(Received for publication, March 6, 1997, and in revised form, May 19, 1997)
From the Department of Biological Chemistry, School
of Medicine, University of California, Davis, California 95616 and the
§ Department of Biochemistry and Biophysics, Washington
State University, Pullman, Washington 99164
We have previously demonstrated that there exist
two distinct genes for the thermostable inhibitor protein of the
cAMP-dependent protein kinase, PKI and PKI
(Van
Patten, S. M., Howard, P., Walsh, D. A., and Maurer, R. A. (1992)
Mol. Endocrinol. 6, 2114-2122). We have also shown that in
the testis, at least eight forms of PKI
exist, differing as a result
of at least post-translational modification and alternate translational
initiation (Kumar, P., Van Patten, S. M., and Walsh, D. A. (1997)
J. Biol. Chem. 272, 20011-20020). We now report that
in the testis, there is a unique cellular distribution of protein
kinase inhibitor forms, with PKI
being essentially (if not
exclusively) a germ cell protein and PKI
being expressed primarily
in Sertoli cells. Furthermore, there is a progressive change in the
forms of PKI
that are present within germ cells with development
that is initiated in testis tubules and continues as the germ cells
migrate through the epididymis. These conclusions are derived from
studies with isolated cell populations and with the at/at
germ cell-deficient mouse line, by in situ hybridization,
and by following the developmental expression of these proteins in both
testis and epididymis. We have also shown that follicle-stimulating
hormone (FSH) can increase the expression of both PKI
and PKI
.
The FSH-regulated expression of PKI
in the Sertoli cell likely
occurs via the normal route of second messenger signal transduction. In
contrast, the FSH-dependent PKI
expression must arise by
some form of Sertoli cell-germ cell intercommunication.
The cAMP signal transduction pathway is central to the
physiological function, development, and maturation of both the Sertoli and germ cells of the testis. cAMP is one of the key mediators of the
actions of FSH,1 whose primary, nearly sole
target in males is the Sertoli cell (1). The response of the Sertoli
cell to FSH is complex and changes developmentally, affecting at least
two critical phases of Sertoli cell development (2). In the rat, FSH is
required for normal proliferation of Sertoli cells in utero
and in the first 2 weeks of life, at which time their proliferation
essentially ceases. FSH is essential for proper maturation of the
Sertoli cell and is required for correct formation of the tight
junctions responsible for the "blood-testis" barrier. FSH action on
the Sertoli cell is also necessary for initiation of the first wave of
spermatogenesis (2). cAMP also has a number of important functional
roles in germ cells. In early spermatogenesis, it is a key regulator of
transcription, acting via stimulation of PKA-dependent phosphorylation of the complex family of activator and repressor transcription factors, CREB, CREM, and CREM (3, 4). During the
transit of sperm through the epididymis, an elevation of cAMP is one of
the primary initiation signals for the acquisition of flagellar
movement (5-7). One of the prominent proteins whose cAMP-dependent phosphorylation is correlated with the onset
of sperm motility is a 56,000-Da protein, first called axokinin (8, 9),
but subsequently identified as the PKA RII subunit (10,
11). Later in germ cell function, cAMP appears to be a key mediator to
induce capacitation (12).
Given the extensive role of cAMP-mediated phosphorylation in these
essential processes of germ cell maturation, it is not surprising that
there also exists some complexity of PKA itself. Essentially all of the
different species of PKA subunits (RI,
RI
, RII
, RII
, C
, and
C
) are apparent in these cells, with key differences in their
distribution among the distinct cell types and noted changes with
cellular development (13-16). A complex pattern of PKI isoforms is
also beginning to emerge. It is now established that the PKI isoforms,
as first studied in skeletal muscle (17) and testis (18, 19), arise
from two distinct genes (20, 21) and furthermore that at least eight
forms of PKI
exist that differ due to at least post-translational
modification and alternate translational initiation (22, 23). In this
report, we demonstrate that the PKI
and PKI
isoforms are
differentially localized to Sertoli and germ cells, respectively; that
there is a progressive change in PKI
isoform formation with germ
cell development; that testis PKI
and PKI
are both under
hormonal/developmental regulation; and that Sertoli cell-germ cell
communication likely plays an important role in the regulation of
PKI
expression.
Unless indicated otherwise, for the tissues used for both Northern blot analysis of mRNA species and Western blot analysis of PKI isoforms, adult Harlan Sprague Dawley rats were sacrificed by decapitation; and immediately after dissection, the tissues were freeze-clamped with Wollenberger clamps precooled in liquid nitrogen, and the tissue was powdered. For studies of FSH dependence, immature male rats of the specified age were injected intraperitoneally with either 0.1 ml of phosphate-buffered-saline (PBS) containing 8 µg of ovine FSH (USDA-oFSH-19-SIAFP, National Hormone and Pituitary Program, NIDDK, National Institutes of Health, Bethesda, MD) or PBS alone. The tissues were removed from the animal at the indicated times after injection, and at least three separate animals were used for each data point.
Isolated testis tubules were dissected as described by Parvinen and Ruokonen (24) using transillumination-assisted microdissection. Tubules were dissected into 2-mm segments starting from an interface pale zone (Stages IX-XI) through to the next dark zone (Stage VIII) and staged by the transillumination pattern. The segments were transferred into 50 µl of PBS containing a protease mixture of 0.5 mM (2-aminoethyl)benzenesulfonyl fluoride, 1 µM leupeptin, 2 mM benzamidine, 0.1 mM TPCK, and 20 milliunits/ml aprotinin. The tubular segments were homogenized with an Eppendorf Teflon homogenizer, and the proteins were then extracted and analyzed. The blots shown are representative of three experiments.
Epididymal cauda and caput sperm were obtained by microdissection and extraction as described by Moore et al. (25). The sperm were separated from the epididymal tissue fragments by first suspending the diced tissue in Petri dishes in 5 ml/animal of PBS containing a protease inhibitor mixture of 2 µg/ml aprotinin, 1 mM EDTA, 10 µg/ml benzamidine, 0.28 mM TPCK, 2.1 µM leupeptin, and 1 mM (2-aminoethyl)benzenesulfonyl fluoride (PBS-PIC buffer); gently rocking the dishes for 30 min; filtering the aspirated supernatant through cheesecloth; and then collecting the sperm by centrifugation. The sperm were resuspended in fresh PBS-PIC buffer and washed twice by centrifugation and resuspension. Soluble protein was extracted by incubating the isolated sperm at 4 °C for 5 min in 1 ml/animal of PBS-PIC buffer containing 1% Triton X-100 and then removing sperm fragments by centrifugation at 600 × g for 10 min. Total epididymal tissue PKI extract was obtained by extracting freeze-clamped powdered tissue in PBS-PIC buffer, heating for 10 min at 100 °C, and then removing insoluble/denatured material by centrifugation. Cultured Sertoli cells were prepared as described by Karl and Griswold (26), as an adaptation of the method originally described by Dorrington and Fritz (27). Total germ cells were isolated by the method of Stallard and Griswold (28), as adapted from Bellve et al. (12), and the enriched germ cell populations were obtained and characterized following the procedure of Grootegoed et al. (29) using unit gravity sedimentation. The at/at germ cell-deficient mice, as originally described by Handel and Eppig (30), and the heterozygous wt/at mice, used for controls, were purchased from Jackson Laboratories (Bar Harbor, ME). All animal studies were conducted in accord with the NIH Guide for the Care and Use of Laboratory Animals.
Northern Blot mRNA AnalysesThe preparation of total
RNA from powdered frozen testis, agarose gel electrophoresis, and
procedures for blotting/probing were done exactly as described
previously (21). The probes for PKI and PKI
, prepared as
described (21), correspond to base pairs 10-1000 and 1-945 of the rat
cDNAs, respectively. Following autoradiography of the blots,
quantitation of the mRNA was accomplished either by cutting out the
bands of radioactivity corresponding to the appropriate mRNA
(4.3-kilobase pair band for PKI
and 1.4-kilobase pair band for
PKI
) and counting in a liquid scintillation counter (for the
experiment of Fig. 7) or by scanning the blots using a Bio-Rad GS-250
molecular imager (for the experiment of Fig. 8).
Western Blot Analyses of PKI Isoforms
The procedures for
Western blot analyses were identical to those used in the accompanying
paper (22). Unless indicated otherwise, powdered frozen tissue was
extracted in 1 mM EDTA, pH 7.0, containing 1.6 µg/ml
aprotinin, 2 mM benzamidine, 0.1 mM TPCK, 1 µM leupeptin, and 0.5 mM
(2-aminoethyl)benzenesulfonyl fluoride and heated-treated for 10 min at
100 °C, and insoluble material was removed by centrifugation. One-dimensional electrophoresis (SDS) and two-dimensional
electrophoresis (isoelectric focusing and SDS) were performed as
described (22) using anti-PKI-(5-22)-amide and
anti-PKI
-(5-22)-amide antisera, as noted.
Rats of ages 5-60 days (5-day
intervals) were killed using carbon dioxide asphyxiation followed by
decapitation, and testes from animals were rapidly frozen on dry ice
and stored at 80 °C until sectioned. Cross-sections of testis were
cut to give transverse sections of the seminiferous tubules. Brains
were sectioned sagitally to give a general overview of mRNA
distribution. Sections (10 µm) were cut at
20 °C on a cryostat
(Bright) and freeze-thawed onto
gelatin/poly-L-lysine-subbed slides. Slides were stored at
80 °C until processed for in situ
hybridization.
The full-length cDNAs for
PKI and PKI
are 1183 and 1350 base pairs, respectively (20, 21).
PKI
cDNA was digested with HindIII/NotI,
gel-purified, treated with Klenow DNA polymerase to blunt the DNA ends,
and religated (EcoRI/NotI cDNA fragment 1-364 in pBluescript). PKI
cDNA was digested with
BamHI and gel-purified, and the backbone was religated
(EcoRI/BamHI cDNA fragment 1-356 in
pBluescript). This strategy removed poly(A) sequences present in the
3
-regions of both cDNAs. Riboprobes complementary to each isoform
mRNA were generated from these constructs using in vitro transcription with T7 (PKI
) and T3 (PKI
) RNA polymerases and 35S-UTP. Probes were labeled to a specific activity of
3-5 × 108 Ci/mmol.
In situ hybridization
was performed as described previously (31) with modifications as noted
in the fixation of tissues and hybridization temperatures. Briefly,
sections of brain were post-fixed in 4% paraformaldehyde in PBS
solution for 10 min, and sections of testis were fixed similarly for
1 h. Sections were then rinsed once in PBS and three times in
2 × SSC. All solutions were treated with diethyl pyrocarbonate
(0.02%). 35S-Labeled cRNA probes were denatured by heating
at 70 °C and added to hybridization buffer to give 10 × 106 counts/ml. Hybridization buffer (200 µl) was added to
each slide to cover the sections, and hybridization was carried out
overnight in sealed humid chambers at 45 °C. After hybridization,
slides were rinsed in 2 × SSC, treated with RNase A (30 µg/ml
in 0.5 M NaCl, 10 mM Tris-Cl, pH 7.5, and 1 mM EDTA, pH 8) for 60 min at 37 °C; and washed to a
maximum stringency of 0.1 × SSC at 55 °C for 60 min. Sections
were then dehydrated in 50, 70, and 90% ethanol in 0.3 M
ammonium acetate and air-dried. Sections were exposed to
autoradiographic film (Amersham Hyperfilm -max) for 10 days and then
dipped in K5 nuclear emulsion (Ilford, Cheshire, United Kingdom),
exposed at 4 °C for 2 weeks, developed, and counterstained with
hematoxylin and eosin. Control sections either were pretreated with
RNase A (100 µg/ml) for 60 min at 37 °C prior to hybridization or
were hybridized with a "sense" probe transcribed from the
complementary strand of the same cDNA template. In both tissue
types and with both probes, RNase pretreatment or sense probes gave no
detectable signal.
mRNA levels at each age were determined by estimation of
silver grain number overlying particular fields dependent on tissue. For PKI mRNA in the testis, mature tubules are defined as those containing elongated spermatids. Immature tubules are those containing the stages of developing germ cells up to, but not including, elongated
spermatids. Images of sections were captured on a Macintosh computer
using a video camera attached to a Nikon Optiphot microscope at a
magnification of ×200. Images were then exported to NIH Image (Version
1.52), where silver grains were counted using a threshold slice to
detect silver grains. Area covered by silver grains was calculated as
the pixel number in the field. Several fields were counted per section,
and background counts were subtracted before means were calculated.
Values shown are means ± S.E.
Previous studies, based upon Northern blot analyses (21),
have indicated that a major developmental shift occurs with testis PKI
isoforms. This is also seen by Western blot analysis (Fig. 1, a and b). In the neonate
testis, only the PKI protein isoform is evident, reaching a maximum
level by days 15-20 post-birth, then declining to the much lower rat
adult level. In contrast, none of the isoforms of PKI
are detectable
until days 20-25, when first the low molecular mass forms, PKI
-70
and PKI
-78, develop. This is followed by the appearance by days
35-45 of the higher molecular mass species, PKI
-X and PKI
-Y
(Fig. 1, a and c). (The designation used for the
PKI
forms, as based upon their currently established characteristics
(22), is depicted in Fig. 1d). The developmental profile of
the phospho forms of each of the PKI
isoforms is coincident with
that of their counterpart nonphosphorylated species (Fig.
1c), suggesting that development regulates the protein
species present, but not their phosphorylation. In the adult rat
testis, there is a slight predominance of the higher (PKI
-X and
PKI
-Y) versus lower (PKI
-70 and PKI
-78) molecular
mass species (55% versus 45%, as based upon Western blot
staining intensity (22)) and a greater than 98% predominance of total
PKI
versus total PKI
(as based upon determination of inhibitory activity following separation by DEAE chromatography (20)).
The profile of change observed for PKI isoforms is consistent with
these two forms being located in different cells types. In the immature
rat testis, the predominant cell type from neonate to 20 days of age is
the Sertoli cell. After 20 days of age, as sexual maturation ensues,
germ cells proliferate and Sertoli cells cease proliferating, and the
percentage mass of the testis derived from Sertoli cells markedly
decreases. These distinctive changes in the Sertoli composition of the
testis mirror the developmental profile exhibited by PKI
, suggesting
that it may be primarily a Sertoli cell product. Results observed with
PKI
are in noted contrast. Germ cells do not begin to differentiate
until day 20 with the appearance of pachytene spermatocytes, followed
by round spermatids by about day 30 and elongated spermatids by about
day 40. The profile of PKI
development therefore suggests that it is
primarily a germ cell protein and further that the higher molecular mass PKI
species may be constituents of the more developed
spermatocyte.
A parallel can also be noted between the appearance of the higher
molecular mass species of PKI protein species (shown here, Fig. 1)
and that of a lower molecular mass PKI
mRNA (as identified previously (21)). In all rat tissues examined other than testis, only a
single 1.4-kb PKI
message is evident (21). In the testis, the 1.4-kb
PKI
message first becomes evident by day 20, coincident with the
first appearance of PKI
protein, but by day 30, a second smaller
0.7-kb PKI
message is detected (21), the timing of which is
coincident with the first appearance of PKI
-X and PKI
-Y proteins
(Fig. 1). In the adult testis, both sizes of PKI
messages are
abundant (21), as are also both the lower and higher molecular mass
forms of PKI
protein (Fig. 1). Alternate (and smaller) mRNA species are evident for several germ cell proteins, including, in the
cAMP signal transduction pathway, the CREM transcriptional regulator
(32) and PKA subunits RI
, RII
,
RII
, and C
(14). It has been suggested that the
reason for the shift to smaller messages may be their greater
stability. As with the CREM message, a shorter form of PKI
message,
possibly as a result of an alternate polyadenylation site at base pairs
664-671, would have eliminated two or more destabilizing 3
-downfield
"AUUUA" elements (20, 33).
The specific localization of the PKI and PKI
isoforms to germ cells and Sertoli cells, respectively, has been
specifically examined by in situ hybridization. The
35S-labeled cRNA probes specific for each PKI form were
generated as described in under "Experimental Procedures." PKI
mRNA expression, as evidenced by in situ hybridization,
was undetectable in the early neonate (>20 days), became well evident
by postnatal day 30, and by day 55 was notably abundant (Fig.
2, a-c). When first detectable, PKI
mRNA expression was quite uniform across the entire testis section,
with only one or two tubules showing more pronounced mRNA levels
(Fig. 2b, arrows). By day 55, however, it was
quite apparent that some tubules showed very high levels of PKI
expression (arrowheads), and others showed much lower amounts (Fig. 2c). Microautoradiography of testis sections
confirmed these findings. As illustrated in Fig. 2d, silver
grains clearly delineated the individual seminiferous tubules and were
highly concentrated in them, and there was a much more intense signal is some tubules than in others. This observed variation between tubules
clearly became more accentuated with increasing age and increasing
testicular maturity and therefore suggested that the level of PKI
mRNA expression might be related to the stage of the seminiferous
cycle. In any given cross-section of testis, the different tubules
would contain germ cells at different stages of development (34).
Examination of the germ cell types in those tubules with high levels of
PKI
mRNA expression showed that they were indeed at a later
developmental stage (Fig. 3). A high signal level of
expressed PKI
mRNA (Fig. 3b) was coincident with
tubules having an extensive number of elongated spermatids (Fig.
3a), and in these tubules, a high density of silver grains
ringed the inner area of the tubule colocalized with the abundant
elongated spermatids. At higher magnification, a specific localization
of dense silver grains over elongated spermatids is very clearly evident (Fig. 3, c and d). The profile of PKI
developmental expression was quantitated by silver grain count, with
the data reported as pixel number per tubule, and was determined over
the full age range of 5-60 days of development (Fig.
4a). Prior to day 45, the counts reported in
Fig. 4a for PKI
are for the full complement of tubules.
At postnatal day 45 and after, silver grain counts were determined
separately for those tubules that contained elongated spermatids and
those that did not. Elongated spermatid presence was assessed by visual
inspection of stained tissue slices. PKI
mRNA was undetectable
in testes from rats of <20 days of age. Low levels of expression were
detectable from days 25 to 35, following which PKI
mRNA showed a
rapid increase in expression level. After 40 days of age, the separate
counts of those tubules that contained elongated spermatids (Fig.
4a, closed bars) and those without (hatched
bars) clearly documented that the marked elevation in PKI
mRNA with development was associated with the later stage tubules
that contained elongated spermatids. Those tubules that did not contain
elongated spermatids expressed PKI
mRNA at a much reduced level.
These more immature tubules (i.e. with no elongated
spermatids) showed a level of expression similar to that found in
younger animals. The overall pattern of developmental change observed
by the in situ studies is identical to that observed by
Western blot analyses of PKI
protein (Fig. 1) and also to that
previously reported for PKI
mRNA determined by Northern blotting
(21). The full complement of data clearly demonstrate that PKI
is a
germ cell protein whose expression is highest in the later stages of
the seminiferous cycle.
The pattern of PKI expression, as evidenced by in situ
hybridization, was quite different from that of PKI
. PKI
exhibited a uniform distribution over the entire testis (Fig. 3,
e and f). Such a pattern is most consistent with
PKI
being primarily in Sertoli cells. It is notably different from
the germ cell pattern displayed by PKI
(Fig. 3, a-d),
and were PKI
to have been primarily localized to Leydig and/or
peritubule myoid cells, a much more defined nontubular pattern would
have been expected. The changes in PKI
were quantitated by silver
grain count. PKI
was present at readily detectable levels even at
the earliest time point examined (5 days) and then increased slowly
and, following a peak of expression at about day 30, diminished
somewhat to the adult level (Fig. 4b). This pattern is
overall quite similar to that observed by Western blot analyses (Fig.
1). Because the PKI
protein levels by Western blot analyses are
reported for an identical amount of total testis protein applied to the
gel, whereas the in situ results are reported per tubule,
they differ in the day that the maximum level was observed (day 20 versus day 30) and the degree to which the level then
subsequently declined. The total set of data, however, are fully
consistent. Developmentally, rat Sertoli cells differentiate from the
supporting cells and proliferate until about days 15-20 (2, 34), at
which point proliferation ceases, and they remain at a fairly constant
number thereafter. Germ cells, in contrast, represent only a very small
fraction of the total testis tubule until day 20, when very active
proliferation is initiated such that by day 60 they constitute <95%
of testicular mass and testis protein. The increase in PKI
observed
by both Western blotting (Fig. 1) and in situ hybridization
(Fig. 4) between days 5 and 20 is likely associated with the increasing
number of Sertoli cells. Between days 20 and 30, the continuing
increase identified by in situ hybridization per
tubule is most likely due to a continuing increase in total
tubular PKI
mRNA per Sertoli cell. Since this is occurring
during a period of very active germ cell proliferation, the amount of
PKI
per mg of total testis protein (i.e. as
detected by Western blotting) declines. From day 30 on, the total
PKI
per tubule appears to drop only slightly (as evidenced by the
in situ hybridization studies); however, with the massive increase in total testis protein due to germ cell proliferation, the
amount of PKI
per total testis protein markedly diminishes.
Further documentation that the testis PKI and PKI
isoforms have a
different cellular distribution is supported by studies with
at/at germ cell-deficient mice. These mice are homozygous for the recessive atrichosis mutation (at/at) and are
characterized by having small testes essentially devoid of germ cells,
but with apparently normal Sertoli cells (30). Homozygous mutants are easily distinguished as they are nearly hairless. Littermates, which
are not homozygous for the mutant (at/wt or
wt/wt), are phenotypically normal (normal testis size,
mature sperm present in abundance, normal hair growth). The abundance
of PKI forms in the testis and cerebellum of at/at mice was
examined by Western blotting (Fig. 5). In testis
extracts, PKI
is present at similar if not somewhat higher levels in
the germ cell-deficient mice compared with controls (Fig.
5a). This clearly indicates that this isoform of PKI is
expressed in cells other than germ cells. The slightly higher level of
PKI
apparent in the germ cell-deficient animals is as might be
expected. Equal amounts of total testis protein were loaded onto each
gel lane; in the germ cell-deficient animals, Sertoli cells would
constitute a higher percentage of the total tissue and protein of the
testis. In contrast to the results observed with PKI
, a very marked
difference is observed with the expression of PKI
in the
at/at mice. PKI
isoforms are clearly evident in the
testes of control mice, but undetectable in the testes of germ
cell-deficient animals (Fig. 5b). These data support the
conclusion that PKI
in the testis is predominantly (if not
exclusively) a germ cell protein. There was no detectable difference
between the control and at/at mice in the level of either
PKI
or PKI
in the cerebellum, a tissue rich in both species. Thus, the PKI
gene itself is not defective in the at/at
mutant, and the altered profile in the testis is a direct consequence of the germ cell deficiency. The SDS gel profile for the control mice
suggests that, compared with rats, mice have a less complex pattern of
PKI
isoforms. Both PKI
-70 and PKI
-X isoforms are prominent in
the mouse cerebellum and testis, but there is little to no
PKI
-Y.
Northern blot analyses of isolated testis cell populations also support
the conclusion of distinct cellular localization for the PKI and
PKI
isoforms. The 4.3-kb PKI
mRNA was readily detectable in
isolated Sertoli cells (Fig. 6a, first
lane), but undetectable in either a total germ cell population or
enriched fractions of round or elongated spermatids (Fig.
6c). In contrast, no PKI
message was apparent in the
isolated Sertoli cell preparations (Fig. 6, b and
d), but both the 1.4- and 0.7-kb PKI
mRNA messages were readily detected in the total germ cell population as well as in
each of the enriched germ cell fractions (Fig. 6d). In
total, the data of Figs. 1, 2, 3, 4, 5, 6 show that in the testis, PKI
is
predominantly (if not exclusively) a germ cell protein, whereas PKI
is the principal PKI constituent of the Sertoli cell. We have yet to
evaluate the possible presence of the PKI species in the other cell
types of the testis. (The remaining data presented in Fig. 6 are
discussed below.)
PKI
Studies of
the PKI constituents of the epididymis and its constituent sperm both
support and extend the conclusions of the distribution profile of PKI
isoforms in germ cells. PKI was readily detectable in epididymal
extracts, which contained both the epididymal tissue and sperm, but was
absent in the isolated epididymal sperm (Fig. 7,
a and b), thus indicating that PKI
was a
constituent of the cells of the epididymis, but not of their content
sperm cells. This is similar to what is observed for the testis, with PKI
being a constituent of the nurse cells that support the germ cells, but not of the germ cells themselves. PKI
is abundant in
extracts of the caput and cauda regions of the epididymis (containing both the epididymal tissue and sperm cells) and also in the epididymal sperm cells themselves isolated from these epididymal regions (Fig. 7,
c and d). Clearly from these data, PKI
is a
constituent of the epididymal sperm; whether or not it is also a
component of the epididymal tissue itself was not evaluated. The
profile of PKI
isoforms that is observed is of note. In both the
caput epididymal extracts and the sperm isolated from these extracts, the primary forms present are PKI
-X and a form labeled
"PKI
-Z" that migrates at a slightly higher apparent molecular
mass than PKI
-Y. Minimum levels of PKI
-Y and PKI
-70 are
evident. There is a very notable change in the PKI
composition as
the sperm transit the epididymis. In the cauda epididymal extracts and
the sperm isolated from this region, the predominant PKI
species is
PKI
-Z, with a marked reduction in the level of PKI
-X.
We have further examined the nature of PKI-Z. Incubation of both
caput and cauda sperm extracts with alkaline phosphatase resulted in
the elimination of PKI
-Z from the Western blot and the formation of
PKI
-Y (Fig. 8). This would indicate that PKI
-Z is
a phosphorylated form of PKI
-Y. It is, however, different from the
phospho form of PKI
-Y denoted in Fig. 1d since it
migrates in a different location. Presumably, PKI
-Z either
represents a multiple phosphorylated form of PKI
-Y or is
phosphorylated in a different site.
Given the pattern of PKI changes observed in sperm during their
transit through the epididymis, a further evaluation was undertaken of
the possible changes that might be occurring prior to this time point
during the later stages of germ cell development in the testis.
Individual seminiferous tubules were separated from the interstitial
tissue by manual dissection (35) and then separated into the different
defined stages of tubular development by transillumination-assisted
microdissection (24, 36). The PKI
profiles of these segments at
these different stages are illustrated in Fig. 9 and
show a very clear developmental profile. Interpretation of the changes
is complicated since each segment contains germ cells at multiple and
diverse levels of development during the cycle of the seminiferous
epithelium (36). The pattern of change seen in Fig. 9, as supported
also by the data of Figs. 1 and 4, suggests that likely the very early
germ cells up to and including pachytene may have little to no PKI
protein. Later, more at the time of mature round spermatocytes, there
is a presence of both PKI
-70 and PKI
-X (Stages IX-XI), but then
in the more developed spermatid and with the appearance of elongated
spermatids (steps 15-19 associated with Stages I-VIII), there is a
greater abundance of PKI
-Y and possibly the PKI
-Z form, as also
identified in the epididymal sperm.
Thus, overall, there appears to be a continuum of PKI developmental
change in isoform type with germ cell maturation starting in the testis
and continuing with transit through the epididymis. The progression of
developmental change observed for the testis (Figs. 1 and 7-9) showed
initially the presence of the lower molecular mass PKI
-70 and
PKI
-78 species (days 20-30), followed by the appearance of ever
increasing amounts of the higher molecular mass PKI
-X and PKI
-Y
forms, concordant with the increasing maturation of the spermatocyte.
In most developed germ cells of the testis and in the epididymis, the
higher molecular mass species of PKI
are most prominent, and with
progression through the epididymal tract from caput to cauda, there is
a continuing change, with the predominant appearance of the
phospho-PKI
-Z species (Fig. 7, c and d; and
Fig. 8).
Previous
studies by Means and co-workers (18, 37-39) have shown that PKI
expression in the testis is regulated by FSH based upon measurements of
PKA inhibitory activity. The primary site of action of FSH in the male
is the Sertoli cell (1, 2, 40). The elucidation that there are two
distinct genetic forms of PKI, PKI and PKI
(20, 21), which also
have distinctive cellular distribution in the testis (Figs. 1, 2, 3, 4, 5, 6), prompted an extended investigation of this FSH-dependent
control of PKI expression. Sexually immature male rats of 14 days of
age were injected intraperitoneally with FSH, using a protocol similar to that of the initial studies (18, 38), and PKI isoform expression was
monitored by Northern blot analyses. At 14 days of age, the level of
circulating endogenous FSH in the rat is at a nadir, having been high
at birth, declining shortly thereafter, and then increasing again by
days 25-30 (41). Northern blot analyses demonstrated that the
expression of both PKI
and PKI
is stimulated by FSH in the testes
of rats of this age (Fig. 10). For each, there was a
prompt response resulting in a 3-5-fold increase in message by 8-12
h. The level of the PKI
message then returned quite rapidly to
control levels, whereas that for PKI
remained elevated for a longer
period. Exploration of this FSH-dependent sensitivity of
PKI expression was also undertaken at other prepubertal ages. With
PKI
, the greatest increases in response to FSH occurred during the
period (days 12-18) when there were also the highest levels of
endogenous protein in the absence of FSH treatment (Fig. 11). The decrease in FSH responsiveness with age may
reflect a decrease in capacity for PKI
synthesis. In contrast to
what was observed with PKI
, the greatest response of PKI
to
injected FSH occurred when the endogenous control level of proteins was very low (days 14-16). Later (days 18-20), the response to injected FSH was dampened, coincident with an elevated level of endogenous PKI
. It would appear likely that FSH is a major regulator of PKI
expression and that the increase in the level of transcript seen with
development is a consequence of both the beginning presence of
spermatocytes and the concordant increase in circulating levels of
endogenous FSH.
Hormone-dependent regulation of PKI was also
demonstrated with cultured Sertoli cells (Fig. 6a). FSH
treatment resulted in a 1.6-fold increase in PKI
transcript level,
an effect also apparent with dibutyryl cAMP treatment, likely
reflective of the mechanism of FSH action. An increase in PKI
mRNA in the isolated Sertoli cells was also observed in response to
insulin and serum treatment (~2-fold), whereas testosterone
diminished the level of endogenous transcript to ~50%. Phorbol ester
was without apparent effect. It is clear the PKI
is under the
control of a range of hormonal effectors, and further investigation is
warranted. Consonant with its absence in the Sertoli cell, none of
these effectors resulted in a discernible level of PKI
transcript
(Fig. 6b).
These data add another layer of complexity to the already
intricate picture of the role of cAMP in the transduction of events in
developing germ and Sertoli cells and subsequent germ cell maturation.
From extensive studies that have been undertaken by a variety of
approaches including studies with isolated germ cell populations,
testis from different developmental ages, and microdissected testis
fractions to identify germ cells at different stages of development,
Jahnsen and co-workers (13-15, 42) have demonstrated that the PKA
subunits RI, RI
, RII
,
RII
, and C
exhibit cell- and stage-specific
differential patterns of expression. These results are likely
reflective that each of the PKA subunits has specific roles at
different stages of spermatogenesis and in the different cell types.
The cAMP-responsive transcription factors that are key for germ cell
development exhibit a pattern of even greater complexity than that
observed for the PKA subunits. Alternate transcript processing of the
separate genes for CREM and CREB occurs, leading to both activators and
repressors of cAMP-regulated transcription, and as with the PKA
subunits, which form of these factors is present at which time is very
cell- and developmental stage-specific (3, 32, 43, 44). We now observe
with PKI
an intricate pattern of expression, especially evident by
the profile of forms that evolve with germ cell maturation. These forms
arise as a consequence of covalent modification and alternate
translation (22). Left to be unraveled is the unique function/activity
that each of the different PKI
forms manifests, and only from that
knowledge will an understanding be derived of why the transitions among
the forms may be important for the process of germ cell maturation.
What is apparent is that the potential for detailed and specific
regulation within the cAMP signal transduction cascade for the
regulation of germ cell function is immense. There is a growing body of
evidence that one key function of PKI is in the trafficking of the PKA
catalytic subunit (45-47). Whether each of the multiple forms of
PKI
has this as its function and/or manifests some other key
regulatory role remains to be resolved, and such information is
critical to our understanding of the role that PKI
plays in germ
cell maturation. As we have now demonstrated (Figs. 1, 2, 3, 4, 5, 6, 7, 8, 9), PKI
and
PKI
are also specifically segregated between nurse cells and germ cells, respectively, in both the testis and epididymis, and the specialized role of each of these isoforms awaits elucidation.
The transcriptional regulation of both CREM and PKI is
FSH-dependent (Ref. 32 and Figs. 10 and 11). However, in
the male, only Sertoli cells have been established as FSH-sensitive;
germ cells are not directly regulated by FSH as they do not contain FSH
receptors (1, 40). To accomplish the FSH-dependent
regulation of germ cell PKI
transcription therefore requires that
some Sertoli cell-derived FSH-dependent message modulates
germ cell PKI
transcription. A model for such is presented
schematically in Fig. 12. Candidates for the message
between the Sertoli cell and germ cell include one or more of the many
Sertoli cell-derived paracrine factors that have been defined (48, 49)
or some agent, such as cAMP, that may be transmitted through the gap
junctions that exist between these cell types.
The preparation of the anti-peptide antibodies was accomplished by David Harrison, and he and Ho-Yin Chan undertook the first studies of the testis and epididymal developmental patterns of PKI.