in mice at p <dig>  left-ventricular mass normalized to body weight was not significantly different in neonatal mice.
adult cx4326/ <dig> mice had a significantly lower body  and heart weight  as compared to heterozygous  and control mice .
heart rate and left-ventricular ejection fraction did not differ significantly between cx4326/26- , cx4343/26-  and cx4343/43-mice .
compared to their wt-littermates, cx4326/ <dig> mice exhibited prolonged qrs-  and interestingly shorter rate-corrected  qtc-intervals  in the surface ecg.
infra-hisian conduction was impaired  binds to growth hormone receptors in the stromal compartment of the mammary gland and stimulates insulin-like growth factor-i  mrna expression.
gh-induced igf-i is of major importance in ductal morphogenesis, and seems to be necessary for later stages of mammary development  <cit> .
the sparse branching of the milk ductuli could be due to missing prolactin or growth hormone from the anterior pituitary gland which was shown to express cx <dig>  <cit> .
anterior pituitary cx <dig> levels were at maximal level during periods of high prolactin secretion.
however, reduced estrogen or progesterone levels in heterozygous females cannot be excluded as a reason for impaired ductal morphogenesis.
an impaired cooperation between myoepihelial and glandular cells, as it has been proposed for the heterozygous cx4343/ <dig> mice which revealed only milk ejection problems but no dysmorphogenesis of the mammary glands  <cit> , could not be confirmed with cx4343/ <dig> mice.
thus, the replacement of one allele by cx <dig> in the cx <dig> locus seems to play a different role for mammary development during lactation as it has been shown for cx4343/ <dig> mice.
further analyses will be needed to pinpoint the defect in lactating mammary glands of cx4326/ <dig> mice.
decreased levels of growth hormone could also explain our observation that homozygous animals showed a reduced body size without pathological phenotype, even if nourished by foster mothers.
the gonads of both male and female homozygous cx4326/ <dig> mice, however, were hypotrophic compared to other organs.
neither in testes nor in ovaries, complete gametogenesis was found.
due to the smaller gonads and impaired gametogenesis we investigated whether these animals suffer from a defective release of the follicle stimulating hormone from the pituitary gland which regulates both follicle maturation and spermatogenesis.
however, our measurements of fsh did not confirm this hypothesis.
the presence of the androgen receptor in both leydig and sertoli cells is not consistent with impaired spermatogenesis leading to a kind of "sertoli-cell-only" syndrome.
our results indicate that cx <dig>  similar to cx <dig>  <cit> , cannot functionally replace cx <dig> which is abundantly expressed between sertoli cells.
since spermatogonia are present and even start to enter spermatogenesis, this indicates a direct nourishing role for cx <dig> in sertoli cells in this differentiation process.
the finding that functional expression of cx <dig> gap junction channels is necessary for spermatogenesis confirms the investigations of roscoe et al.
<cit> .
these authors recognized a "sertoli-cell-only" syndrome in testes which lacked cx <dig> and were cultivated under the kidney capsule, although the production of testosterone was similar to wild-type testes.
replacing cx <dig> with cx <dig> in ovarian follicles led to an arrested growth of less follicles up to the secondary stage and a cystic transformation.
like in the cx4326/ <dig> mice, the ovaries of cx <dig> deficient mice, when grafted under the kidney capsule  <cit>  did not develop beyond the primary stage and revealed less follicles.
thus, cx <dig> channels between granulosa cells were not able to maintain folliculogenesis, and its expression led to a similar defect as shown in cx <dig> null mouse mutants.
again, this is different from the observation in cx4332/ <dig> mice which showed normal ovarian histology and could become pregnant  <cit> .
thus, in contrast to cx <dig> which cannot replace the function of cx <dig> in folliculogenesis and spermatogenesis, the cx <dig> protein is able to take over the function of cx <dig> in granulosa cells for oocyte maturation.
although replacement of cx <dig> by cx <dig> leads to gonadal dysfunction in male and female, cx <dig> can apparently partially replace cx <dig> in working myocardium, at least in short-term function during several postnatal weeks, similar as previously described for cx43ki <dig> and cx43ki <dig> mice  <cit> .
cx4326/ <dig> mice seemed to develop similar electrophysiological abnormalities, although slower than cx43-/- mice.
we noticed a prolonged qrs complex as a marker for possible bundle branch blocks or longer depolarization phase and a tendency towards ventricular arrhythmia as described in cx <dig> deficient mice  <cit> .
interestingly, the qtc interval was shorter in the present mouse model.
thus, the repolarization phase did not seem to be affected in a similar way.
repolarization characteristics should therefore be further investigated using optical or electrical mapping systems.
despite this, in vivo electrophysiological investigation showed that cx <dig> cannot completely compensate for cx <dig> missing in conductive and working myocardium.
the strongly decreased survival of cx4326/ <dig> mice suggests that the long-term function of the myocardium is impaired.
CONCLUSIONS
finally, we found differences in the ability of cx <dig> and cx <dig> to functionally replace cx <dig> in the lactating mammary gland.
both heterozygous knock-in mice demonstrated impairment of mammary function but in a different way.
replacement of cx <dig> by cx <dig> disturbed milk ejection, whereas replacement of cx <dig> by cx <dig> led to a dysmorphogenesis of gland arboration.
these results extend previous observations with cx <dig> knock-in mice  <cit>  that the different connexin isoforms fulfill individual roles in certain cell types but can at least partially replace each other in other cell types.

 connexins form intercellular conduits permitting diffusional exchange of ions, secondary messenger molecules and metabolites up to dalton. these exchange properties regulate and coordinate several cell biological functions such as cell growth, differentiation and developmental processes. six connexin protein subunits form a hemichannel, also called connexon. two docking connexons, contributed by two adjacent cells, form a gap junctional channel. to date, different connexin genes have been identified in the mouse genome. connexins can assemble into homomeric or heteromeric hemichannels, which in turn can dock to hemichannels of the same or different connexin composition in the plasma membrane of contacting cells to form homotypic or heterotypic channels. gap junction channels composed of different connexin isoforms differ from each other in unitary conductance and permeability to second messenger molecules. this raised the question to which extent the different connexin isoforms limit or support functional specialization of different cell types. of the known connexin genes, cx is most abundantly expressed in many cell types. in mouse heart, cx protein is found in the working myocardium and in purkinje fibers. cx deficient mice die shortly after birth due to obstruction of right ventricular outflow tract of the heart. in reproductive organs cx has been shown to control proliferation of granulosa cells. in the testis, cx containing gap junction channels connect the leydig as well as the sertoli cells and loss of cx seems to impair spermatogenesis. it was described that postnatal lethality of cx deficient mice could be rescued by cx or cx indicating that cx cx and cx share some common functions. nevertheless, cx knock-in cx and cx knock-in cx mice showed functional and morphological differences, compared to wild-type mice. cx is one of the two major gap junction proteins expressed in hepatocytes. it was also found in placenta, in the epidermis, and in several other organs, including support cells of the inner ear, and in the uterine epithelium during pregnancy in response to embryo recognition. in mammary tissue, cx and cx are present in the secretory epithelium and cx is localized in the myoepithelium of the mammary gland as well as in several cell lines suggesting that these connexins might be important for development during pregnancy and function during lactation. cx and cx have been co-localized in gap junctions of mouse mammary epithelium during lactation and both homomeric and heteromeric connexins have been identified. the unique expression patterns of cx and cx suggest distinct and overlapping roles in mammary development and function. cx mrna and protein were first detected in mouse mammary tissue on day of pregnancy which was followed by a steady increase and maximal expression during lactation. in contrast, a significant induction of cx expression occurred within a few hours after parturition. the analysis of connexin knock-in mice is a useful strategy to identify unique features of different connexins and to distinguish them from properties shared by several connexin channels. especially the cx locus was used several times for the generation of knock-in mice. the phenotypes of cx43ki cx43ki and cx43ki mice have been investigated. the regulatory consequences of cx phosphorylation were investigated in several laboratories. here we have replaced the coding region of cx by that of cx the non-phosphorylated cx protein is of special interest, since it is possible to gain insights into the function of connexin channels independent of the regulation by phosphorylation. furthermore, we wanted to clarify whether the replacement of cx by cx causes a similar phenotype as observed with homozygous cx43ki mice, i.e. severe impairment of spermatogenesis. in addition, heterozygous cx43ki females exhibited problems with lactation. it had been hypothesized that this phenotype could be due to defects in cooperation between myoepithelial and glandular cells.  generation and transgenic expression analysis of cx43ki26neo mice in order to generate cx4326/ mice, we replaced the cx coding dna by cx coding dna followed by a neomycin resistance cassette in hm- embryonic stem cells. the coding region of cx was cloned behind the translational start codon of cx for this purpose an additional ncoi restriction site was created by insertion of an additional cytosine residue using modified primers. three correctly recombined es cell clones gave rise to chimeras that transmitted the mutant allele through the germ line when injected into blastocysts. heterozygous, male cx4343/ mice were crossed with female flp recombinase expressing mice. the flp-mediated recombination led to excision of the pgk-neomycin cassette. the heterozygous mice gave birth to homozygous mutant offspring when intercrossed, as demonstrated by southern blot and pcr analyses. a multiplex pcr was established for genotyping using a common reverse primer directed to the 3' utr of cx a forward primer directed to the coding region of cx and another one to the coding region of cx were employed. a amplicon was derived from the wild-type allele and a bp amplicon was derived from the cx knock-in cx allele. in the southern blot hybridization, the cx external probe recognized a kb wild-type fragment and a kb knock-in fragment. intron spanning rt-pcr analyses were used to demonstrate spliced isoforms, transcribed from the different cx alleles. the mrna from brain, heart, liver and testis was transcribed in cdna, and the pcrs were carried out with different primers for cx cx and β-actin. as expected, the primers yielded an amplicon of bp for cx an amplicon of bp for cx and an amplicon of bp for β-actin. the cx amplicon was found in brain, heart and testis of wild-type and heterozygous mice, but only weakly in liver where ito cells and cholangiocytes but not hepatocytes express cx cx was expressed in brain, liver as well as testis of all mice due to endogenous expression and in particular in hearts of heterozygous cx4343/ as well as homozygous cx4326/ mice. as expected, wild-type mice cx4343/ did not express cx mrna in their hearts. expression of the cx protein was confirmed by western blot analysis in the hearts of heterozygous cx4343/ and homozygous cx4326/ mice. no cx protein was detected in hearts of homozygous cx4343/ mice. dominant impairment of mammary function in cx4343/ heterozygous mice only about 17% of the homozygous cx4326/ mice instead of 25%, expected according to mendelian inheritance, were born and only 6% survived to day post partum and longer. preferential loss of homozygous cx4326/ animals coincided with general high mortality in the offspring of heterozygous cx4343/ parents. this high mortality correlated with the maternal heterozygous cx4343/ phenotype. the surviving heterozygous adult mice were indistinguishable in their appearance from wild type litter mates, whereas homozygous males and females weighed about 30% less. the generally high mortality among the pups of heterozygous cx4343/ mothers was accompanied by decreased postnatal growth, and continuous loss of the pups, suggesting that the pups died from starvation this phenomenon was already observed with heterozygous cx4343/ mothers, but was not reported for heterozygous cx43-deficient mice, confirming the dominant and specific mode of inheritance of the cx4343/ allele. maternal behaviour of heterozygous cx4343/ mothers did not differ from controls. when newborn litters of cx4343/ mothers were raised by wild type cx4343/ foster mothers, survival of the pups was highly improved. this procedure had to be carried out, in order to expand the cx4343/ mouse colony but it was not quantitated due to occasional loss of pups not accepted by the foster mothers. these observations suggested an insufficient nutrition due to an impaired mammary gland function during lactation. in comparison to wild-type mice, histological analysis of heterozygous cx4343/ mothers revealed that the glandular alveoli were less dilated and the ductuli were obviously less branched resulting in lower amount of secretory alveoli. instead, extended areas of adipose tissue were observed between the glandular units. higher magnification confirmed secretory transformation of alveolar epithelium with dilated alveoli and the typical apocrine protrusions as well as milk droplets in the alveoli. both the presence of milk droplets found in alveoli and the histological features suggested that reduced development of the glands rather than milk production was impaired in heterozygous cx4343/ mothers. infertility of homozygous cx4326/ mice histological analysis of lung, thymus, pancreas, spleen, gastrointestinal tract, liver, kidney and brain did not reveal any pathological differences between heterozygotes and homozygotes when compared to wild-type controls. however, the testes of homozygous cx4326/ mice weighed only about one fourth of those from wild-type males and cx4326/ male as well as female mice were infertile as demonstrated by mating them with wild-type animals. histological investigations revealed that, compared to the wild-type controls, the differentiated stages of spermatogenesis were absent in knock-in mice, although tubuli seminiferi were developed. spermatogonia were still present, and sporadically primary spermatocytes were observed but neither spermatozoa nor other stages of spermatogenesis were found. as a consequence, no sperms were detected in the lumen of the ductuli epididymidis. since it was shown that the leydig cells are connected by cx, and impaired spermatogenesis could be due to a decreased number of testosterone producing cells and the presence of the androgen receptor. immunolabeling of the androgen receptor revealed that specifically stained leydig cells were present in a similar amount as in the controls. most of the seminiferous tubules were only lined by sertoli cells but like in the wild-type controls, sertoli cells of the cx4343/ testes expressed the androgen receptor. finally, intratesticular testosterone levels were normal in homozygous cx4326/ mice as compared to controls. compared to the ovaries of wild-type animals the ovaries of homozygous cx4326/ mice were obviously smaller and exhibited only few follicles. the full range of follicle stages was missing and corpora lutea were absent. the maturation of the few follicles appeared to be arrested at the early secondary stage. in some cases the morphology of the follicles was impaired, i.e. in most follicles the oocytes were missing, and instead of an oocyte and follicle cells, a cyst like structure was observed. in an extreme case, the complete ovary was displaced by one big cyst. since this effect could be ascribed to an impaired release of follicle stimulating hormone of the anterior pituitary gland, we investigated the fsh levels in homogenates of the pituitary gland. fsh hormone levels showed high interindividual variations but did not significantly differ between pituitary glands of cx4326/ mice when compared with heterozygous or wild-type animals. cardiac effects of replacing cx with cx26 cx is the most abundantly expressed connexin isoform in atrial and ventricular working myocardium of mice. its loss in cx43-deficient mice resulted in dysmorphogenesis and obstruction of the right ventricular outflow tract, leading to early postnatal death. the hearts of adult heterozygous and homozygous cx4326/ mice showed no structural defects like those in cx deficient mice except of the small size of homozygous cx4326/ hearts, in comparison to control animals. cx deficient neonates revealed significantly reduced heart rates as compared to littermates. ecg parameters revealed no significant differences. p durations were ± ms, ± ms and ± ms, respectively. pr intervals were ± ms, ± ms and ± ms, respectively. qrs durations were ± ms, ± ms and ± ms. qt and qtc intervals were ± ms and ± ms, ± ms and ± ms, ± ms and ± ms, respectively. no significant differences were obtained for left-ventricular mass, ± mg, ± mg, respectively) and ejection fraction, ± %, ± %, respectively) in mice at p left-ventricular mass normalized to body weight was not significantly different in neonatal mice. adult cx4326/ mice had a significantly lower body and heart weight as compared to heterozygous and control mice. heart rate and left-ventricular ejection fraction did not differ significantly between cx4326/26-, cx4343/26- and cx4343/43-mice. compared to their wt-littermates, cx4326/ mice exhibited prolonged qrs- and interestingly shorter rate-corrected qtc-intervals in the surface ecg. infra-hisian conduction was impaired binds to growth hormone receptors in the stromal compartment of the mammary gland and stimulates insulin-like growth factor-i mrna expression. gh-induced igf-i is of major importance in ductal morphogenesis, and seems to be necessary for later stages of mammary development. the sparse branching of the milk ductuli could be due to missing prolactin or growth hormone from the anterior pituitary gland which was shown to express cx. anterior pituitary cx levels were at maximal level during periods of high prolactin secretion. however, reduced estrogen or progesterone levels in heterozygous females cannot be excluded as a reason for impaired ductal morphogenesis. an impaired cooperation between myoepihelial and glandular cells, as it has been proposed for the heterozygous cx4343/ mice which revealed only milk ejection problems but no dysmorphogenesis of the mammary glands, could not be confirmed with cx4343/ mice. thus, the replacement of one allele by cx in the cx locus seems to play a different role for mammary development during lactation as it has been shown for cx4343/ mice. further analyses will be needed to pinpoint the defect in lactating mammary glands of cx4326/ mice. decreased levels of growth hormone could also explain our observation that homozygous animals showed a reduced body size without pathological phenotype, even if nourished by foster mothers. the gonads of both male and female homozygous cx4326/ mice, however, were hypotrophic compared to other organs. neither in testes nor in ovaries, complete gametogenesis was found. due to the smaller gonads and impaired gametogenesis we investigated whether these animals suffer from a defective release of the follicle stimulating hormone from the pituitary gland which regulates both follicle maturation and spermatogenesis. however, our measurements of fsh did not confirm this hypothesis. the presence of the androgen receptor in both leydig and sertoli cells is not consistent with impaired spermatogenesis leading to a kind of "sertoli-cell-only" syndrome. our results indicate that cx similar to cx, cannot functionally replace cx which is abundantly expressed between sertoli cells. since spermatogonia are present and even start to enter spermatogenesis, this indicates a direct nourishing role for cx in sertoli cells in this differentiation process. the finding that functional expression of cx gap junction channels is necessary for spermatogenesis confirms the investigations of roscoe et al.. these authors recognized a "sertoli-cell-only" syndrome in testes which lacked cx and were cultivated under the kidney capsule, although the production of testosterone was similar to wild-type testes. replacing cx with cx in ovarian follicles led to an arrested growth of less follicles up to the secondary stage and a cystic transformation. like in the cx4326/ mice, the ovaries of cx deficient mice, when grafted under the kidney capsule did not develop beyond the primary stage and revealed less follicles. thus, cx channels between granulosa cells were not able to maintain folliculogenesis, and its expression led to a similar defect as shown in cx null mouse mutants. again, this is different from the observation in cx4332/ mice which showed normal ovarian histology and could become pregnant. thus, in contrast to cx which cannot replace the function of cx in folliculogenesis and spermatogenesis, the cx protein is able to take over the function of cx in granulosa cells for oocyte maturation. although replacement of cx by cx leads to gonadal dysfunction in male and female, cx can apparently partially replace cx in working myocardium, at least in short-term function during several postnatal weeks, similar as previously described for cx43ki and cx43ki mice. cx4326/ mice seemed to develop similar electrophysiological abnormalities, although slower than cx43-/- mice. we noticed a prolonged qrs complex as a marker for possible bundle branch blocks or longer depolarization phase and a tendency towards ventricular arrhythmia as described in cx deficient mice. interestingly, the qtc interval was shorter in the present mouse model. thus, the repolarization phase did not seem to be affected in a similar way. repolarization characteristics should therefore be further investigated using optical or electrical mapping systems. despite this, in vivo electrophysiological investigation showed that cx cannot completely compensate for cx missing in conductive and working myocardium. the strongly decreased survival of cx4326/ mice suggests that the long-term function of the myocardium is impaired.  finally, we found differences in the ability of cx and cx to functionally replace cx in the lactating mammary gland. both heterozygous knock-in mice demonstrated impairment of mammary function but in a different way. replacement of cx by cx disturbed milk ejection, whereas replacement of cx by cx led to a dysmorphogenesis of gland arboration. these results extend previous observations with cx knock-in mice that the different connexin isoforms fulfill individual roles in certain cell types but can at least partially replace each other in other cell types. 
furthermore, connexin knock-in connexin male and female mice were infertile and exhibited hypotrophic gonads. only about 17% of the homozygous connexin knock-in connexin mice instead of 25% expected according to mendelian inheritance, were born and only 6% survived to day post partum and longer. in testes, tubuli seminiferi were developed and spermatogonia as well as some primary spermatocytes were present, but further differentiated stages of spermatogenesis were absent. heterozygous mothers showed impaired mammary gland development responsible for decreased lactation and early postnatal death of the pups which could be partially rescued by wild type foster mothers.
 in order to further distinguish unique from general functions of connexin we have generated mice in which the coding region of connexin was replaced by that of connexin  heterozygous mothers showed impaired mammary gland development responsible for decreased lactation and early postnatal death of the pups which could be partially rescued by wild type foster mothers. only about 17% of the homozygous connexin knock-in connexin mice instead of 25% expected according to mendelian inheritance, were born and only 6% survived to day post partum and longer. neonatal and adult connexin knock-in connexin mice exhibited slowed ventricular conduction in their hearts, i.e. similar but delayed electrophysiological abnormalities as connexin deficient mice. furthermore, connexin knock-in connexin male and female mice were infertile and exhibited hypotrophic gonads. in testes, tubuli seminiferi were developed and spermatogonia as well as some primary spermatocytes were present, but further differentiated stages of spermatogenesis were absent. ovaries of female connexin knock-in connexin mice revealed only few follicles and the maturation of follicles was completely impaired.  the impaired gametogenesis of homozygous males and females can explain their infertility. 