G Protein-Coupled Receptor Transactivation: From Molecules to Mice - Methods in Cell Biology - page 434

brief, we used sodium citrate antigen retrieval, a blocking buffer of 10% normal goat
serum, antibody diluent of 0.05% goat serum in PBS and primary antibody concen-
trations of 1:100. For a wash buffer, we used PBS and secondary antibodies of Alexa
488 to visualize eCFP and Alexa 594 to visualize RFP, both at 1:500. To visualize
nuclei, we conducted the last wash step in PBS-Triton (0.1%) and counterstained the
nuclei using 4 0 ,6-diamidino-2-phenylindole dihydrochloride (DAPI). Analysis of
whole testis revealed that the expression of LHCGR B and LHCGR S was confined
to the Leydig cells, with no off-target expression, meaning that any functional rescue
of the LuRKO animal phenotype could be directly correlated with LHCGR B /
LHCGR S transactivation in the correct cellular compartments ( Fig. 23.4 (II)). To
visualize the structural and cellular components of the testis, we utilized hematoxylin
and eosin staining. The most important histological analysis for determining effects
of LHCGR transactivation on the functional rescue of the LuRKO animals is the size
of the seminiferous tubules and Leydig cell islets and the progression/resumption of
spermatogenesis to beyond arrest at the round spermatid stage as observed in the ho-
mozygous LuRKO animals. Histological analysis of the testis of the homozygous
LuRKO/LHCGR B /LHCGR S animals revealed the presence of mature sperm, in-
dicating that spermatogenesis was restored by transactivation, and that the sizes of
the seminiferous tubules and Leydig cell islets were comparable to WT animals.
Taken together, these data suggested that LHCGR transactivation was sufficient
to restore the gonadal function of the LuRKO to that of WT animals.
To determine the expression level of the LHCGR B and LHCGR S , we conducted
qPCR to look at mRNA expression of the mutant receptors. Although the mRNA ex-
pression levels were higher for the homozygous LuRKO/LHCGR B /LHCGR S ani-
mals, analysis of the serum LH and testosterone showed that the circulating
concentration of LH was slightly elevated in comparison to WT (but lower than in
LuRKO animals). Ligand-binding assays (carried out as previously described in
Rivero-M¨ller et al., 2010 ) to assess the number of LHCGR S showed no significant
difference to that of WT LHCGR. This indicates that the sensitivity of LHCGR B /
LHCGR S may be somewhat decreased in comparison toWTLHCGR. The restoration
of testosterone production in the homozygous LuRKO/LHCGR B /LHCGR S supports
this notion, as although not statistically different fromWT animals due to the large bi-
ological variation, it was slightly lower, most likely resulting from decreased negative
feedback to the pituitary and hence the higher circulating concentration of LH in the
transactivational model. Quantitative analysis of key LHCGR-dependent Leydig
cell-specific steroidogenic factors, StAR and CYP17a1 , which showdiminished expres-
sion in LuRKO animals, was also restored to expression levels approximating that of
WT animals in the homozygous LuRKO/LHCGR B /LHCGR S animals. This also
suggests that LHCGR transactivation is sufficient tomediate the downstream signaling
requirements for the transcription/translation of LH-dependent genes.
23.2.3.4 Fertility
The ultimate proof that transactivation of LHCGR B /LHCGR S could fulfill the
functional requirement of the LHCGR in vivo is to test the fertility of the animals
through mating experiments. For our studies, we set up breeding cages crossing