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Department of Human Genetics, Mount Sinai School of Medicine, New York, New York, USA
1Correspondence: Department of Human Genetics, Mt. Sinai School of Medicine, 1425 Madison Avenue, New York, NY 10029, USA. E-mail: edward.schuchman{at}mssm.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: apoptosis • oocytes • ceramide
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3
4)
. Growing interest in the biology of this and other ceramidases stems from the fact that these enzymes play a central role in ceramide metabolism. Ceramide is a signaling lipid that is produced in response to various stimuli (5
, 6)
. Normally present in low amounts, in response to these factors ceramide is rapidly produced at the cell surface, leading to membrane reorganization and downstream signaling that results in apoptosis. After stimulation, AC and/or other ceramidases may then hydrolyze ceramide into the individual fatty acid and sphingosine components (7
8
9)
. Because ceramide degradation is the only source of intracellular sphingosine (10)
, these enzymes may also be rate-limiting steps in determining the intracellular levels of this compound. Importantly, a derivative of sphingosine, sphingosine-1-phosphate (S1P), can counteract the apoptotic effects of ceramide (11)
, leading to the suggestion that ceramidases can be "rheostats" that maintain a proper balance between cell growth and death (12)
.
Ovulated eggs undergo molecular changes characteristic of apoptosis unless successful fertilization occurs (13
, 14)
. While multiple factors, including ceramide, have been characterized as proapoptotic elements involved in this process (15
16
17
18)
, little is known about factors that sustain egg or embryo survival. Herein we provide evidence demonstrating that AC is one such factor required for early embryo survival. Several years ago our laboratory used gene targeting to inactivate the AC gene (Asah1) in mice (19)
. Initial characterization of these animals revealed that heterozygous mice (Asah1±) had a progressive lipid storage disease phenotype, and that a complete loss of AC activity led to the absence of mutant individuals. It remained unclear, however, whether the Asah1–/– embryos were formed, or, alternatively, if they were formed, whether they died during early embryogenesis.
We have now used a combination of molecular, biochemical, and morphological methods to follow the development of individual embryos obtained from Asah1 ± intercrosses. From these analyses we found that Asah1–/– embryos could be formed, but underwent apoptotic death at the 2-cell stage. Importantly, these embryos could be rescued by adding S1P to the culture media, permitting their survival to at least the 4–8 cell stage. We also demonstrate that Asah1 is one of the earliest genes expressed in newly formed embryos. Lastly, we show that AC is a predominant protein in unfertilized eggs and that expression of this protein and gene is decreased during egg aging unless fertilization occurs. Overall, these results demonstrate that C is an essential component of newly formed embryos and is required for their survival beyond the 2-cell stage.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. For 2-cell embryo collection, superovulated females were caged with males of proven fertility and sacrificed 46 h after injection of hCG. Embryos were isolated from the oviducal ampullae and cultured at 37°C in a humidified atmosphere of 5% CO2 and 95% air.
Single-cell long nested (SCLN) PCR genotyping
DNA from individual embryos was subjected to PCR amplification using a mixture of two sets of long and short (nested) primers. Long1st and short2nd PCR amplification of the wild-type Asah1 allele was performed using forward and reverse primers (5'-ACCCAGGTTCCATCGTTGCACATTTCATC-3', 5'-ATGCCACATGGGAATACTGTCCAAA-GCAGAA-3' and 5'-CACACAAACACATGTATGTG-CACACGTGAA-3', 5'-GCTGCCCTGGAACTCACTCACTCT-3') to produce 
9-kb and 180-bp DNA fragments, respectively. Amplification of the mutated Asah1 allele using forward and reverse primers (5'-ATGCCACATGGGAATACTGTCCAAAGCAGAA-3', 5'-GAGGAGTAGAAGGTGGCGCGAA-GGGG-3' and 5'-GCTGCCCTGGAACTCACTCACTCT-3', 5'-GGTGGATGTGGAATGTGTG-CGA-3') produced 7-kb and 255-bp DNA fragments, respectively.
S1P treatment
Two-cell embryos from heterozygous mating pairs were isolated from the oviducal ampullae (as described above) and cultured in M2 media for 36 h in the presence of 2 µM S1P (Biomol International, Plymouth Meeting, PA, USA) at 37°C in a humidified atmosphere of 5% CO2 and 95% air. SCLN PCR was then performed as described above.
Western blot analysis
Eggs and embryos were subjected to lysis in buffer containing 50 mM Tris-HCL, 150 mM NaCl, 2 mM EDTA, 1% Nonidet P-40, 1 mM Vanadate, 5 mM Naf, and 10 µg/ml aprotinine, pH 7.4. Proteins were separated by SDS-PAGE using 10% or 12% precast Nupage Bis/Tris gels under reducing conditions and MES running buffer (Invitrogen, Carlsbad, CA, USA) and transferred onto a nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ, USA) using a semidry transfer apparatus (Bio-Rad, Hercules, CA, USA) and Nupage-MOPS transfer buffer. For immunoblot analysis, blots were blocked with TBS/Tween containing 5% dry milk and then were incubated with Goat IgG against AC (specific for the ß-subunit). Bound antibodies were recognized by secondary antibodies conjugated to horseradish peroxidase. Detection was performed by an enhanced chemiluminescence (ECL) detection reagent (Amersham Biosciences). Approximate molecular masses were determined by comparison with the migration of prestained protein standards (Bio-Rad).
AC activity assay
Eggs were subjected to lysis in 0.25% sucrose solution. Total cell extracts were incubated for 22 h at 37°C with 0.1 ng/µl BODIPY conjugated C12-ceramide in 0.1 M citrate/phosphate buffer, pH 4.5, 150 mM NaCl, 0.05% BSA, and 0.1% Igepal CA-630. After the reactions were complete, 5 µl of the assay mixtures was removed and added into 95 µl of ethanol, mixed, and then centrifuged for 5 min for 10,000 g. The supernatants were then transferred to a Waters glass sampling vial, and 5 µl (2.5% of the original reaction mixture) was autosampled by a WIPS 712 (Waters) autosampler onto a high-performance liquid chromatograph equipped with a reverse-phase column (BetaBasic-18, 4.6x30 mm, Keystone Scientific Inc., Bellefonte, PA, USA) and eluted isocratically with methanol/water (95:5 v/v) at a flow rate of 1 ml/min. Fluorescence was quantified using a Waters 474 fluorescence detector set to excitation and emission wavelengths of 505 and 540 nm, respectively. The undigested substrate and product (i.e., BODIPY-conjugated C12-ceramide and fatty acid, respectively) peaks were identified by comparing their retention times with standards, and the amount of product was calculated using a regression equation that was established from a standard curve using BODIPY-conjugated C12 fatty acid.
Immunohistochemistry
Eggs were isolated and fixed in 3% paraformaldehyde. Zonae pellucidae (ZP) were removed postfixation by Pronase (Sigma), and the ZP free eggs were permeabilized by Nonidet P-40. The eggs were then incubated with different primary and secondary antibodies (20)
. The entire fluorescent reagent were visualized and photographed with a Ziess confocal laser-scanning microscope (CLSM). For apoptosis detection, live 2-cell embryos were labeled using an Annexin V Apoptosis Detection Kit (Santa Cruz Biotechnology, Santa Cruz, CA, USA).
mRNA quantification by PCR
Total mRNA was extracted from equal numbers of eggs and embryos and was reverse-transcribed according to the manufacturer’s instruction (Invitrogen). Mac990 (5'-TTACCGCAGAACACCGG-CC-3'), mac1137r (5'-TTGACCTTTGGTAACATCCATC-3') were used for murine AC PCR amplification with QuantiTect SYBR Green PCR kit (Qiagen, Valencia, CA, USA). Changes in AC mRNA levels in old eggs and 2-cell embryos were assessed relatively to the level of AC mRNA in young eggs using the formulas 2^(Ctyoung-Ctold) and 2^(Ctyoung-Ct2-cell embryo), respectively. Housekeeping proteins, actin beta (Actb), glyceraldehyde-3-phosphate dehydrogenase (G3), and ribosomal proteins S11 (RPS 11) were used as internal controls for embryonic mRNA expression.
Data presentation and statistical analysis
All experiments were independently replicated at least three times with different mice. The combined data from the replicate experiments were subjected to a t-test analysis, and results were considered statistically significant at P < 0.005. Graphs represent the mean ± SEM of combined data from the replicate experiments. Representative photomicrographs are presented for the egg morphology, Annexin V labeling and immunohistochemistry.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). This technique allowed us to genotype individual embryos immediately after fertilization. Two- to 8-cell stage embryos were collected from Asah1± intercrosses 36–60h after hCG injection. Genotyping of 196 embryos from these intercrosses revealed that Asah1–/– embryos could be formed (Fig. 1A
). However, no Asah1–/– embryos were identified beyond the 2-cell stage (Fig. 1B
), suggesting that the lack of AC activity led to embryo death during the 2- to 4-cell transition.
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ACKO–/– embryos undergo apoptotic death during the 2-cell stage
Ceramide-mediated signaling often leads to apoptosis (6)
. Therefore, one consequence of inactivating the Asah1 gene might be the increase of ceramide in embryos, leading to cell-cycle arrest or apoptosis (5
, 6)
. Recent publications have shown that ceramide levels in eggs are increased during in vivo and in vitro aging (15)
. These data support our hypothesis that AC plays an important role in egg/embryo survival by removal of ceramide.
To further investigate the involvement of AC during development and to characterize the mechanism leading to death of Asah1–/– embryos, the possibility of apoptotic death was assessed by Annexin V staining (21)
. To perform this analysis, 86 live, 2-cell embryos from Asah1 ± intercrosses were collected and designated numbers. Each embryo was examined independently for apoptotic morphology and Annexin V binding using laser-scanning confocal microscopy, and then genotyped by SCLN PCR. The outcome of these analyses revealed that all of Asah1–/– embryos had apoptotic morphology (Fig. 2
D) and positive Annexin V staining (Fig. 2E
), while wild-type embryos had normal morphology (Fig. 2A
) and no Annexin V staining, except of the apoptotic polar body (Fig. 2B
). The percentages of apoptotic wild-type and heterozygous embryos were negligible, compared to Asah1–/– embryos (11% and 5% vs. 100%, respectively, t test, P < 0.00001, Fig. 2G
).
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Thus, these findings revealed that the absence of functional AC causes apoptotic death during the 2-cell stage and provide direct in vivo evidence that AC activity is essential for the 2- to 4-cell transition. This developmental period marks the beginning of embryonic genome activation (EGA).
S1P treatment of embryos from Asah1 ± intercrosses
To confirm that the death of Asah1–/– embryos at the 2-cell stage was due to ceramide-induced apoptosis, we examined the effect of S1P, a ceramide antagonist and downstream product of AC activity, on the survival of these embryos. For this purpose we collected early 2-cell embryos from Asah1 ± mating pairs and followed their development in the presence of 2 µM S1P during 36 h of culture. In the presence of S1P we observed that 14 out of 15 embryos reached the 4- to 8-cell stage after 36 h. Genotyping of these embryos showed that 70% (10/14) were Asah1 ±, 2 were Asah1+/+, and 2 were Asah1–/–. As noted above, in the absence of S1P no Asah1–/– embryos were ever found among 53 4- to 8-cell embryos studied (Fig. 1B
). Thus, these results revealed that S1P treatment could rescue Asah1–/– embryos from ceramide-induced apoptosis, and permitted their survival to the 4- to 8-cell stage.
AC expression in unfertilized oocytes
Since AC appeared to be required for the earliest stages of embryo development, we hypothesized that the enzyme must be provided to newly formed embryos by the donor egg prior to EGA in order for these embryos to survive. To examine this aspect of our hypothesis, cell extracts were prepared from 400 pooled, unfertilized MII eggs (collected 16 h after hCG injection), and analyzed by Western blot to identify the AC protein. As can be seen in Fig. 3
A, the AC precursor (55 kDa) and ß-subunit (40 kDa) are expressed in the egg before fertilization. The presence of the processed ß-subunit indicated that some of the AC was likely to be active. Cell extracts were therefore prepared from an additional 65 pooled, unfertilized eggs and subjected to AC activity assays. These analyses revealed a high enzymatic activity (t test, P<0.005, Fig. 3A
), confirming the Western blotting results.
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To obtain information about the subcellular location of AC in eggs, we next preformed immunohistochemistry using anti-AC specific antibodies combined with anti-LAMP1 staining for late endosome/lysosome detection. The fluorescence distribution of the AC and LAMP-1 signals was visualized at the equator and cortex of the egg and photographed with a Ziess confocal laser-scanning microscope (Fig. 3B, E
). These studies revealed that AC is localized mainly at the egg cortex (Fig. 3B
and 3B'), and colocalized with LAMP-1 in the late endosomes/lysosomes (Fig. 3C
, 3C’ and 3D, 3D’).
Normal embryos express AC at EGA
The death of AC-deficient embryos during the 2-cell stage implies that in normal embryos AC gene expression occurs as early as the EGA to sustain survival. To confirm this hypothesis, changes in AC mRNA levels in old, unfertilized MII eggs and 2-cell embryos (both collected 46 h after hCG injection) were assessed relative to the levels in young, unfertilized eggs (collected 16 h after hCG injection) using semiquantitative PCR. Housekeeping proteins, ß-actin (Actb), glyceraldehyde-3-phosphate dehydrogenase (G3), and ribosomal protein S11 (RPS 11) were used as internal controls for embryonic mRNA expression. As shown in Fig. 4
A, a significant decrease of AC mRNA was found in old vs. young unfertilized eggs (t test, P<0.0003). This would predictably result in ceramide increase and apoptotic cell death. On the other hand, in fertilized, healthy 2-cell embryos, AC mRNA levels were enhanced (t test, P<0.0005, Fig. 4A
), suggesting AC gene activation during EGA.
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Western blot analysis of 140 pooled, unfertilized eggs and 2-cell embryos, followed by densitometry, reinforced the PCR findings; i.e., the level of AC precursor was increased in 2-cell embryos as compared to unfertilized eggs, consistent with the mRNA findings (Fig. 4B
). As expected, the levels of Actb protein and mRNAwere decreased in the 2-cell embryo stage (Fig. 4A, B, D
, respectively), serving as a useful control. Ribosomal protein S11 (RPS 11), one of the first genes expressed immediately after fertilization, also was used as a control, marking the initiation of EGA. Glyceraldehyde-3-phosphate dehydrogenase (G3) represented a negative control (Fig. 4A
). Overall, the fact that AC was expressed in young eggs prior to fertilization and that these levels were decreased during the aging process, together with the fact that there was enhanced expression during EGA, highlights the importance of this enzyme for embryo survival.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 16)
. Other physiological changes in unfertilized eggs and early embryos, including Ca2+ oscillations, are also important components of this regulatory decision. On fertilization, young, healthy eggs must supply sufficient antiapoptotic proteins and mRNA to newly formed embryos to overcome the default apoptosis pathway. After, the newly formed embryo must supply these factors through expression of it’s own genome at EGA. In the mouse, EGA begins during the 2-cell stage (22)
, whereas in humans the major activation event occurs between the 4- and 8-cell stages (23)
. Although antiapoptotic factors should be among the genes/proteins expressed at EGA, surprisingly, very few such factors have been identified to date.
Consistent with prior evidence showing that increased ceramide levels in aging eggs leads to apoptosis (15
, 16)
, it is reasonable to hypothesize that AC, an enzyme responsible for the hydrolysis of ceramide and production of sphingosine (the precursor of S1P), might be an essential factor required for embryo survival. We also hypothesized that in the absence of AC activity, ceramide levels in 2-cell Asah1–/– embryos increase, leading to apoptosis. This hypothesis was strongly supported by data showing that S1P, a ceramide antagonist, led to rescue of Asah1–/– embryos and permitted their survival to the 4- to 8-cell stage. We also attempted to directly determine ceramide levels in individual embryos by immunocytochemistry. However, such analysis requires fixation of the embryos, which precludes subsequent PCR genotyping due to poor yield and quality of the extracted DNA. Even under optimal conditions, reliable, PCR genotyping of 2- to 8-cell embryos is extremely difficult. In combination with fixation, this analysis was not feasible.
Nonetheless, our studies clearly show that embryo-derived AC is one of the first proteins expressed during the 2-cell stage of development and that its activity is necessary for the subsequent expression of the normal developmental program. In the absence of this activity, embryos undergo apoptotic death. The fact that S1P permits survival of Asah1–/– embryos beyond the 2-cell stage also strongly supports our hypothesis that the apoptotic death is due to the accumulation of ceramide.
It is also important to point out that AC activity is not only essential during embryonic development, but during postnatal life as well. In humans, reduced AC activity leads to the lipid storage disease, FD. Farber disease is an extremely rare and fatal lipid storage disorder, and at least two cases of fetal death have been reported (24
, 25)
. Mutation analysis carried out on surviving FD patients has shown that subtle point mutations account for most of the abnormalities, rather than large gene deletions, rearrangements, or frameshift mutations that are likely to cause complete loss of function. Indeed, even these subtle point mutations often lead to a severe clinical condition (26)
, providing further evidence that AC activity is essential for normal postnatal development. Herein we demonstrate that mice homozygous for the complete loss-of-function Asah1 allele undergo apoptotic death at the 2-cell stage. These findings predict that complete loss-of-function mutations in FD individuals would lead to early embryonic lethality, and are consistent with the fact that only patients with subtle point mutations survive.
Historically, AC was classified as a "lysosomal enzyme" because of the appearance of lipid storage vacuoles in FD patients that were reminiscent of lysosomes, as well as the enhanced in vitro activity at acidic pH. Our study documents for the first time the subcellular location of AC in unfertilized eggs and shows the presence of this protein both inside and outside of lysosomes. Although several reports have suggested that ceramide produced in lysosomes does not participate in cell signaling (e.g., 27), it is important to recognize that AC may contribute to the hydrolysis of nonlysosomal, as well as intralysosomal, ceramide pools. In fact, the related lipid hydrolase, acid sphingomyelinase, can hydrolyze sphingomyelin in both lysosomal and nonlysosomal compartments and rapidly relocates to the cell surface following various stimuli (27)
. In the future it will be important to evaluate changes in the trafficking of AC following fertilization, as well as during egg and embryo development.
Finally, several practical implications of the work reported here deserve mention. First, the development of a single-cell, PCR genotyping method for AC could potentially facilitate preimplantation diagnosis of FD embryos for at-risk couples. While this method would have to be adapted from mice to humans, since the genes are highly conserved, this should not be problematic. Furthermore, based on our findings, physicians could potentially use AC to prolong egg/embryo survival during IVF procedures, facilitating the identification and selection of healthy embryos for reimplantation, especially for older women. In conclusion, these data reveal a new and important role for AC in the earliest stages of mammalian embryogenesis and suggest that this enzyme and/or gene may be used to facilitate egg/embryo survival in vitro and/or in vivo.
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ACKNOWLEDGMENTS |
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Received for publication August 23, 2006. Accepted for publication December 14, 2006.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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