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Ovary |
Monash Institute of Reproduction and Development,3 Monash University, Clayton 3168, Victoria, Australia
Department of Obstetrics and Gynaecology,4 University of Adelaide, Adelaide 5005, South Australia, Australia
Prince Henry's Institute of Medical Research,5 Clayton 3168, Victoria, Australia
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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female reproductive tract, ovary, ovulation, pregnancy, uterus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adamts-1 expression has been detected in a wide range of mammalian tissues, so the gene may be important for the development and function of a number of biological systems. Expression has been documented in the developing mouse urogenital system, with Adamts-1 transcripts detected in the kidney and the developing ovary and uterus [6]. In adult rodents, Adamts-1 has been shown to be expressed in the kidney [6] and the granulosa cell layer of mature follicles in the ovary [7]. Although normally barely detectable in adult ovaries, Adamts-1 expression was induced in rats 4 h after the administration of human chorionic gonadotropin (hCG, an LH analogue), and peaked after 12 h, the time when the follicles start to rupture. This expression of Adamts-1 was shown to be dependent on normal synthesis of progesterone in the ovary at the time of ovulation because Adamts-1 was barely detectable at the same time after treatment in mice lacking the progesterone receptor (PR)[8]. Mice deficient for PR fail to ovulate because their follicle walls do not rupture and are therefore infertile. Furthermore, PR is expressed in the granulosa cells of preovulatory follicles destined to ovulate, as is Adamts-1, implying a role for Adamts-1 in the ovulatory process downstream of PR [8, 9]. Also, uteri from mice deficient in PR fail to decidualize [10], and thus Adamts-1 may act downstream of PR in this uterine process and be involved in embryo implantation.
To investigate the biological role of Adamts-1, we generated an Adamts-1 null mouse. We show that Adamts-1 null mice have a kidney defect and that null females are subfertile (as reported earlier by Shindo et al. [see Ref. 21]). Furthermore, we clearly demonstrate that, as predicted from the study of PR-deficient mice, the reason for the subfertility of Adamts-1 null females is an impaired ability to ovulate. We show that the kidney defect is apparent only at birth and may explain the perinatal lethality that we find associated with the absence of the Adamts-1 gene.
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MATERIAL AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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Oligonucleotides used in this study are shown in Table 1.
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The Adamts-1 gene spans a total of 9.2 kb and consists of nine coding exons, with exons 2, 3, and 4 encoding the metalloproteinase domain [11, 12]. To disrupt the Adamts-1 gene, exon 2 was deleted. Three arms of homology were generated by polymerase chain reaction (PCR) from 129/Sv mouse genomic DNA, corresponding to an intron 1 fragment (using oligonucleotides Adam-ko5'F and Adam-ko5'R), an intron 1 to intron 2 fragment (using oligonucleotides Adam-komidF(HindIII) and Adam-komidR(EagI)), and an intron 2 to intron 7 fragment (using oligonucleotides Adam-ko3'Fb and Adam-ko3'R). Specific restriction endonuclease sites were added to the oligonucleotides to facilitate cloning of the arms. All PCR products were cloned and sequenced. The positive selection marker neo, (driven by the pMC1 promoter) flanked by two loxP sites, was inserted in intron 1, between the first and second arms, and a third loxP site was inserted in intron 2, between the second and third arms. Exon 2 was therefore flanked by loxP sites so that in the presence of Cre recombinase, the modified Adamts-1 allele, with exon 2 deleted, should represent a functionally null allele. The negative selection marker tk (driven by the hsv promoter) was cloned at the end of the 3' arm of homology. The Adamts-1 targeting vector is illustrated in Figure 1A. The DNA was linearized with SalI before being electroporated into 129/sv ES cells. After positive and negative selection, four clones that had undergone homologous recombination were identified by PCR and Southern blot screening (Fig. 1B). Positive ES cell clones were transfected by electroporation with a plasmid encoding Cre recombinase, together with a puromycin resistance-encoding plasmid. After selection with puromycin, surviving ES cell clones having undergone a complete recombination between the two most external loxP sites were identified by PCR and Southern blot screening (Fig. 1C). In these clones, the Neo cassette has been removed, together with exon 2 and parts of the flanking introns. Deleting exon 2 not only removes half the metalloproteinase domain but also, if normal splicing events still occur, results in a frame-shift that will lead to the production of a prematurely truncated protein, harboring a pre-/pro-domain only. Recombined ES cells were microinjected into 129/sv blastocysts, and the chimeric founder males obtained were bred to C57BL/6 and 129/sv mice.
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Genotyping of the progeny confirmed germline transmission of the exon 2 deletion. Genotyping of mice was performed by PCR on ear clip DNA as follows: genomic DNA was released from mouse ear clips by a 60-min incubation at 55°C in ear clip buffer (10 mM Tris-Cl [pH 8.3], 50 mM NaCl, 0.2% [w/v] Tween 20, 10 µg/ml proteinase K), and the reaction was stopped by incubation at 95°C for 10 min; the DNA was then used as template for a multiplex PCR using oligonucleotides Adam-in1F1, Adam-ex2F, and Adam-ex3R (Fig. 1D). Disruption of the Adamts-1 gene was then confirmed by Northern blot analysis and immunohistochemistry (Fig. 1, E and F).
All experiments were conducted on mice of mixed genetic background (C57BL/6 x 129/Sv), except for the in vitro oocyte to blastocyst development experiment, which was carried out using females on the pure 129/Sv genetic background. Females on the pure 129/Sv genetic background and the mixed C57BL/6 x 129/Sv genetic background present the same reduction in fertility (data not shown). All procedures were conducted following approval by the Monash Medical Centre Animal Ethics Committee.
Estrous Cycle Testing
Vaginal smears were collected using a cotton tip moistened in PBS and gently rolled into the vagina of the mice. The cotton tip was then rolled onto a slide to deposit the cells, which were stained using Diff-Quik (Lab Aids, Sydney, NSW, Australia). The stage of the estrous cycle was determined by the presence and relative abundance of leukocytes and epithelial cells in the vaginal smears [13].
Induction of Ovulation
Each female was injected i.p. with 4 IU eCG (Intervet, Castle Hill, NSW, Australia), followed 48 h later by 5 IU hCG (Intervet). Mice were killed either 48 h after eCG administration or between 12 and 48 h after hCG administration, and either the ovaries were excised for examination or the oocytes were collected from the oviducts.
Collection and Culture of Fertilized Oocytes
Adamts-1 null and wild-type females (129/Sv pure genetic background) were paired with wild-type and null males, respectively (natural ovulation), to generate heterozygous embryos. This was done so that the genotypes of the embryos would be identical and hence not influence the results of the experiment because we were testing the capacity of the null females to produce normal embryos. Upon detection of a vaginal plug the next morning, females were killed and oocytes harvested and incubated in M2 medium [14] with 300 U/ml hyaluronidase to detach the oocytes from the surrounding granulosa cells. Fertilization of the oocytes was assessed immediately by expulsion of the polar body. Fertilized oocytes were cultured in KSOM medium [15] in a 37°C, 5% CO2 incubator and left for 7 days to develop to blastocysts.
Uterine Implantation
Adamts-1 null and wild-type females were mated with wild-type and null males, respectively, such that all resultant embryos were of the same heterozygous genotype. Females were killed and their uteri collected at Day 4.5 or 6.5 after mating (day of plug = Day 0.5) and fixed in formalin. Implantation site morphology was examined after hematoxylin and eosin (H&E) staining.
Uterine Decidualization
Wild-type and Adamts-1 null females were ovariectomized. They then received s.c. injections of estradiol 17ß on Days 6, 7, and 8 after ovariectomy (100 ng) and on Days 12, 13, and 14 (7.5 ng) and progesterone (P) on Day 12 (0.5 mg). P was subsequently administered from a P implant inserted on Day 12 [16, 17]. To induce decidualization, sesame oil (0.02 ml) was injected into the right uterine horn on Day 14. Uteri were collected on Day 16 and each uterine horn was weighed separately.
Histology, Immunohistochemistry and In Situ Hybridization
Ovaries and uteri were fixed in formalin or 4% paraformaldehyde overnight, embedded in paraffin, sectioned, and stained with H&E or periodic acid Schiff following standard protocols. In situ hybridization for cholesterol side-chain cleavage cytochrome P450 (P450scc) in ovaries was performed as previously described [18]. Antisense and sense riboprobes were labeled with [35S]deoxyuridine 5-triphosphate by in vitro transcription. Sections pretreated with proteinase K and 0.1 M triethanolamine/acetic anhydride were incubated with labeled probes overnight at 55°C in the presence of 50% formamide. Sections were then treated with RNase A and washed to a final stringency of 0.1x saline sodium citrate before being coated in liquid photographic NTB-2 emulsion (Kodak, Rochester, NY). Following a 2-day exposure, slides were developed in Kodak D-19 developer and counterstained with hematoxylin. Immunostaining for Adamts-1 was performed on a number of wild-type and null animals using rabbit polyclonal antibodies as previously described [19].
Statistical Analysis
Data were analyzed using unpaired two-tailed t-tests, using GraphPad Prism version 2.0 (GraphPad Software Inc., San Diego, CA). Data are expressed as mean ± SEM. A value of P < 0.05 was considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adamts-1 null mice were screened for nonfunctionality of the Adamts-1 gene. By Northern blot analysis, a 4.6-kb band corresponding to the Adamts-1 wild-type transcript was present in wild-type and heterozygous mice. A very weak band of similar size (deletion of exon 2 results in a transcript shorter by only 347 bp) was detected in Adamts-1 null mice, indicating that nonsense-mediated mRNA decay [20] was most likely occurring (Fig. 1E). The absence of Adamts-1 protein in the Adamts-1 null mice was confirmed by immunohistochemistry using an Adamts-1 specific antibody (Fig. 1F).
An early lethality appeared to affect our Adamts-1 null mice. Progeny of heterozygous crosses were genotyped at various ages, and although the expected Mendelian ratio of Adamts-1 null mice was present on the day preceding birth, only 55% remained on the day after birth. Of those null pups surviving after Day 1, most survived to adulthood with 40% surviving at the time of weaning (Table 2). The only gross abnormality apparent upon examination of null pups on the days before and after birth was a structural defect of the kidney manifest as a marked reduction in the width of both the cortex and medulla with an enlarged caliceal space. Although kidneys of Embryonic Day (E)18.5 null embryos were not anatomically distinguishable from kidneys of wild-type embryos, on the day following birth the Adamts-1 null pups presented a very obvious shrinkage of both layers of the kidney, resulting in the appearance of an enlarged caliceal space (Fig. 2). The null pups did not appear to have difficulties in feeding or breathing (data not shown), and because kidney function begins perinatally, we propose that the cause of premature death of a large proportion of the Adamts-1 null pups was due to inadequate kidney function.
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Fertility Is Impaired in Adamts-1 Null Mice
The fertility of Adamts-1 null male mice was not affected, but the fertility of null females was reduced. Females were mated naturally with fertile males and subsequently checked for the presence of vaginal plugs. Of 13 Adamts-1 null females presenting a vaginal plug, only five (38%) gave birth to pups, whereas 10 of 13 (77%) wild-type females presenting a vaginal plug gave birth. Also, the average number of pups born to females that became pregnant was significantly fewer for null females (3 ± 0.3, n = 5), compared with wild-type females (5.9 ± 0.7, n = 10; t-test, P < 0.05).
To investigate the reasons for the reduction in female fertility observed, we examined the processes involved. We first examined the status of the estrous cycle in null females and then determined their capacity to ovulate and produce functional oocytes and finally examined their potential to sustain uterine decidualization and embryo implantation.
The Estrous Cycle Is Normal in Adamts-1 Null Mice
The status of the estrous cycle was investigated by examination of vaginal smears collected daily from adult wild-type and Adamts-1 null females over 14 days. The succession of the four stages of the estrous cycle: estrus, postestrus, diestrus, and proestrus, was observed in the smears taken from Adamts-1 null females, and their duration was not different from wild-type animals (data not shown), indicating that Adamts-1 null females have a normal estrous cycle.
Ovulation Is Impaired in Adamts-1 Null Mice
To examine the capacity of Adamts-1 null females to ovulate, Adamts-1 wild-type and null females aged 21–23 days were superovulated and oocytes and ovaries examined. Forty-eight hours after eCG administration, ovaries from both Adamts-1 null and wild-type females contained normal preovulatory follicles (Fig. 3A). The ovaries were then examined 12 h after hCG administration when expression of Adamts-1 is at its highest in the granulosa cell layer of mature follicles in wild-type ovaries [7]. Follicles with some expansion of cumulus oocyte complexes (COCs), were observed in null ovaries, whereas in wild-type ovaries, most follicles had released the oocyte (Fig. 3B). Histological assessment of cumulus matrix expansion in follicles about to ovulate indicated that some matrix formation does occur in the COCs of null ovaries. However, the degree of expansion consistently appeared less extensive in the null mice, compared with wild-type littermates. Interestingly, 16 h after hCG administration, whereas no oocytes remained in the follicles of wild-type ovaries, oocytes could still be found in large follicles of Adamts-1 null ovaries (Fig. 3C). Consistent with this, a significantly decreased number of oocytes were found in the oviducts of null females, compared with wild-type females (41.2 ± 9.2 from wild-type females, n = 6 vs. 12.2 ± 1.9 from null females, n = 5; t-test, P < 0.05; Fig. 4) when oocytes were collected 16 to 24 h after hCG administration.
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When examining postovulatory follicles at 24 and 48 h after hCG administration, corpora lutea (CL), the structures formed by the follicles after ovulation, were observed in both wild-type and Adamts-1 null ovaries. Significantly, trapped oocytes were frequently observed in the CL of null females, whereas none were ever observed in the normal CL of wild-type females (Fig. 5A). Interestingly, expression of the CL functional marker, P450ssc, was normal in null ovaries, demonstrating the normal differentiation of these unovulated structures (Fig. 5B). This is consistent with observations in PR null ovaries.
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To test the maturation potential and development capability of oocytes produced by null females, their capacity to be fertilized and develop into embryos was assessed. Wild-type and null females were mated naturally to null and wild-type males, respectively, so that all resulting embryos would be of the same heterozygous genotype. Oocytes were collected from the oviducts and allowed to develop in vitro. Fertilized oocytes from both wild-type and null females developed up to the blastocyst stage, indicating that no obvious early developmental defect is found in embryos produced by null females (Fig. 6).
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Uterine Function Is Maintained in Adamts-1 Null Mice
It was also plausible that compromised endometrial function contributed to the decreased female fertility observed in our mice. No gross morphological abnormalities were detected in the uteri of the Adamts-1 null mice generated in this study. Nevertheless, we examined the presence and number of implantation sites as an additional measure of the uterine function. Implantation sites were present in the uteri of wild-type and null females at 4.5 and 6.5 days post coitum (dpc), with no apparent morphological abnormality detected in the implantation sites of Adamts-1 null females (Fig. 7A). Consistent with the reduced number of oocytes released by Adamts-1 null females (70% reduction), the number of implantation sites in null females was significantly reduced, compared with wild-type females at both 4.5 dpc (43% reduction: 9.8 ± 0.8 for wild-type females, n = 4 vs. 5.6 ± 1.1 for null females, n = 5; t-test; P < 0.05) and 6.5 dpc (50% reduction: 7.5 ± 0.7 for wild-type females, n = 4 vs. 3.8 ± 1.1 for null females, n = 4; t-test; P < 0.05; Fig. 7B; only mice presenting implantation sites were included in these results). Also, established pregnancies in plugged females, as detected by the presence of implantation sites, were observed in a smaller proportion of the null females, compared with the wild-type females, and the pregnancy rate was found to decrease during gestation at greater rate in null females (83% at 4.5 dpc to 50% at 15.5 dpc), compared with wild-type females (100% at 4.5 dpc to 83% at 15.5 dpc; Fig. 7C), suggesting a loss of embryos as the pregnancy progressed.
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The capacity of the uterus to support implantation was also examined by artificially inducing decidualization. Decidualization normally occurs in response to an implanting blastocyst, but the process can be induced in the entire uterus by injection of oil into the lumen. Oil was injected into one uterine horn, whereas the other horn served as a control. No significant difference was observed for the weights of the non-stimulated uterine horns between Adamts-1 null females (weight range 14–21 mg) and wild-type females (weight range 16–24 mg; Fig. 8A). Examination of the weights of the stimulated horns revealed a range of weights for both null and wild-type females (Fig. 8B), and although the mean was slightly lower in null females (weight range 26–84 mg, mean = 43 ± 7), compared with wild-type females (weight range 32–123 mg, mean = 65 ± 13), the difference was not statistically significant (t-test; P > 0.05). A similar result was observed when comparing the weights of stimulated versus non-stimulated horns. In this case, although more than half the wild-type females presented an increase in uterine horn weight of at least 3-fold after induction of decidualization, only a quarter of null females presented such a response (Fig. 8C). These results suggest that a lack of Adamts-1 might be contributing to a reduced decidualization response but indicate that the gene is not absolutely required for decidualization to occur. No obvious gross morphological or histological changes in either the decidualized uterine horn or control horn were observed between wild-type and Adamts-1 null mice (Fig. 9).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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We show here that the basis for reduced fertility in Adamts-1 null females is primarily due to impaired ovulation. We observed, similar to Shindo et al. [21], that only a proportion of null females became pregnant after mating and that the number of embryos/pups they carried was reduced, compared with wild-type mice. We conducted a detailed analysis of the stages of the female reproductive process to explain the decrease in fertility. Although the estrous cycle occurred normally in Adamts-1 null females, the release of oocytes from follicles was reduced. Indeed, several oocytes were found to be trapped in the CL after ovulation. The fertilization potential of those oocytes successfully released by Adamts-1 null females was not proven, although our qualitative results showed that oocytes that were released by Adamts-1 null females were able to be fertilized and were capable of developing to the blastocyst stage in vitro. The capacity of Adamts-1 null females to support implantation was tested by examination of embryo implantation after natural mating and by artificially inducing decidualization. In both cases, Adamts-1 null females were capable of a decidualization response.
Our results indicate that Adamts-1 plays an important nonredundant role in the process of ovulation. For a follicle to mature and eventually release its oocyte at ovulation, a sequence of events must occur within a specific time frame, following hormonal signaling. At the preovulatory stage, the granulosa cells are composed of two distinct populations: the cumulus cells, which surround the oocyte, and the mural cells, which line the wall of the follicle. Recently Adamts-1, secreted by the mural granulosa cells, has been shown to localize to the matrix of expanding COCs and cleave versican during this process [19]. Our morphological and histological analysis of follicles of Adamts-1 null females determined that some expansion of the COC matrix persists. In the mural granulosa cells, the LH surge triggers expression of a number of genes, an early one being PR, a member of the nuclear receptor superfamily of transcription factors. Adamts-1 expression follows this initial event, peaking later on (Fig. 10A) [22], and this induction is dependent on progestin/PR signaling [7, 8]. Interestingly, PR null females show complete failure to ovulate because of a failure of the follicle to rupture. Disruption of the Adamts-1 gene results in a similar, although not as drastic a phenotype. Although some oocytes are released, a significant proportion of them remain trapped in the follicles in Adamts-1 null mice. This observation is consistent with the notion that Adamts-1 mediates some, but not all, of the biological functions of PR (Fig. 10, B–D). It remains to be determined whether PR acts directly on the Adamts-1 promoter because computer-based searches failed to identify PR sites in the putative Adamts-1 promoter region [8], or whether it regulates Adamts-1 indirectly as reported for other genes [23]. Compensation to a certain level by other Adamts proteases, such as the closely related Adamts-4, could also explain why some ovulation still occurs in null females.
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The question remains as to what molecular role Adamts-1 plays in ovulation. Based on what is known about the ovulatory process and the potential functions of the Adamts-1 protein, Adamts-1 could be acting as a key player in two main events (schematically represented in Fig. 10, B–D). The first is that Adamts-1 may cleave, and hence activate, other proteinases, in particular matrix metalloproteinases (MMPs) such as MMP13 and MMP14 [22]. The ECM of the thecal cell layer must be digested at the time of ovulation, to allow the COC to be released from the ovary, and MMPs could play a role in the proteolytic cleavages required for follicular rupture to occur (although so far no ovulation deficiency has been reported in mice deficient for MMPs [24–27]). The second is that Adamts-1 may be acting to cleave a range of proteoglycans. For instance, Adamts-1 has been shown to cleave aggrecan, brevican, or versican [28–31]. Versican is present in the COC matrix [32] and is a substrate for Adamts-1 during COC expansion and ovulation [19]. Numerous recent studies indicate a link between expansion of the COC matrix and ovulatory success [33–36]. Our observations suggest that a more detailed assessment of COC expansion in the Adamts-1 null mice is warranted. Other proteoglycans, such as perlecan, present in the thecal layer, or syndecan or glypican, present on the cell surfaces of granulosa, cumulus, or thecal cells [37], may also be substrates of Adamts-1 processing. Proteoglycans have been shown to inhibit the binding of gonadotrophin to their receptors on granulosa cells, so by cleaving proteoglycans, Adamts-1 could facilitate the hormonal response of granulosa cells [22]. Also, the activity of certain bioactive factors is known to be blocked or altered by the presence of proteoglycans, so it is possible that Adamts-1 (by cleaving these proteoglycans) could modulate the release or efficacy of these factors.
Although we may have expected Adamts-1 to be important for uterine function because it is expressed in both pregnant (GenBank EST 3594533) and nonpregnant uterus [38], and the appearance of cysts and uterine thickening was reported by Shindo et al. [21] in their Adamts-1 null mice, we observed no gross morphological changes in the uteri of the Adamts-1 null mice generated in this study. Again, this might be due to a difference in targeting strategy. A lack of Adamts-1 in either the embryo or the mother did not seem to adversely affect the implantation process. Indeed, Adamts-1 null embryos were found in the expected Mendelian ratio up to E18.5 and embryos had implanted in both Adamts-1 wild-type and null uteri, albeit in reduced numbers in the null. Our data nevertheless suggest that null mothers may be less capable of maintaining pregnancy than wild-type mothers because the rate of pregnancy in null females decreased as gestation progressed at a greater rate than that of wild-type females (Fig. 7C). A contribution of Adamts-1 may hence be required to ensure normal progression of the pregnancy and adequate placentation in later stages of gestation. The lack of Adamts-1 had no measurable effect on the decidualization process either be it when inducing decidualization naturally by embryonic implantation or artificially by injection of oil into the uterine lumen. Although Adamts-1 might not be required for decidualization to occur, it is also possible that other molecules may compensate for its absence. Indeed, studies on individual MMPs such as MMP-3 and MMP-7 have shown that compensatory overexpression of other MMPs occurs when these are knocked out in mice [24].
In summary, a lack of Adamts-1 results in a perinatal kidney defect and a decrease in female fertility. It does not alter the female estrous cycle, nor does it markedly affect the implantation process. Subfertility of Adamts-1 null females resides in the impaired ovulation process, leading to a reduced number of oocytes being released. This is the first identification of a critical nonredundant protease required in the process of ovulation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: M.A. Pritchard, Monash Institute of Reproduction and Development, Level 2, 27-31 Wright St., Clayton 3168, Victoria, Australia. FAX: 61 3 9594 7211; melanie.pritchard{at}med.monash.edu.au
Received: 20 October 2003.
First decision: 7 November 2003.
Accepted: 25 November 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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