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are through functional, morphological, and histological analysis of the adult male
gonads and reproductive tract.
The first step we undertook to assess the effects of LHCGR
B
/LHCGR
S
trans-
activation in homozygous LuRKO animals was to examine the testes and reproduc-
tive tracts of the males. When euthanizing the animals, we used terminal anesthesia
and cardiac puncture to collect cardiac blood for measurement of circulating concen-
trations of LH and testosterone in serum. The reproductive tract, inclusive of epidid-
ymides, seminal vesicles, and testes, were dissected and photographed, as the
anabolic effects of testosterone on testis size and development of the reproductive
tract are key indicators of LHCGR function. Following this, the reproductive tract
and one testis were fixed using paraformaldehyde in (4% in PBS) for histological
and spatiotemporal expression analysis of the LHCGR
B
/LHCGR
S
, and the other
testis was snap frozen using liquid nitrogen for assessment of the effects of LHCGR
transactivation on LH-dependent gene expression and LHCGR binding activity. As
with the gross morphology of the testis and reproductive tract, the weights of these
tissues are also indirect indicators of the concentration of circulating testosterone and
hence LHCGR function. Examination of the dissected reproductive tract and testes
revealed that expression of the single LHCGR
B
or LHCGR
S
in a homozygous
LuRKO background had no restorative effect on the gross morphology of the repro-
ductive tract and testis weights, despite clear detection of the expressed LHCGR
B
or LHCGR
S
in Leydig cells. However, coexpression of LHCGR
B
and LHCGR
S
in a homozygous LuRKO background showed surprising results, with the gross mor-
phology, size, and weights of the reproductive tract and testis approximating those of
WT animals (
Fig. 23.4
(II)). These data are indicative that transactivation of LHCGR
can rescue the hypogonadal phenotype of the LuRKO animals.
For detailed histological analysis of testicular structures, paraformaldehyde-fixed
testes were dehydrated, paraffin embedded, and 5
m sections mounted onto poly-
D
-
lysine-coated slides for use in staining protocols. We conducted immunohistochem-
istry and histology staining on serial sections so that the images could be directly
compared. In order to interpret the functional/morphological findings of the func-
tional rescue of LuRKO phenotype by LHCGR transactivation, it was important
to undertake careful examination of the spatial/temporal expression pattern of
LHCGR
B
/LHCGR
S
to determine whether the expression was detected in the cor-
rect cellular compartments of the testis, that is, the Leydig cells. We determined the
expression pattern of LHCGR
B
and LHCGR
S
using the IRES fluorescent proteins.
It should be noted that although these proteins should be easily visualized without the
need for immunological-based detection, the testis, as a steroidogenic tissue with a
high cholesterol content, generates a high background fluorescent signal due to auto-
fluorescence making the direct visualization of the dsRed and eCFP proteins chal-
lenging. To circumvent this, we used immunohistochemistry to detect the
expression pattern of the IRES proteins, using differentially raised primary anti-
bodies to red fluorescent protein (RFP) and green fluorescent protein (GFP) (anti-
GFP antibodies recognize eCFP due to sequence similarity). We found that standard
immunohistochemical methods are sufficient for visualization of RFP and eCFP. In
m
