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Departments of Pathology (T.R.K., S.V., M.M.M.), Molecular and Cellular Biology (T.R.K., M.M.M.), and Molecular and Human Genetics (M.M.M.), Baylor College of Medicine, Houston, Texas 77030; and Department of Anatomy (N.G.W., N.M.T.) and Monash Institute of Reproduction (D.M.D.) Monash University, Melbourne, Victoria 3168, Australia
Address all correspondence and requests for reprints to: Dr. T. Rajendra Kumar, Department of Pathology, One Baylor Plaza, Baylor College of Medicine, Houston, Texas 77030. E-mail: tkumar{at}bcm.tmc.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunit is shared among other members of
the family, LH and TSH, whereas the ß-subunit is hormone specific
(1, 2). The noncovalent interaction between the two
subunits is required for biological activity of each of these hormones
(1, 2). Expression of the FSH ß-subunit is positively
regulated by the activins (3, 4). FSH receptors are
localized to Sertoli cells within the testis, and FSH is a well known
mitogen for Sertoli cells (5, 6). Activins are members of the TGFß superfamily, synthesized as precursor proteins and cleaved to mature forms before secretion (3, 4). They were originally discovered as homo- or heterodimeric gonadal peptides involved in positively regulating FSH biosynthesis and secretion from the pituitary (7, 8). Physiologically, three types of activins exist: activin A (ßA:ßA), activin B (ßB:ßB), and activin AB (ßA:ßB); however, activins A and B are the most characterized activins (3, 4). Activins are also expressed in multiple tissues outside the reproductive axis, where they act in both an autocrine and paracrine manner (9, 10).
Within the reproductive axis, activin B is predominantly expressed in the pituitary, whereas both activins A and B are expressed in Sertoli cells of the testis (11). Several in vivo and in vitro studies have shown that activins influence testis development and function directly or indirectly (12, 13, 14, 15, 16). However, it is not known whether activins can act directly within the testis in vivo independently of their effects on FSH homeostasis.
Activins bind to two types of serine/threonine kinase receptor isoforms, type IIA and type IIB (ActRIIA, ActRIIB), and the genes encoding them are localized to distinct chromosomes (17, 18). ActRIIA is more ubiquitously expressed than ActRIIB, is evolutionarily conserved, and is the major component in the activin signaling pathway in the adult (17, 18, 19). Rat, mouse, and human ActRIIA are also localized to pituitary gonadotropes and Sertoli cells in the testis, similar to activins A and/or B (20). Additionally, ActRIIA is localized to germ cells, in particular to pachytene spermatocytes and A-type spermatogonia, in the testis of rats and mice (21, 22). The coordinated expression of the activin ligands and their type II receptors to multiple cell types within the testis suggests that they may act locally in an autocrine/paracrine manner.
Gene knockout studies from our laboratory have previously confirmed the
in vivo role of ActRIIA signaling in the mouse reproductive
axis (23). The majority of ActRIIA-null mice are viable
(
22% die embryonically), and demonstrate reproductive defects
(24). Mutant males are fertile, have suppressed serum and
pituitary FSH levels, and demonstrate a decrease in testis size
(23). It is not known whether these phenotypes are
secondary to suppressed FSH levels or are a direct result of impaired
ActRIIA signaling locally within the testis. Previously, we have also
generated FSHß-null (and hence FSH-deficient) mice to study the
consequences of isolated FSH deficiency in reproductive physiology
(24). Similar to ActRIIA-null male mice, FSHß-null male
mice are fertile and demonstrate reduced testis size and reduced
epididymal sperm number and motility (24).
To examine the local role of ActRIIA signaling within the testis independent of its effects on FSH homeostasis, we initially intercrossed FSHß-null males and ActRIIA heterozygous female mice (ActRIIA-null females are infertile) and eventually generated FSHß/ActRIIA (FAR) double homozygous mutant male mice. Here, we report the male reproductive phenotypes in these double mutant mice that lack both FSH and ActRIIA.
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Materials and Methods |
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Morphological and histological analysis
Testes were collected from adult mice at 42 d [five
wild-type (WT) and five FAR double mutant mice] and weighed. For
histological analysis, testes samples were fixed in Bouin’s reagent
overnight at room temperature and thereafter rinsed extensively for
48 h in LiCO3-saturated 70% ethanol
(23, 24). Paraffin-embedded 4-µm sections were later
stained with periodic acid-Schiff/hematoxylin as previously described
(23, 24). Apoptotic cells were stained using a variation
of the terminal transferase-mediated deoxy-UTP nick end labeling method
as described by Gavrieli et al. (25). The
labeled DNA was detected using an alkaline phosphatase-labeled sheep
antidigoxigenin Fab conjugated with alkaline phosphatase. The chromogen
substrate mixture used was Fast Red TR/napthol (Sigma, St.
Louis, MO).
Stereological analysis
Stereological analysis of the testis (from five mice per group)
was performed on 20-µm thick methacrylate sections stained with
periodic acid-Schiff reagent as described previously
(26).
Epididymal sperm quantitation
Epididymides from both sides were collected from mice at 42
d (five or six per group) into 1 ml M2 (modified Whitten’s) medium
(PGC Scientifics, Gaithersburg, MD) to release sperm after a 15-min
incubation at 37 C. The released sperm were counted using a
hemocytometer as previously described (24, 27, 28).
Estimation of intratesticular testosterone content
Freshly isolated testis samples from 42-d-old mice (five or six
per group) were decapsulated, and homogenates (1 ml) were prepared
according to the method of Meistrich et al.
(29). The testosterone content in the supernatants was
quantitated by a solid phase testosterone RIA (Diagnostics Systems Laboratories, Inc., Webster, TX) according to the
protocols provided by the manufacturer using calibrated hormone
standards. The values are expressed as nanograms per mg testis. The
sensitivity of the assay is 0.08 ng/ml, with an intraassay coefficient
of variation of 8.6% and an interassay coefficient of variation of
9.6%.
RNA isolation and Northern blot analysis
Total RNA from testes was extracted (three mice per group) using
RNA STAT-60 (Leedo Medical Laboratories, Houston, TX) as previously
described (30). Fifteen micrograms of denatured RNA
samples were separated on 1.4% agarose gels containing 13%
formaldehyde, transferred to nylon membranes, vacuum-baked at 80 C,
hybridized with [32P]deoxy-CTP-labeled random
primed probes, washed, and exposed to autoradiography films as
previously described (31). The autoradiography films were
scanned by densitometry, and the ratio of the individual probe signal
to 18S signal was calculated and analyzed by one-way ANOVA for
statistical significance. Northern blot analysis was performed twice
under exactly the same conditions. The cDNA probes were PCR-generated
and sequenced to confirm the identity of the sequences, or they were
obtained as gifts.
Statistical analysis
All the data are presented as the mean ± SEM
and were analyzed by t test or one-way ANOVA using an Excel
version 6.0 software package (Microsoft Corp., Seattle,
WA). P < 0.05 was considered statistically
significant. Stereological data were analyzed using SigmaStat version
2.0 software (San Rafael, CA). The SE of the
ratios were calculated using the formula given by Kendall and Stuart
(32).
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Results |
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) involving
initial matings between FSHß-null male and AtRIIA heterozygous female
mice (step 1, Fig. 1). This intercross resulted in double heterozygous
male and female mice, which were subsequently intercrossed to generate
FAR double homozygous mutant mice (step 2A, Fig. 1). As a result of
this genetic cross, we also obtained male mice that were homozygous
mutant at the FSHß locus and heterozygous at the ActRIIA locus, or
vice versa. These male mice were also mated to double
heterozygous female mice (step 2B, Fig. 1) to increase the frequency of
generating FAR double mutant mice. Male mice lacking FSHß or ActRIIA
alone are fertile and produced comparable number of pups when mated to
the corresponding heterozygous female mice (data not shown) (23, 24). To evaluate the fertility of FAR double mutant male mice, 6
double mutant males were mated to double heterozygous female mice (1
female/1 male) beginning at 42 d over a period of 1 yr. All of the
6 double mutant males were fertile and sired offspring. However, the
mean litter size was significantly decreased compared with that from
matings between age-matched double heterozygous male and female mice
[2.8 ± 0.2 pups (31 litters) vs. 5.0 ± 0.1 pups
(49 litters); P < 0.001]. Although 22% of mice die
embryonically due to deficiency of ActRIIA alone, the average litter
size from ActRIIA-null male and heterozygous ActRIIA female
matings was significantly higher than that obtained from matings
between FAR double mutant male and FAR double heterozygous female mice
[5.2 ± 0.6 pups (14 litters) vs. 2.8 ± 0.2 pups
(31 litters); P < 0.001]. Furthermore, there were no
statistically significant differences between single and double mutants
when their average ages at siring the first litters (mated to
corresponding heterozygous females) were compared
(FSH-/-, 70 d;
ActRIIA-/-, 78 d;
FAR-/-, 73 d;
P > 0.05; n = 6–7/group). Thus, double mutant
male mice lacking both FSH and ActRIIA are fertile, but they produce
small litters when mated to double heterozygous female mice.
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) (23, 24) with no apparent effect on the ability to achieve a
pregnancy, although litter size is decreased in the double homozygous
mutants vs. double heterozygous matings at 42 d or at
later time points (data not shown). Thus, it is unlikely that the male
reproductive phenotypes of double homozygous mutants at 42 d
represent a delay in puberty. To further characterize the apparently
decreased fertility in FAR double mutant male mice, testicular
phenotypes were morphologically, stereologically, and functionally
analyzed. First, we measured testis size in FAR double mutant mice at
42 d and compared these to testis size in FSHß- or ActRIIA-null
mice at the same age. As shown in Table 1, the testis size in double
mutants was not significantly different from that in FSHß- or
ActRIIA-null mice, although the FAR double homozygous mutant mice
lacked both FSH and ActRIIA. Similarly, routine histological analysis
of testes from FAR double mutant male mice at 42 d revealed no
differences compared with sections from either FSHß- or ActRIIA-null
mice (Fig. 2) (23, 24). At
42 d all of the stages of spermatogenesis appeared normal in the
testis of FAR double mutant mice, and there were normal Leydig cell
islands present between the tubules (Fig. 2). Although spermatogenesis
appeared grossly normal, and there were no differences in epididymal
weights (data not shown), epididymal sperm number was significantly
reduced in FAR double mutant mice at 42 d compared with the number
in single mutants (Table 1) (23, 24). Furthermore, despite
the wide variation, there was a trend toward an increase in the mean
intratesticular testosterone levels in FAR double mutant mice (Table 2). Together, these results suggest that
the decreased fertility in FAR double mutant male mice is due to
decreased sperm production.
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), which is very similar to
the 30% decrease seen in the FSHß and the 39% decrease seen in the
ActRIIA knockout mice. Comparing the functional activity of the Sertoli
cell in terms of its germ cell-carrying capacity, the ratio of round
spermatids to Sertoli cells in the double knockouts was 4.93 ±
0.2 compared with 9.7 ± 0.6, 5.44 ± 0.21, and 6.94 ±
0.47 in the controls, FSHß mutants, and ActRIIA knockouts,
respectively. Similarly, there were substantial reductions in all germ
cell types from spermatogonia to elongated spermatids in single and
double knockouts (Fig. 3) (34) compared with WT controls.
The reduction in type A and I spermatogonia was particularly marked
(Fig. 3). The numbers of preleptotene and B spermatogonia were less
marked in their reduction compared with type A and I spermatogonia.
More advanced germ cell types showed a gradual attrition in numbers
compared with wild-type mice. The ratio of round spermatids to
pachytene spermatocytes was not different in the knockout and wild-type
mice. These data suggest that despite the absence of both FSH and
ActRIIA, the double mutants demonstrate only a moderate effect on
testis cell populations and a significant reduction in type A and I
spermatogonia.
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showed that there were no
significant changes (P > 0.05, by single factor ANOVA)
in the expression profiles of cell-specific markers such as inhibin
(Sertoli cell-specific), LH receptor (Leydig cell-specific), protamine
1 (spermatid-specific), and germ cell nuclear factor (germ
cell-specific) among the three groups. However, the significant
differences in expression were only in protamine 2
[spermatid-specific; 2.6 (wild-type), 2.3
(ActRII-/-)
vs. 1.6 (FAR double mutant); P < 0.05, by
single factor ANOVA] and CREM (spermatocyte-specific; 2.5 (wild-type),
2.2 (ActRII-/-)
vs. 1.1 (FAR double mutant); P < 0.05, by
single factor ANOVA] expression when ActRIIA null and WT groups were
compared with FAR mutants. To further verify the possibility that the
unaltered expression of most of these genes was due to a compensatory
increase in ActRIIB, the other activin type II receptor, we also
hybridized the Northern blot with an ActRIIB-specific cDNA probe (Fig. 4, left, top panel). Although ActRIIB appeared to
be reduced in the testis of ActRIIA-null mice compared with that in WT
testis, the absence of both FSH and ActRIIA did not result in any
compensatory increase in ActRIIB mRNA expression [0.8 (WT), 0.8
(ActRII-/-), 0.7 (FAR
double mutant); P > 0.05, by single factor ANOVA] in
the testis of double mutant mice. Hence, these expression data indicate
that the male reproductive phenotypes in the absence of FSH and
ActRIIA signaling are not due to compensatory mechanisms involving
up-regulation of ActRIIB or other marker gene-encoded mRNA expression
in the testis of FAR double mutant mice.
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20%) of the seminiferous tubules
(data not shown). A similar pattern was observed in the double mutants.
In both groups the cells affected tended to be in the layers
corresponding to spermatogonia and primary spermatocytes (data not
shown). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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22%); all of the viable mice have suppressed FSH levels and
demonstrate reproductive defects (23). Hence, FAR double
mutant mice are an important model to examine the local effects of
activins in the testis in the absence of both ActRIIA signaling and
FSH.
Male fertility is not impaired in single mutant mice lacking either FSH
or ActRIIA. Similarly, FAR double mutant mice are fertile despite the
absence of both FSH and ActRIIA; however, their fertility is greatly
reduced. This reduced fertility in FAR double mutant mice may be
explained by reduced epididymal sperm number compared with that in mice
lacking either FSH or ActRIIA alone. The absence of a direct effect of
ActRIIA signaling within the epididymis (in addition to the indirect
effects of FSH) in double mutant mice may result in significantly
reduced epididymal sperm counts. There is evidence to demonstrate that
in the rat epididymis, mRNAs encoding , ßA,
and ßB subunits are expressed, and these
observations would support a direct role of ActRIIA signaling within
the epididymis (de Kretser, D., unpublished data). The absence of any
noticeable defect in the testis and the presence of qualitatively
normal spermatogenesis in the absence of both FSH and ActRIIA (in the
double mutants) suggest that a variety of other growth factor signaling
pathways are important for testis development and function.
Stereological data indicate a decrease in type A and I spermatogonia, which is the only noticeable defect in the testis of FAR double mutants compared with the single mutants (34). The comparison is consistent with the proposition that the double knockout has some additive effects, although the final resolution of this issue requires an FSH replacement study. Despite the marked reduction in germ cell production, expression profiles of important marker genes are unaltered in the testis of FAR double mutant mice. The data from Northern blot analysis support our hypothesis that ActRIIB, the other activin type II receptor (not up-regulated in the absence of ActRIIA), whose expression was barely detectable by in situ hybridization (data not shown), cannot compensate for ActRIIA function in the testis. We cannot, however, rule out the possibility that the absence of FSH and ActRIIA leads to changes in gene expression profiles of other testis-specific genes not analyzed in the present study. Those genes may be downstream of ActRIIA signaling and are stage-specifically expressed during spermatogenesis. Within the testis, ActRIIA mRNA and protein are localized to Sertoli cells, pachytene spermatocytes (21, 22), and round spermatids. The signal transducer, Smad2, which is implicated downstream of ActRIIA signaling, has also been shown to be coexpressed in these testis cell types (41), suggesting the existence of a full complement of activin-signaling components in these cells. It will be important to analyze in the future the alterations in this signaling pathway in FAR double mutants.
Our genetic analysis with FAR double mutant mice confirms an important
local role of ActRIIA signaling (in the testis) independent of its
effects on FSH homeostasis. Based on our analyses with mice lacking
FSH, ActRIIA (34), and both FSH/ActRIIA, a hypothetical
model can be formulated, as shown in Fig. 5. In ActRIIA-null mice, FSH levels are
suppressed, leading to a reduction in Sertoli cell number accompanied
by reduced germ cell and sperm number. In this model the direct effects
of ActRIIA signaling within spermatogenic cells are masked by the
FSH-mediated effects secondary to the absence of ActRIIA signaling.
This is further confirmed by a comparable reduction in Sertoli cells,
germ cells, and sperm number in FSH-null mice, in which ActRIIA
signaling is intact. The local effects of ActRIIA signaling directly on
the spermatogenic cells are readily apparent in the FAR double mutant
mice (absence of both FSH and ActRIIA), which have a further reduction
in type A and I spermatogonia and sperm, but not in Sertoli cell
number. More quantitative analyses and mechanistic studies are required
to clearly establish this model supporting the local role of ActRIIA
in vivo.
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Acknowledgments |
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Footnotes |
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Abbreviations: ActRIIA, Activin receptor IIA; FAR, FSHß/ActRIIA double homozygotes; WT, wild-type.
Received February 6, 2001.
Accepted for publication April 30, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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