| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
ANZAC Research Institute and Andrology Laboratory (C.M.A., M.H., S.S., J.S., A.K., M.J., D.J.H.), Department of Medicine, University of Sydney, New South Wales 2006, Australia; Department of Reproductive Medicine (P.I.), Westmead Hospital, University of Sydney, New South Wales 2145, Australia; and Department of Physiology (M.P., J.L., I.H.), University of Turku, 20520 Turku, Finland
Address all correspondence and requests for reprints to: Charles M. Allan, ANZAC Research Institute, Concord Hospital, Sydney, New South Wales 2139, Australia.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
- and ß-subunit of human FSH, under the
rat insulin II promoter, expressed biologically active heterodimers at
serum levels, by immunoassay, equivalent to circulating FSH
concentrations in fertile humans (0.1–25 IU/liter). Transgenic mice
were crossed into the hpg mouse genotype to obtain
LH-deficient animals secreting FSH alone. Testis weights of adult
FSHxhpg mice were increased up to 5-fold, relative to
nontransgenic hpg controls (P <
0.001). However, only transgenic males with serum FSH levels more than
1 IU/liter showed testis weights increased relative to
hpg controls, indicating a physiological FSH threshold
for the testicular response. Histology of enlarged
FSHxhpg testes revealed round spermatids and sparse
numbers of elongated spermatids, demonstrating that the
testosterone-independent FSH response targeting the Sertoli cell can
facilitate completion of meiosis and minimal initiation, but not
completion, of spermiogenesis. Transgenic FSH also induced inhibin B
secretion in FSHxhpg mice, but showed a distinct sexual
dimorphism with only females exhibiting a strong FSH dose-dependent
increase in serum inhibin B levels (r2 = 0.84). In
addition, ovaries of FSHxhpg females were enlarged up
to 10-fold (P < 0.001), characterized by increased
follicular recruitment and development to type 7 antral follicles.
Thus, these findings show that the transgenic FSHxhpg
mouse provides a unique model for detailed investigations of the
definitive in vivo actions of FSH alone. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-subunit and has related ß- subunits and cell surface
receptors. Furthermore, gonadotropins can share the same ultimate
cellular sites of action, such as ovarian granulosa cells that express
FSH and LH receptors (7), and testicular Sertoli cells
with receptors for both FSH (8) and the androgens
(9) synthesized in response to LH. These intimate
connections make it very difficult to separate the in vivo
effects of each gonadotropin. Previous research models have evaluated FSH action by hypophysectomy followed by exogenous FSH administration, or by inhibiting the FSH response using GnRH receptor antagonists, steroidal negative feedback, or immunoneutralization (10, 11, 12, 13, 14). Though these classical paradigms have provided useful information, they have fundamental limitations that do not allow unequivocal interpretation of FSH-specific actions, such as incomplete or nonselective suppression of FSH activity, and impurity of early nonrecombinant FSH preparations. Therefore, these models fall short of the ideal of specific, durable, and complete FSH deficiency or replacement. The uncertainties of such models can now be resolved by more decisive animal models, using carefully constructed, targeted, transgenic approaches to disrupt and/or express selected gonadotropin activities.
We previously used the hypogonadal (hpg) mouse (15), as a model of congenital and functionally complete gonadotropin deficiency, to investigate the role of each gonadotropin in testicular function (6, 16, 17, 18). The gonads of hpg mice fail to develop postnatally because of a major deletion in the GnRH gene (19). Therefore, the hpg mouse differs from hypophysectomy in that its gonads have never been exposed to gonadotropins, yet retain other pituitary hormones (e.g. PRL, GH) that influence gonadal development (20, 21). We showed that the undeveloped, infantile-like hpg testes remain functionally responsive as androgen treatment alone induced all spermatogenic stages, testicular growth, and fertility (6, 16). Subsequent mouse models with targeted disruptions of the FSH ß-subunit (5) or FSH receptor genes (22, 23, 24) confirmed our findings that FSH is not an absolute requirement for male mouse fertility, although the greatly diminished testis size (30–50%) in all these models demonstrated that FSH has an important contribution to rodent spermatogenesis. Furthermore, FSH seems to have a more significant role in human male fertility, although its absolute requirement remains in question (4, 5, 25).
To further investigate the actions of FSH in vivo, and to avoid enigmas associated with indirect loss-of-function approaches (such as the preserved and potentially complementing activities of LH in the FSH ß-subunit or receptor-deficient models), we have focused on the converse gain- of-function approach using the hpg mouse model to selectively express FSH in the absence of LH. Our laboratory initially treated the hpg mouse with exogenous recombinant (r) human (h) FSH (17, 18). However, rhFSH treatment had several limitations, including the inability to deliver FSH prenatally and the immunogenicity of exogenous hFSH (17, 18). To establish a better model to study the definitive in vivo actions of FSH, we have generated novel transgenic animals that express heterodimeric hFSH. The combination of transgenic FSH mice and the hpg genetic background has provided a novel paradigm that will allow, for the first time, an investigation of the specific testicular actions of FSH in the absence of LH effects, both during and after the perinatal period. The FSHxhpg model also provides the opportunity to examine perinatal actions of FSH alone on the ovarian follicle population. We now describe the characterization of these transgenic mice and the initial examination of the gonadal physiology of hpg males and females expressing transgenic FSH.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
- (26) and ß-subunit minigenes,
kindly provided by Dr. I. Boime (Wahington University, St. Louis, MO),
and Dr. L. Jameson (Northwestern University, Chicago, IL),
respectively, were modified by PCR to incorporate a 5'-NotI
site and Kozak consensus sequence (27) adjacent to the
start codon, and a KpnI ( gene) or ClaI (ß
gene) site after the stop codon. The 5'-flanking promoter sequences of
the modified FSH and ß minigenes were replaced with the RIP
sequence by separate cloning into pRSV-RIP, forming pRSV-RIP or
pRSV-RIPß. Nucleotide sequences of the modified - and
ß-minigenes were confirmed by automated Dye-deoxy sequencing
(PE Applied Biosystems, Foster City, CA) at SUPAMAC
(Sydney University Prince Alfred Molecular Analysis Centre). A
MluI-BssHII fragment containing the RIPß sequence and SV40
polyadenylation sequence was subcloned into the MluI site of
pRSV-RIP. The resulting tandem RIP-RIPß DNA construct was
digested with BssHII to remove most vector and all RSV sequences, then
purified by agarose gel electrophoresis for microinjection into mouse
oocytes as described below. For cell transfection experiments the
RIP-, RIPß- or RIP-RIPß-constructs were each subcloned into
PCR-script plasmid (Stratagene) to remove RSV
sequences.
Cell culture and transfection
Rat insulinoma RINm5F cells were grown at 37 C in
75-mm3 flasks in supplemented RPMI 1640 growth
medium (Trace Biosciences, Noble Park, Victoria, Australia) containing
10% FCS. RINm5F cells were transfected at 50–80% confluency with
hFSH subunit DNA vectors (pRIP plus pRIPß, or pRIP-RIPß)
using lipofectamine reagent (Life Technologies, Inc.,
Rockville, MD) as recommended. A plasmid encoding green fluorescent
protein (28) was included as a control for transfection
efficiency, determined by visualization of green cells using UV-light
microscopy. Culture media was removed, 48 h after transfection,
for direct analysis of hFSH levels using the two-site hFSH immunoassay
described below.
Generation of transgenic mice
Transgenic animals were generated as described
(29), using the inbred FVB/N strain (lines ß.043,
045, 113) or the hpg strain (19), originally
derived from C3H/HeHx101/H F1 hybrids (lines ß.1 to 7). The
RIP-RIPß construct was microinjected at 2–3 ng/µl in 10
mM Tris-HCl (pH 7.4), 0.1
mM EDTA buffer. Transgenic founders and offspring
were identified by PCR analysis of genomic DNA, using oligonucleotides
5'-AATGCTCAGCCAAGGACAAAGA-3', 5'-TAAATTCCATGGGTGGCCTC-3' for the
transgene, and 5'-AATGCTCAGCCAAGGACAAAGA-3',
5'-AACTTAATGAAACCGGCCTAAT-3' for the ß transgene. Expression of the
transgene in vivo was confirmed by immunoassay analysis of
serum hFSH levels as described below. Animals were housed under
standard conditions, with ad lib access to food and water.
Transgenic animals expressing hFSH on a mouse gonadotropin-deficient
background (denoted hFSHxhpg) were obtained by
cross-breeding with fertile animals carrying the heterozygote GnRH gene
deletion. Mice of the hpg genotype were identified by PCR as
described (17).
Quantitation of serum hFSH, mouse inhibin B, and testosterone
levels
Serum levels of transgenic hFSH were determined using a two-site
immunofluorometric hFSH assay kit (DELFIA; Wallac, Inc.,
Turku, Finland) as recommended by the manufacturer, with minor
modification of the supplied standards. Serial dilution of the
32-IU/liter standard in pooled nontransgenic mouse serum provided
0.125-, 0.25-, 0.5-, 1.0-, 2.0-, 4.0-, 8.0-, and 16.0-IU/liter
standards. Serum samples were assayed in duplicate, and the hFSH
detection limit was 0.05 IU/liter. Relative serum levels of mouse
inhibin B were determined by a human inhibin B immunoassay
(30), validated for detection of mouse inhibin B using
serial dilutions of conditioned mouse preantral follicle medium as
reference standards in each assay (pooled aliquots stored at -20 C).
The diluted standards showed parallel detection with curves of diluted
murine ovarian or testicular extracts (i.e. containing
inhibin B), and PMSG-stimulated female serum (Illingworth and Wang,
unpublished observations). Detection of mouse inhibin B was expressed
as arbitrary units (AU/ml), with 1000 AU/ml set to 1000 pg/ml human
inhibin B. The detection limit was 8–14 AU/ml, and analysis of male
serum inhibin B was limited because of the lowered sensitivity of the
human assay for mouse inhibin B, as reported elsewhere
(22). Serum testosterone levels were measured in duplicate
by RIA as previously described (18).
Serum and tissue collection
Serum was collected from rompun/ketamine anesthetized animals,
by retroorbital or cardiac puncture procedures, and aliquots were
stored at -20 C. Fixed tissues were collected and weighed after left
ventricle perfusion of anesthetized mice with 30 ml heparinized (10
IU/ml) saline (prewarmed to 37 C) followed by 30 ml Bouins fixative
(2% glutaraldehyde, BDH; 2% paraformaldehyde (Merck,
Darmstadt, Germany; 0.1% picric acid, in 0.2 M sodium
phosphate buffer, pH 7.4). Testes or ovaries were fixed overnight, then
transferred to 70% ethanol and reweighed. The tissues were dehydrated
in 100% ethanol (3 x 30 min) and butanol (2 x 1 h)
and infiltrated and embedded in hydroxymethylmethacrylate resin
(Technovit 7100; Kulzer and Co., Friedrichsdorf, Germany) according to
the manufacturers instructions. Tissue sections (3–30 µm) were cut
using a Polycut S microtome (Reichert Jung, Nossloch, Germany) and
stained with 0.5% toluidine blue (Amerecso, Solon, OH). Age-matched
hpg littermates were used as nontransgenic control animals.
All procedures were approved by the Animal Ethics Committee within the
national guidelines for animal experimentation.
Data analysis
Statistical significance (P < 0.05) was
determined using the unpaired t test. Results are expressed
as mean ± SEM.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-RIPß
construct-RIPß DNA construct (Fig. 1
). The RIP sequence directs pancreatic
expression of heterologous transgenes (31, 32) and was
selected over native FSH gene promoters to avoid the requirement of
GnRH in hpg mice as well as inhibitory feedback actions of
androgens on pituitary FSH expression (33). hFSH was
selected for construct preparation because human heterodimers could be
readily differentiated from mouse FSH by selective immunoassay and was
previously shown to be bioactive in the mouse (5, 17). The
tandem RIP-RIPß construct was predicted to confer simultaneous
expression of both - and ß-minigenes in the same cell and,
consequently, improve the efficiency of secretion of bioactive hFSH
heterodimer, as suggested by in vitro studies
(26), and avoid the cross-breeding of separate RIP- and
RIPß-transgenic lines. Nucleotide sequence analysis confirmed that
the human - and ß-transgene coding sequences matched those
previously reported. Analysis of the 0.7-kb RIP fragment revealed two
single base differences, compared with the original sequence
(34), a previously reported A-to-C substitution at -211,
resulting in an extra TAAT-motif (35), and an extra G at
-529 (not reported to date), which is located outside the active
promoter region downstream of -448 (35).
|
-RIPß-transgene construct was able to direct expression of both
FSH subunits in the rat pancreatic RINm5F cell line. A two-site
immunofluorometric assay that requires epitopes on both subunits for
any detection of hFSH was used to verify the presence of an assembled
hFSH heterodimer complex. The RIP-RIPß-construct produced hFSH
levels equivalent or higher than combined pRIP- and
pRIPß-transfection (0.37 vs. 0.29 IU/liter),
supporting the use of tandem RIP-RIPß-transgenes to generate
transgenic animals.
Several transgenic hFSH founders were obtained using fertilized oocytes
of the C3H mouse strain (15) carrying the hpg
genotype. Three male founders, selected to generate F1 offspring,
transmitted hFSH transgenes at the expected 50% frequency, and one
(designated ß.6) produced offspring expressing detectable serum
hFSH. Comparison of nontransgenic hpg and normal male mouse
serum confirmed that the human-specific assay exhibited little or no
cross-reactivity to mouse gonadotropins (data not shown), and previous
studies showed that endogenous serum FSH is undetectable in
hpg mice (15, 18, 33). Three additional
transgenic founders were prepared and expanded to independent
transgenic lines (designated ß.043, ß.045, and ß.113)
using FVB/N mice, which all expressed detectable serum hFSH. To
generate offspring expressing transgenic hFSH on a mouse
gonadotropin-deficient background (i.e. FSHxhpg
mice), transgenic animals were bred with fertile mice of the C3H strain
carrying heterozygote GnRH gene deletions.
Transgenic hFSH lines expressing a range of serum hFSH
levels
The analysis of serum hFSH in the independent transgenic lines
revealed distinct differences in circulating FSH levels. Hemizygous
(+/-) transgenic hpg mice of line ß.6 expressed
consistent FSH levels (3.86 ± 0.30 IU/liter, n = 14) with no
significant difference between males and females (P >
0.9), shown in Table 1. By comparison,
line ß.045 (+/-) hpg mice secreted approximately
10-fold lower levels of hFSH (0.37 ± 0.11 IU/liter, n = 7),
again with no difference between sexes (Table 1), and line ß.043
expressed levels comparable with those of ß.6 (4.17 ± 0.43
IU/liter, n = 9). In contrast, line ß.113 animals expressed a
range of FSH concentrations from 0.1 to >10 IU/liter (shown in Fig. 2), with both high- and low-level
hFSH-secreting mice present in the same litter. Conveniently, the hFSH
levels remained low or high for individual ß.113 mice, which
provided the opportunity to examine the effects of a range of different
FSH levels in a single transgenic line.
|
|
ß.6 and ß.113).
There was no difference in the serum testosterone levels of transgenic
FSHxhpg males (2.2 ± 0.3 nM,
n = 11) compared with nontransgenic hpg males (2.4
± 0.3 nM, n = 11). Thus, the presence of
transgenic FSH did not seem to alter or induce androgen synthesis on
the hpg background. The absence of an androgen response was
also consistent with the undeveloped, androgen-dependent epididymal and
seminal vesicle organs of FSHxhpg males, which were
unchanged compared with those of hpg controls for all
transgenic lines. The effect of transgenic FSH on inhibin B levels was
determined, using a human inhibin B immunoassay, in transgenic
FSHxhpg mice expressing a range of serum hFSH levels. The
relative mouse serum inhibin B levels showed a distinct gender
dimorphism with 9- to 10-week-old female FSHxhpg mice
having increased inhibin B levels that were strongly positively
correlated with hFSH levels (r2 = 0.84), as shown
in Fig. 2
. In comparison, there was little correlation
(r2 = 0.04) between inhibin B and FSH levels of
transgenic FSHxhpg males, noting inhibin B of male samples
with moderate serum hFSH concentrations (4.06, 4.88, 5.16 IU/liter)
were below the detection limit of the assay (Fig. 2). The serum inhibin
B levels of all 9- to 10-week-old nontransgenic hpg males
were consistently undetectable (n = 12).
Testicular phenotype of transgenic hFSH mice
The testicular actions of hFSH in the gonadotropin-deficient
hpg males were determined by comparison of testis weights
(relative to body weight) and histology with those of nontransgenic
hpg littermates. In the FSHxhpg males of line
ß.6, with serum levels of 3.89 ± 0.48 IU/liter hFSH, there
was a 5-fold increase in testis-to-body weight ratios, relative to
nontransgenic controls (Table 1; P < 0.001). We
further examined the testicular effects of different hFSH levels in
lines ß.043, 045, and 113 mice, which were all generated on a
FVB/N x C3H mouse background (compared with the C3H strain of
ß.6). Nontransgenic littermate controls were necessary to avoid
strain-specific differences in gonad weights. In contrast to the larger
testes of ß.6 mice, there was no change in the testes ratios of
ß.045 FSHxhpg males, exhibiting hFSH levels
approximately 10-fold lower (<0.5 IU/liter) than the ß.6 animals
(Table 1). Similar analysis of the ß.043 and ß.113 transgenic
lines revealed enlarged testes (i.e. above the weight range
of nontransgenic hpg control mice) only in males expressing
over 1 IU/liter hFSH. Analysis of ß.113 hpg males that
expressed a range of distinct serum hFSH concentrations demonstrated
that the increased testis size was strongly positively correlated with
hFSH levels (Fig. 3, r2 = 0.9).
|
, A compared with B). The tubules of
transgenic FSHxhpg testes were also found to contain
disorganized epithelium with no distinct lumen formation (Fig. 4, C and
D). However, approximately 50% of tubules of larger testes contained
round spermatids, as well as sparse numbers of elongated spermatids
(Fig. 4, C and D). Fully differentiated spermatozoa were not observed
in tubules of any transgenic FSHxhpg testes examined.
The Leydig cell morphology of transgenic testes seemed similar to those
of nontransgenic hpg males, supporting the proposal
that FSH alone does not alter Leydig cell development or function
(18, 36). Therefore, these observations suggest that the
in vivo actions of FSH, presumably via the Sertoli cell, can
induce the completion of meiosis, although the progress of subsequent
spermiogenesis is minimal. The exact nature of FSH actions on germ and
Sertoli cell populations awaits more detailed and extensive
stereological examination.
|
The specific actions of hFSH in the absence of LH were examined in
ovaries isolated from FSHxhpg animals. The ovary-to-body
weight ratios of ß.6 FSHxhpg mice, which expressed
serum levels of 3.86 ± 0.30 IU/liter hFSH, were (on average)
10-fold larger than those of nontransgenic control hpg
animals (Table 1; P < 0.001). Transgenic hFSH induced
larger ovaries in other transgenic lines; and, like the dose-dependent
hFSH effect on testicular weight, there was a strong positive
correlation (r2 = 0.9) between the circulating
levels of hFSH and ovary weight (Fig. 3). Histological examination of
the small ovaries of hpg animals showed the expected
gonadotropin-independent development of preantral follicles
(39), which failed to develop beyond the type 5 preantral
stage (Fig. 5A). By comparison, the
ovaries of transgenic FSHxhpg mice showed increased
follicle recruitment and development to at least type 7 antral
follicles, characterized by a single enlarged cavity of follicle fluid
(Fig. 5B). There was no evidence of corpus luteum formation in any
transgenic ovary examined, and there was no significant difference in
uteri weights of transgenic and nontransgenic hpg controls
(P = 0.89), suggesting that an estradiol response to
transgenic hFSH was minimal or absent.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
An important feature of our transgenic model is that the in vivo actions of hFSH suggested that its circulating levels were within, or did not exceed, a physiological range of FSH concentration. Serum hFSH levels did not disrupt the reproductive function of normal (non-hpg) females or males and were without the confounding effects of infertility and abnormal gonadal hyperplasia reported in other transgenic models expressing supraphysiological FSH levels (37, 38). In addition, transgenic hFSH in normal males with a functional hypothalamus-pituitary-gonadal axis did not alter testicular weight or spermatid numbers (Allan and Haywood, unpublished observations), whereas earlier studies using high FSH doses (40) or supraphysiological levels of transgenic FSH (37) reported testis weights exceeding those of normal animals. Abnormally high levels of transgenic FSH enhanced serum testosterone levels and seminal vesicle growth (37), suggesting that such high FSH concentrations cross-reacted with the LH receptor or aberrantly stimulated Sertoli cell paracrine factors capable of stimulating Leydig cell steroidogenesis, which vitiates a physiological FSH-specific phenotype. In the current FSHxhpg model, the selective expression of hFSH did not increase circulating testosterone levels in hpg males, and other androgen-dependent tissues remained undeveloped, which is consistent with previous work showing that FSH treatment of hpg mice had no effect on intratesticular testosterone levels (36). Thus, circulating transgenic hFSH concentrations in the present model, which (by immunoassay) are equivalent to circulating FSH levels found in fertile humans, did not interfere with normal gonadotropin function or induce aberrant gonadal phenotypes.
The FSHxhpg transgenic mouse provides a uniquely constructed model that allows, for the first time, the in vivo investigation of specific FSH actions on Sertoli cell development and function. Our laboratory previously showed that neonatal treatment of hpg mice with rhFSH produced a modest response, doubling Sertoli cell numbers with only a 43% increase in testis size (18), whereas rhFSH treatment of weanling and adult hpg animals had no effect on final Sertoli cell numbers (17). The finding of much larger testes observed in transgenic FSHxhpg mice supports the proposal that perinatal FSH expression plays an important role in determining the magnitude of the testicular FSH response. This FSH-specific effect is likely to reflect its postulated mitogenic role on Sertoli cell proliferation during the perinatal period of testicular development (41). The FSH-induced increase in testicular size was dose-dependent; however, enlarged testes were only observed in FSHxhpg males with serum FSH levels more than 1 IU/liter. Presumably circulating hFSH levels less than 1 IU/liter were below the FSH threshold for a testicular response. The presence of postmeiotic spermatids in the enlarged FSHxhpg testes also revealed that the FSH-specific response promoted germ cell development. Thus, although not supporting full spermatogenesis, transgenic FSH stimulated the progression of meiosis beyond the arrested pachytene stage of the FSH-deficient hpg mouse. Together with our earlier observations of increased spermatogonia and spermatocyte populations after neonatal rhFSH treatment (17), which were consistent with the reduction of spermatogonial populations after FSH-specific immunoneutralization (12), these findings demonstrate that the LH- independent FSH response can effect a range of testicular germ cell types. Because FSH receptor expression is exclusive to the Sertoli cell (8), these FSH-mediated effects are most likely to involve pathways or trophic factors initiated by or derived from Sertoli cells.
We have previously shown that androgens alone, without any requirement for aromatization to estradiol, stimulated meiotic progression and qualitatively complete spermatogenesis in hpg testes (6, 16). Surprisingly, estradiol alone was recently shown to induce spermatogenesis in hpg males, in the absence of detectable androgens (42). Unexpectedly, circulating FSH levels, which are undetectable in untreated hpg mice (15, 18, 33), were reported to be detectable after estradiol administration, and FSH was proposed to be a stimulatory effector for spermatogenesis in the testes of estrogen-treated hpg mice. Our current findings indicate that FSH alone is unlikely to explain this effect of estradiol, because circulating transgenic FSH does not stimulate spermatogenesis to the extent observed in the estrogen-hpg mice, even at higher levels. Alternatively, it is possible that estradiol effects are mediated via cross-reactivity with androgen receptors in the testis, or undefined estradiol receptor pathways that may converge on and stimulate the same intracellular processes as androgens. A full stereological examination of testicular cell types in the FSHxhpg mouse, complemented by analysis of androgen- or estrogen-treated FSHxhpg animals (to examine synergistic FSH and steroidal actions) will enable a more complete dissection of the specific FSH response.
The transgenic FSHxhpg model enables further examination of isolated perinatal FSH exposure on ovarian development (in particular, the absolute effects of FSH on whole ovarian follicle populations and the rate of growth and recruitment of preantral follicles). In our initial analysis, transgenic hFSH increased preantral follicle recruitment and development to the late antral stage. These histological observations were consistent with the increased inhibin B levels in transgenic FSH females. Inhibin B is thought to be the predominant form of inhibin produced by the preovulatory follicles, with elevated inhibin B levels occurring in the early follicular phase (30, 43). The current findings support an association between inhibin B synthesis and FSH-induced follicle recruitment.
Inhibin secretion is a well established FSH-induced response in both the ovary and testis, which normally acts to negatively regulate pituitary FSH production (44, 45). The pituitary-independent FSH production of the FSHxhpg mouse provides a unique paradigm in which to study FSH-regulated inhibin secretion. Our initial analysis of FSHxhpg animals revealed a distinct gender-dimorphism in the inhibin B response to FSH, with a strong positive correlation between serum inhibin B and hFSH levels of transgenic females, contrasting with the relatively low-level inhibin B response of transgenic males. The LH-independent inhibin B response in females may reflect increased follicle recruitment and development, which results in larger numbers of differentiating granulosa cells that secrete inhibin B (43), and pituitary-independent transgene expression that is free of inhibin B negative-feedback modulation. The lower serum inhibin B response of FSHxhpg males suggests a more limited testicular capacity to synthesize inhibin B, relative to the ovary. The physiologically important form of inhibin in the male is inhibin B derived from Sertoli cells (46, 47). In normal males, the pituitary-regulation of FSH secretion results in serum FSH levels negatively correlated with inhibin B levels (46). It is possible that the lower inhibin B response of FSHxhpg males is linked to the fixed numbers of adult Sertoli cells that reach maximal levels during neonatal development, which may maintain a threshold inhibin B response despite the continual FSH exposure. Alternatively, FSHxhpg testes may require additional factors to increase inhibin B synthesis, including the proposed requirement of later-stage spermatogenic cell types (48), which may be lacking in the incomplete spermatogenic cycle of the FSHxhpg testis. Leydig cells could also play a role in the production of inhibin B or its regulation. However, the FSH-induced and LH-independent production of inhibin B agrees with recent reports of individuals with disrupted FSH activity. A hypogonadal man with FSH ß-subunit deficiency (49), a female with primary amenorrhea caused by a mutated inactivating FSH receptor (50), as well as men homozygous for a different inactivating FSH receptor mutation (3) were all found to have greatly diminished plasma inhibin B levels despite the presence of LH.
In summary, we have described a new transgenic model to examine the gonadal actions of FSH independently of LH effects. This model provides the unique opportunity to examine the biochemical pathways of the specific FSH response. Animals expressing FSH alone represent unique models that will enable further investigation into the unknown target genes and molecular downstream actions that mediate the FSH response. In addition, these models can be expanded to examine the elements of the LH response required to integrate with FSH actions to initiate and maintain gonadal development and function.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Received October 4, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-, ß A-, and ß B-subunits in ovarian cell types during
the rat estrous cycle. Mol Endocrinol 2:1352–1363[Abstract]
This article has been cited by other articles:
 |
|
 |
|
  S. Mhaouty-Kodja, A. Lozach, R. Habert, M. Tanneux, C. Guigon, S. Brailly-Tabard, J.-P. Maltier, and C. Legrand-Maltier Fertility and spermatogenesis are altered in {alpha}1b-adrenergic receptor knockout male mice J. Endocrinol., November 1, 2007; 195(2): 281 - 292. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. J. McTavish, M. Jimenez, K. A. Walters, J. Spaliviero, N. P. Groome, A. P. Themmen, J. A. Visser, D. J. Handelsman, and C. M. Allan Rising Follicle-Stimulating Hormone Levels with Age Accelerate Female Reproductive Failure Endocrinology, September 1, 2007; 148(9): 4432 - 4439. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. A. Walters, C. M. Allan, M. Jimenez, P. R. Lim, R. A. Davey, J. D. Zajac, P. Illingworth, and D. J. Handelsman Female Mice Haploinsufficient for an Inactivated Androgen Receptor (AR) Exhibit Age-Dependent Defects That Resemble the AR Null Phenotype of Dysfunctional Late Follicle Development, Ovulation, and Fertility Endocrinology, August 1, 2007; 148(8): 3674 - 3684. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K.R. Barnett, C. Schilling, C.R. Greenfeld, D. Tomic, and J.A. Flaws Ovarian follicle development and transgenic mouse models Hum. Reprod. Update, September 1, 2006; 12(5): 537 - 555. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. M. Allan, A. Garcia, J. Spaliviero, M. Jimenez, and D. J. Handelsman Maintenance of Spermatogenesis by the Activated Human (Asp567Gly) FSH Receptor During Testicular Regression Due to Hormonal Withdrawal Biol Reprod, May 1, 2006; 74(5): 938 - 944. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C M Allan, Y Wang, M Jimenez, B Marshan, J Spaliviero, P Illingworth, and D J Handelsman Follicle-stimulating hormone increases primordial follicle reserve in mature female hypogonadal mice. J. Endocrinol., March 1, 2006; 188(3): 549 - 557. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Young, R. Salomon, W. Au, C. Allan, P. Russell, and Q. Dong Ornithine Decarboxylase (ODC) Expression Pattern in Human Prostate Tissues and ODC Transgenic Mice J. Histochem. Cytochem., February 1, 2006; 54(2): 223 - 229. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Wang, H. Newton, J. A. Spaliviero, C. M. Allan, B. Marshan, D. J. Handelsman, and P. J. Illingworth Gonadotropin Control of Inhibin Secretion and the Relationship to Follicle Type and Number in the hpg Mouse Biol Reprod, October 1, 2005; 73(4): 610 - 618. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. P Meehan, B. G Harmon, M. E Overcast, K. K Yu, S. A Camper, D. Puett, and P. Narayan Gonadal defects and hormonal alterations in transgenic mice expressing a single chain human chorionic gonadotropin-lutropin receptor complex J. Mol. Endocrinol., April 1, 2005; 34(2): 489 - 503. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Jimenez, J.A. Spaliviero, A.J. Grootenhuis, J. Verhagen, C.M. Allan, and D.J. Handelsman Validation of an Ultrasensitive and Specific Immunofluorometric Assay for Mouse Follicle-Stimulating Hormone Biol Reprod, January 1, 2005; 72(1): 78 - 85. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. M. Allan, A. Garcia, J. Spaliviero, F.-P. Zhang, M. Jimenez, I. Huhtaniemi, and D. J. Handelsman Complete Sertoli Cell Proliferation Induced by Follicle-Stimulating Hormone (FSH) Independently of Luteinizing Hormone Activity: Evidence from Genetic Models of Isolated FSH Action Endocrinology, April 1, 2004; 145(4): 1587 - 1593. [Abstract] [Full Text] [PDF]
|
)
transgenic hFSH mice on a gonadotropin-deficient
hpg background, determined as described in
Materials and Methods. Serum samples were obtained from
9- to 10-week-old animals of the 
