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Department of Physiology (T.P., L.N. M.P., I.H.), University of Turku, Fin-20500 Turku, Finland; Department of Physiology (F-P.Z.), University of Helsinki, Biomedicum, Fin-00014 Helsinki, Finland; and Institute of Reproductive and Developmental Biology (I.H.), Imperial College London, London W12 0NN, United Kingdom
Address all correspondence and requests for reprints to: Professor Ilpo Huhtaniemi, Institute of Reproductive and Developmental Biology, Imperial College London, Du Cane Road, London W12 0NN, United Kingdom. E-mail: ilpo.huhtaniemi{at}imperial.ac.uk.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Further information about the specific effects of gonadotropins on gonadal function has recently been obtained from phenotypes of women with inactivating mutations of FSHß subunit, FSHR, and LHR (4), as well as from knockout (KO) mice for the gonadotropin subunits and receptors (5, 6, 7, 8). The human mutations and respective genetically modified animals demonstrate that, although the early stages of follicular growth are responsive to FSH, they may also mature independently of gonadotropin action until the preantral stage (9). LH action seems to become essential for follicular development from the antral stage onward, and for ovulation (7). Proper granulosa cell estrogen production is dependent on FSH action, whereas LH action is indirectly necessary by stimulating androgen production in theca cells. Surprisingly, the theca cell layers of follicles appear normal also in mice devoid of LH or LHR (5, 7).
Earlier studies on hypophysectomized rats and mice have demonstrated that it is possible to induce ovulation by recombinant FSH (rFSH) without LH (10, 11, 12, 13). In addition to follicular maturation, these studies indicated a role for FSH in ovulation and luteinization and showed that ovulation is possible without an LH surge. Despite these results, it still remains uncertain whether the FSH-stimulated ovulation was mediated purely via FSHR or whether some level of LHR activation is necessary. Furthermore, evidence for the essential role of the epidermal growth factor (EGF) family members, amphiregulin, epiregulin, and ß-cellulin, as mediators of the LH action in follicle maturation, has recently been shown (14). These proteins have been demonstrated to mediate the LH actions by acting as paracrine mediators between the mural granulosa and cumulus cells and to be necessary for the formation of cumulus-oocyte complex and oocyte maturation.
In the present study, we analyzed the possibility to complete follicular maturation and induce ovulation in LHR KO (LuRKO) mice (7) by different combinations of gonadotropin treatments. We also analyzed whether estradiol (E2) priming before gonadotropin stimulation could enhance the gonadotropin response of LuRKO mice. The expression of certain members of the EGF family, reported recently to be important mediators of LH action (14), were also analyzed in LuRKO mice shortly after rFSH injections. The total lack of LH action in LuRKO mice causes severe disturbances in the development of reproductive organs and leads to female infertility through low estrogen production and anovulation (7). Thus, these mice provide a good in vivo model in which to study the consequences of missing LH action in vivo, and to revisit the possibility that high doses of FSH alone are able to compensate for missing LH action and trigger ovulation.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). In WT ovaries, pregnant mare serum gonadotropin (PMSG) stimulation (48 h) induced follicular maturation, and a number of preovulatory follicles could be detected but, as expected, only sporadic corpora lutea could be detected. Additional injections of hCG or rFSH at 48 h induced further follicular maturation and multiple ovulations in the WT ovaries when studied 24 h later. In contrast to WT mice, no response to any of the gonadotropin treatments could be observed in the LuRKO ovaries, where the most mature follicles remained at the early antral stage. In contrast, several of the oocytes showed deformed structures, and granulosa cells were disordered and pyknotic, indicating rupture of apoptotic follicles (Fig. 2). Follicular maturation did not proceed beyond the antral stage either, when the LuRKO mice were primed with E2 before PMSG + rFSH stimulation (Fig. 2).
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). Furthermore, the uterine weights showed clear response to PMSG + rFSH stimulation in WT mice (7.1 ± 1.03 vs. 25.8 ± 1.37 mg), whereas the LuRKO mice showed no response to these treatments (3.2 ± 0.30 vs. 3.6 ± 0.46 mg). Similarly, the ovarian weights responded to gonadotropins in WT mice (1.9 ± 0.11 vs. 5.8 ± 0.36 mg), but no changes occurred in LuRKO mice after the hormone injections (1.5 ± 0.07 vs. 1.6 ± 0.08 mg).
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Quantitative RT-PCR Analysis
The mRNA expression of selected genes known to be important for follicular development, ovulation, and function of the corpus luteum was analyzed to confirm the findings observed upon histological analysis. Growth differentiation factor 9 (GDF-9), a gene expressed in oocytes and essential for the early stages of follicular development (15), was highly expressed in LuRKO ovaries (Fig. 3). The expression level was higher than in WT ovaries, apparently because the oocytes composed a larger proportion of the ovarian volume in LuRKO mice. A similar reduction in GDF-9 expression was observed in LuRKO and WT ovaries after the hormone treatments.
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). However, after the other hormone stimulations (24 h after hCG/rFSH injections), P450-arom expression returned to control levels. In LuRKO mice, no response of aromatase mRNA expression to any hormone stimulation could be detected. P450 side chain cleavage (P450-scc) is known to be most highly expressed after ovulation, representing a marker of luteinization. Accordingly, the mRNA for this enzyme showed highest expression in the ovaries with corpora lutea, being approximately 20- to 25-fold induced after PMSG + hCG/rFSH stimulations as compared with nonstimulated WT ovaries (Fig. 3). A similar expression pattern was found for another marker of luteinization, i.e. the prolactin receptor (PRLR), for which all the isoforms were analyzed simultaneously (Fig. 3). Neither the preovulatory marker (P450-arom), nor the luteinization markers (P450-scc and PRLR), showed any expression after any of the hormone treatments in LuRKO mice.
Interestingly, progesterone receptor (PR), normally expressed shortly after the LH surge, and cyclooxygenase-2 (COX-2), essential for ovulation, were both expressed in WT and in LuRKO mice (Table 2). The former was expressed at lower level in nonstimulated LuRKO than WT ovaries, but neither showed consistent response to the gonadotropic stimulations. In WT mice the PR levels decreased slightly after hormone stimulations, but COX-2 expression did not change in either group of mice. In addition to the ovulation markers, we also analyzed the expression of certain other genes related to ovarian endocrine function (Fig. 3 and Table 2). Of these markers, FSHR expression was similar in unstimulated WT and LuRKO ovaries, increased transiently with PMSG in WT ovaries, but did not respond to gonadotropins in LuRKO ovaries. Estrogen receptors 
(ER) and -ß (ERß) displayed similar basal expression in both types of ovaries, with decreasing tendency of expression in WT ovaries after the hormone treatments. The expression level of 3ß-hydroxysteroid dehydrogenase type 1 (3ß-HSD 1) was similar at basal levels and was clearly up-regulated by gonadotropins in WT ovaries. IGF binding protein (IGFBP) 4, a marker of follicular atresia (16, 17, 18, 19), showed a higher level of expression in LuRKO ovaries but did not respond clearly to gonadotropins in either type of ovaries.
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and Table 2), i.e. those of the steroidogenic acute regulatory protein (StAR), 17ß-hydroxysteroid dehydrogenase type 1 (17ß-HSD 1), and TNF- -stimulated gene-6 (TSG-6). StAR expression in LuRKO mice was close to the detection limit, and no response to any hormone treatment could be detected, whereas the WT mice showed a clear response to the gonadotropins. The expression of 17ß-HSD 1 was significantly higher in LuRKO ovaries as compared with WT mice, but a significant transient increase in response to PMSG was only found in WT mice. TSG-6, expressed in ovulating follicles involving cumulus-oocyte complex formation (20, 21), displayed a similar basal level of expression in WT and LuRKO ovaries, and a positive response to gonadotropin treatments was observed in WT mice. The results of the real-time mRNA analyses are summarized in Table 3.
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), further indicating that the genes are tightly controlled by LH action.
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). However, in the WT mice, after hCG or rFSH stimulation, only a few apoptotic follicles were observed, when the ovaries were strongly luteinized. Contrary to this, numerous apoptotic follicles were detected in LuRKO ovaries. Immunohistochemistry of P450-arom confirmed the real-time RT-PCR findings, and expression of this enzyme was only detected in granulosa cells of preovulatory WT follicles after PMSG stimulation (Fig. 5). The lack of P450-arom immunostaining confirmed the lack of preovulatory follicles in the treated LuRKO ovaries.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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There are several caveats why the hypophysectomized rat cannot be considered totally free of gonadotropin action. The hypophysial pars tuberalis is not destroyed by hypophysectomy, and it has been shown to have the capacity to synthesize and secrete gonadotropins (24). Moreover, using an ultrasensitive immonometric assay for LH, we have shown that the level of LH in hypophysectomized rat circulation is about 5% of the control level (25). The cross-reaction of FSH with LHR is minimal (26), and although major constitutive activity of the unliganded LHR has not been documented (27), this is a common feature of G protein-coupled receptors (27). Finally, there are reports on the synthesis of gonadotropin subunits in gonads (28, 29, 30, 31, 32). In summary, it is reasonable to conclude that a surgical hypophysectomy model may not necessarily be totally free of LH/LHR effects. A permissive role of very low LH activity in FSH-stimulated ovulation still remains a possibility. It is therefore warranted to reassess this question in a model in which all LH activity is excluded through targeted inactivation of the LHR.
The LuRKO mice (7), in which LHR has been inactivated by targeted deletion of exon 11, allow us to study selectively the effects of FSH on folliculogenesis without confounding effects of the persistent LHR expression, residual serum LH levels, and the hypothetical involvement of ovarian LH. The LuRKO mice also offer an excellent model by which to analyze LHR dependence of factors important for follicle maturation and the relation of LH and FSH action in ovulation. These observations clearly indicated that ovulation does not occur in the absence of functional LHR. Histological evaluation showed that gonadotropin stimulations, either alone or after E2 priming, were unable to advance follicular growth from the antral to the preovulatory stage, and to induce ovulation. Furthermore, the fact that ovarian weights did not change during gonadotropin stimulations refers to a total resistance of follicular development to gonadotropins in LuRKO ovaries. However, it should be emphasized that the LuRKO females have about 2-fold elevated levels of serum FSH and their ovaries express FSHR (7). These effects are apparently maximally stimulated by the endogenous hormone and cannot be advanced with PMSG or rFSH. The near-total unresponsiveness of ovaries to PMSG in the absence of functional LHR has been previously briefly reported using another LHRKO model (33, 34), but these studies did not address in detail the specific question of whether ovulation can be induced by FSH.
Interestingly, we were able to show that FSH triggered ovulation in the WT mice, because the number of ovulated oocytes after PMSG + rFSH treatment was close to that induced by PMSG + hCG. This could not be a delayed effect of LH in the PMSG injection, because only few oocytes had ovulated at 72 h after the PMSG injection alone.
In contrast to LHß and LHRKO mice (5, 7, 33), folliculogenesis in FSHR and FSHß KO mice is arrested at the preantral stage (6, 8, 35). Hence, folliculogenesis develops further in LuRKO mice, apparently due to normal FSH action, allowing follicular maturation to progress to the antral stage. It has been previously shown with other transgenic mice that folliculogenesis until the antral follicle stage is gonadotropin independent (9), although gonadotropin responsive, whereas local paracrine factors are responsible for the initiation of follicular development and growth. One such factor, necessary for early follicle growth, is the oocyte-specific GDF-9 (36), the importance of which for folliculogenesis has been emphasized by the observations on GDF-9 KO mice (15). The elevated expression of GDF-9 in LuRKO ovaries is likely due to the increased proportion of oocytes in the absence of large preovulatory follicles.
The analyses of the steroidogenic pathway in LuRKO ovaries (Table 3) demonstrated severe disruption in steroid biosynthesis, confirming the previous data on dramatically decreased E2 levels in LuRKO females (7). StAR, P450-scc, and P450-arom mRNAs were not detected in LuRKO ovaries either basally or after hormone treatments. As expected, P450-arom and P450-scc, being markers of preovulatory follicles and luteal cells, respectively, responded to gonadotropins only in WT mice. Immunohistochemical staining also confirmed the lack of preovulatory follicles in LuRKO ovaries by the absence of P450-arom immunostaining. Interestingly, the expression of 3ß-HSD 1 was similar in WT and LuRKO ovaries, confirming a previous finding in LuRKO testes (37) on independence of this steroidogenic enzyme on LH regulation. Furthermore, the expression of 17ß-HSD 1 was similarly LH independent in LuRKO ovaries. The lack of expression of the two luteal markers, P450-scc and PRLR, confirmed the histological findings on absence of corpora lutea in LuRKO ovaries. Finally, disruption of ovarian steroidogenesis in the KO mice was further illustrated by the fact that uterine weights did not show any response to gonadotropin stimulations, due to defective estradiol production. Ovarian steroidogenesis of the LuRKO females was apparently already disrupted before puberty, because their uterine weights were significantly smaller than in WT mice at this age. Furthermore, basal LH levels in prepubertal LuRKO mice were clearly elevated as a sign of defective steroid feedback.
PR expression is required for follicular rupture before the LH-induced ovulation (38). The level of PR mRNA was low and unresponsive to gonadotropin stimulations in LuRKO ovaries. Furthermore, the expression levels of COX-2 and hyaluronan binding protein TSG-6, essential for the complex inflammatory-like reactions during ovulation and cumulus expansion after the LH surge (20, 21, 39, 40), did not respond to hormone treatments in LuRKO mice. TSG-6 expression has been shown to be essential for ovulation in previous studies with TSG-6-deficient mice, which indicated sterility due to failing in cumulus expansion and anovulation in the absence of this protein (41). Moreover, COX-2 KO mice also present with incompetence to ovulate; their otherwise healthy follicles fail to ovulate in the absence of the protein (42). However, this defect could be overruled by exogenously administrated prostaglandin E2, the product of COX-2 catalysis (43). The basal expression levels of these two genes were similar in WT and LuRKO mice, and only TSG-6 showed a moderate increase in response to hCG/rFSH in WT ovaries. The small responses were most likely due to the long time lag (24 h) between the last hormone injection and sample collection (22, 40). The treatment regimen was chosen because of the documented slower response of follicles to rFSH than hCG/LH (13). Also, previous studies have indicated only transient expression of PR after a hCG surge (44, 45), which is likely to explain the low PR expression in WT mice. Thus, it remains open whether these genes, necessary for ovulation, responded acutely to hCG/rFSH in LuRKO mice. However, such eventual responses were not able to advance follicular maturation in the anovulatory LuRKO mice beyond the early antral stage.
FSHR is expressed in the ovary exclusively in granulosa cells, and it stimulates follicular maturation and the expression of LHR in granulosa cells. On the basis of the phenotypes of inactivating human mutations and KO mice for FSHR and FSHß, FSH action is essential for normal follicular maturation (4, 6, 8). PMSG and rFSH stimulations up-regulate, and ovulatory doses of hCG or rFSH suppress, FSHR expression (46, 47). In accordance, in the present study, we found approximately 3-fold increase in FSHR levels in WT mice after PMSG stimulation, which were suppressed after hCG/rFSH injections. However, in LuRKO mice the FSHR levels remained unaltered after FSH stimulation, which indicated a permissive role for LH in the autoregulation of FSHR. A typical LH-dependent suppression in ERß expression was observed in WT mice after PMSG injection, but not in LuRKO ovaries. In contrast to a previous report (48), we observed similar down-regulation of ER expression in WT mice both after PMSG and PMSG + hCG/rFSH stimulations, but no effect in LuRKO mice.
Recent data about the role of the EGF family members, epiregulin, amphiregulin, and ß-cellulin, in mediating LH effects in granulosa cells have provided a new concept about the mechanisms of ovulation and cumulus expansion (14). The expression of these genes is highly increased in cumulus cells shortly after the LH/hCG surge, and we confirmed this finding in WT mice. However, in LuRKO mice, no such response was observed, showing the lack of activation of this regulatory cascade normally involved in LH-triggered ovulation. The finding was expected because of the absence of preovulatory follicles in LuRKO ovaries.
The presence of developing follicles only until the antral stage, with lack of preovulatory follicles and corpora lutea, raises the possibility of increased apoptosis in LuRKO ovaries. Accordingly, ovarian histology showed ruptured, malformed oocytes, surrounded by disordered and pyknotic granulosa cells, indicating increased apoptosis. In many species, follicular atresia is associated with increased expression of IGFBP 4 in granulosa cells (16, 17, 18, 19). Most of the follicles in normal ovaries undergo atresia instead of developing to mature follicles and achieving ovulation. Hence, atretic follicles at different stages of development are normally present in the ovaries. However, elevated IFGBP 4 expression in LuRKO ovaries both basally and after gonadotropin stimulation, along with enhanced immunostaining for cleaved caspase 3 (49, 50), indicate abundant apoptosis of LuRKO ovaries.
The differences in basal expression, and responses to gonadotropin stimulations, of the genes studied were in harmony with the information on their regulation (Table 3). The treatment with PMSG alone for 48 h induced a state of preovulatory follicular maturation, and all gene markers of this stage were up-regulated. The PMSG + rFSH and PMSG + hCG combinations were both able to induce ovulation, as demonstrated by histological analysis, and the luteinization-specific genes were found to be up-regulated. When the gene expression of LuRKO ovaries was evaluated, the only consistent response to the hormonal stimulations observed was the suppression of GDF-9 expression, which apparently represents a response to the high FSH stimulus and reflects an increase in proportion of the stromal and somatic component and reduced proportion of oocytes of the total ovarian volume.
In conclusion, we have demonstrated in the present study that functional LHR expression is essential for the later stages of follicular maturation preceding ovulation. Moreover, and in contrast to studies previously carried out on hypophysectomized rodents, our study indicates that ovulation cannot be induced with high doses of FSH in the absence of functional LHR. Thus, it is likely that LH action permits the final stages of follicular maturation to the preovulatory stage. Such effects, and even ovulation, can be induced by FSH only in the presence of functional LHR. E2 priming was not able to render the LuRKO ovaries responsive to FSH stimulation, confirming further the necessity of functional LHR. The clinical relevance of these observations is that they provide experimental confirmation for the empiric observations that LH priming is important during gonadotropin induction of follicular maturation in in vitro fertilization cycles for the optimal follicle maturation, induction of ovulation, and oocyte quality (51, 52). Further investigations will be needed to further characterize the para/autocrine factors essential for follicular maturation under the influence of LH.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Hormone Stimulations and Sample Collection
Four groups of prepubertal 19- to 23-d-old female LuRKO and WT mice were stimulated by ip injections of: 1) 10 IU of PMSG (Sigma Chemical Co., St. Louis, MO) for 48 h, followed by 15 IU of recombinant human FSH (rFSH; National Hormone & Peptide Program, Torrance, CA) for 24 h; 2) 10 IU PMSG for 48 h; 3) 10 IU PMSG for 48 h followed by 5 IU hCG (Organon, Oss, The Netherlands) for 24 h; and 4) vehicle injections without hormones. After stimulations the mice were killed and the ovaries were collected. The number of oocytes in oviductal ampullae after ovulation was calculated by stereomicroscope, and the weights of uteri in rFSH-stimulated and nonstimulated control mice were recorded. To test whether the ovulation rate would increase during longer PMSG treatment, one group of WT mice was stimulated by 10 IU PMSG, and the oocytes were calculated in oviducts 72 h later.
In another experimental setting, we analyzed whether E2 priming (10, 12) before PMSG + rFSH stimulation would lead to further improvement of the follicular maturation. For this experiment, 21-d-old LuRKO and WT mice were anesthesized with 100–300 µl of 2% tribromoethanol [Avertin; Aldrich, Milwaukee, WI, (53)] and a SILASTIC (Dow Corning Corp., Midland, MI) implant tubes, 8 mm in length (inner diameter 1.58 mm, outer diameter 2.41 mm), filled with E2 powder (Sigma Chemical Co.) and sealed at both ends with SILASTIC adhesive (Elastosil RTV-1 Silicone Rubber; Wacker-Chemie GmbH, Munich, Germany), were implanted subdermally into each mice. Buprenorphine (Temgesic, Schering-Plough, Brussels, Belgium; 3–5 µg/mouse sc) was used as postoperative analgesia. After 5 d of treatment, the mice were injected with 10 IU PMSG ip followed by 15 IU rFSH ip 48 h after the PMSG injection. The mice were killed 24 h later and the ovaries were collected for histological analysis.
For analysis of expression of the EGF family members (see below), adult 2-month-old LuRKO and WT mice were stimulated with 10 IU PMSG ip followed by 5 IU hCG ip 48 h later. The mice were killed 3 h after the hCG injection, and the ovaries were collected. Nonstimulated LuRKO and WT littermates were used as controls. Identical injections of saline were given in each experiment to control animals.
Histological Analysis and Immunohistochemistry
For histological and immunohistochemical analyses, ovaries were fixed in 4% paraformaldehyde at 20 C for 10–15 h. Fixed ovaries were dehydrated, embedded in paraffin, and cut in 5 µm-thick sections. For histological analysis, sections were stained with hematoxylin and eosin (Delafield’s).
For immunohistochemical detection of apoptotic cells in the ovaries, a rabbit polyclonal antibody against cleaved activated caspase-3 (1:200 dilution in PBS; Cell Signaling Technology, Inc., Beverly, MA) was used as the primary antibody. Visualization of the antigen-antibody complexes was performed using the immunoperoxidase technique (Histostain-Plus kit; Zymed Laboratories, Inc., South San Francisco, CA) following the manufacturer’s instructions. P450-arom was detected with a mouse monoclonal antibody (1:50 dilution in TBS; Serotec Ltd, Kidlington, Oxford, UK), and antigen-antibody complexes were visualized using the immunoperoxidase technique by applying the M.O.M. Kit (Vector Laboratories, Inc., Burlingame, CA). The ovarian sections stained against P450-arom were also counterstained with Mayer’s hematoxylin.
RNA Analysis
Snap-frozen ovaries from three mice were pooled, total RNA was isolated using the RNeasy Mini Kit (QIAGEN, Valencia, CA), and RNA samples were DNase treated by DNase I Amplification Grade Kit (InVitrogen, Life Technologies, Paisley, Scotland, UK). Three RNA pools, collected from a total of nine mice, were used for PCR analyses. Real-time RT-PCR analyses were performed using the QuantiTect SYBR Green RT-PCR Kit (QIAGEN) according to the manufacturer’s instructions. Either 60 ng or 100 ng DNase-treated total RNA was used for the reactions.
For the analysis of amphiregulin, epiregulin, and ß-cellulin expression, RNA was isolated from five mice per experimental group. RNA isolation and DNase treatment were performed as described above, and mRNA levels were analyzed by real-time RT-PCR from 100 ng of total RNA. All samples and standard curves in the real-time RT-PCR analyses were analyzed in triplicate, and the results were presented as relative values compared with the expression of ß-actin. The primer pairs and annealing temperatures used in PCR are presented in Table 4.
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Statistical Analysis
Statistical analyses were performed using SigmaStat program (SPSS, Inc., Chicago, IL), and Kruskal-Wallis test and Wilcoxon rank sum tests were used for analyses. Significance was set as P < 0.05, and the values are presented as mean ± SE.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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First Published Online June 7, 2005
Abbreviations: CG, Chorionic gonadotropin; COX-2, cyclooxygenase 2; E2, estradiol; EGF, epidermal growth factor; ER, estrogen receptor; FSHR, FSH receptor; GDF-9, growth differentiation factor 9; HSD, hydroxysteroid dehydrogenase; IGFBP, IGF-binding protein; KO, knockout; LHR, LH receptor; LuRKO, LHR knockout; P450-arom, P450 aromatase; P450-scc, P450 side chain cleavage; PMSG, pregnant mare serum gonadotropin; PR, progesterone receptor; PRLR, prolactin receptor; rFSH, recombinant FSH; StAR, steroidogenic acute regulatory protein; TSG-6, TNF- -stimulated gene-6; WT, wild type.
Received for publication February 7, 2005. Accepted for publication May 31, 2005.
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0.05). n = 3/group. Mean ± SE.
