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Endocrinology Vol. 143, No. 2 700-707
Copyright © 2002 by The Endocrine Society

REPRODUCTION-DEVELOPMENT

Embryo Transfer Experiments and Ovarian Transplantation Identify the Ovary as the Only Site in Which Nuclear Receptor Interacting Protein 1/RIP140 Action Is Crucial for Female Fertility

Göran Leonardsson, Mary Ann Jacobs, Roger White, Rosemary Jeffery, Richard Poulsom, Stuart Milligan and Malcolm Parker

Institute of Reproductive and Developmental Biology, Faculty of Medicine, Imperial College (G.L., R.W., M.P.), London, United Kingdom W12 1ONN; Imperial Cancer Research Fund, Clare Hall Laboratories (M.A.J.), South Mimms, Potters Bar, United Kingdom EN6 3LD; Imperial Cancer Research Fund, In Situ Hybridization Service (R.J., R.P.), London, United Kingdom WC2A 3PX; and School of Biomedical Sciences, Kings College London, Guy’s Campus (S.M.), London, United Kingdom SE1 1UL

Address all correspondence and requests for reprints to: Dr. Malcolm Parker, Institute of Reproductive and Developmental Biology, Faculty of Medicine, Imperial College, Du Cane Road, London, United Kingdom W12 ONN. E-mail: m.parker{at}ic.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Spatial and temporal regulation of gene expression by a number of different nuclear receptors is critical in female reproduction. In this study we investigated whether the nuclear receptor corepressor nuclear receptor interacting protein 1 (Nrip1)/RIP140, which is essential for ovulation, is also required for postovulatory events, leading to pregnancy and parturition. Expression analysis indicated that Nrip1 is present in the uterus in stromal and glandular epithelial cells, primary decidual cells, and subsequently in differentiating decidual cells at the anti-mesometrial side of the implantation site. It also indicated a temporal regulation of Nrip1 in the corpora lutea at different stages of pregnancy, with increased levels at midgestation at approximately d 9.5 postcoitum (pc). By performing both embryo and ovarian transfer experiments we demonstrate that, provided the block to ovulation is by-passed, Nrip1-/- mice are capable of establishing and maintaining pregnancies. However, although the majority of offspring derived from ovarian transplantation survived, approximately 50% of embryos were resorbed by d 13.5 pc after embryo transfer, and the majority of pups were stillborn or died soon thereafter. Thus, although Nrip1 is differentially expressed in the reproductive tract, we conclude that the ovary is the only site in which its action is essential for fertility, with a crucial role in ovulation and a secondary role in the maintenance of pregnancy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
NUCLEAR RECEPTOR interacting protein 1 (Nrip1), formerly known as receptor interacting protein 140 (RIP140), was identified by means of its estrogen-dependent interaction with the mouse ER{alpha} (1). However, the protein is capable of interacting with other nuclear receptors in a ligand-dependent manner, and functional analysis suggests that it acts as a corepressor, because it inhibits their ability to stimulate transcription from reporter genes in transfected cells (2, 3, 4, 5, 6). Interestingly Nrip1 not only inhibits transcriptional activation, but also overcomes the ability of the GR to transrepress NF-{kappa}B activity (7). Recent studies suggest that its function as corepressor may be mediated by the recruitment of histone deacetylases (8) and/or the repressor C-terminal binding protein (9).

To investigate the role of Nrip1 in vivo, we generated mice devoid of the Nrip1 gene (10). The mice are viable and morphologically normal, but are 15–20% smaller than their littermates. Mature female Nrip null mice are completely infertile because of the failure of mature follicles to release oocytes at ovulation. Luteinization occurs, however, resulting in a phenotype closely resembling that of luteinized unruptured follicle syndrome, which is frequently associated with infertility in women (11). In this study we performed embryo transfer and reciprocal ovarian transfer experiments to determine whether the anovulation observed in Nrip1-/- mice is accompanied by additional reproductive defects, which may involve other sites of action, such as the hypothalamic-pituitary-gonadal axis, or postovulatory events in the reproductive tract.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Generation of Nrip1-null mice has previously been described (10). Briefly, a 3.6-kb fragment containing the entire coding region of Nrip1, except the first 27 amino acids, was replaced with an IRESßNEO cassette containing an internal ribosome entry site and a lacZ-neo fusion gene. This allowed the analysis of ß-galactosidase expression as a marker of Nrip1 promoter activity. Mice were maintained on a 12-h light cycle and had free access to food and water. Genotyping of mice was determined by PCR as previously described (10) using Nrip1 (MR9 and MR22)- and neomycin (173 and 630)-specific primers: MR9, CAA AAT TCC GCC ACG TTC AGT G; MR22, GGG GGCGCT CTT GGC ATC GTG; 173, ATG AAC TGC AGG ACG AGG CA; and 630, GCC ACA GTC GAT GAA TCC AG. All animal experiments were performed at the Transgenic Services Unit at Imperial Cancer Research Fund in accordance with Home Office approval.

Embryo transfer
Embryo transfer experiments were performed as previously described (12). Briefly, 1-d-old wild-type embryos were collected from oviducts of superovulated wild-type donor female mice that were kept with wild-type males to obtain fertilized embryos. Collected embryos were incubated in prewarmed medium until transfer to pseudopregnant recipients. Recipient wild-type, heterozygous, and Nrip1-/- females (8–12 wk old) were anesthetized, and the oviduct and top of the uterus were carefully exposed. Pools of 16 embryos were then transferred into the oviduct of each recipient (8 on each side of the uterus) using a mouth pipette with a hand-pulled transfer needle. After transfer, the body wall was sewn up, and the skin was closed using wound clips, after which the anesthetic was reversed by analgesic. For histology and expression analysis, uteri and ovaries were collected at different stages of pregnancy; the day of transfer was referred to as d 0.5 postcoitum (pc). Offspring from the mice left to term were monitored daily and were subsequently genotyped.

Ovary transplantation
Reciprocal ovarian transplantation was performed using pairs of 4-wk-old littermates as described previously (12), except that the donor was not killed, and both ovaries were replaced in all cases. Briefly, ovarian and uterine tissues in anesthetized mice were exposed, followed by removal of one ovary via a small incision of the bursa surrounding it. The ovary was then kept in prewarmed culture medium while the recipient female was treated in the same way. The donor ovary from each mouse was then placed back into the empty bursa of the other (recipient) female, after which the body wall was sewn up. After reciprocal replacement of the first ovary, the whole procedure was repeated so that both ovaries were replaced. Finally, the skin was closed using wound clips, and the anesthetic was reversed by analgesic. After operation, mice were kept for 3–4 wk before they were mated with proven wild-type males. Offspring from the breeding were recorded daily and were subsequently genotyped.

Superovulation
To induce ovulation, immature (25–26 d old) mice were injected (ip) with 5 IU serum gonadotropin (PMSG, Folligon, Intervet UK Ltd., Milton Keynes, UK), and after 48 h 10 IU hCG (Chorulon, Intervet UK Ltd.) were administered. Ovarian tissues for expression analysis were then collected at different time points during the ovulatory process.

Histology and in situ hybridization
Tissue for histology and in situ hybridization were fixed in neutral buffered formalin and sectioned (4 µm paraffin). For analysis of uteri and ovarian morphology, sections were stained with hematoxylin and eosin. For in situ hybridization, slides were pretreated, hybridized, washed, and dipped in Ilford K5 for autoradiography (Ilford Imaging Ltd., Knutsford, UK) (13). Autoradiography was carried out at 4 C before developing in Kodak D19 (Rochester, NY) and counterstaining with Giemsa. Sections were examined under conventional or reflected light darkfield conditions that allowed individual autoradiographic silver grains to be seen as bright objects on a dark background.

Antisense RNA probes specific for ERß (10), 17ß-hydroxysteroid dehydrogenase/17-ketosteroid reductase type 7 (17HSD/7KSR; a gift from P. Vihko) (14), LH receptor (LHR) (15), and tissue inhibitor of metalloproteinase 3 (TIMP-3; LHR and TIMP-3 were gifts from T. Ny) (16) were generated for in situ hybridization as previously described (10).

PR and cycloxygenase-2 (COX-2) cDNAs were isolated from mouse ovarian RNA by RT-PCR using specific primers (PR forward, AGC AGA GGA TGA AGG AGC TG, PR reverse, AAA TTC CAC AGC CAG TGT CC; COX-2 forward, TGT ACA AGC AGT GGC AAA GG; and COX-2 reverse, GCT GTG GAT CTT GCA CAT TG) and were subcloned into PCR-B II Blunt TOPO plasmids (Invitrogen, San Diego, CA). These plasmids were then used to generate antisense RNA probes, as previously described (10), to detect PR and COX-2 expression.

Analysis of ß-galactosidase expression
Nrip1 promoter activity in uterine and ovarian tissue was assessed by monitoring ß-galactosidase activity as previously described (10). Tissues were collected in isopentane and then stored at -70 C until use. For sectioning, tissue was immersed in Tissue-Tek OCT compound (Sakura Finetek Europe BV, Zoeterwoude, The Netherlands), followed by cryostat sectioning (10 µm). ß-Galactosidase activity was detected in sections that were fixed with 4% paraformaldehyde in PBS for 10 min at 4 C and thereafter washed with PBS and 2 mM MgCl2, followed by incubation in detergent solution (PBS, 2 mM MgCl2, 0.01% sodium deoxylate, and 0.02% Nonidet P-40) twice for 2 min each time. Finally, after a 2-min incubation in preincubation buffer [20 mM K3Fe(CN)6, 20 mM K4Fe(CN)6·6H2O, 2 mM MgCl2, 0.01% sodium deoxycholate, and 0.02% Nonidet P-40 in PBS], the sections were incubated in the same buffer in the presence of 1 mg/ml X-galactosidase at 30 C overnight, followed by staining with Nuclear Fast Red.

TaqMan real-time PCR
Total RNA was isolated using TRIzol (Life Technologies, Inc., Gaithersburg, MD) according to the manufacturer’s instructions. To obtain first strand cDNA for further analysis, 2 µg total RNA were treated with deoxyribonuclease, and cDNA was prepared using the Superscript First-Strand Synthesis System for RT-PCR according to the manufacturer’s instructions (Life Technologies, Inc.). Real-time PCR was performed with the ABI PRISM 7700 Sequence Detection System (PE Applied Biosystems, Foster City, CA) using specific primers and TaqMan probes for Nrip1 and the constitutively expressed L19 gene as an internal control. Primers and probes were designed with the Primer Express software accompanying the system. TaqMan primer sequences were: Nrip1TM forward, GAA CCT GGG CTT TTG AAT GG; Nrip1TM reverse, GTT TTG GTC AGT CTT GGA GAG TCT T; L19TM forward, GGA AAA AGA AGG TCT GGT TGG A; L19TM reverse, TGA TCT GCT GAC GGG AGT TG; progesterone receptor (PR)TM forward, TTC TAC TCG CTG TGC CTT ACC A; PRTM reverse, CCA AAG GAA TTG TGT TAA GAA GTA GTA AGA; COX-2TM forward, GGT GTC CCT TCA CTT CTT TCA ATG; and COX-2TM reverse, TCT GGA GTG GGA GGC ACT TG. TaqMan probe sequences were: Nrip1TM probe, TAT CTG TGA TGA CCC ACT TAA TGG GTC CCT T; L19TM probe, CCC AAT GAG ACC AAT GAA ATC GCC A; PRTM probe, TGG CAA ATC CCA CAG GAG TTT GTC AAA CT; and COX-2TM probe, AAG ATC CAC AGC CTA CCA AAA CAG CCA CC.

Hormone measurements
Progesterone was measured in 25 µl plasma from terminal blood samples using the DSL3900 Active-Progesterone Coated-Tube RIA Kit (Diagnostics Systems Laboratories, Inc., Webster, TX), as described by manufacturer, with a reported sensitivity of 0.12 ng/ml and inter- and intraassay coefficients of variation of 4.8% and 9.2%, respectively.

Statistical analysis
Values are presented as the mean ± SEM. Significance between experimental groups was analyzed by t test, with P < 0.05 considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Embryo transfer experiments
As Nrip1-/- mice fail to ovulate, we investigated the role of Nrip1 in postovulatory events by performing embryo transfer experiments. Implantation was successful in both wild-type and Nrip1-/- recipients (Table 1Go). Although the proportion of Nrip1-/- recipients with implantation sites appeared to be lower than that in wild-type animals at each stage of pregnancy, there was no significant difference in the total number of implantation sites per pregnant female in early pregnancy (d 6.5–8.5 pc). Histological examination confirmed that implantation appeared to be normal in mice on d 6.5 pc However, at later stages of pregnancy there was a progressive reduction in the number of surviving embryos in Nrip1-/- mice. This reduction was associated with a proportion of the implantation sites becoming hemorrhagic, with excess blood at the embryo/uterine tissue boundary and less well developed embryos (Fig. 1AGo); resorption of the embryos and implantation sites followed. This was apparent by gross observation on d 9.5 pc, and by d 12–13.5 pc more than half of the embryos were resorbing. At term, 14 of 19 pups from Nrip1-/- mothers were dead at birth or within 24 h. In contrast, the majority (96%) of pups produced by either wild-type or heterozygous control mice survived.


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  		Table 1. Wild-type embryo transfer to WT, heterozygous, and Nrip-/- recipients

 


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  		Figure 1. Uterine and ovarian histology after embryo transfer. Representative hematoxylin and eosin (H & E)-stained sections are shown. A, A d 13.5 pc Nrip-/- uterus showing a healthy embryo (left) and a resorbing implantation site (right). Arrowheads indicate hemorrhagic blood. Scale bar, 5000 µm. B, Ovaries from pregnant wild-type (left) and Nrip1-/- (right) females showing multiple large corpora lutea on d 9.5 pc (upper) and 13.5 pc (lower). The arrow indicates a retained oocyte within a corpora lutea of an Nrip-/- ovary. Scale bars, 500 µm.

 
There was also a clear trend for Nrip1-/- females to have a longer gestation period of 21.5 ± 0.5 d (mean ± SEM) compared with 19.9 ± 0.4 d for wild-type and 20.0 ± 0.4 d for heterozygous control females, respectively (P = 0.08 for wild-type/heterozygous vs. Nrip1-/-).

As progesterone is essential for the maintenance of pregnancy, we compared the circulating levels of the hormone in wild-type and Nrip1-/- females at different stages of pregnancy (Table 1Go). Progesterone levels increased during the early stages of pregnancy to similar levels in both genotypes, consistent with the appearance of morphologically normal corpora lutea (Fig. 1BGo). The rise in progesterone levels that normally occurs at midgestation was not as pronounced in Nrip1-/- females compared with wild-type mice. The corpora lutea present in Nrip1-/- mice on d 13.5 pc, when many embryos were resorbing, were still large and apparently healthy. A high proportion of the corpora lutea in Nrip1-/- mice contained retained oocytes (Fig. 1BGo), consistent with the failure of Nrip1-/- mice to ovulate (10).

Regulation of uterine Nrip1 expression demonstrated by ß-galactosidase activity
Northern blot and TaqMan real-time PCR analysis demonstrated that Nrip1 mRNA was expressed in the mouse uterus (data not shown), as reported for other species (rat, sheep, cow, and pig) (17, 18). As the Nrip1 gene has been replaced by the lacZ gene, we could investigate Nrip1 promoter activity in tissues by monitoring ß-galactosidase activity as an indication of Nrip1 expression. In nonpregnant heterozygotes (Nrip+/-) and null (Nrip1-/-) mice, ß-galactosidase activity was detected in the uterine glandular epithelial and stromal cells, but not in the luminal epithelium (Fig. 2Go). On d 6.5 pc, ß-galactosidase activity was apparent in the primary decidual cells that surround the embryo, indicating that Nrip1 may be involved in regulation of the decidual response. Expression was also apparent in subepithelial stromal and glandular epithelial cells in uterine tissue separating implantation sites. On d 9.5 pc, expression was lower than on d 6.5 pc and was detected primarily in differentiating decidual cells at the antimesometrial side of the implantation site. No ß-galactosidase activity was observed in implanted wild-type embryos, demonstrating the specificity of the assay. It is noteworthy that ß-galactosidase expression in tissues from Nrip1-/- mice with two copies of the lacZ gene is proportionally higher than expected compared with that in Nrip-/+ mice with one copy of the gene, suggesting that transcription from the Nrip1 promoter is subject to feedback repression by the corepressor.



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  		Figure 2. Sites of Nrip1 expression in uterine tissue revealed by ß-galactosidase activity. Nrip1 promoter activity was assessed by monitoring ß-galactosidase activity in sections from Nrip1-/- (KO) and heterozygous (HET) mice at different stages of pregnancy. Cryosections of uterine tissue show ß-galactosidase activity in glandular epithelial cells (GEC) and stromal cells (SC), but not luminal epithelial cells (LEC), in both nonpregnant and pregnant (d 6.5 pc) mice. Analysis of implantation sites on d 6.5 pc indicates expression in primary decidual cells (PDC) surrounding the embryo (E) and subsequently in antimesometrial (AM) decidual cells on d 9.5 pc. Note the complete lack of ß-galactosidase activity expressed by wild-type embryos in both d 6.5 pc and 9.5 pc implantation sites. Scale bars, 500 µm.

 
Expression of uterine marker genes
The maintenance of pregnancy is dependent on the actions of progesterone on the uterus mediated by PRs (19, 20). To investigate whether the many failing pregnancies in Nrip1-/- mice were due to inappropriate uterine PR expression, we studied PR expression by in situ hybridization. On d 6.5 pc, high levels of PR mRNA were found throughout the decidual cell layer in both wild-type and Nrip1-/- mice (Fig. 3Go). Subsequently on d 9.5 pc, PR expression in residual decidual cells surrounding the embryo was similar in wild-type and Nrip1-/- mice in addition to pronounced expression in the developing placentas (Fig. 3Go). Thus, inadequate PR expression is unlikely to underlie embryonic failure.



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  		Figure 3. Expression of uterine marker genes. Comparison of PR and TIMP-3 mRNA expression in uterine tissue of wild-type (left) and Nrip1-/- (right) mice determined by in situ hybridization. PR expression was localized throughout the decidual cell layer on d 6.5 pc and subsequently on d 9.5 pc in the remaining decidual cells surrounding the embryo, in the developing placenta (PL), and in the anti-mesometrial (AM) decidual cells in both wild-type and Nrip1-/- mice. Similar to ß-galactosidase activity (Nrip1 promoter activity), TIMP-3 was expressed in primary decidual cells (PDC) surrounding the embryo (E) on d 6.5 pc. Scale bars, 500 µm.

 
Matrix metalloproteinases and their inhibitors (TIMPs) are thought to play an important role in the substantial tissue remodeling in the uterus that accompanies and follows implantation (21). Impaired regulation of proteolytic activity caused by alterations in TIMP expression at the uterine/embryonic tissue border could potentially lead to early pregnancy loss. As TIMP-3 is highly expressed transiently in mouse uterine decidual cells on d 6.5–7.5 pc and in human endometrial stromal cells treated with progesterone (22, 23, 24, 25), we analyzed whether its expression was altered in Nrip1-/- mice. Expression of TIMP-3 was detectable in both wild-type and Nrip1-/- mice in the primary decidual cells that surround the embryo on d 6.5 pc (Fig. 3Go), suggesting that the signaling mechanism(s) regulating TIMP-3 expression was intact in Nrip1-/- mice and was unlikely to contribute to the observed pregnancy failures.

Ovarian transfer experiments to establish the primary site of Nrip1 action in female fertility
To determine whether the anovulation in Nrip1-/- mice (10) and the fetal losses described here depend primarily on the action of Nrip1 in the ovary itself, or involve other sites of action (e.g. the hypothalamic-pituitary-ovarian axis), we performed reciprocal ovarian transfer experiments. Ovaries from 4-wk-old immature Nrip1-/- mice were transferred into control (wild-type or heterozygous) littermates and vice versa. After a recovery period, all mice were mated with proven wild-type males for a 13-wk continuous breeding period, and their litter sizes were monitored (Table 2Go). Control mice receiving control ovaries produced a mean of 3.8 litters/mouse and 5.5 pups/litter, all but 1 of which survived to maturity. In contrast, control mice bearing Nrip-/- ovaries only produced 1.2 litters/mouse with 1 pup/litter, all of which were dead at birth or died within 72 h. Nrip-/- mice with control ovaries produced a mean of 2.8 litters/mouse and 4.0 pups/litter, most of which survived. As the fertility of Nrip-/- mice could be appreciably rescued by replacing their ovaries with those from control mice, we conclude that the only essential site of Nrip1 action in female fertility is in the ovary itself.


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  		Table 2. Ovarian transplantation, followed by 13 wk continuous breeding with wild-type males

 
Regulation of ovarian Nrip1 expression demonstrated by ß-galactosidase activity
As the ovary itself was found to be the primary site of Nrip1 action in female fertility, we monitored ß-galactosidase activity as a marker of Nrip1 expression in ovarian sections from different stages of pregnancy. ß-Galactosidase activity was detectable in thecal cells at all stages of pregnancy and in the granulosa cells of medium to large follicles, but it was not apparent in the early stages of follicle growth (Fig. 4Go). ß-Galactosidase activity was highly regulated in the corpora lutea during pregnancy, with negligible activity on d 6.5 pc that increased at midgestation (d 10.5 pc) and was maintained until at least d 13.5 pc; it declined to negligible levels on d 17.5 pc, correlating with initiation of the regression of corpora lutea. This pattern of ovarian Nrip1 expression was also supported by TaqMan real-time PCR analysis. As shown in Fig. 4BGo, there was an increase in Nrip1 mRNA levels on d 9.5 pc which then declined during the later stages of pregnancy, reaching a basal level after birth. Note that the induction of Nrip1 expression in the corpora lutea is underestimated as a consequence of the expression in thecal cells and granulosa cells of midsized follicles.



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  		Figure 4. Regulation of Nrip1 expression in ovarian tissue revealed by ß-galactosidase activity and TaqMan real-time PCR. A, Ovarian sections demonstrate high levels of ß-galactosidase activity in corpora lutea (CL) at midgestation (d 10.5, 11.5, and 13.5 pc), but low levels during early and late stages (d 6.5 pc and 17.5 pc, respectively). B, Nrip1 mRNA levels in ovarian samples in wild-type mice collected at the indicated stages (d pc) of pregnancy. np, Nonpregnant; P1, 1 d postpartum. Scale bars, 500 µm.

 
Expression of ovarian marker genes
To determine whether the morphological similarity between ovaries from pregnant Nrip1-/- and wild-type mice (Fig. 1BGo) was also accompanied by similar changes in a number of marker genes, we used in situ hybridization to analyze the expression of 17ßHSD/KSR7, LHR, and ERß. High levels of 17ßHSD/KSR7 mRNA, which is highly expressed in mouse and rat corpora lutea during pregnancy (14), was detected on d 9.5 and 13.5 pc, with no apparent difference between wild-type and Nrip1-/- ovaries (Fig. 5Go). LHR expression, which is important for triggering the production of the steroidogenic cytochrome side-chain cleavage enzyme P-450 and subsequent synthesis of progesterone after the LH surge (26) was also similar in Nrip1-/- and wild-type mice on d 9.5 pc, approximately the stage when embryo failure becomes evident. ERß was detectable in granulosa cells of small follicles, but not in the corpora lutea of either wild-type or Nrip1-/- mice, in contrast to human corpora lutea where it was shown to colocalize with steroidogenic cells (27).



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  		Figure 5. Expression of ovarian marker genes. Comparison of ovarian gene expression in wild-type (upper) and Nrip1-/- (lower) mice. Expressions of 17HSD/7KSR, LHR, and ERß were analyzed by in situ hybridization in ovaries collected on d 9.5 and 13.5 pc as indicated. Scale bars, 500 µm.

 
Finally, we examined the expression of PR and COX-2, because mice devoid of these genes exhibit an anovulation phenotype similar to that of Nrip-/- mice (28, 29). Both genes were induced in ovarian follicles with similar kinetics in response to superovulation, although the levels of COX-2 in Nrip1-/- were 30–40% of those in wild-type mice, as judged by quantitative TaqMan real-time PCR (Fig. 6Go).



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  		Figure 6. PR and COX-2 mRNA expression during the periovulatory period. Quantitative and qualitative analysis of PR (A) and COX-2 (B) mRNA expression in ovarian tissue from gonadotropin-treated wild-type and Nrip1-/- mice. mRNA was quantitated by TaqMan real-time PCR (left), and cellular localization was determined by in situ hybridization (right) in samples collected at the indicated time points. Mice were treated with PMSG for 48 h, followed by hCG treatment for the indicated time (3, 6, 10, and 24 h). Scale bars, 500 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous work established that the overt cause of infertility in female Nrip1-/- mice is the failure of follicles to release oocytes at ovulation (10). The present study demonstrates that, provided the block to ovulation is by-passed, Nrip1-/- mice are capable of establishing and maintaining pregnancies. Given the ability of Nrip1-/- mice bearing control ovaries to become pregnant through natural mating, we conclude that ovarian expression of Nrip1 is the primary determinant of oocyte release and that other sites of expression, such as the hypothalamic-pituitary axis, are not essential for ovulation. Moreover, as such mice produce viable offspring, Nrip1 expression does not appear to be essential in the uterus for implantation, uterine decidualization, placentation, or pregnancy maintenance or in the mammary gland for lactation.

Nrip1-null mice are also capable of becoming pregnant after embryo transfer, but their fertility and the survival of their pups (26%) are appreciably less than those in Nrip1-/- mice bearing control ovaries (73%). The high incidence of fetal loss in midpregnancy and the death of the majority of pups at or shortly after birth following embryo transfer suggest that the Nrip1-/- maternal environment is suboptimal for the maintenance of pregnancy and the subsequent survival of offspring.

The ability of control mice bearing Nrip1-/- ovaries to produce offspring, albeit a very small number, was surprising given the complete infertility of Nrip1-/- females, although occasional ovulations have been observed in intact mice (10). The basis for this apparent discrepancy is unclear, but it is conceivable that nonovarian cells may contribute to ovarian functions.

Although Nrip1 expression in the uterus is not essential for successful pregnancy, the observed pattern of ß-galactosidase activity, corresponding to Nrip1 gene promoter activity, would be consistent with Nrip1 playing a role in the remodeling of decidual tissue that occurs after implantation. Interestingly, studies of gene-deficient mice indicate that the PR and the PPAR{delta} are important for the decidual process in the mouse uterus (19, 30). Both PRs and PPAR{delta} are expressed in uterine decidual cells (30, 31), thereby overlapping with the distribution of Nrip1, so they may provide potential targets for repression by Nrip1 in the mouse uterus.

It is noteworthy that the anovulation phenotype of Nrip-/- mice resembles that of PR-null (29) and COX-2-null mice ( 28), both of which exhibit luteinized unruptured ovarian follicles. Although Nrip1 is expressed in thecal cells, we postulate that it is the expression in granulosa cells, which increases as follicles mature before the LH surge, that is more likely to be critical for subsequent ovulatory events. Given that both PR and COX-2 are expressed later than Nrip1, in response to LH surge, we have considered the possibility that anovulation in Nrip-/- mice might be caused by aberrant PR and/or COX-2 expression. Interestingly, preliminary expression analysis suggests that PR expression in Nrip-/- ovaries is normally induced by LH, but that the level of COX-2 mRNA is reduced; thus, we are now in the process of determining whether there is a link between Nrip1 and PG signaling pathways.

The restoration of fertility in Nrip1-/- animals with transplanted control ovaries and the survival of the majority of their pups suggest that lack of expression in the uterus is unlikely to be the primary cause of the suboptimal maternal environment, but, instead, it may be attributable to an inappropriate endocrine support by the Nrip1-/- ovary. It is known that fetal survival in midpregnancy is very sensitive to endocrine disruption. Experimental studies using slow release implants revealed that both the amounts of and the balance between progesterone and E2 are critical for the maintenance of pregnancy from about d 10 of pregnancy in mice (32, 33, 34, 35). In view of these observations, any disturbance in luteal or ovarian endocrine function caused by the Nrip1 deletion would be expected to be detrimental to embryo survival. It is noteworthy that progesterone levels at midpregnancy are not increased in Nrip1-/- mice to the same extent as in wild-type mice, but, on the other hand, the reduction may result from, rather than represent the cause of, the pregnancy failure. In any event, disturbances in ovarian endocrine function during pregnancy may provide an explanation for the slightly longer pregnancies in Nrip1-/- mice and the neonatal losses of pups from mothers with Nrip1-/- ovaries. It is interesting to note that Nrip1-/- expression, as reflected by increased mRNA levels and ß- galactosidase activity, is turned on in the corpora lutea toward midpregnancy, just at the time when fetal losses become apparent. Thus, we suggest that Nrip1 may have two functions in the ovary, an essential role in ovulation and a secondary role in the corpora lutea involved in the maintenance of pregnancy.


   Acknowledgments
 
We thank C. Young, R. Peak, P. Hagger, T. Crafton, J. MacDonald, J. Bee, G. Hutchinson, and the staff of Biological Resources; Sara Chalk for excellent assistance with ovarian transplantations; J. Longcroft and T. Hunt for in situ hybridization studies; and George Elia and the staff of the histopathology unit. We also thank P. Vihko for the 17HSD/7KSR plasmid, and T. Ny for LHR and TIMP-3 plasmids. Finally, we are grateful to J. White as well as members of the Molecular Endocrinology Laboratory for advice and comments on the manuscript.


   Footnotes
 
This work was supported by a Marie Curie Fellowship provided by the European Commission (to G.L.). The work was also supported in part by the Imperial Cancer Research Fund and partly by the Wellcome Trust, Grant No. 061930.

Abbreviations: COX-2, Cycloxygenase-2; 17ßHSD, 17ß-hydroxysteroid dehydrogenase; 7KHR, 17-ketosteroid reductase type 7; LHR, LH receptor; Nrip, nuclear receptor interacting protein; pc, postcoitum; PRTM, progesterone receptor TM; RIP, receptor interacting protein; TIMP-3, tissue inhibitor of metalloproteinase 3.

Received July 25, 2001.

Accepted for publication October 25, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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