Final Report Summary - MIGBTB (Molecular mechanisms of germ cell migration across the mammalian blood-testis barrier)
Project context and objectives
The study focuses in particular on the mechanism of germ cell migration during early postnatal testicular development and later spermatogenesis. Early postnatal migration comprises of gonocytes migrating to the basal part of seminiferous tubule to produce spermatogonia. Second migratory events take place throughout spermatogenesis where differentiating spermatocytes pass through the blood-testis-barrier (BTB).
The study focuses on the mechanism of germ cell migration at these two time points using conditional knock-out mouse models (cKO) for CAR (the coxsackie virus and adenovirus receptor) (CAR's official symbol is CXADR).
The overall objectives of this study are as follows:
- to investigate the role of CAR in germ cell migration across blood-testis-barrier (BTB) and early postnatal migration of gonocytes to basal membrane;
- the effects of cytotoxic agents on BTB integrity;
- exploring the possible interacting partner that helps CAR in this process.
Work performed
We knocked out CAR at two time points: the adult and early postnatal period. Characterisation of the morphological and functional aspect of testicular function was carried out. We found that the down-regulation of CAR does not affect the morphology of the testis in adults; the mice were fertile. BTB was intact and was fully functional as determined both by the use of a biotin-labelled tracer and a Lanthanum tracer. These tracers were retained in the interstitial space and were restricted on entering the seminiferous tubules due to the presence of functional BTB. The distribution of other junctional proteins like occludin, ZO-1, JAMA, claudin 3 remained normal as judged by immunofluorescence staining. The ultra-structural examination by transmission electron microscopy revealed that the junctions appeared normal. When CAR was knocked out in early postnatal mice, there was efficient floxing of the CAR gene and the CAR protein level was also decreased. We did not observe any morphological differences in the testis of six-day-old mice as compared to the control. The testis from these mice showed a normal appearance of differentiating spermatocytes at postnatal day 24. The search for an interacting partner for CAR showed that JAML protein was found to be highly expressed in the adult testis. Also JAML interacts with CAR in the testes based on immuno-precipitation experiments. The integrity of the BTB and the expression and localisation of BTB components were analysed as part of the collaborative project. The results showed that the effect of doxorubicin is age-dependent and the early pubertal mice showed mis-localisation of the junctional proteins. In conclusion these results indicate that CAR might not have a direct role in the migration of the germ cells at these two critical periods of testicular development. The other explanation could be that a small amount of remaining CAR protein is enough to help migrating germ cells. The model can be further improved by using a Sertoli-cell CAR knock-out in order to knock out CAR efficiently.
The first event of migration takes place immediately after birth, constituting movement of prospermatogonia from the lumen of the seminiferous tubules toward the peripheral region where the spermatogonial stem cells find their niche. The second migration comprises actively dividing mitotic spermatocytes across blood-testis-barrier (BTB) towards the lumen of seminiferous tubules during active spermatogenesis. The BTB is constituted largely by inter-Sertoli cell tight junctions. It divides the seminiferous tubule into basal and adluminal compartment. Migration through the BTB has been extensively studied and the maintenance of the barrier integrity during migration has been greatly emphasised. One of the widely accepted theories suggests a transit intermediate compartment between the adluminal help germ cell migration through the BTB based on morphological evidence. Several members of junctional protein families are implicated in the movement of germ cells; among these are claudins, occludin and CTX family members, like Coxsackie and Adenovirus Receptor (CAR) and Junction Adhesion Molecules (JAMs). CAR and JAM-C have been shown to be crucial for normal heart development and germ cell differentiation, respectively. Interestingly, JAML and CAR interaction is important in leukocyte migration across endothelial and/or epithelial barriers and we have also shown that JAML is expressed in the cultured Sertoli cells. In this context similar interactions might be important in germ cell migration. In testis CAR is expressed in high amounts during the early postnatal period (up to day 12) and then its expression becomes stage-dependent with the highest concentration at stage VII-IX where the germ cells are migrating through BTB. This expression pattern thus suggests a role in these two migration events.
The overall objective of this study was to explore the participation of the CTX family of proteins, in particular CAR, in facilitating the migration process at two major time points of testicular development: early postnatal migration of prospermatogonia to the peripheral region of seminiferous tubules and later, the migration of spermatocytes towards the lumen of seminiferous tubules across the blood-testis-barrier (BTB). We also explored the interacting partner and downstream signalling pathways of CAR contributing to its functions in testis. As a collaborative endeavour we have also studied BTB junctional components like Pg1 and its correlation with the protection of testis against toxic effects of cytotoxic agents. In this context, the effects of cytotoxic agents on formation and integrity of BTB is explored.
The work is divided into three packages. A brief summary of all the packages and main results achieved in each work package (WP) is described below.
1. WP 1: CAR and germ cell migration
The objectives of WP 1 were to assess the role of CAR in the two germ cell migratory events occurring during postnatal testicular development. The first is the early postnatal migration event of gonocytes from the centre of the lumen towards the peripheral region where gonocytes or prospermatogonia are differentiated to spermatogonia and germ stem cells find their niche. The second migration event is initiated during adulthood where the differentiating spermatocytes move from the basal to the adluminal compartment of the seminiferous tubules to become differentiated sperms. This process involves trans-epithelial migration through BTB and ensures continual spermatogenesis. The initial characterisation of the second phase of migration in the adult animal was done in year 1 and completed in the second year. This part largely focused on characterising early postnatal gonocyte migration and the embryonic testicular development after E13.5. Three major findings are reported in this WP:
a. Adult CAR cKO testis displayed normal spermatogenesis and fertility. Distribution of other junctional proteins and BTB integrity was unaffected;
b. CAR down-regulation in the early postnatal period does not affect gonocyte migration and later spermatogenesis, followed up to the age of P24 in these mice;
c. The embryonic development of testis after E13.5 is not affected by the CAR down-regulation.
The conclusions from these results, however, should be treated with caution as minimal amounts of CAR protein is still present, which might be enough for the migration of germ cells at the phases analysed. This notion is also supported by our data on normal appearance and expression of other junctional proteins like claudin 3 and 11, occludin and JAMA, which are not changed as compensatory mechanisms.
2. WP 2: BTB dynamics: effects of cytotoxic agents
The main objective of this work package was characterising the intermediate compartment that exists at the BTB during the transit of spermatocytes to the luminal compartment of the seminiferous tubules. The nature of the compartment is dynamic and short lived, appearing at stage VIII of the seminiferous tubules as suggested in the studies by our group. The approach was to construct the 3-D images of this compartment by utilising several microscopes, like dual-beam microscopes, and a transmission electron microscope (TEM), together with a confocal microscope and laser micro-dissection to finally analyse the components of the compartments. Our analyses were limited basically to electron microscopes and the use of confocal microscopes. Due to the failure of the application of both laser micro-dissection and the dual beam electron microscopy to characterise the intermediate compartment, alternative approaches were utilised and the dynamics of BTB was studied instead. Three model systems were designed in the form of collaborative projects as outlined below:
- using CAR cKO mouse models to study the effect of the absence of CAR on BTB formation and maintenance;
- analysis of ontogeny and expression of drug transporter Pg1, an integral component of BTB in young, early pubertal and adult rats;
- age-dependent effects of cytotoxic drug (doxorubicin) on BTB formation and integrity in rats.
The above-mentioned studies are designed as collaborative projects and are still ongoing. The results obtained to date from these studies can be summarised as follows:
a. Pg1 is an integral part of BTB and is found to be age-dependently regulated with increasing expression from PP6 and onwards. The level of cytotoxicity of doxorubicin is negatively correlated with the expression level of the Pg1. PP6 was found to have the lowest expression of Pg1, hence the maximum toxicity effect by doxorubicin is observed;
b. the expression and localisation of junctional proteins is changed in early pubertal rats after three and six days of doxorubicin treatment. No such effects were observed in immature and adult rat testis.
3. WP 3: Interacting partners of CAR
The main objectives of this work package were to characterise interacting partners and the downstream signalling of CAR in testis. For this purpose, primary Sertoli-cell cultures were established to utilise it as in vitro model systems to analyse cells from CAR cKO testis. The aim was to knock-out CAR in Sertoli-cell culture and study the effects on the downstream signalling of CAR. CAR has been shown to interact with other junctional proteins and forms both haemophilic and heterophilic dimers. Such interactions were observed with CAR-CAR, CARJAMA and CAR-JAML. In particular, the CAR-JAML interaction is important in trans-endothelial migration and could be important in testis too in trans-epithelial migration ongoing at BTB. In order to analyse the interaction of CAR and JAML, constructs were generated and immuno-precipitation experiments on CAR and JAML from testis was performed. Several commercially available antibodies for JAML were screened for this purpose. The main results achieved to date are:
a. the establishment of the in vitro primary Sertoli-cell culture from 14-day-old mice. The Sertoli-cell culture from control and CAR cKO mice has been set up and CAR has been down-regulated by adding Tamoxifen. RNA isolation and microarray experiments are forthcoming;
b. CAR-JAML interaction has been shown to be present in testis by immuno-precipitation experiments.
The study is an attempt to explore the complex mechanism of germ cell migration during the two critical periods of testicular development and spermatogenesis using a transgenic model for one of the important junctional proteins, CAR. Failure to migrate at these two steps results in the testicular dysgenesis syndrome that includes cryptochidism and Sertoli-cell-only syndrome and infertility. The phenomenon has been explained by many theories and a number of players required for this process has been identified, a few with functional evidence. The study exploits the use of transgenic technology to explore the in vivo function of a protein. The other aspect of the project is studying the dynamics of BTB, which revealed important information regarding the harmful effects of anti-cancer drugs on BTB formation. Other studies have concentrated on the direct toxic effect of this drug on germ cells. Our report points towards the effects of this drug on Sertoli-cell function in relation to BTB disturbances, thus suggesting a cautious use of this drug in early pubertal boys.
Socioeconomic impact of the project so far
Apart from presenting these results in the European testis workshop in May 2010, the results of the cKO model characterisation has been submitted for publication and is currently in the review process. The above results on testicular phenotype characterisation in young and adult mice are now being compiled as a manuscript. The research fellow has also substantially participated in the cytotoxic effects on BTB and the results are also being compiled in a manuscript. Taken together, the Marie Curie fellowship has helped the research fellow to develop her skills and greatly increased scientific understanding.
The study focuses in particular on the mechanism of germ cell migration during early postnatal testicular development and later spermatogenesis. Early postnatal migration comprises of gonocytes migrating to the basal part of seminiferous tubule to produce spermatogonia. Second migratory events take place throughout spermatogenesis where differentiating spermatocytes pass through the blood-testis-barrier (BTB).
The study focuses on the mechanism of germ cell migration at these two time points using conditional knock-out mouse models (cKO) for CAR (the coxsackie virus and adenovirus receptor) (CAR's official symbol is CXADR).
The overall objectives of this study are as follows:
- to investigate the role of CAR in germ cell migration across blood-testis-barrier (BTB) and early postnatal migration of gonocytes to basal membrane;
- the effects of cytotoxic agents on BTB integrity;
- exploring the possible interacting partner that helps CAR in this process.
Work performed
We knocked out CAR at two time points: the adult and early postnatal period. Characterisation of the morphological and functional aspect of testicular function was carried out. We found that the down-regulation of CAR does not affect the morphology of the testis in adults; the mice were fertile. BTB was intact and was fully functional as determined both by the use of a biotin-labelled tracer and a Lanthanum tracer. These tracers were retained in the interstitial space and were restricted on entering the seminiferous tubules due to the presence of functional BTB. The distribution of other junctional proteins like occludin, ZO-1, JAMA, claudin 3 remained normal as judged by immunofluorescence staining. The ultra-structural examination by transmission electron microscopy revealed that the junctions appeared normal. When CAR was knocked out in early postnatal mice, there was efficient floxing of the CAR gene and the CAR protein level was also decreased. We did not observe any morphological differences in the testis of six-day-old mice as compared to the control. The testis from these mice showed a normal appearance of differentiating spermatocytes at postnatal day 24. The search for an interacting partner for CAR showed that JAML protein was found to be highly expressed in the adult testis. Also JAML interacts with CAR in the testes based on immuno-precipitation experiments. The integrity of the BTB and the expression and localisation of BTB components were analysed as part of the collaborative project. The results showed that the effect of doxorubicin is age-dependent and the early pubertal mice showed mis-localisation of the junctional proteins. In conclusion these results indicate that CAR might not have a direct role in the migration of the germ cells at these two critical periods of testicular development. The other explanation could be that a small amount of remaining CAR protein is enough to help migrating germ cells. The model can be further improved by using a Sertoli-cell CAR knock-out in order to knock out CAR efficiently.
The first event of migration takes place immediately after birth, constituting movement of prospermatogonia from the lumen of the seminiferous tubules toward the peripheral region where the spermatogonial stem cells find their niche. The second migration comprises actively dividing mitotic spermatocytes across blood-testis-barrier (BTB) towards the lumen of seminiferous tubules during active spermatogenesis. The BTB is constituted largely by inter-Sertoli cell tight junctions. It divides the seminiferous tubule into basal and adluminal compartment. Migration through the BTB has been extensively studied and the maintenance of the barrier integrity during migration has been greatly emphasised. One of the widely accepted theories suggests a transit intermediate compartment between the adluminal help germ cell migration through the BTB based on morphological evidence. Several members of junctional protein families are implicated in the movement of germ cells; among these are claudins, occludin and CTX family members, like Coxsackie and Adenovirus Receptor (CAR) and Junction Adhesion Molecules (JAMs). CAR and JAM-C have been shown to be crucial for normal heart development and germ cell differentiation, respectively. Interestingly, JAML and CAR interaction is important in leukocyte migration across endothelial and/or epithelial barriers and we have also shown that JAML is expressed in the cultured Sertoli cells. In this context similar interactions might be important in germ cell migration. In testis CAR is expressed in high amounts during the early postnatal period (up to day 12) and then its expression becomes stage-dependent with the highest concentration at stage VII-IX where the germ cells are migrating through BTB. This expression pattern thus suggests a role in these two migration events.
The overall objective of this study was to explore the participation of the CTX family of proteins, in particular CAR, in facilitating the migration process at two major time points of testicular development: early postnatal migration of prospermatogonia to the peripheral region of seminiferous tubules and later, the migration of spermatocytes towards the lumen of seminiferous tubules across the blood-testis-barrier (BTB). We also explored the interacting partner and downstream signalling pathways of CAR contributing to its functions in testis. As a collaborative endeavour we have also studied BTB junctional components like Pg1 and its correlation with the protection of testis against toxic effects of cytotoxic agents. In this context, the effects of cytotoxic agents on formation and integrity of BTB is explored.
The work is divided into three packages. A brief summary of all the packages and main results achieved in each work package (WP) is described below.
1. WP 1: CAR and germ cell migration
The objectives of WP 1 were to assess the role of CAR in the two germ cell migratory events occurring during postnatal testicular development. The first is the early postnatal migration event of gonocytes from the centre of the lumen towards the peripheral region where gonocytes or prospermatogonia are differentiated to spermatogonia and germ stem cells find their niche. The second migration event is initiated during adulthood where the differentiating spermatocytes move from the basal to the adluminal compartment of the seminiferous tubules to become differentiated sperms. This process involves trans-epithelial migration through BTB and ensures continual spermatogenesis. The initial characterisation of the second phase of migration in the adult animal was done in year 1 and completed in the second year. This part largely focused on characterising early postnatal gonocyte migration and the embryonic testicular development after E13.5. Three major findings are reported in this WP:
a. Adult CAR cKO testis displayed normal spermatogenesis and fertility. Distribution of other junctional proteins and BTB integrity was unaffected;
b. CAR down-regulation in the early postnatal period does not affect gonocyte migration and later spermatogenesis, followed up to the age of P24 in these mice;
c. The embryonic development of testis after E13.5 is not affected by the CAR down-regulation.
The conclusions from these results, however, should be treated with caution as minimal amounts of CAR protein is still present, which might be enough for the migration of germ cells at the phases analysed. This notion is also supported by our data on normal appearance and expression of other junctional proteins like claudin 3 and 11, occludin and JAMA, which are not changed as compensatory mechanisms.
2. WP 2: BTB dynamics: effects of cytotoxic agents
The main objective of this work package was characterising the intermediate compartment that exists at the BTB during the transit of spermatocytes to the luminal compartment of the seminiferous tubules. The nature of the compartment is dynamic and short lived, appearing at stage VIII of the seminiferous tubules as suggested in the studies by our group. The approach was to construct the 3-D images of this compartment by utilising several microscopes, like dual-beam microscopes, and a transmission electron microscope (TEM), together with a confocal microscope and laser micro-dissection to finally analyse the components of the compartments. Our analyses were limited basically to electron microscopes and the use of confocal microscopes. Due to the failure of the application of both laser micro-dissection and the dual beam electron microscopy to characterise the intermediate compartment, alternative approaches were utilised and the dynamics of BTB was studied instead. Three model systems were designed in the form of collaborative projects as outlined below:
- using CAR cKO mouse models to study the effect of the absence of CAR on BTB formation and maintenance;
- analysis of ontogeny and expression of drug transporter Pg1, an integral component of BTB in young, early pubertal and adult rats;
- age-dependent effects of cytotoxic drug (doxorubicin) on BTB formation and integrity in rats.
The above-mentioned studies are designed as collaborative projects and are still ongoing. The results obtained to date from these studies can be summarised as follows:
a. Pg1 is an integral part of BTB and is found to be age-dependently regulated with increasing expression from PP6 and onwards. The level of cytotoxicity of doxorubicin is negatively correlated with the expression level of the Pg1. PP6 was found to have the lowest expression of Pg1, hence the maximum toxicity effect by doxorubicin is observed;
b. the expression and localisation of junctional proteins is changed in early pubertal rats after three and six days of doxorubicin treatment. No such effects were observed in immature and adult rat testis.
3. WP 3: Interacting partners of CAR
The main objectives of this work package were to characterise interacting partners and the downstream signalling of CAR in testis. For this purpose, primary Sertoli-cell cultures were established to utilise it as in vitro model systems to analyse cells from CAR cKO testis. The aim was to knock-out CAR in Sertoli-cell culture and study the effects on the downstream signalling of CAR. CAR has been shown to interact with other junctional proteins and forms both haemophilic and heterophilic dimers. Such interactions were observed with CAR-CAR, CARJAMA and CAR-JAML. In particular, the CAR-JAML interaction is important in trans-endothelial migration and could be important in testis too in trans-epithelial migration ongoing at BTB. In order to analyse the interaction of CAR and JAML, constructs were generated and immuno-precipitation experiments on CAR and JAML from testis was performed. Several commercially available antibodies for JAML were screened for this purpose. The main results achieved to date are:
a. the establishment of the in vitro primary Sertoli-cell culture from 14-day-old mice. The Sertoli-cell culture from control and CAR cKO mice has been set up and CAR has been down-regulated by adding Tamoxifen. RNA isolation and microarray experiments are forthcoming;
b. CAR-JAML interaction has been shown to be present in testis by immuno-precipitation experiments.
The study is an attempt to explore the complex mechanism of germ cell migration during the two critical periods of testicular development and spermatogenesis using a transgenic model for one of the important junctional proteins, CAR. Failure to migrate at these two steps results in the testicular dysgenesis syndrome that includes cryptochidism and Sertoli-cell-only syndrome and infertility. The phenomenon has been explained by many theories and a number of players required for this process has been identified, a few with functional evidence. The study exploits the use of transgenic technology to explore the in vivo function of a protein. The other aspect of the project is studying the dynamics of BTB, which revealed important information regarding the harmful effects of anti-cancer drugs on BTB formation. Other studies have concentrated on the direct toxic effect of this drug on germ cells. Our report points towards the effects of this drug on Sertoli-cell function in relation to BTB disturbances, thus suggesting a cautious use of this drug in early pubertal boys.
Socioeconomic impact of the project so far
Apart from presenting these results in the European testis workshop in May 2010, the results of the cKO model characterisation has been submitted for publication and is currently in the review process. The above results on testicular phenotype characterisation in young and adult mice are now being compiled as a manuscript. The research fellow has also substantially participated in the cytotoxic effects on BTB and the results are also being compiled in a manuscript. Taken together, the Marie Curie fellowship has helped the research fellow to develop her skills and greatly increased scientific understanding.
