Androgens are essential for the maintenance of male health and wellbeing. A disturbance in
androgen signalling has been associated with a number of clinically relevant disorders such
as cardiovascular disease, diabetes and metabolic disorders as well as infertility. Primarily
produced in the testis in males, the actions of androgens are mediated through binding to
androgen receptor (AR), a member of the nuclear receptor superfamily of ligand-activated
transcription factors. The somatic cells of the testis are known to have a number of key roles
in both testis function and development and the Sertoli, Leydig and Peritubular Myoid cells
are known to express AR in adulthood. It is through AR that some testicular functions are
mediated; for example, the Sertoli cells support of complete spermatogenesis with Sertoli
cell androgen receptor knockout (SCARKO) testis demonstrating a halt of spermatogenesis
before meiosis. However, how androgen signalling is impacting testicular function through
each of the somatic cell types is not yet fully understood.
Currently, treatments for male reproductive disorders such as hypogonadism (low
androgens) and infertility are limited to treatment of the symptoms; using androgen
replacement therapy and in vitro fertilisation techniques. This has been, up until recently, a
result of a lack of understanding of the causes of these conditions and a lack of resources
able to treat them, with research suggesting that a genetic component may be responsible
in a number of cases. However, due to the limited genetic investigation diagnosis of men with
male reproductive disorders, the wider understanding of the genetics underpinning male
hypogonadism and infertility is incomplete.
Developments in technology for the investigation and editing of the genetic code are
triggering a surge in the exploration of genetic disorders and, in parallel, into the fields of
gene delivery vectors and editing technologies. These technologies will allow an expansion
into the knowledge and understanding of genetic disorders whilst simultaneously affording
the opportunity to exploit this understanding for the development of therapeutics.
There have been a small handful of previous studies using technologies such as viral vectors
to target the testicular somatic cells and deliver exogenous transgenes with the purpose of
both gene editing and repair, all with varying degrees of success.
Here, techniques to introduce and target the Leydig and Sertoli cells were investigated to
determine the most appropriate methodology for gene delivery to and manipulation of the
testis. Refinement of injections into the interstitial compartment were carried out before
introducing lentiviral vectors and targeting of Leydig cells was validated and optimised.
Lentiviral vectors are able to permanently integrate into the host cell. Surprisingly, analysis
of testis post lentiviral injection determined that the lentiviral targeted Leydig cells began to
undergo apoptosis one week post injection and were subsequently cleared from the testis
after ten days. Contrastingly, this was not the case when adenoviral vectors were introduced
into the interstitial compartment, with Leydig cells continuing to express the delivered
reporter transgene and, importantly, not expressing markers of apoptosis, ten days post
injection. This would suggest that using adenoviral vectors to target the Leydig cell
population in the adult testis would be more appropriate than using lentiviral vectors.
Previous studies have successfully used lentiviral vectors to target the Sertoli cells in the adult
testis via the introduction of the particles through the efferent duct. However, this can result
in damage to efferent duct, resulting in blockages and subsequently the seminiferous
tubules. To circumvent this, introduction of the lentiviral particles through the rete
compartment of the testis at a range of lower injection pressures was examined and injecting
at a lower pressure through the rete testis was found to reduce the likelihood of introducing
negative impacts on testicular histology when targeting the seminiferous tubules. Using
these refined methods of introducing lentiviral vectors, targeted Sertoli cells stably expressed
the delivered transgene for up to one year post injection.
Using viral vector delivered transgenes for both the investigation of testicular genetic
disorders and for the development of therapeutics has great potential. To explore this
potential, we first generated a mouse model in which AR was ablated from both the Leydig
and Sertoli cells using Cre/LoxP technology, termed the SC-LC-ARKO. Alongside providing a
potential model to ‘repair’ with viral vectors, the SC-LC-ARKO model also provided an
additional model for comparison with other models exhibiting ablation of AR from both single
somatic cell types and double somatic cell types. This further enabled a characterisation of
the roles of AR in adult testicular function, with results suggesting that loss of AR from more
than one cell type results in an additive phenotype when compared to single cell knock outs.
Despite providing further insight into the roles of AR in the testis, further analysis of the Cre
line used to generate the SC-LC-ARKO model indicated that a small number of Leydig cells
were expressing the Cre recombinase, resulting in only a small population of Leydig cells with
ablated AR.
Considering this, to explore the potential of rescuing Sertoli cell AR using lentiviral vectors,
we then utilised an already well characterised Sertoli Cell AR knockout (SCARKO) model.
Lentiviral vectors expressing mouse AR and monomeric GFP (moeGFP) downstream of a CMV
promoter were generated and injected into the rete testis of WT and SCARKO adult (day 100)
males at low pressure. The contralateral testis was injected with a lentiviral vector expressing
moeGFP alone (also downstream of a CMV promoter) using the same technique. Analysis of
testis sections revealed a reintroduction of AR to Sertoli cells in 100% of SCARKO testis
injected with lentivirus expressing mouse AR. As a result of this re-expression of AR in Sertoli
cells, 66% of the testis injected with lentivirus expressing mouse AR had evidence of
morphologically mature elongated spermatids, indicative of ongoing spermatogenesis. These
results suggest that a rescue of the infertility phenotype reported in previous studies of
SCARKO testis. Also demonstrated is the reversal of the SCARKO testicular phenotype in
tubules targeted by the mAR expressing lentiviral vector. This suggests that absence Sertoli
cell AR throughout development does not have a permanent impact on the Sertoli cells
capacity to support spermatogenesis in adulthood following rescue of SC AR expression in
adulthood.
In summary, the results of these studies have provided a refinement in the methodologies
for targeting the Sertoli and Leydig cells of the adult testis with viral vectors as well as
demonstrating successful rescue of a previously reported mouse model exhibiting infertility
through reintroduction of a functional gene. Alongside this, comparisons of AR knockout
models have afforded insight into maintenance of testis function through AR