| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
Leland J. and Dorothy L. Olson Center for Women’s Health, Departments of Obstetrics and Gynecology (S.K.R., L.A.), and Physiology and Biophysics (S.K.R.), University of Nebraska Medical Center, Omaha, Nebraska 68198-4515
Address all correspondence and requests for reprints to: Shyamal K. Roy, BH 3040, Departments of Ob/Gyn and Physiology and Biophysics, University of Nebraska Medical Center, 984515 Nebraska Medical Center, Omaha, Nebraska 68198-4515. E-mail: skroy{at}unmc.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
18%) by a single injection of eCG on
the first day of life. Delaying antibody treatment during late
gestation caused a time-dependent block in granulosa cell
differentiation, with a consequent proportional increase in the
percentage of primordial follicles. This indicates that FSH-induction
of primordial follicle development begins at a critical time of ovarian
development. On the other hand, shortening the postnatal duration of
eCG exposure reduced the degree of reversal, suggesting that prolonged
perinatal FSH action is essential for developing the full gamut of
primordial follicles. These results provide the first direct evidence
that FSH action during fetal ovarian development is critical for the
onset of primordial follicle formation. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Generation and determination of specificity and efficacy of
polyclonal anti-FSH antibody
An anti-FSH antibody was raised in New Zealand white rabbits
using ovine FSH-20 with 5% and 2% LH and TSH contamination,
respectively (20). Blood was collected, before antigen
injection, for preimmune serum preparation. After a satisfactory titer
was reached, the specificity of the antiserum was verified, first by an
enzyme-linked immunosorbent assay using ovine-FSH-20 and
ovine-LH-25, and then by Western immunoblotting and chemiluminescence
(21) using 1 µg each of ovine-FSH-20, ovine-LH-25, ovine
PRL-19, and equine CG (eCG).
To verify the ability of the antibody to neutralize FSH action in vivo, cyclic hamsters were injected sc with 200 µl of the anti-FSH-antiserum, on proestrus, at 1600 h, after the periovulatory LH surge but before the second FSH surge at 2200 h (20); the latter induces preantral follicle growth (15) and recruitment for ovulation (for review, see Ref. 1). Animals were checked the next morning for the estrous vaginal discharge and again on estrus of the next cycle; ovaries were collected and processed for morphological evaluation of follicle development. Because pure hamster FSH does not exist, the cross-reactivity of the FSH antiserum with hamster FSH was assessed by 1) competition assay; and 2) Western immunoblotting of hamster pituitary lysate. For the former protocol, [125I]-ovine FSH was incubated at 4 C with an optimal dilution of the antiserum in the presence of 0.1, 0.5, 1, 10, 100, and 1000 ng ovine-FSH or LH, 10, 20, 40, 80, and 100 µl of serum from proestrous (day 4: 0900 h) or long-term (10 days) hypophysectomized hamsters for 20 h. The antibody-[125I]-FSH complex was precipitated with goat-antirabbit-IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) and counted in a Packard (Packard Instrument Co., Meriden, CT) auto- gamma counter. The results were expressed as percentage binding against the amounts of FSH or volume of serum. For Western immunoblotting of hamster pituitary FSH, fresh pituitary glands were obtained from proestrous (D4: 0900 h) hamsters and were homogenized in ice-cold 1x RIPA buffer [PBS (pH 7.4), 0.1% SDS, 1% NP-40, and 0.5% deoxycholate] containing a protease inhibitor cocktail (Sigma, St. Louis, MO). After 30 min on ice, the mixture was centrifuged for 20 min at 12,000 rpm in a refrigerated IEC microfuge, the protein content of the supernatant was determined by the BCA protein assay kit (Pierce Chemical Co., Rockford, IL), and 40 µg of hamster pituitary protein was electrophoresed under a reducing condition, transferred to a nitrocellulose membrane, and probed with the FSH-antiserum, as described previously (21). Positive controls were 0.25 and 0.5 µg of ovine-FSH-20.
Determination of the optimal window of FSH action during primordial
folliculogenesis
Hamsters were mated on proestrous evening, and the presence of
sperm in the vagina next morning was considered as day 1 of gestation
(1DG). Pregnant hamsters were injected sc with 200 µl anti-FSH-serum
either on 12DG, 13DG, or 14DG; or pups were injected sc with 20 µl of
the antiserum on D1pn. Some animals received preimmune serum or saline
as controls. On D8pn, pups were anesthetized with nembutal, blood was
collected for FSH RIA, and ovaries were saved for morphometric
quantitation of primordial and primary follicle development.
Based on the results, in the next experiment, the antiserum was injected into pregnant hamsters on 10DG. Because ovarian structures consisting of germ cells and somatic cells are present by day 12 of fetal life, the objective was to deprive somatic cells of any FSH action during ovary formation so that primordial follicle formation could be stopped completely. Ovaries were collected on D8pn, and follicle development was quantitated morphometrically.
Determination of the presence of functional FSH receptor in fetal
hamster ovaries
Because [125I]-FSH autoradiography is
inconclusive for fetal ovaries and RRA is often not feasible because of
limited ovarian materials, production of cAMP, in response to FSH
in vitro, was assessed, following (with modification) a
protocol described by Sokka and Huhtaniemi (22). Ovaries
were collected from 13-day-old fetal hamsters and were cultured in 0.5
ml DMEM, as described previously (23), in the presence of
0.5 mM methylisobutylxanthine (MIX,
Calbiochem, San Diego, CA) and in the presence or absence
of 50 ng/ml ovine-FSH-19 (NIH) or 50 µM
forskolin (Calbiochem, San Diego, CA) for 24 h. After
termination, the medium was placed in a boiling water bath for 5 min,
clarified by centrifugation, and stored at -80 C for cAMP RIA.
eCG reversal of the antibody inhibition of primordial follicle
development
To determine whether the effect of the antiserum was
attributable to the inactivation of endogenous FSH or antibody-mediated
toxicity, pregnant hamsters received 200 µl antiserum on 12DG, and
pups received an sc injection of 20 IU eCG (Sigma) or
saline on D1pn; ovaries were collected on D8pn for follicular
morphometry. The rationale for using eCG was 1) its longer half-life;
2) no cross-reactivity with the anti-FSH-antiserum; and 3) binding to
the FSH receptor and induction of FSH-like activity in the hamster
(24).
To determine the time-course of eCG rescue of primordial follicle formation, pregnant females were injected with 200 µl of the anti-FSH-antiserum on 12DG, and pups received 20 IU eCG either on D1, D4, or D6pn, with ovaries retrieved on D8pn.
Reversibility of the antiserum inhibition by endogenous FSH
Whether inactivation of FSH during pregnancy delays primordial
follicle development or makes the ovary refractory to subsequent FSH
signaling was determined by injecting pregnant hamsters with 200 µl
of the antiserum sc on 12DG or 13DG and ovaries collected on D12pn, at
a time when ovaries of untreated hamsters contain primordial through
secondary follicles with 4 layers of granulosa cells
(5).
Morphometric quantitation of primordial, primary, and secondary
follicle development
The percentage of follicles in different stages of development
was assessed from 2-µm-thick hematoxylin and eosin-stained plastic
sections, under 650x magnification, using the Image Pro Plus image
analysis software (Media Cybernetics, Silver Spring, MD).
Because primordial oocytes (i.e. oocytes in clusters and
without any somatic cell partners) dominate the ovaries of 8-day-old
hamsters (5, 19), the total number of oocytes with
nucleolus, regardless of their follicular association, was counted in a
given optical field. Next, the number of follicles, at various stages,
corresponding to those oocytes was determined. The fields were chosen
at random from the entire ovary, and 250 or more oocytes for each ovary
were counted. The proportion of follicles in an ovary was then
expressed as percentage of oocytes. An oocyte surrounded by the
cytoplasmic processes of at least one flattened parenchymal cell was
considered a primordial follicle, whereas a primary follicle was
defined as one surrounded by a complete layer of parenchymal cells, of
which the majority were cuboidal. Primordial follicles were identified
by a double-blind screening of coded slides, and the code was revealed
after completion of follicle counting.
Determination of serum levels of FSH and rabbit IgG
Sera from anti-FSH-serum-exposed animals were treated overnight
with protein A-agarose with a binding capacity of 
19 mg IgG/ml to
remove any trace of the antibody and were used for FSH RIA using
[125I]-rat FSH, rat FSH standards, and antirat
FSH serum (NIH, Ref. 25).
Whether the antibody entered fetal blood was evaluated by injecting 200 µl anti-FSH serum into pregnant females, on 12DG, and the presence of rabbit IgG in the serum of 1-day-old hamsters was detected by Western immunoblotting (21). The stability of FSH-antiserum in the plasma of postnatal hamsters was evaluated by immunodetection of rabbit IgG in the sera of 8-day-old pups that received the antiserum either on 12DG or D1pn.
Determination of cAMP
cAMP was determined by using an RIA kit (Biomedical Research Instruments, Inc., Stoughton, MA) with a sensitivity of
0.05 pmol cAMP after acetylation. The data were presented as pmol cAMP
per ml medium per 24 h.
Data analysis
Morphometric evaluation of folliculogenesis was done on at least
3 ovaries, which were collected from 3 different pups born to different
mothers. Moreover, 250 or more oocytes from each ovary were counted to
determine the percentage of follicles at each size class. Follicles
were classified (26) as follows: stage 0, primordial
follicles with 1 or more flattened parenchymal cells; stage 1, primary
follicles with unlaminar granulosa cells, of which the majority were
cuboidal; stage 2, secondary follicles with 2 layers of granulosa
cells; stage 3, secondary follicles with 3 layers of granulosa cells;
and stage 4, secondary follicles with 4 layers of granulosa cells. All
data were analyzed by 2-way ANOVA and Scheffé’s test with
5% level of significance.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
), under a nonreducing condition, and
cross-reacted with an approximately 23-kDa protein in ovine FSH
preparation and in hamster pituitary lysate under a reducing condition
(Fig. 2]) The electrophoretic pattern of
FSH corresponded well with that provided by the NIH. Moreover,
[125I]-ovine FSH binding to the antiserum was
inhibited by proestrous hamster serum but not by the serum from
long-term hypophysectomized hamsters or LH (Fig. 3). The antiserum also completely blocked
antral follicle development in adult cyclic hamsters when administered
before the second FSH peak (Fig. 4, A
and B).
|
|
|
|
); however, only a trace amount of
rabbit IgG was present by D5pn when the antiserum was injected on 12DG
(data not shown). The antiserum did not interfere with the gestation
period, delivery time, placental weight, litter size, or litter weight.
Moreover, the pups showed normal phenotype, milk intake, and postpartum
weight gain, indicating that antiserum did not induce any adverse
change in the phenotype that could be construed as a potential cause of
retarded ovary development. No significant difference in the ovarian
weight was noted between the control and antiserum-exposed pups by day
D8pn (data not shown).
|
and 7), which was similar to untreated
controls. However, antiserum exposure on 12DG dramatically reduced the
proportion of primordial follicles to 2.4% and completely blocked
primary follicle formation (Figs. 6B and 7). No further reduction in
the proportion of primordial follicles was noted when the antiserum was
injected on 10DG (data not shown). A gradual increase in primordial
follicle formation was noted when the antiserum treatment was delayed
to 13DG or 14DG (Fig. 7), but the values were still significantly lower
than controls. No further improvement was noted when the antiserum was
injected on D1pn. However, a small percentage of primary follicles
developed (Fig. 7), suggesting that FSH-dependent morphogenesis of
primordial follicles continues after birth, but the onset of FSH
signaling may occur around 12DG.
|
|
), thus corroborating
previous findings (5, 9). FSH levels were significantly
(P < 0.05) low in 8-day-old hamsters exposed to the
antiserum on 12DG; however, no FSH was detected when the antiserum was
injected on D1pn (Fig. 8). FSH levels reached normal values (9 ±1
ng/ml) by D12pn after the antiserum exposure on 12DG, indicating a
gradual reversal of the availability of endogenous FSH (hence, its
action). Conversely, rabbit IgG was detectable in pups receiving the
antiserum postnatally.
|
). cAMP production increased more than
2-fold (P < 0.05) in response to FSH and more than
4-fold (P < 0.05) in response to forskolin (fig. 9).
|
and 10). The efficacy of eCG,
however, declined significantly as the treatment was delayed by 4 or 6
days (Fig. 10), suggesting that primordial folliculogenesis is a
relatively long FSH-dependent process.
|
). In fact, the rate of
primordial-to-primary follicle transition seemed to have accelerated,
because there was no difference in the percentage of primary follicles
between the control and antiserum-exposed pups despite a significantly
lower percentage of primordial follicle development (Fig. 11). However,
the percentage of stages 2 and 3 follicles (2 and 3 layers of granulosa
cells, respectively) was significantly lower than in controls, and
stage 4 follicles were completely absent (Fig. 11). This indicated that
antiserum did not affect FSH receptors, given that eCG was able to
function. Therefore, when clearance and degradation adequately reduced
the serum antibody titer around D8pn (only a trace amount of rabbit IgG
was present by D5pn), functional FSH levels began to increase,
resulting in a compensatory induction of primordial follicles by D12pn
and the formation of primary follicles. The delay in folliculogenesis
beyond the primordial stage was more pronounced when the antiserum was
administered on 13DG (Fig. 11).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Lack of FSH (8) by an inactivating mutation of either FSH-ß-subunit gene (10) or FSH-receptor gene (11) blocks preantral-to-antral follicle transition in homozygous mice, and genetic inactivation of growth differentiation factor-9 results in follicular arrest beyond the late primary stage (31). Because none of these studies has quantified primordial or primary follicle development, the results do not fully address the importance of FSH in primordial follicle development. It is likely that silencing the FSH gene in the mouse may activate other FSH-mediated compensatory mechanism(s), which induce follicular morphogenesis. In fact, overexpression of cyclin D2, a granulosa cell-specific mitotic cyclin (32), has been reported in FSH receptor knockout mice (11). Nevertheless, women with inactivating mutation in the extracellular domain of the FSH receptor have streak gonads (12, 13, 14) with few follicles. Although primordial follicles have been identified in ovaries with inactivating FSH-receptor mutation, it is unclear whether their number matches normal ovaries. Aittomaki et al. (12) have further documented that the mutation responsible for the development of hypoplastic ovaries in women occurs at the extracellular domain of the receptor and leads to reduced hormone binding and cAMP production capacity without affecting the affinity (12). These lines of evidence clearly indicate that a low, but residual, FSH-receptor activity may persist in the ovaries of women with FSH-receptor mutation, which can provide adequate signal for primordial follicle development. The development of a few primordial follicles in 8-day-old hamsters, after gestational exposure to the FSH-antiserum, confirms that low FSH activity may be adequate for the initiation of primordial follicle formation, but a prolonged FSH action is needed for the development of a full cohort of primordial follicles.
The inhibition of primordial follicle formation by the anti-FSH serum given during the critical window of FSH action on the fetal ovary, and its reversal by eCG, suggests that perinatal FSH plays an important role in the onset of somatic cell differentiation into granulosa cells, which is essential for the development of the very first cohort of primordial follicles. Moreover, FSH action seems to begin long before primordial follicles appear, i.e. by D7pn–D8pn of postnatal life. This notion is supported by the development of approximately 2.4% primordial follicles in pups exposed to the antiserum on 12DG. It is likely that only a few somatic cells are capable of processing the FSH signal by 12DG; and once committed, they differentiate into granulosa cells to form primordial follicles despite a subsequent block in FSH action. This is further evident by the progressive increase in primordial follicle development when antiserum treatment is delayed.
The failure to develop a full cohort of primordial follicles by D8pn, even when the antiserum treatment was delayed until D1pn, suggests that sustained FSH signaling is essential until all presumptive granulosa cells are differentiated and become associated with the oocyte to constitute the finite pool of primordial follicles. Interruption of FSH action, at anytime during this period, blocks further cell differentiation while cells that have already received FSH stimulus undergo differentiation, resulting in a final low number of primordial follicles. The presence of serum FSH in 8-day-old hamsters exposed to the antiserum on 12DG correlates with the decline in serum antiserum concentration and suggests that antiserum-induced inactivation of FSH is a reversible process. The temporal eCG action in reversing the antiserum inhibition supports this contention. Moreover, eCG reversal also indicates that the antiserum inhibition is specifically caused by inactivation of endogenous FSH and not a nonspecific antibody-mediated toxicity. This is further evident from the failure of the preimmune rabbit serum to block primordial follicle formation. Because LH-receptors do not appear in the hamster ovary before 15 days of age (33), eCG effect cannot be attributed to any inherent LH-like activity.
It seems that FSH-mediated differentiation of somatic cells into granulosa cells is a slow process, spanning through several days of the perinatal period (i.e. late prenatal through early postnatal) and can be halted at any time during this period without any permanent adverse consequence. Therefore, no differentiation induction may occur when FSH action is blocked on 10DG but will resume when FSH is available; and consequently, some primordial follicles will be formed by D8pn. On the other hand, if the initiation of FSH-mediated signaling occurs by 12DG, as evident in the present study, some cells will differentiate before the antibody, given on 12DG, completely neutralizes serum FSH and will form some primordial follicles by D8pn. This mechanism may explain why the development of primordial follicles cannot be blocked completely, even when the antiserum is injected before 12DG. The degree of initial differentiation (hence, the percentage of primordial follicles developed) by D8pn will depend on the timing of initiation of FSH inactivation.
A rapid induction of primordial follicles within 4 days after D8pn in gestationally antiserum-exposed hamsters suggests the possibility that 1) FSH availability increases after D8pn; and 2) somatic cell responsiveness to FSH heightens because of the increased presence of FSH receptors. Serum FSH levels in postnatal hamsters start to increase after D9pn (5, 9), and serum FSH levels are closer to the untreated values by D12pn in hamsters exposed to the antiserum on 12DG. If the antiserum inactivates FSH, a possible reduction in FSH-induced receptor down-regulation is likely, which may subsequently accelerate the process of differentiation. Because the presence of functional FSH receptors is a prerequisite for FSH action, factors inducing FSH receptors must work in conjunction with FSH for granulosa cell differentiation. Mayerhofer et al. (6) have suggested that cAMP inducers, such as vasoactive intestinal peptide, may be responsible for FSH receptor induction in neonatal rat ovary. Whether such a mechanism exists in the hamster needs further evaluation; however, the production of cAMP in response to FSH in vitro has been demonstrated in the present study.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Received May 15, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  D. Adhikari and K. Liu Molecular Mechanisms Underlying the Activation of Mammalian Primordial Follicles Endocr. Rev., August 1, 2009; 30(5): 438 - 464. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Wang and S. K. Roy Expression of Bone Morphogenetic Protein Receptor (BMPR) during Perinatal Ovary Development and Primordial Follicle Formation in the Hamster: Possible Regulation by FSH Endocrinology, April 1, 2009; 150(4): 1886 - 1896. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. A McLaughlin and S. C McIver Awakening the oocyte: controlling primordial follicle development Reproduction, January 1, 2009; 137(1): 1 - 11. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.L.H. Ireland, D. Scheetz, F. Jimenez-Krassel, A.P.N. Themmen, F. Ward, P. Lonergan, G.W. Smith, G.I. Perez, A.C.O. Evans, and J.J. Ireland Antral Follicle Count Reliably Predicts Number of Morphologically Healthy Oocytes and Follicles in Ovaries of Young Adult Cattle Biol Reprod, December 1, 2008; 79(6): 1219 - 1225. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Wang, E. R. Prossnitz, and S. K. Roy G Protein-Coupled Receptor 30 Expression Is Required for Estrogen Stimulation of Primordial Follicle Formation in the Hamster Ovary Endocrinology, September 1, 2008; 149(9): 4452 - 4461. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Morimoto, Y. Oku, M. Sonoda, A. Haruki, K. Ito, S. Hashimoto, and A. Fukuda High oxygen atmosphere improves human follicle development in organ cultures of ovarian cortical tissues in vitro Hum. Reprod., December 1, 2007; 22(12): 3170 - 3177. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Wang and S. K. Roy Development of Primordial Follicles in the Hamster: Role of Estradiol-17{beta} Endocrinology, April 1, 2007; 148(4): 1707 - 1716. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Wang and S. K. Roy Expression of Growth Differentiation Factor 9 in the Oocytes Is Essential for the Development of Primordial Follicles in the Hamster Ovary Endocrinology, April 1, 2006; 147(4): 1725 - 1734. [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]
|
|
|
|
|
|
T. Steckler, J. Wang, F. F. Bartol, S. K. Roy, and V. Padmanabhan Fetal Programming: Prenatal Testosterone Treatment Causes Intrauterine Growth Retardation, Reduces Ovarian Reserve and Increases Ovarian Follicular Recruitment Endocrinology, July 1, 2005; 146(7): 3185 - 3193. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Yang, J. Wang, Y. Shen, and S. K. Roy Developmental Expression of Estrogen Receptor (ER) {alpha} and ER{beta} in the Hamster Ovary: Regulation by Follicle-Stimulating Hormone Endocrinology, December 1, 2004; 145(12): 5757 - 5766. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y.-M. Zhang and S. K. Roy Downregulation of Follicle-Stimulating Hormone (FSH)-Receptor Messenger RNA Levels in the Hamster Ovary: Effect of the Endogenous and Exogenous FSH Biol Reprod, June 1, 2004; 70(6): 1580 - 1588. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Wang and S. K. Roy Growth Differentiation Factor-9 and Stem Cell Factor Promote Primordial Follicle Formation in the Hamster: Modulation by Follicle-Stimulating Hormone Biol Reprod, March 1, 2004; 70(3): 577 - 585. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Balla, N. Danilovich, Y. Yang, and M. R. Sairam Dynamics of Ovarian Development in the FORKO Immature Mouse: Structural and Functional Implications for Ovarian Reserve Biol Reprod, October 1, 2003; 69(4): 1281 - 1293. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
-subunit of LH.
